EA046161B1 - VACCINES BASED ON PEPTIDES, METHODS OF THEIR PRODUCTION AND APPLICATION FOR INDUCING AN IMMUNE RESPONSE - Google Patents

Description

This application claims priority to 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 of which are entitled as follows. : Peptide-based vaccines, methods of their manufacture and use for inducing an immune response, the contents of which are incorporated herein by reference in their entirety.

The present invention was made pursuant to a Research Cooperative Agreement with the National Institutes of Health, an agency of the Department of Health and Human Services. The Government of the United States of America has certain rights to this invention.

Field of technology to which the invention relates

The present invention relates to novel peptide vaccines, methods for making novel peptide vaccines, and their use to induce an immune response, and in particular a T cell response, in a subject. Embodiments of the peptide vaccines of the present invention can be used to prevent or treat infectious diseases and cancer, as well as to induce tolerance or modulate immunity to prevent or treat autoimmune diseases and allergic reactions.

Prerequisites for creating the invention

Vaccines containing immunogenic antigen compositions can be used to induce an immune response in a subject, including for the treatment or prevention of cancer or infectious diseases, or even to induce tolerance and/or immune suppression for the treatment or prevention of autoimmune diseases or allergic reactions. Vaccine compositions for inducing immune responses for the treatment or prevention of cancer and infectious diseases contain antigens and specific types of adjuvants that induce cytotoxic T cell responses and/or antibodies directed to the antigen that mediate the clearance of pathogens or the destruction of virus-infected cells or cancer cells. However, vaccine compositions for inducing tolerogenic or suppressive responses may contain an antigen and a carrier (eg, a delivery system such as a particle carrier) and/or immunosuppressive compounds such as mTOR inhibitors, but will lack specific adjuvants that induce cytotoxic T cells, but instead, they may induce T cell tolerance or activation of regulatory cells, such as regulatory T cells, which suppress or modulate the qualitative characteristics of the response.

There is growing recognition that a patient's genetic background, in particular the composition of major histocompatibility complex (MHC) alleles, and a patient's environment can unfavorably influence their susceptibility to cancer, infectious diseases, autoimmune diseases and allergic reactions, as well as influence their response to vaccines used to treat such health conditions. The continuous development of new diagnostic tools and informatics that offer insight into the molecular basis of disease has resulted in patient-specific information becoming increasingly available. Thus, there is increasing interest in using information about an individual's genes, proteins, and environment to guide the individual's treatment process, including the selection of specific immunotherapeutic mechanisms, including vaccines for the treatment or prevention of cancer, infectious diseases, autoimmune diseases, and allergic reactions.

In cancer therapy, specific information about an individual's tumor can be used to assist in diagnosis, treatment planning, to determine the effectiveness of treatment, or to provide prognosis for the disease. For example, specific information about an individual's genes, proteins, and environment can be used to develop a personalized preventive approach to cancer treatment by developing vaccines based on such information. Vaccines used to treat immunological diseases or to prevent certain cancers must induce an immune response to tumor-associated antigens. One preferred immune response is a CD8 T cell response and/or a CD4 T cell response that recognizes a tumor-associated antigen.

Likewise, individualized approaches to the treatment of autoimmune diseases and allergic reactions are possible through the identification of specific self-antigens or foreign antigens, respectively, that are the cause of immune-mediated pathology. Although some autoantigens and allergens are known and common in patients, some autoantigens and allergens may be patient specific and therefore treatment for these patients must be individualized. Antigens identified as the cause of the pathology can be administered in peptide form as a vaccine that is capable of inducing tolerance to autoantigens (i.e., self-antigens). In an alternative embodiment of the present invention, the immune response to a foreign antigen that causes allergic reactions may be a particular type of immune response that leads to pathology. Thus, a vaccine against such a foreign antigen can be proposed as a vaccine based on

- 1 046161 ve peptides to shift the immune response to a qualitatively different type of immune response that does not lead to pathology.

Role of T cells in treating or preventing cancer

T cells are known to mediate tumor regression and improve patient survival, as has been shown through adoptive T cell therapy and immune checkpoint inhibitor-based immunotherapeutic mechanisms. Some 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). Additionally, treating certain cancers with drugs that block or reverse suppression of T cells, such as monoclonal antibodies that block CTLA-4, PD-1 and/or PD-L1 (called checkpoint inhibitors), results in tumor regression and improved overall survival (see: D.M. Pardoll, Nat Rev Cancer, 12: 252-264, 2012; F.S. Hody et al., N Engl J Med, 363:711-723, 2010; and M Reck et al., N Engl J Med, 375: 1823-1833, 2016; JE Rosenberg et al., Lancet 387: 1909-1920, 2016). PD-1/PD-L1 is a receptor-ligand pair, with PD-1 expressed on T cells and PD-L1 expressed on cells in the tumor. The PD-1/PD-L1 interaction blocks T cells from performing their effector functions, which would otherwise lead to slower growth or elimination of tumor cells expressing the tumor-associated antigen recognized by the T cell. Blocking the interaction of PD-1/PD-L1 with a monoclonal antibody results in the termination of the suppressive signal, thereby freeing T cells to perform effector function, resulting in slower growth of tumor cells or death of tumor cells (D. M. Pardoll, Nat Rev Cancer, 12 : 252-264, 2012).

The role of T cells in promoting tumor clearance in patients is supported by human studies showing that checkpoint inhibitors have higher efficacy (lower mortality) in patients with higher levels of T cell infiltration present in their tumor biopsy at the time of initiation treatment (see: J.E. Rosenberg et al., Lancet 387: 1909-1920, 2016). In addition, checkpoint inhibitors have also been shown to have higher efficacy in patients whose tumors have a higher mutation rate (see: A. Snyder et al., N Engl J Med 371, 2189-2199 2014 and N.A. Rizvi et al., Science, 348: 124-128, 2015) presumably because an increase in the number of mutations leads to an increase in the number of mutant proteins, thereby providing a greater number of potential targets for T cells to recognize and target tumor cells for elimination.

However, the main problem is that many common tumors (for example, prostate, breast and pancreatic cancer) have a low mutational load (see: T. N. Schumacher, R. D. Schreiber, Science, 348: 69- 74, 2015), and patients with these cancers receive little or no benefit from checkpoint inhibitors.

Moreover, even among tumors that have a high mutational load, only a subset of patients benefit from checkpoint inhibitor therapy because most patients lack the necessary precursor tumor-specific T-cell response that immunotherapeutic mechanisms require to mediate effect.

Vaccines targeting tumor-associated antigens for the treatment or prevention of cancer

Generating immune responses, in particular T cell responses, for the treatment or prevention of cancer requires the identification of suitable tumor-associated antigens. Tumor-associated antigens include self-antigens that are present on certain healthy cells but are predominantly expressed by tumor cells; pathogen-mediated antigens derived from oncogenic microbial pathogens; and/or neoantigens, which are aberrant proteins that are specific to tumor cells and are often, but not always, unique to individual patients.

Suitable self-antigens may be antigens derived from proteins that are no longer expressed in subjects who are in a postnatal state. For example, suitable self-antigens may include antigens that are predominantly expressed by tumor cells, such as NY-ESO-1 and Mage-Az (see: N. N. Hunder et al., N Engl J Med, 358:26982703, 2008 ). In an alternative embodiment of the present invention, self-antigens may include antigens that are expressed in healthy tissue where the T cell response to that healthy tissue is not unduly harmful to the patient, for example, prostatic acid phosphatase (PAP) (see: P V. Kantoff et al., N Engl J Med 363:411-422, 2010).

The tumor-associated antigen may be a pathogen-mediated antigen, such as a virus or other pathogen that is a major driver of the neoplastic process, such as proteins from human papillomavirus (HPV) (see: G. G. Renter et al., N Engl J Med, 361: 1838-2 046161

1847, 2009).

Neoantigens are antigens resulting from mutations that are present only in tumor cells and not in healthy cells (see: T. N. Schumacher, R. D. Schreiber, Science, 348: 69-74, 2015). Neoantigens can be created by nucleotide polymorphisms that result in changes in non-conserved amino acids. Neoantigens can be created through insertions and/or deletions, which can result in peptide antigens containing an insertion or deletion, or a frameshift mutation. Neoantigens can be created by introducing a stop codon that, in its new context, is not recognized by the stop codon complex, causing the ribosome to skip the codon and generate a peptide containing a single amino acid deletion. Neoantigens can be created through mutations at splice sites, resulting in incorrectly spliced mRNA transcripts. Neoantigens can be created by inversions and/or chromosomal translocations that result in peptide fusions.

Finally, a tumor-associated antigen may contain novel post-translational modifications of non-mutant or mutant peptides, while post-translational modifications are not present in healthy tissue.

Thus, a tumor-associated antigen is any antigen produced by a tumor cell that, when targeted by a T cell response, ideally results in significant regression of tumor growth or prevents tumor formation with limited effect on healthy, non-cancerous cells.

Challenges in the field of vaccination

For the treatment of certain cancers, the preferred immune response is the CD8 T-cell response (see E. Tran et al., N Engl J Med 375:2255-2262, 2016) and/or the CD4 T-cell response (and see : E. Tran et al., Science 344: 641-645, 2014; and N. N. Hunder et al., N Engl J Med, 358:2698-2703, 2008), which recognizes tumor-associated antigen, which may preferentially be sustainable, high intensity and high functional (high quality) (L. Gattinoni et al., Nature Medicine, 17:1290-1297, 2011).

One mechanism for inducing a T cell response to antigens, such as tumor-associated antigens, in a subject is vaccination. A variety of approaches are currently being explored to induce CD8 and/or CD4 T cell responses to tumor-associated antigens, including DNA/RNA, viral vector, and peptide-based approaches (e.g., see: L. M. Kranz et al., Nature 534 , 396-401, 2016; and S. J. Melieff, S. H. van der Burg, Nat Rev Cancer 8:351-360, 2008). However, a major challenge in generating effective T-cell immunity against cancer is that most current vaccine approaches are limited by poor immunogenicity for generating CD 8 T-cell immunity and have a limited breadth of antigenic responses to most predicted neoantigens (see: C Kreuter et al., Nature 520, 692-696, 2015).

Enormous efforts aimed at inducing antitumor T-cell immunity have focused either on the use of synthetic peptide antigens, simply mixed with various immunostimulants, and/or carriers (adjuvants) (see: S.J. Melieff, S.H. van der Burg, Nat Rev Cancer 8:351-360, 2008; and M. S. Bijker et al., Eur J Immunol 38, 1033-1042, 2008) or RNA (see: L. M. Kranz et al. Nature 534 (7607):396-401, 2016 and Kreuter S, et al. Nature 520(7549):692-696, 2015).

However, a major challenge in generating effective T cell immunity against cancer is the fact that most current vaccine approaches based on peptide antigens or RNA have been hampered by the low intensity and limited antigenic breadth of responses to tumor-associated antigens, including neoantigens. For example, regarding the breadth of the antigenic spectrum, current conventional standard peptide vaccine approaches based on peptide antigens (e.g., mixing synthetic long peptides of 25 amino acids with a poly IC:LC adjuvant) or RNA induce T cell responses in less than 10% of predicted neoantigens (see: S. Kreuter et al., Nature 520, 692-696, 2015). Thus, improved vaccine approaches are needed, in particular peptide-based vaccine approaches.

A full explanation of the low intensity and narrow antigenic breadth of T cell responses by current vaccine technologies remains to be seen, and thus a continuing problem is that there is currently no consensus on optimal peptide delivery parameters. antigens to ensure reliable priming of T-cell immunity for the treatment and prevention of cancer, as well as for other applications, including the treatment and prevention of infectious diseases. Likewise, the optimal parameters of peptide-based vaccines to induce suppression and tolerance remain unknown.

Current issues regarding peptide-based T-cell vaccines

A major challenge in developing vaccines to treat or prevent infectious diseases and cancer is that there is currently no consensus on

- 3 046161 information on how best to design a peptide-based vaccine to produce highly intense T-cell immunity against most antigens. Likewise, there is no consensus on how best to design a peptide-based vaccine to induce immune tolerance or to shift the immune response toward a harmless response away from those that cause allergic reactions. For example, there is still considerable debate regarding the optimal length of peptide antigen, the physical format of peptide antigen delivery (eg, soluble versus particulate), and the type of natural immune stimulation (eg, choice of adjuvant) required to induce optimal T cell response. immunity for the treatment or prevention of cancer and infectious diseases, as well as for the treatment or prevention of autoimmune diseases and allergic reactions. Indeed, the influence of many parameters of peptide-based vaccines, including the influence of amino acids flanking CD4 and CD8 T-cell epitopes, used linker chemistries, charge, and others on immune responses remains largely unexplored. This problem is particularly acute when considering that individualized vaccine approaches must be developed as unique to each patient and, therefore, universal peptide-based vaccine approaches are needed to reliably produce immunity, particularly T-cell immunity, against patient-specific antigens.

Another major challenge facing current peptide vaccine approaches is that they do not take into account the wide range of possible physical and chemical characteristics of peptide antigens. For example, personalized approaches to cancer vaccines will require that a unique set of peptide antigens be generated for each patient, which may have a wide range of possible physical and chemical characteristics. With regard to autoimmune diseases, several different antigens can be identified as the cause of the pathology. Thus, tolerance-inducing vaccines must contain a set of peptide antigens that are unique to each patient. The problem is that the variability of the peptide antigen composition can lead to the formation of several peptide-based antigens that are difficult or impossible to produce as a native peptide antigen, which is estimated to constitute approximately 10-30% of peptide-based antigens in length. at least 25 amino acids. Thus, many antigens cannot be targeted using current vaccine approaches because the native peptide antigen cannot be efficiently produced or isolated once synthesized. In addition, variability in the peptide antigen composition, affecting charge and solubility, which is not controlled in current peptide-based vaccine approaches, may affect various properties of the vaccine composition, including loading of peptide materials, and/or lead to adverse interactions of the peptide antigen with either other peptide antigens, or with other vaccine components.

Thus, to overcome the limitations of current approaches to peptide-based vaccines, new compositions and methods for preparing peptide-based vaccines are needed that (i) take into account the variability in the physical and chemical properties of peptide antigens during the manufacturing process, as well as the variability in vaccine composition for providing optimal delivery of peptide antigens to (ii) reliably induce an immune response, and in particular a T cell response in a subject, to most antigens. Such vaccines and methods that take into account the variability of the peptide antigen and therefore can be generalized to any peptide antigen would be particularly useful in the field of personalized cancer treatment, however, the characteristics of the peptide antigens used in a personalized cancer vaccine may differ in each individual patient. However, the utility of peptide vaccine compositions for any peptide antigen means that the compositions may also be used in other individualized immunological therapies, such as those used to induce tolerance or to modulate the immune response to allergens for the treatment of autoimmune diseases and allergic reactions. respectively.

Summary of the invention

The inventors of the present invention have developed new compositions and methods for making peptide-based vaccines that overcome at least one of the limitations of current approaches to peptide-based vaccines. The novel peptide vaccine compositions and methods for their preparation disclosed herein take into account the variability in the physical and chemical properties of peptide antigens and therefore can be generalized to any peptide antigen. In addition, the novel peptide vaccine compositions disclosed herein provide optimal delivery of a variety of different peptide antigens to induce an immune response in a subject.

The new compositions disclosed herein relate to immunogenic compositions containing peptide antigen conjugates. Peptide antigen conjugates contain a peptide antigen (A) that is linked either to a hydrophobic molecule (H) that forms the particles or to a preformed particle (P), either directly or indirectly through

- 4 046161 optional Linker (L) and/or optional N- or C-terminal extension (B1 or B2) that is linked to either the N- or C-terminus of the peptide antigen, respectively.

In some embodiments of the present invention, particles formed by peptide antigen conjugates further contain an optional charged molecule (C). In some embodiments of the present invention, the charged molecule is linked to a peptide antigen conjugate to stabilize the particles under aqueous conditions. In other embodiments of the present invention, the charged molecule (C) is provided on a separate molecule and included in particles formed by peptide antigen conjugates.

The addition of specific N- and/or C-terminal extensions (B1 and/or B2) and/or charged molecules (C) leads to unexpected improvements in the production of peptide-based vaccines by solid-phase synthesis, as well as more convenient purification due to improved solubility in an organic solvent, as well as unexpected improvements in controlling the size and stability of particles formed by peptide antigen conjugates. These compositions demonstrate unexpected improvements in T cell immunity and tumor clearance, particularly in the intensity and breadth of T cells generated against tumor-associated antigens.

Embodiments of the present invention include a peptide antigen conjugate comprising: peptide antigen (A); and either a hydrophobic molecule (H) or a particle (P), wherein the peptide antigen (A) is linked to either the hydrophobic molecule (H) or the particle (P), directly or indirectly, through an N-terminal extension (B1) , which is linked to the N-terminus of the peptide antigen (A), or by a C-terminal extension (B2), which is linked to the C-terminus of the peptide antigen (A). Additional embodiments of the present invention include an immunogenic composition containing peptide antigen conjugates. Other embodiments of the present invention include a method of treating a patient suffering from a disease, comprising administering to the patient suffering from the disease a peptide antigen conjugate or immunogenic composition.

In some embodiments of the present invention, a vaccine used to treat cancer may be used alone or in combination with other immunotherapy and cancer treatment methods, including, but not limited to, chemotherapy, checkpoint inhibitors, radiation therapy, and any other technology used to treat cancer. fight cancer.

Thus, also disclosed herein is a method of treating a patient suffering from a disease, comprising administering to the patient suffering from the disease a peptide antigen conjugate or immunogenic composition of the present invention.

Further disclosed herein is the use of a peptide antigen conjugate or immunogenic composition of the present invention in the manufacture of a medicament for treating a disease.

Unexpected results 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 sequence extensions, that is, N- or C-terminal extensions (B1 and/or B2) and/or optionally charged molecules (C), which facilitate the manufacture of peptide-based vaccines;

(iii) how the characteristics of the hydrophobic molecule (H) containing the peptide antigen conjugates affect processability, as well as particle size and stability, and how these parameters affect biological activity;

(iv) the composition of charged molecules (C) and the net charge of peptide antigen conjugates necessary to induce and stabilize particles of optimal size to stimulate T-cell immunity;

(v) a composition of enzymatically degradable peptide sequences that promotes efficient processing of minimal epitopes delivered in the context of a peptide antigen conjugate; and/or (vi) how the activity and quality characteristics of ligands with adjuvant properties (eg, PRR agonists) delivered on peptide antigen conjugates affect the breadth, intensity and quality of T cell responses.

Brief description of drawings

Fig. 1: Drawing diagram of certain embodiments of peptide antigen conjugates;

fig. 2: 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), which links the peptide antigen (A) to a hydrophobic molecule (H);

fig. 3: synthesis scheme of Compound 1;

fig. 4: molecular weight and hydrodynamic behavior of peptide antigens (A), and peptide

- 5 046161 antigen conjugates delivering peptide antigens (A) consisting of either synthetic long peptides (SLP or LP) or minimal (Min) CD8 T cell epitopes derived from the MC38 tumor cell line. Peptide antigens (A) consisting of LP were linked to the X1 linker precursor, azido-lysine (K'), which either remained unlinked or was linked to a hydrophobic molecule (H) consisting of either DBCO-W3 or DBCO -2BXy ₃ . Peptide antigens (A) consisting of LP were linked to an extension (B1) that was linked to the X1 linker precursor, azido-lysine (K'), which either remained unlinked or was linked to a hydrophobic molecule (H) consisting either from DBCO-W3 or from DBCO-2ВХу ₃ ;

fig. 5: Effect of peptide antigen (A) length, hydrodynamic behavior, and co-delivery of TLR-7/8 agonist (TLR-7/8a) on the immunogenicity of peptide-based neoantigen (Irgq). Mice (N=5 per group) were immunized with various immunogenic compositions containing peptide antigens (A) based on either the synthetic long peptide (LP, sometimes referred to as SLP) or the minimal CD8 T cell epitope (Min, sometimes referred to as ME) Irgq antigen, obtained from the MC38 tumor cell line, or containing only peptide antigen (A) added to a mixture with the TLR-7/8a adjuvant; containing a peptide antigen (A) linked to a hydrophobic molecule (H), W3 and added to a mixture with a TLR-7/8a adjuvant; or containing a peptide antigen (A) linked to a hydrophobic molecule (H) co-delivering the TLR-7/8a adjuvant, 2BXy ₃ . Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (%IFNg+ of total CD8 T cells) were assessed relative to whole blood on days 0 and 25. Open circles indicate that peptide antigen (A) was soluble (that yes, not particulate), and the filled circles indicate that the mice received the immunogenic particulate peptide antigen (A) composition;

fig. 6: Effect of peptide antigen length (A), hydrodynamic behavior, and TLR-7/8a adjuvant co-delivery on the immunogenicity of a peptide-based neoantigen (Cpne1). Mice (N=5 per group) were immunized with various immunogenic compositions containing peptide antigens (A) based on either a synthetic long peptide (LP), the minimal CD8 T cell epitope (Min) antigen Cpne1, derived from the MC38 tumor cell line, or containing only peptide antigen (A) added to the mixture with the adjuvant IbK-7/8a; containing a peptide antigen (A) linked to a hydrophobic molecule (H), W ₃ and added to a mixture with a TLR7/8a adjuvant; or containing a peptide antigen (A) linked to a hydrophobic molecule (H) co-delivering the TLR-7/8a adjuvant, 2BXuz. Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (%IFNg+ of total CD8 T cells) were assessed relative to whole blood on days 0 and 25. Open circles indicate that peptide antigen (A) was soluble (that yes, not particulate), and the filled circles indicate that the mice received the immunogenic particulate peptide antigen (A) composition;

fig. 7: molecular weight and hydrodynamic behavior of peptide antigens (A), and peptide antigen conjugates delivering peptide antigens (A), consisting of either synthetic long peptides (SLP or LP) or minimal (Min) CD8 T cell epitopes derived from the B16 tumor cell line. Peptide antigens (A) consisting of LP were linked to the X1 linker precursor, azido-lysine (K'), which either remained unlinked or was linked to a hydrophobic molecule (H) consisting of either DBCO-W3 or DBCO -VKhUZ. Peptide antigens (A) consisting of LP were linked to an extension (B1) that was linked to the X1 linker precursor, azido-lysine (K'), which either remained unlinked or was linked to a hydrophobic molecule (H) consisting either from DBCO-W3 or DBCO-2BXy ₃ ;

fig. 8: Effect of peptide antigen length (A) and TLR-7/8a activity coupled to a hydrophobic molecule (H) on the immunogenicity of peptide antigen conjugates. Mice (N=5 per group) were immunized with various immunogenic compositions containing peptide antigen conjugates delivering either synthetic long peptide (LP), minimal CD8 T cell epitope (Min), or both SLP and Min peptide antigens (A), obtained from the B16 tumor cell line linked to either the hydrophobic molecule (H) DBCO-2BXy ₅ or DBCO-2B5. Mice were immunized on days 0 and 14, and antigen-specific CD4 and CD8 T cell responses (% IFNg+ total) were assessed relative to whole blood on day 24. Histograms show the sum of the mean responses for each peptide antigen (M47, M44, M30, M25 , M33, M27 and M08);

fig. 9: Impact of hydrophobic molecule (H)-linked TLR-7/8a activity on the immunogenicity and breadth of T cell responses generated against a library of peptide neoantigens. Mice (N=5 per group) were immunized with immunogenic compositions containing 4 unique peptide antigen conjugates delivering detectable minimal CD8 T cell epitopes derived from the MC38 tumor cell line (total number of epitopes - 40), which were linked to either a hydrophobic molecule ( H) 2BXy5, or 2B5. Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (% IFNg+ of total CD8 T cells) were assessed relative to whole blood on day 24. CD8 T cell responses to each of the 40 epitopes are shown. Proportion of peptide antigens (A) containing minimal epitopes of CD8 T cells that led to

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CD8 T cell responses, shown in pie chart;

fig. 10: Effect of the number and dose of peptide antigens (A), as well as the number of vaccination sites on the immunogenicity of peptide-based neoantigens. Mice (N=5 per group) were immunized with either peptide antigen conjugates containing a single minimal CD8 T cell epitope (Adpgk) or peptide antigen conjugates containing 4 other minimal CD8 T cell epitopes (Dpagt, Cpne1, Adpgk and Aatf). The dose of peptide antigen (A) and the number of vaccination sites differed between groups. Mice were immunized on days 0 and 14, and antigen-specific CD8 T cell responses (% IFNg+ of total CD8 T cells) were assessed relative to whole blood on day 24;

fig. 11: diagram of a peptide antigen conjugate of formula V;

fig. 12: Effect of N- and C-terminal extensions (B1 and B2) on in vitro activity to activate CD8 T cells. Cultures of splenocytes with APC and neoantigen (Cpne1)-specific CD8 T cells were stimulated in vitro with peptide antigens (A) with a minimal CD8 T cell epitope (Cpne1) linked to various extensions (B1 and B2) and assessed for the ability to stimulate the production of IFNg Cpne1-specific CD8 T cells;

fig. 13: Peptide antigen conjugates evaluated in FIG. 12. Peptide antigen (A), consisting of the minimal epitope Cpne1 (Ser-Ser-Pro-Tyr-Leu-His-Tyr-Leu SEQ ID NO: 1), was linked to any or both of the B1 and /or B2 containing cathepsin, and/or with an immunoproteasomal cleavage site, wherein, additionally, the peptide antigen (A) was linked either directly or by extension of B2 to the X1 linker precursor, azido-lysine (K'), which was linked with a DBCO molecule that was linked to a hydrophobic molecule (H), 2ВХУз;

fig. 14: Effect of charged molecule (C) and extensions (B1 and/or B2) on the in vivo immunogenicity of peptide antigen conjugates delivering a peptide antigen (A) consisting of a minimal CD8 T cell epitope. Mice (N=5 per group) were immunized with peptide antigen conjugates containing various charged molecules (C) and extension(s) (B1 and/or B2) on days 0 and 14, and antigen-specific CD8 T cell responses (%IFNg+ of total CD8 T cell counts) were assessed relative to whole blood at day 10 (upper panel) and day 24 (lower panel);

fig. 15: Effect of N- and C-terminal extensions (B1 and B2) on in vitro activity to activate CD8 T cells. The minimal CD8 T cell epitope (Adpgk) of peptide antigen (A) was linked to various extensions (B1 and B2) and assessed for the ability to stimulate IFNg production from Adpgk-specific CD8 T cells;

fig. 16: Effective concentration at half maximum activity (EC50) is shown for the various peptide antigen conjugates evaluated in FIG. 15. Lower EC50 indicates higher activity;

fig. 17: Effect of N- and C-terminal extensions (B1 and B2) on in vitro activity to activate CD8 T cells and on in vivo activity to generate new CD8 T cell responses. (A) The minimal CD8 T cell epitope (Adpgk) of the peptide antigen (A) was linked to various extensions (B1 and B2) and assessed for the ability to stimulate IFNg production from Adpgk-specific CD8 T cells (B). The ability of peptide antigen conjugates with various N- and C-terminal extensions (B1 and B2) linked to a hydrophobic molecule (H) 2B ₃ W ₂ was assessed on day 13 after a single immunization;

fig. 18: Effect of net charge of peptide antigen conjugate and hydrophobic molecule composition on hydrodynamic behavior. The hydrodynamic behavior of a number of different peptide antigen conjugates of Formula V at a concentration of 0.1 or 0.5 mg/ml in PBS at pH 7.4 was assessed by dynamic light scattering. The total charge of the peptide antigen conjugates and the number or mass average diameter of the particles are noted. Turbidity was determined by measuring the absorbance of the solution at 490 nm. Turbidity > 0.04 indicates aggregation;

fig. 19: Effect of net charge of peptide antigen conjugate on hydrodynamic behavior. (A) The effect of net charge on the hydrodynamic behavior of a range of different Formula V peptide antigen conjugates suspended at 0.1 or 0.5 mg/ml in PBS at pH 7.4 was assessed by dynamic light scattering. (B) Panel B of the figure shows the current list of data shown in panel (A) of the figure. (C) Frequency distribution of GRAVY and (D) charge frequency distribution of antigens delivered as peptide antigen conjugates. (E) Particle size of corresponding LP neoantigens (n=39) prepared as Formula V peptide antigen conjugates that self-assemble into nanoparticles co-delivering TLR-7/8a (SNP-7/8a) with a net charge of either +6 or > +8. (F) Effect of net charge of peptide antigen conjugate and overall average hydrophobicity value (GRAVY) on hydrodynamic behavior. The hydrodynamic behavior of a range of different peptide antigen conjugates suspended at a concentration of 0.1 mg/ml in PBS at pH 7.4 was assessed by dynamic light scattering. The number-average particle diameter is reported as a function of the total charge and the value

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GRAVY;

fig. 20: Effect of net peptide antigen conjugate charge and hydrophobic molecule length (H), and composition on the hydrodynamic behavior of peptide antigen conjugates of Formula V. Hydrodynamic behavior of various peptide antigen conjugates suspended at a concentration of 0.1 mg/ml in PBS at pH 7, 4, with varying net charge and compositions with a hydrophobic molecule delivering a neoantigen-based minimal epitope (Min) model, AlaSer-Met-Thr-Asn-Met-Glu-Leu-Met-Ser-Ser (SEQ ID NO: 2) ( Adpgk), assessed by dynamic light scattering. (A) The number-average particle diameter is plotted as a function of the net charge of the peptide antigen conjugate for 4 different hydrophobic molecules (2B ₂ W8, 2BXy5, 2B5, W ₅ ). (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 conjugated peptide antigen delivered by the same peptide antigen (A) but with total charge 0, which is collected into microparticles/aggregates (Min-MP-7/8a);

fig. 21: Effect of net charge on the hydrodynamic behavior of a Formula V peptide antigen conjugate delivering a hydrophobic peptide antigen. (A) Overall average of the hydrophobicity distribution plot (GRAVY) of 1,377 LP neoantigens among 4 mouse tumor models (B16.F10, MC38, 3123, and Panc02). (B) The most hydrophobic LP neoantigen identified in panel (A) of the figure (red filled circle) was prepared as LP and Min delivered as a Formula V peptide antigen conjugate that self-assembles into TLR-7/8a co-delivering nanoparticles (SNP -7/8a) and assessed for the effect of net charge (absolute value) on particle size. Particle size was assessed by dynamic light scattering using a peptide antigen conjugate suspended at a concentration of 0.5 mg/ml in PBS at pH 7.4. The mass average particle diameter is reported as a function of the net charge of the peptide antigen conjugate;

fig. 22: Effect of composition with a charged molecule (C or C-block) and a hydrophobic molecule (H or H-block) on immunogenicity to generate CD8 T cell responses to a neoantigen minimal epitope, Adpgk, delivered as a peptide antigen conjugate of Formula V. ( AE) Peptide antigen conjugates with a composition containing molecules with a changing charge (either based on poly(K), poly(C), poly(O), or in their absence) and with a composition with a hydrophobic molecule (or based on W ₅ , 2BXy ₃ W2, either based on 2B3W2) delivering the minimal epitope, Adpgk, was administered to mice (N=3 per group per dose) at various doses on days 0, 14 and 28, and neoantigen (Adpgk)-specific CD8 T cells were assessed responses regarding whole blood at successive time points;

fig. 23: Effect of a charged molecule (C or C-block) composition on immunogenicity to generate CD8 T cell responses to a neoantigen minimal epitope, Adpgk, delivered as a peptide antigen conjugate of Formula V. (A) Peptide antigen conjugates with a C-block on based on either poly(K), poly^) or PEG (polyethylene glycol), delivering the minimal epitope, Adpgk, was administered to mice (N=3 per group per dose) at various doses on days 0, 14 and 28, and neoantigen (Adpgk) was assessed )-specific CD8 T cell responses relative to whole blood at day 36 (B);

fig. 24: Effect of route of administration on CD8 T cell responses generated by peptide antigen conjugates ('^No-7/8a'). Peptide antigenic conjugate with a charged molecule (C) poly(K) and with a hydrophobic molecule (H) 2B3W2, delivering a neoantigenic minimal epitope (Adpgk = Ala-Ser-Met-Thr-Asn-Met-Glu-Leu-Met (SEQ ID NO : 77)) were administered to mice on days 0, 14 and 28, and neoantigen(Adpgk)-specific CD8 T cell responses were assessed against whole blood on day 36;

fig. 25: Effect of hydrophobic molecule (H) composition and hydrodynamic behavior of the peptide antigen conjugate on CD4 and CD8 T cell responses. Mice (N=5 per group) were immunized with immunogenic formulations containing minimal CD8 T cell epitope and CD4 T cell epitope (PADRE epitope) either in the form of microparticles (MP) (>500 nm in diameter) or nanoparticle micelles (NP). (<200 nm in diameter) carrying varying amounts and activity 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 relative to whole blood on day 24;

fig. 26: drawn chemical diagram of peptide antigen conjugates with a charged molecule (C) of poly(K) and with a hydrophobic molecule (H) based on 2B3W2, delivering either LP or Min epitope forms of peptide antigens;

fig. 27: Particulate delivery of peptide neoantigens enhances CD8 T cell responses directed to neoantigen-based peptide antigen (A). (A) Turbidity of various neoantigens as native LPs, LPs linked to a hydrophobic molecule (2B3W2) that aggregate into microparticles (MP-7/8a), or as LPs synthesized as a Formula V peptide antigen conjugate that self-assembles into nanoparticles (SNP-7/8a). Turbidity > 0.05 OD indicates aggregate

- 8 046161 tion. (B-E) Mice were immunized with native LP supplemented with poly-ICLC, or LP delivered as MP-7/8a or SNP-7/8a on days 0 and 14, and CD8 T cell responses were assessed relative to whole blood on day 28. Data on a logarithmic scale are marked as geometric mean with color-coded 95% CI (confidence interval); Comparisons between multiple groups for statistical significance were made using one-way or two-way ANOVA (analysis of variance); ns = not significant; *, p = 0.05; **, p = 0.01;

fig. 28: Mechanism to account for the improved immunogenicity of peptide antigen conjugates that self-assemble into TLR-7/8a co-delivering nanoparticles (SNP-7/8a). (A-C) A minimal epitope, Adpgk, was fluorescently labeled and then injected subcutaneously into the hind footpads of mice on day 0 or as a soluble peptide added to a mixture with low molecular weight TLR-7/8a (7/8a), a soluble peptide added to a mixture with MP-7/8a, either covalently linked to MP-7/8a, or to SNP-7/8a. Lymph nodes (N=5 per time point per group) draining the immunization site were then collected at successive time points and assessed for peptide abundance (E), cell-based lymph node APC (antigen-presenting cell) peptide uptake (F), and production cytokine IL-12p40 (G). Data are marked with mean ± SEM (Standard Error of the Mean). Comparisons between multiple groups for statistical significance were performed using one-way or two-way ANOVA; ns = not significant; *, p = 0.05; **, p = 0.01;

fig. 29: Immunogenicity of peptide-based neoantigens delivered as either LP or Min as Formula V peptide antigen conjugates that self-assemble into TLR^^ co-delivering nanoparticles (SNP-7/8a). Various peptide-based neoantigens have been prepared as either LP or Min, delivered as peptide antigen conjugates, wherein the charged molecule (C) is poly(lysine) and the hydrophobic molecule (H) is 2B3W ₂ which self-assembles into nanoparticles co-delivering TLR-7/8a (referred to as SNP-7/8a). Mice (N=7 per group) were immunized with either the LP or Min form of SNP-7/8a-linked peptide neoantigens on days 0 and 14. (A) CD8 T cell responses and (B) CD4 T cell responses assessed relative to whole blood on day 28. Data on a logarithmic scale are marked as geometric mean with color-coded 95% CI (confidence interval); Comparisons between multiple groups for statistical significance were made using one-way or two-way ANOVA (analysis of variance); ns = not significant; *, p = 0.05; **, p = 0.01;

fig. 30: Immunogenicity of peptide-based neoantigens delivered as Formula V LP-based peptide antigen conjugates, wherein the charged molecule (C) is poly(lysine) and the hydrophobic molecule (H) is 2B ₃ W ₂ which self-assembles into nanoparticles co-delivering TLR-7/8a (referred to as SNP-7/8a) compared to native LPs added to the poly-ICLC mixture. Mice were immunized on days 0 and 14, and CD8 T cell responses were assessed against whole blood on day 28;

fig. 31: Relationship between immunogenicity and predicted binding affinity. (A) Immunogenicity of minimal epitopes (N=179) to generate CD8 T cell responses when delivered as peptide antigen conjugates, where the hydrophobic molecule (H) is polymer-TLR-7/8a, plotted against predicted binding affinity using the Immune Epitope Database (IEDB) consensus algorithm. (B) Number of correctly classified positive objects versus number of incorrectly classified negative objects (ROC curves) for sensitivity and specificity of different MHC-I binding prediction algorithms based on a consensus score cutoff of 0.5 or 500 nm as binder;

fig. 32: Self-antigens and neo-antigens delivered as peptide antigens (A) on Formula V peptide antigen conjugates that self-assemble into TLR-7/8a co-delivering nanoparticles (SNP-7/8a) produce mediated CD8 and CD4 T cells clearance of developed melanoma. Mice that developed B16.F10 tumors were treated with PD1 and/or with SNP-7/8a delivering Adpgk LP (CD8 T-cell epitope not present in B16.F10), self-antigen, Trp1, minimal CD8 T-cell epitope, or via a neoantigen whose M30 LP has a CD4 T cell epitope, on days 2, 9 and 16;

fig. 33: Neoantigens delivered as Formula V peptide antigen conjugates that self-assemble into TLR-7/8a co-delivering nanoparticles (SNP-7/8a) administered by the subcutaneous and intravenous routes generate sustained CD8 T cell-mediated clearance of established tumor. Mice that developed MC38 tumors were vaccinated with Adpgk LPSNP-7/8a either by the subcutaneous or intravenous route on days 9 and 16, and tumor volume was then assessed at successive time points;

fig. 34: Neoantigens delivered as Formula V peptide antigen conjugates that self-assemble into TLR-7/8a co-delivering nanoparticles (SNP-7/8a) generate CD8 T cell-mediated clearance of established melanoma. Mice that developed B16.F10 tumors were treated with PDL1 and either SNP-7/8a delivering Med12 (MC38 neoan

- 9 046161 antigen with CD8 T-cell epitope), self-antigen, Trp1 (B16 self-antigen with CD8 T-cell epitope), or via M39 (B16 neoantigen with CD8 T-cell epitope) on days 1, 8 and 15. (A) Tumor growth was monitored and (B) CD8 T cell responses were assessed at day 17;

fig. 35: Viral (HPV) antigens delivered as Formula V peptide antigen conjugates that self-assemble into nanoparticles co-delivering TLR-7/8a (SNP-7/8a) generate CD8 T cell-mediated rejection of HPV+ tumors. (A and B) Mice were immunized with various HPV-derived E6 and E7 peptide minimal epitopes (min), delivered as Formula V peptide antigen conjugates, wherein the charged molecule (C) is poly(lysine) and the hydrophobic molecule ( H) is a 2B ₃ W ₂ that self-assembles into nanoparticles co-delivering TLR-7/8a (referred to as SNP-7/8a) on days 0 and 14. (A) CD8 T cell responses were assessed on day 28, and mice were infected with an HPV+ cancer cell line (TC1), and (B) tumor volume was assessed at day 14 postinfection;

fig. 36: Particle size and stability of multi-antigen particles. (A) Neoantigenic LPs as peptide antigens (A) (A1-A9) with charge range (-6 to +6) and hydropathy (GRAVY -2 to +2) (B) were synthesized as peptide antigen conjugates of Formula V , wherein the charged molecule (C) is poly(K), B1 is VR, B2 is SPVZ, the linker precursor X1 is azido-lysine (X, also called K'), and the hydrophobic molecule (H) is is 2B ₃ W ₂ , which self-assembles into nanoparticles that co-deliver TLR7/8a (SNP-7/8a). Peptide antigen conjugates suspended at a concentration of 0.5 mg/ml in PBS at pH 7.4 and assessed for turbidity (OD 490 nm) and particle size (diameter, nm). (C) Multiantigen particles containing several different peptide antigen conjugates (A1-A9) were mixed together in various ratios in DMSO solution (scenarios 1-7) and then suspended at a concentration of 0.5 mg/ml in PBS at pH 7 ,4, and were assessed for turbidity (OD 490 nm) and particle size (diameter, nm). The table suggests the percentage of each peptide antigen conjugate containing multiantigen particles for each scenario;

fig. 37: shows T cell responses induced by Formula V peptide antigen conjugates delivering tumor-associated self, infectious disease (HIV) and foreign antigens;

fig. 38: Shows the particle size and biological activity of Formula V peptide antigen conjugates consisting of various charged molecules (C) or various hydrophobic molecules (H) based on poly(amino acids) of Formula II coupled with adjuvants of Formula III;

fig. 39: Shows the particle size and biological activity of Formula V peptide antigen conjugates consisting of various Formula II poly(amino acid) hydrophobic molecules (H) linked to various PRR agonists;

fig. 40: shows the particle size and biological activity of peptide antigen conjugates of Formula V, wherein the peptide antigen (A) is linked to a hydrophobic molecule (H) using various linker chemical compounds (amide ester and thioester);

fig. 41: Shows the particle size and biological activity of Formula V peptide antigen conjugates consisting of various hydrophobic molecules (H) based on fatty acids, lipids and cholesterol;

fig. 42: shows the particle size and biological activity of peptide antigen conjugates of Formula V, consisting of various hydrophobic molecules (H) based on diblock copolymers of type AB;

fig. 43: shows the particle size and stability of peptide antigen conjugates of Formula V based on peptide antigens (A) linked to preformed particles (P);

fig. 44: shows the particle size and biological activity of peptide antigen conjugates delivering autoantigens used to induce tolerance;

fig. 45: shows the particle size and biological activity of particles consisting of A-H + C-H or A-H(C) (Formula VI).

Detailed Description of Embodiments of the Invention

A detailed description of the terms and methods is provided below to provide greater clarity regarding the compounds, compositions, methods and their use(s) for inducing an immune response in a subject of the present invention as methodological material for those of ordinary skill in the art. The terminology of the present invention is understood to be used for the purpose of offering a better description of specific embodiments of the present invention, and should not be construed as limiting the present invention.

Approximately: In the context of the present invention, approximately means plus or minus 5% of the prescribed amount. For example, approximately 10 refers to 9.5-10.5. A ratio of approximately 5:1 refers to a ratio of 4.75:1 to 5.25:1.

Adjuvant: Any material added to vaccines to enhance or modify the immunogenicity of an antigen. Adjuvants can be delivery systems such as particles based on inorganic salts (for example, aluminum hydroxide or phosphate salts called alum),

- 10 046161 water-in-oil or oil-in-water emulsions or polymer particles (eg PLGA) in which the antigen is simply added to the mixture or is adsorbed, incorporated or linked, indirectly or directly, through covalent interactions. In an alternative embodiment of the present invention, adjuvants can be chemically defined molecules that bind to established receptors and induce downstream signaling pathways, including pattern recognition receptor (PRR) agonists, such as synthetic or naturally occurring Toll-like receptor agonists ( TLR), stimulator of interferon genes (STING), nucleotide-binding oligomerization domain-like receptors (NLR), retinoic acid-inducible gene-1-like receptors (RLR) or C-type lectin receptors (CLR), and biological molecules (biological adjuvant) , such as, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, GCSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL. Low molecular weight nucleotide base analogues, such as hydroxyadenine and imidazoquinolines, which bind to Toll-like receptors-7 (TLR-7) and TLR-7/8A, respectively, as well as agonists of TLR-2/6, TLR-4, STING and NODs are used in the present invention as exemplary PRR agonists. Adjuvants are known to one of ordinary skill in the art (see Perry et al., Int J Pharm 364:272-280, 2008 and Brito et al., Journal of Controlled Release, 190C:563-579, 2014). In general, any PRR agonist or biological adjuvant listed herein can be coupled to the peptide antigen conjugate of the present invention through any suitable mechanisms.

Introduction:

To offer or provide to a subject an agent, for example, an immunogenic composition comprising a peptide antigen conjugate as described herein, by any effective means.

Exemplary routes of administration include, but are not limited to, oral, injectable (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), transdermal (eg, topical), intranasal, vaginal, and inhalation routes.

Administration and use of a compound should be understood as offering a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition may be administered to the subject by another person, or the subject may self-administer it.

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

Antigen:

Any molecule containing an epitope that binds to a T cell or B cell receptor and can stimulate an immune response, particularly a B cell response and/or a T cell response, in a subject. Epitopes may consist of peptides, glycopeptides, lipids, or any suitable molecules containing an epitope that can interact with components of specific B-cell and T-cell proteins. Such interactions can generate a response from the immune cell. An epitope refers to the region of a peptide antigen with which B cell and/or T cell proteins, that is, B cell receptors and T cell receptors, interact.

Antigens used in embodiments of the present invention may be selected from pathogens, cancer cells, self-antigens, or allergens. In some embodiments of the present invention, the antigen may be a peptide-based antigen, which may include a region of a polypeptide or protein from a pathogen (such as a virus, bacteria or fungus) or tissue of interest (such as a cancer cell). In other embodiments of the present invention, the antigen may be a whole protein or glycoprotein derived from a pathogen, or a peptide or glycopeptide fragment of a protein or glycoprotein. In other embodiments of the present invention, the antigen may be a protein or peptide fragments of a protein that is expressed predominantly by tumor tissue (but not healthy tissue) and is a tumor-associated antigen. In other embodiments of the present invention, the antigen is a protein or peptide that is associated with an autoimmune disease. In other embodiments of the present invention, the antigen is a protein or glycoprotein that is associated with allergic reactions.

Many such antigens can be used in accordance with embodiments of the present invention, and they are discussed in more detail herein.

Aromatic compounds:

Aromatic compounds are unsaturated cyclic rings with an odd number of pairs of Pi orbital electrons that are delocalized between the carbon or nitrogen atoms forming the ring. Aromatic amino acids include amino acids whose side chain contains an aromatic group, such as phenylalanine, tyrosine or tryptophan. Benzene, a 6-carbon ring containing three double bonds, is a prototypical aromatic compound. Phenylalanine (Phe) and tryptophan (Trp) are the prototype aromatic amino acids. Aryl may refer to an aromatic substituent, and arylamine may refer to an aromatic group.

- 11 046161 pe containing amine. An example of an aromatic amine is aniline. Aromatic heterocycles refer to aromatic rings containing cyclic ring structures containing carbon and another atom such as nitrogen, oxygen or sulfur. Nucleotide bases such as adenine and cytosine are exemplary aromatic heterocycles.

Biocompatible:

Materials are considered biocompatible if they have minimal destructive effects or cause a body response when in contact with biological fluids, cells or tissues.

A biocompatible group may contain chemicals, fragments, including from the following classes: aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl or heteroaryl. However, depending on the molecular composition, such fragments are not always biocompatible.

The term biocompatibility is alternatively understood to mean either minimal interactions with recognition proteins and/or other components of biological systems (eg, naturally occurring antibodies, cellular proteins, including glycoproteins, or cells); or substances and functional groups specifically designed to cause interactions with components of biological systems (for example, drugs and prodrugs), such that the result of the interactions is not inherently negative or destructive.

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 express CD4, and these cells are commonly referred to as T helper cells.

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 express CD8, and these cells are commonly referred to as cytotoxic T cells or killer T cells.

Charge:

A physical property of a substance that affects its interactions with other atoms and molecules, including solutes and solvents. A charged substance experiences electrostatic force from other types of charged substance, as well as from molecules that do not contain a full integer charge, such as polar molecules. Two charged molecules with the same charge repel each other, while two charged molecules with different charges attract each other. Charge is often described in positive or negative integer units.

Charged molecule (C):

Charged molecule (C) refers to any molecule that has at least one functional group that is positively or negatively charged. The functional groups containing the charged molecule can be partial or full integer charge values. A charged molecule may be a molecule with a single charged functional group or with multiple charged functional groups. Functional groups may be permanently charged, or functional groups containing a charged molecule may have a charge depending on pH. A charged molecule may consist 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 molecular structure and conventional methods known to those skilled in the art. Charge can be caused by inductive effects, for example atoms bonded together having differences in electron affinity can result in a polar covalent bond resulting in an atom with a partial negative charge and an atom with a partial positive charge. For example, nitrogen bonded to hydrogen results in a partial negative charge on the nitrogen and a partial positive charge on the hydrogen atom. Alternatively, an atom may be considered to have a full integer charge when the number of electrons inherent to that atom is less than or equal to that atom's atomic number. The charge of a functional group is determined by summing the charge of each atom containing the functional group. The total charge of a charged molecule (C) is determined by summing the charge of each atom containing the molecule. Those skilled in the art are familiar with the process of estimating the charge of a molecule or individual functional groups by summing the formal charge of each atom in the molecule or functional group, respectively.

Charged molecules (C) may contain negatively charged functional groups, such as those that occur as a conjugate acid base at physiological pH (e.g., functional groups with a pKa of less than about 6.5), e.g. component of approximately 7.4. These include, but are not limited to, molecules bearing carboxylates, sulfates, phosphates, phosphoramidates and phosphonates. Charged molecules may contain positive

- 12 046161 but charged functional groups, such as groups that occur as a conjugate acid base at physiological pH (for example, functional groups where the pKa of the conjugate acid base is greater than about 8.5). These include, but are not limited to, molecules bearing primary, secondary and tertiary amines, as well as ammonium, guanidinium. Charged molecules may contain functional groups with charge independent of pH, including quaternary ammonium, phosphonium, and sulfonium functional groups. In some embodiments of the present invention, the charged molecule is a poly(amino acid) containing negatively or positively charged amino acids, or both negatively and positively charged amino acids. In some embodiments of the present invention, the negatively charged amino acid is glutamic acid or aspartic acid. In other embodiments of the present invention, the positively charged amino acid is lysine or arginine. Those skilled in the art will appreciate that many such embodiments of the present invention are possible.

Click chemical reaction:

May refer to a bioorthogonal reaction that brings two compounds together under mild conditions in a highly efficient reaction that generates minimal, biocompatible and/or harmless byproducts. An example of a click reaction used in the present invention is the reaction of the azide group proposed on the linker precursor X1 with the alkyne proposed on the linker precursor X2, which forms a triazole Linker (L) via strain-promoted [3+2] azide-alkyne cycloaddition .

Effective amount:

The amount required to induce the desired response. For example, the amount of an agent, either alone or with at least one additional agent, necessary to induce an immune response, for example, to a peptide antigen conjugate.

Extension(s):

The term extension is used herein to describe molecules linked to the N or C terminus of a peptide antigen (A) that are composed of amino acids, non-natural amino acids; hydrophilic ethylene oxide monomers (ie, PEG); hydrophobic alkane chains; or combinations thereof and function to modulate the rate of degradation of peptide antigen (A). Extensions linked to the N-terminus of the peptide antigen are referred to as B1, and extensions linked to the C-terminus of the peptide antigen are referred to as B2. The extensions (B1 and B2) primarily function to control the rate of degradation of the peptide antigen, but they may have at least one additional function. In some embodiments of the present invention, extensions (B1 and/or B2) can be linked to another molecule, such as a charged molecule (C) or a hydrophobic molecule (H), and function as a linker, as well as control the rate of release of the peptide antigen (A) from another molecule. In additional embodiments of the present invention, the extensions (B1 and/or B2) function to provide distance, that is, space, between any two heterologous molecules. In other embodiments of the present invention, extensions (B1 and/or B2) function to impart hydrophobic or hydrophilic properties to the peptide antigen conjugate. In other embodiments of the present invention, extension compositions (B1 and/or B2) used as a linker may be selected to impart rigidity or flexibility between the peptide antigen (A) and the heterologous molecule. In preferred embodiments of the present invention, extensions (B1 and/or B2) are peptide sequences selected for recognition and hydrolysis by enzymes such as proteases. It should be noted that extensions B1 and B2 may also be referred to as linkers B1 and B2 or _B1 and _B2 , respectively. Specific extension compositions that would be suitable for the practice of the present invention have been described throughout.

Graft polymer:

May be described as a polymer resulting from the adhesion of a polymer of one composition to the side chains of a second polymer of another composition. The first polymer linked via comonomers to the second polymer is a graph copolymer. The first polymer end-linked to the second polymer may be described as a block polymer (eg, an A-B diblock) or a end-linked graft polymer.

Hydropathy index/GRAVY value:

Is a number representing the hydrophobic or hydrophilic characteristics of an amino acid. There are many scales that can be used to describe the relative hydrophobic and hydrophilic characteristics of amino acids containing peptides. In the present invention, the Kyte and Doolittle Hydropathy Score (Kyte J, Doolittle RF, J. Mol. Biol, 157: 105-32, 1983) is used to calculate the overall mean hydropathy value (GRAVY), sometimes referred to as the GRAVY score, of the sequence of amino acids containing peptide antigen conjugates, including a peptide antigen (A), N- and C-terminal optional peptide-based extensions (B1 and B2), and an optional charged molecule (C). The GRAVY value of a peptide is the sum of the Hydropathy values

- 13,046,161 of all amino acids containing the peptide divided by the length (i.e., number of amino acids) of the peptide. The GRAVY value is a relative value. The higher the GRAVY value, the more hydrophobic the peptide sequence is considered, while the lower the GRAVY value, the more hydrophilic the peptide sequence is considered.

Hydrophilic:

Refers to the tendency of a material to disperse freely in aqueous environments. A material is considered hydrophilic if it preferentially interacts with another hydrophilic material and avoids interacting with a hydrophobic material. In some cases, hydrophilicity may be used as a relative term, for example, the same molecule could be described as hydrophilic or whatever it is being compared to. Hydrophilic molecules are often polar and/or charged, and they have good solubility in water, for example, they are soluble up to a concentration of at least 0.1 mg/ml.

Hydrophobic:

Refers to the tendency of a material to avoid contact with water. A material is considered hydrophobic if it preferentially interacts with another hydrophobic material and avoids interacting with a hydrophilic material. Hydrophobicity is a relative term; the same molecule could be described as hydrophobic or whatever it is compared to. Hydrophobic molecules are often non-polar and uncharged, and they have poor solubility in water, for example, they are insoluble down to a concentration of 0.1 mg/ml or less.

Hydrophobic ligand:

It is a molecule that binds to biological receptors and has hydrophobic characteristics. In some embodiments of the present invention, hydrophobic ligands are located along the polymer backbone, thereby imparting hydrophobic properties to the polymer to which it is linked. In some embodiments of the present invention, the hydrophobic ligand is a pattern recognition receptor agonist that has limited solubility in water and therefore may be described as hydrophobic. In additional embodiments of the present invention, the hydrophobic ligand is a TLR-7 or TLR-7/8 agonist, such as imidazoquinoline.

Hydrophobic molecule (H):

In the present invention, the term hydrophobic molecule (H) is used as a general term to describe a molecule with limited water solubility or amphiphilic characteristics that can be linked to peptide antigens, resulting in the formation of a peptide antigen conjugate that forms particles under aqueous conditions. The hydrophobic molecule (H) in the present context promotes particle aggregation due to its poor solubility or tendency to aggregate into particles under aqueous conditions at certain temperature and pH ranges.

Hydrophobic molecules (H) described herein include amphiphilic molecules that can form supramolecular structures, such as micelles or bilayer lamellar or multilamellar structures (for example, liposomes or polymersomes), as well as compounds that are completely insoluble and form only units. The hydrophobic characteristics of the molecule may be sensitive to temperature and/or pH. In some embodiments of the present invention, the hydrophobic molecule (H) is a polymer that is soluble in water at low temperatures, but insoluble or forms micelles at temperatures above, for example, 20°C, such as 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 of the present invention, the hydrophobic molecule (H) is a polymer that is soluble in water at low pH, for example, at a pH below 6.5, but insoluble, for example, at a pH above 6.5. Examples of hydrophobic molecules (H) include, but are not limited to, fatty acids, cholesterol and its derivatives, long-chain aliphatic hydrocarbons, lipids, and various polymers such as polystyrene, poly(lactic-co-glycolic acid) (PLGA), and poly(amino acids), consisting predominantly of hydrophobic amino acids. In some embodiments of the present invention, the hydrophobic molecule (H) is a hydrophilic polymer with several hydrophobic ligands attached. Disclosed herein are a variety of hydrophobic molecules useful in the practice of the present invention.

Immune response:

A change in the activity of an immune system cell, such as a B cell, T cell, or monocyte, as a result of a stimulus, either directly or indirectly, such as through a cellular or cytokine mediator. In one embodiment of the present invention, the response is specific for a particular antigen (antigen-specific response). In one embodiment of the present invention, the immune response is a T cell response, such as a CD4 T cell response or a CD8 T cell response. In one embodiment of the present invention, the immune response results in the production of additional T cell progeny. In one embodiment of the present invention, the immune response results in the movement of T cells. IN

- 14 046161 In another embodiment of the present invention, the response is a B cell response and results in the production of specific antibodies or the production of additional B cell progeny. In other embodiments of the present invention, the response is an antigen presenting cellular response. Enhancement of the immune response refers to the co-administration of an adjuvant and an immunogenic agent, such as a peptide antigen, as part of a peptide antigen conjugate, wherein the adjuvant increases the desired immune response to the immunogenic agent, compared to administering the immunogenic agent to the subject in the absence of the adjuvant. In some embodiments of the present invention, the antigen is used to stimulate an immune response resulting in the activation of cytotoxic T cells that kill virus-infected cells or cancer cells. In some embodiments of the present invention, the antigen is used to induce tolerance or immune suppression. A tolerogenic response may result from the T cell or B cell becoming unresponsive to the antigen. Suppression of the immune response can result from the activation of regulatory cells, such as regulatory T cells, which suppress the immune response, that is, weaken the immune response. Antigens administered to a patient in the absence of an adjuvant are typically tolerogenic or suppressive, while antigens administered with an adjuvant are typically stimulating and result in the recruitment, expansion, and activation of immune cells.

Immunogenic composition:

A composition of materials containing an antigen and, if necessary, an adjuvant that induces a measurable immune response to the antigen.

Ligand:

Is a general term to describe any molecule that binds to a biological receptor. A pattern recognition receptor agonist is a specific type of Ligand that binds to a pattern recognition receptor and may also be referred to as an adjuvant or a Ligand with adjuvant properties. For example, a PRR agonist is a Ligand that binds to a PRR, such as a TLR. The Ligand that binds to the PRR (or PRRa) may also be referred to as an adjuvant, molecular adjuvant, adjuvant molecule, or Ligand with adjuvant properties. A ligand that has limited solubility in water may be referred to as a hydrophobic Ligand, while a ligand that is soluble in water may be referred to as a hydrophilic Ligand. A hydrophobic ligand or a hydrophilic ligand that has adjuvant properties may be referred to as a hydrophobic adjuvant or a hydrophilic adjuvant, respectively.

Linked or connected: The term linked or connected means connected together, directly or indirectly. The first fragment may be covalently or non-covalently linked to the second fragment. In some embodiments of the present invention, the first molecule is covalently linked to another molecule. In some embodiments of the present invention, the first molecule is electrostatically linked to another molecule. In some embodiments of the present invention, the first molecule is linked through dipole-dipole forces (eg, hydrogen bonding) to another molecule. In some embodiments of the present invention, the first molecule is linked through van der Waals forces (also known as London forces) to another molecule. The first molecule may be linked through any and all combinations of such bonds to another molecule.

The molecules can be linked indirectly, for example, through a linker. Molecules can be linked indirectly by the interposition of a component that non-covalently binds to both molecules independently.

As used herein, the term linked and its variations refer to the maintenance of molecules in chemical or physical association, including after immunization, at least until they come into contact with a cell, in particular an immune cell.

In some embodiments of the present invention, the adherent components are associated such that the components are not freely dispersed from each other at least until they come into contact with a cell, such as an immune cell. For example, two components may be covalently linked to each other such that the two components are not capable of separate dispersion or diffusion. In preferred embodiments of the present invention, peptide antigen conjugates consist of peptide antigens (A) that are covalently linked to a hydrophobic molecule (H) or particle (P), directly or indirectly through an extension (B1 or B2). Peptide antigen conjugates containing a hydrophobic molecule (H) aggregate into particles under aqueous conditions, wherein at least two peptide antigen conjugates associate to form a stable compound, while individual peptide antigen conjugates and components containing peptide antigen conjugates, unable to disperse or diffuse before encountering a cell, such as an immune cell.

Linkage differs in a specific way from a simple mixture of antigen and adjuvant, such as can be observed, for example, in a conventional vaccine, for example, in a vaccine containing a soluble

- 15 046161 peptide antigen mixed with adjuvant in water. In a simple mixture, the components can be independently dispersed inside and outside the vaccinated tissue.

Linkers, linker precursors and linker (L): A linker is a molecule or group of atoms that links or binds or joins together at least two moieties. Peptide antigen conjugates disclosed herein are complex molecules containing several different functional components (peptide antigen (A), hydrophobic molecule (H) or particle (P), optional extensions (B1 and/or B2), optionally charged molecule (C), optional linker (L) or optional adjuvant(s), etc.), which may be linked or joined together through any suitable mechanisms. For example, a peptide antigen (A) linked to a hydrophobic molecule (H) may employ a linker between the peptide antigen (A) and the hydrophobic molecule (H). The peptide antigen (A) can be linked to a hydrophobic molecule (H) or particle (P), either directly or indirectly, through a linker (L), extensions (B1 or B2) or a charged molecule (C), through any suitable mechanisms, including any suitable linker. In some embodiments of the present invention, the linker is covalently attached to both linked moieties. In some embodiments of the present invention, the linkers are bifunctional, that is, the linker includes a functional group at two sites, and the functional groups are used to link the linker to two moieties. The two functional groups may be the same (which would be considered a homobifunctional linker) or different (which would be considered a heterobifunctional linker). For example, in some embodiments of the present invention, a linker precursor X2 containing a heterobifunctional linker further comprising an alkyne and an acid is used to link a hydrophobic molecule (H) bearing an amine and a peptide antigen (A) linked to a linker precursor X1 carrying an azide ; the acid and alkyne of the X2 linker precursor react to form amide and triazole bonds with the amine and azide, respectively, thus linking two heterologous molecules. In some embodiments of the present invention, the X2 linker precursor containing the heterobifunctional linker is an acid-linked dibenzocyclooctine (DBCO) molecule. In other embodiments of the present invention, the linker precursor is an acid linked to a maleimide, which links an amine and a thiol, or a bis(carboxylic acid), which links two amines. In other embodiments of the present invention, a tri- or multi-functional linker can be used, wherein the linkages are the same or different. In other embodiments of the present invention, a cleavable extension (B1 or B2) of the N- or C-terminal peptide is used to link the peptide antigen (A) to a hydrophobic molecule (H). In some embodiments of the present invention, the cleavable extension (B1 or B2) of the peptide is heterobifunctional, e.g., the N-terminal amine of extension B2 is linked to the C-terminus of the peptide antigen (A), and the C-terminal carboxyl group of extension B2 is linked directly to the hydrophobic molecule (H). An extension (B1 or B2) can function as a linker, but not all linkers are extensions.

Linkers (L) are specific subsets of linkers that result from the reaction between a linker precursor X1 and a linker precursor X2, and function in a specific manner to connect a peptide antigen (A) and a hydrophobic molecule (H) or particle (P) either directly or indirectly , by extension (B1 or B2) or a charged molecule (C). Linkers perform the specific function of site-selective bonding, that is, joining together or linking a peptide antigen (A) and a hydrophobic molecule (H) or particle (P). The X1 linker precursor can be linked to a peptide antigen, directly or indirectly, by extension (B1 or B2), typically during solid-phase peptide synthesis. It should be noted that the X1 linker precursor linked directly to the N- or C-terminus of the peptide antigen (A) is not considered an extension because it does not specifically function to modulate the rate of degradation of the peptide antigen. Although the X1 linker precursor may have some effect on the rate of degradation of the peptide antigen (A), the X1 linker precursor is not selected to modulate the rate of degradation of the peptide antigen (A) or its release from other molecules, but instead functions in a specific manner to link the peptide antigen (A). antigen (A) and hydrophobic molecule (H) or particle (P).

In some embodiments of the present invention, the X1 linker precursor may be linked to peptide antigen (A) during solid-phase peptide synthesis; linkage may be direct or indirect by extension (B1 or B2) including a degradable peptide linker. Typically, an X1 linker precursor linked directly or indirectly to a peptide antigen (A) is selected to promote a bioorthogonal reaction with an X2 linker precursor proposed on a hydrophobic molecule (H) or Particle (P). Bioorthogonal reactions allow site-selective coupling of a peptide antigen (A) to a hydrophobic molecule (H) or particle (P) without modifying any amino acids containing the peptide antigen (A). Preferred precursors of X1 linkers that stimulate

- 16 046161 lyte bioorthogonal reactions include linker precursors carrying azides and alkynes. Additional X1 linker precursors that provide site-selective reactivity, depending on antigen composition, include thiols, hydrazines, ketones, and aldehydes. In some embodiments of the present invention, the linker precursor has an azide functionality. In some embodiments of the present invention, the X1 linker precursor is a non-natural amino acid bearing an azide, for example, azido-lysine Lys(N3). In such embodiments of the present invention, a peptide antigen (A) linked to a linker precursor X1 bearing an azide functionality can react with a linker precursor X2 bearing an alkyne proposed on a hydrophobic molecule (H), resulting in the formation of a triazole linker, which connects a peptide antigen (A) and a hydrophobic molecule (H). Various linker precursors (X1 and X2) and Linkers are described throughout.

As used herein, both the linker and the linker precursor X1 may be referred to as a Mark (T), although the context of the Mark (T) is used to determine whether the Mark (T) is a linker or a linker precursor (X1). A tag (T) linked to a peptide antigen (A) either directly or indirectly through an optional extension (B1 or B2) or an optional charged molecule (C), but not linked to a hydrophobic molecule (H) or Particle (P), also may be referred to as the X1 linker precursor. A tag (T) that links a peptide antigen (A) to a hydrophobic molecule (H) or particle (P) may also be referred to as a linker (L). Linker precursor X2 reacts with linker precursor X1 to form Linker. The linker precursor X1 may sometimes be referred to as a tag, and the linker precursor X2 may be referred to as a reactive tag moiety or a reactive tag molecule containing a functional group that is specific or reactive to the tag.

Net Charge: The sum of the electrostatic charges carried by a molecule or, where specified, a region of a molecule.

Particle: Nano- or micro-sized supramolecular structure consisting of an accumulation of molecules. The peptide antigen conjugates of the present invention contain either peptide antigens (A) linked to preformed Particles (P) or hydrophobic molecules (H) that aggregate into micelles or other supramolecular structures. Particles containing peptide antigen conjugates can be introduced into cells (eg, immune cells such as antigen presenting cells). In some embodiments of the present invention, the peptide antigen conjugate forms a particle in an aqueous solution. In some embodiments of the present invention, the formation of particles by a peptide antigen conjugate is pH or temperature dependent. In some embodiments of the present invention, nanoparticles consisting of peptide antigen conjugates have an average diameter of from 5 nanometers (nm) to 500 nm. In some embodiments of the present invention, nanoparticles consisting of peptide antigen conjugates may be greater than 100 nm. In some embodiments of the present invention, nanoparticles consisting of peptide antigen conjugates are incorporated into larger particle structures that are too large for uptake by immune cells (e.g., particles larger than 5000 nm), and they slowly release smaller nanoparticles containing the peptide antigen conjugate .

In some embodiments of the present invention, peptide antigen conjugates containing a hydrophobic molecule (H) form nanoparticles. Nanoparticles are formed by the association of peptide antigen conjugates through hydrophobic interactions and can therefore be considered as a supramolecular aggregate. In some embodiments of the present invention, the nanoparticle is a micelle. In preferred embodiments of the present invention, the nanoparticle micelles have a diameter of from 5 to 50 nm. In some embodiments of the present invention, the peptide antigen conjugate forms micelles, and the formation of micelles is dependent on temperature, pH, or both temperature and pH. In some embodiments of the present invention, the disclosed nanoparticles comprise peptide antigen conjugates that consist of peptide antigens (A) coupled to a hydrophobic molecule (H) consisting of polymers coupled to a ligand with adjuvant properties, for example, a PRR agonist; the coupling of the peptide antigen to the PRR agonist in the nanoparticles prevents the PRR agonist from freely dispersing after administration to the subject, thereby preventing systemic toxicity.

The particle may be formed by an aggregation of individual molecules containing peptide antigen conjugates, or, in the case of a peptide antigen conjugate consisting of a peptide antigen (A) linked to a preformed particle (P), the particle may be cross-linked through covalent or non-covalent interactions.

Preformed Particle (P)/Particle (P) Formula: Preformed Particle (P) or simply Particle (P) describes a Particle that has already been formed prior to binding to the peptide antigen (A). Thus, Particle (P) is used to describe a particle of the formula and distinguish

- 17 046161 is from particles formed by the accumulation of at least two peptide antigen conjugates containing a hydrophobic molecule (H). For clarity, particles formed by the accumulation of peptide antigen conjugates are different from preformed particles (P) or particles (P) of the formula. In some embodiments of the present invention, the peptide antigen (A) can be linked directly or indirectly to the particle (P) to form a peptide antigen conjugate, and the peptide antigen conjugate can be a particle under aqueous conditions.

To distinguish between particles formed by peptide antigen conjugates and preformed particles (P), the letter p is always capitalized in the word Particle - Particle, followed by an introductory word consisting of a capital letter P, that is, Particle (P), when we are talking about a pre-formed Particle or Particle (P) of the formula. In some embodiments of the present invention, the Particle (P) may be a PLGA Particle (P) that is formed under aqueous conditions and then couples to a peptide antigen (A) to form a peptide antigen conjugate that remains particulate under aqueous conditions. In some embodiments of the present invention, the particle (P) may be composed of lipids, such as a liposomal Particle (P), which is formed under aqueous conditions and then couples to peptide antigens (A) to form a peptide antigen conjugate that remains particulate in water conditions.

Pattern recognition receptors (PRR): receptors expressed by various cell populations, particularly natural immune cells, that bind to a diverse group of synthetic and naturally occurring molecules called pathogen-associated molecular patterns (PAMPs), as well as molecular patterns associated with damage (DAMP). PAMPs are conserved molecular motifs present on certain microorganisms and viruses. DAMPs are cellular components that are released or expressed during cell death or injury.

PAMP activation or DAMP activation of pattern recognition receptors induces an intracellular signaling cascade leading to changes in host cell physiology. Such physiological changes may include changes in the transcriptional profile of the cell to induce the expression of a range of pro-inflammatory and pro-survival genes. The coordinated expression of these genes may enhance adaptive immunity.

There are several classes of PRR. Non-limiting examples of PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), Stimulatory Interferon Gene (STING) receptor, and C-type lectin receptors (CLRs). Agonists of such PRRs can be used to enhance the immune response to a target antigen.

PRR agonists are adjuvants and may be referred to as Ligands or Ligands with adjuvant properties. In some embodiments of the present invention, PRR agonists are used as adjuvants to enhance the immune response to a peptide antigen.

Toll-like receptors (TLRs) 1–13 are transmembrane PRRs that recognize a diverse range of PAMPs. There are two broad categories of TLRs: some localized to the cell surface, and others localized to the endosomal lumen. TLRs, which are present on the cell surface, are generally important for bacterial recognition. TLRs that are localized in the endosomal lumen, such as TLRs 3, 7, 8, and 9, serve to recognize nucleic acids and are thus generally important for recognizing viruses and therefore promoting antiviral immune responses. Polyinosinic polycytidylic acid is a ligand for TLR3. TLR-7 and TLR-8 recognize single-stranded RNA, as well as nucleotide base analogues and imidazoquinolines. TLR-9 recognizes unmethylated deoxycytidylate phosphate deoxyguanylate (CpG) DNA found primarily in bacteria.

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

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

Peptide or polypeptide: at least two natural or non-natural amino acid residues that are linked together by an amide bond. Amino acid residues may contain post-translational modification(s) (eg, glycosylation and/or phosphorylation). Such modifications may mimic post-translational modifications of natural origin in vivo, or of non-natural origin. Any at least one component of a peptide antigen conjugate may consist of peptides.

There is no conceptual upper limit on peptide length. The length of the peptide is usually selected depending on the application. In some embodiments of the present invention, the guide

- 18 046161 rophobic molecule (H) consists of a peptide, the length of which can range from 3 to 1000 amino acids, but usually no more than 300 amino acids. In some embodiments, the N and/or C-terminal extension (B1 and/or B2) is a peptide of about 1 to 8 amino acids in length. In some embodiments of the present invention, the charged molecule (C) is a peptide consisting of positively, negatively, or both positively and negatively charged amino acids, and is typically no more than 16 amino acids in length.

In preferred embodiments of the present invention, the peptide antigen (A) is a peptide containing from 5 to about 50 amino acids, typically from about 7 to 35 amino acids, for example, 7, 8, 9, 10, 11, 12, 13 , 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids. In other embodiments of the present invention, the peptide antigen (A) is at least about 50 amino acids in length. Thus, in some embodiments of the present invention, the peptide antigen (A) may be considered a protein.

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 (sometimes referred to as LP or SLP) that contains a minimal epitope. Therefore, when the minimal epitope or long peptide is said to be delivered as a peptide antigen conjugate, the minimal epitope or long peptide is meant to be a peptide antigen (A), unless otherwise stated.

In some embodiments of the present invention, the optional charged molecule (C), antigen (A), optional extensions (B1 and B2), and linker precursor X1 are amino acids and can be produced by solid-phase peptide synthesis as a continuous peptide sequence, sometimes referred to as a peptide fragment antigen. It should be noted that the calculation of the net charge or GRAVY of a peptide antigen fragment does not include the X1 linker precursor.

Peptide sequences related to the peptide antigen are designated as PA, peptide sequences related to the N-terminal extension (B1) are designated as PN, and peptide sequences related to the C-terminal extension (B2) are designated PC. The sequences of amino acids containing peptide antigens (A) are represented by the formula PA1...PAn, wherein PA is any amino acid residue containing a peptide antigen (A), and n is an integer value. For example, an 8 amino acid (A) peptide antigen may be represented as PA1-PA2-PA3-PA4-PA5-PA6-PA7-PA8. The sequences of amino acids containing N-terminal extensions (B1) are represented by the formula, PN...PNn, wherein PN is any amino acid residue containing an N-terminal extension, and n is an integer value. The sequences of amino acids containing C-terminal extensions (B2) are represented by the formula, PC1...PCn, wherein PC is any amino acid residue containing a C-terminal extension, and n is an integer value.

Peptide Modifications: Peptides may be modified or otherwise synthesized with at least one modification as specified below. In addition, analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules derived originally from the peptide), and variants (homologs) of these peptides can be used in the methods described herein. The peptides described herein consist of a sequence of amino acids, analogs, derivatives and variants, which may be L and/or D versions. Such peptides may contain peptides, analogs, derivatives and variants of natural origin and others.

Peptides can be modified through various chemical methods to produce derivatives having substantially the same activity as the unmodified peptides, and optionally having other desired properties. For example, the carboxylic acid groups of a peptide, either on the carboxyl terminus or on the side chain, can be offered in the form of a pharmaceutically acceptable cation salt or esterified to form a CCi-CCi ₆ ester, with CC referring to the carbon chain (and thus CC1 refers to a single carbon, and CC ₁₆ refers to 16 carbons) or converted to an amide. The amino groups of the peptide, either at the amino terminus or on the side chain, may be in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetic, trifluoroacetic, formic, benzoic, toluenesulfonic, maleic, tartaric and other organic salts, or they can be modified or converted to an amide.

The amino acid can be modified to contain, by covalent linkage, a PRR agonist, such as a TLR agonist, such as an imidazoquinoline-based TLR-7 or TLR-7/8 agonist.

Peptides can be modified to contain substituent groups that carry a positive charge, a negative charge, or both. The positive and/or negative charge can be influenced by the pH at which the peptide is present.

Hydroxyl groups of peptide side chains can be converted to CC1-CC16 alkoxy or _CC1 - _CC16 ester using well known methods, or hydroxyl groups can be

- 19 046161 transformed (eg, sulfated or phosphorylated) to introduce a negative charge. The phenyl and phenolic rings of the peptide side chains may be substituted with at least one halogen atom, such as fluorine, chlorine, bromine or iodine, or C.'C.'|-C.'C.'| ₆ alkyl, CC1-CC ₁₆ alkoxy, carboxylic acids and their esters or amides of such carboxylic acids. The methylene groups of peptide side chains can be extended to homologous CC2- _CC4 alkylenes. Thiols can be used to form disulfide bonds or thioesters, for example by reaction with maleimide. Thiols can be protected by any of a number of well-known protecting groups, such as acetamide groups. Those skilled in the art will also be able to identify methods for introducing cyclic structures into the peptides of the present invention, whereby conformational constraints on the structure leading to enhanced stability can be selected and proposed. By reference, Green et al., Green's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, Inc. 2006, for more information on additional modifications that can be made to the functional groups.

Peptidomimetic and organomimetic embodiments of the peptide antigen (A) are provided, whereby the three-dimensional arrangement of the chemical components of such peptidomimetic and organomimetics mimics the three-dimensional arrangement of the peptide backbone and side chains of the constituent amino acid, resulting in the creation of such peptidomimetic and organomimetic immunogenic peptide having the ability to measure to induce tolerance or immune suppression, or an enhanced ability to generate a stimulatory immune response, such as a cytotoxic T-cell response or antibody response.

Pharmaceutically Acceptable Transporters: Pharmaceutically acceptable carriers (transporters) suitable for the present invention are standard. In Pharmaceutical Sciences of Remington, E.W. Martin, Mack Publishing Co., Easton, Pennsylvania, 15th edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of at least one therapeutic composition, such as at least one therapeutic cancer vaccine and an additional pharmaceutical agent.

In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically contain injectable liquids that include pharmaceutically and physiologically acceptable liquids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a transporter. For solid compositions (eg, in the form of a powder, pill, tablet or capsule), standard non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions for administration may contain small amounts of non-toxic excipients such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Polar: description of the properties of a substance. The term polar means a relative concept and can describe a molecule or part of a molecule that has a partial charge resulting from differences in electronegativity between atoms bonded together in a 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 specific, non-limiting cases, the polar group may contain a hydroxyl group or an amino group, or a carboxyl group, or a charged group. In certain non-limiting cases, the polar group may prefer to react with a polar solvent such as water. In specific, non-limiting cases, the introduction of additional polar groups can increase the solubility of part of the molecule.

Polymer: a molecule containing repeating structural units (monomers). As described in more detail in the present invention, polymers can be used for any number of components of a peptide antigen conjugate and can be natural or synthetic. In preferred embodiments of the present invention, a hydrophobic or amphiphilic polymer is used as the hydrophobic molecule (H) and drives the aggregation of the peptide antigen conjugate particles. In some embodiments of the present invention, the peptide antigen (A) is a polymer containing amino acids. In some embodiments of the present invention, the extensions (B1 and B2) contain polymers, such as, for example, PEG, poly(amino acids), or combinations thereof. The polymers included in the disclosed embodiments of the present invention can form polymeric nanoparticles that can be administered to a subject without causing adverse side effects. The polymers included in the disclosed embodiments of the present invention can form polymeric nanoparticles that can be administered to a subject to induce an immune response or to treat and/or ameliorate a disease. Polymers included in the disclosed embodiments of the present invention may include a side chain with a functional group that can be used, for example, to facilitate coupling to an adjuvant or molecule used to induce immune suppression

- 20 046161 or tolerance, such as macrolides, for example rapamycin. In several embodiments of the present invention, the polymer may comprise at least two polymer blocks linked by a linker to create a block copolymer, such as an amphiphilic diblock copolymer. In several embodiments of the present invention, the polymer block may be predominantly hydrophobic in nature. In several embodiments of the present invention, the polymer consists of peptides, analogs, derivatives and variants thereof. Various polymer compositions suitable for the practice of the present invention are discussed in more detail elsewhere.

Polymerization: A chemical reaction, usually carried out with a catalyst, heat or light, in which monomers combine to form a chain-linked or cross-linked macromolecule (polymer). In addition, the chains can be joined by additional chemical synthesis using appropriate substituent groups and chemical reactions. Monomers may contain chemically active substances. Polymerization usually occurs by addition or condensation. Addition polymerization occurs when an initiator, usually a free radical, reacts with a double bond in a monomer. A free radical is added to one side of the double bond, producing a free electron on the other side. This free electron then reacts with another monomer and the chain becomes self-propagating, 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 the elimination of a water molecule. In other forms of polymerization, monomer is added one at a time to the growing chain through stepwise introduction of activated monomers, such as during solid-phase peptide synthesis.

Purified: A composition that is generally free of impurities or substances that degrade or contaminate the substance. The term purified is a relative concept and does not imply absolute purity. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein found in its natural environment, such as a cell. In one embodiment of the present invention, the drug is purified such that the peptide antigen conjugate constitutes at least 50% of the total content of the drug. Essential purification means purification from other proteins or cellular components. Substantially purified protein has a purity of at least 60%, 70%, 80%, 90%, 95%, 98% or 99%. Thus, in one specific non-limiting example, the substantially purified protein is 90% free of other proteins or cellular components or contaminating peptides.

Soluble: Capable of becoming molecularly or ionically dispersed in a solvent to form a homogeneous solution. When referring to a peptide, a soluble peptide refers to a single molecule in solution that does not assemble into multimers or other supramolecular structures as a result of hydrophobic or other non-covalent interactions. By soluble molecule we mean that it is freely dispersed in solution in the form of single molecules. In some embodiments of the present invention, the peptide antigen may be a soluble peptide antigen that dissolves to a concentration of at least 0.1 mg/ml in phosphate buffered saline pH 7.4 at room temperature. In other embodiments of the present invention, the peptide antigen conjugate may be soluble in dimethyl sulfoxide and/or other organic solvent(s) at room temperature, but may not be soluble in aqueous solvent(s), such as phosphate-buffered saline, with pH 7.4 at room temperature. The hydrophobic molecules described herein are insoluble up to a concentration of approximately 0.1 mg/ml. Solubility can be determined by visual inspection, turbidity measurements, or dynamic light scattering.

Subject: Refers to both human 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), domestic animals (e.g. , domestic dogs and cats), livestock (such as pigs, sheep, cows, llamas and camels), and non-domestic animals (such as big cats).

Supramolecular: Refers to at least two molecules associated through non-covalent interactions. In some embodiments of the present invention, molecules associate due to hydrophobic interactions. In some embodiments of the present invention, molecules associate due to electrostatic interactions. The association endows the supramolecular complex with a new property that none of the constituent molecules has, such as increased size, which affects the interaction of the materials with the immune system and various immune responses. For example, peptide antigen conjugates can aggregate to form supramolecular complexes.

T Cell: A type of white blood cell that is part of the immune system and can participate in the immune response. T cells include, but are not limited to, CD4 T cells and CD8 T cells. CD4 T cells display CD4 glycoprotein on their surface, and these cells often

- 21 046161 are called T-helpers. These cells often coordinate immune responses, including antibody responses and cytotoxic T cell responses, but CD4 T cells can also suppress immune responses, or CD4 T cells can act as cytotoxic T cells. A CD8 T cell displays the CD8 glycoprotein on its surface, and these cells are often referred to as cytotoxic or killer T cells, but CD8 T cells can also suppress immune responses.

Telechelic: Used to describe a polymer that has one or two chemically active ends, which may be the same or different. The word comes from telos and chele, the Greek words for end and claw, respectively. A semi-telechelate polymer describes a polymer with only one end group, such as a reactive functional group, that can undergo additional reactions such as polymerization. A heterotelechelate polymer describes a polymer with two end groups, such as reactive functional groups, that have different reactive properties.

As used herein, hydrophobic molecules (H) may consist of polymers with reactive groups at one or both ends. In some embodiments of the present invention, the adjuvant is placed at one end of the polymer, and the other end of the polymer may be reacted with a linker that is linked to the peptide antigen directly or indirectly through an extension (B1 or B2) or Linker (L). In this example, the polymer is semi-telechelic to the adjuvant, meaning that the adjuvant is attached to only one end of the polymer chain containing the hydrophobic molecule (H).

Treatment, prevention, or reduction of disease: Treatment 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, treatment of a disease may result in a reduction in tumor burden, which means a reduction in the number or size of tumors and/or metastases, or treatment of a disease may lead to immune tolerance, which reduces systems associated with an autoimmune disease. Disease prevention refers to inhibiting the full-blown progression of disease. The full development of the disease can be prevented. The severity, degree or type of disease can be prevented. Amelioration refers to a decrease in the number or severity of signs or symptoms, or a marker of a disease, such as cancer.

Reduction of a sign or symptom, or marker of a disease, or pathological condition associated with a disease, refers to any observed benefit of treatment and/or any observed effect on a proximal, surrogate endpoint, for example, refers to tumor volume, whether or not whether this effect is symptomatic or not. Reduction of a sign or symptom associated with a tumor or viral infection may be inferred, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject (such as a subject having a tumor that has not yet metastasized, or a subject who may be susceptible to viral infection ), by reducing the severity of some or all of the clinical symptoms of the disease, by slower progression of the disease (for example, by prolonging the life of a subject having a tumor or viral infection), by reducing the number of relapses of the disease, by improving the general health or well-being of the subject, or by other parameters that are well known in the art (eg, specific to a particular tumor or viral infection). Prophylactic treatment is a treatment administered to a subject who is not showing signs of a disease or showing only early signs, with the goal of reducing the risk or severity of developing a disease.

In one example, the desired response is the induction of an immune response that results in a reduction in the size, volume, growth rate, or number (eg, metastases) of tumors in a subject. For example, the agent or agents may induce an immune response that reduces the size, volume, or number of tumors by a desired amount, e.g., by at least 5%, by at least 10%, by at least 15%, by at least at least by 20%. by at least 25%, by at least 30%, by at least 50%, by at least 75%, by at least 90%, or by at least 95%, according to compared to the response in the absence of an agent.

Tumor or cancer or neoplastic: abnormal cell growth that may be benign or malignant, often, but not always, causing clinical symptoms. Neoplastic cell growth refers to the growth of cells that do not respond to physiological stimuli such as growth factors and inhibitory factors.

A tumor is a collection of neoplastic cells. In most cases, the tumor refers to a collection of neoplastic cells that form a solid mass. Such tumors may be called solid tumors. In some cases, neoplastic cells may not form a solid mass, such as is the case with some leukemias. In such cases, a collection of neoplastic cells may be called hemoblastosis.

Cancer refers to the malignant growth of neoplastic cells that are solid masses or hematologic malignancies. Signs of cancer that define it as malignant

- 22 046161 levanie include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory substances at abnormal levels and suppression or exacerbation of inflammatory or immunological response(s), invasion of surrounding or distant tissues or organs such as lymph nodes, etc. Further.

A tumor that does not have significant adverse clinical symptoms and/or grows slowly is called benign.

Malignant means causing or likely to cause significant clinical symptoms in the future. A tumor that penetrates into surrounding tissues and/or metastasizes and/or produces significant clinical symptoms through the production and secretion of chemical mediators that affect nearby or distant body systems is called malignant.

The metastatic process refers to cancer cells that have left the original tumor site and migrated to other parts of the body, such as through the bloodstream, through the lymphatic system, or through body cavities such as the peritoneal cavity or chest cavity.

The amount of tumor an individual has is the tumor load. Tumor burden can be measured as the number, volume, or weight of a tumor and is often assessed by physical examination, x-ray imaging, or pathological examination.

An established or existing tumor is a tumor that exists at the time of initiation of therapy. Once a tumor has developed, it can often be detected through diagnostic tests. In some embodiments of the present invention, the developed tumor may be palpable. In some embodiments of the present invention, the developed tumor has a size of at least 500 mm ³ , such as at least 600 mm ³ , at least 700 mm ³ , or at least 800 mm ³ . In other embodiments of the present invention, the tumor is at least 1 cm in length. For a solid tumor, the established tumor typically has a newly developed and robust blood supply and may have induced regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC).

One of ordinary skill in the art will appreciate that the definitions proposed above are not intended to include unacceptable substitution patterns (eg, substitution of methyl with 5 different groups, and the like). A person of ordinary skill in the art will be able to readily recognize such unacceptable substitution patterns. Any functional group disclosed herein and/or defined above may be substituted or unsubstituted unless otherwise indicated herein. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as would normally be understood by a person of ordinary skill in the art to which the present invention relates. The terms a, an and the, when used in the singular, include the plural unless the context clearly indicates otherwise. The term contains means includes. Therefore, containing A or B includes A, includes B, or includes both A and B. It is further understood that all base sizes or amino acid sizes, and all molecular weights or molecular weights given for nucleic acids or polypeptides, are approximate and presented for descriptive purposes. Although methods and materials similar or equivalent to the methods and materials described herein may be practiced or tested in connection with the present invention, suitable methods and materials are described herein. In the event of a conflict, this specification, including explanations of terms, will control. In addition, the materials, methods and examples are illustrative only and do not limit the present invention.

Immunogenic compositions

Disclosed herein are novel immunogenic compositions comprising particles that comprise a peptide antigen conjugate that further comprises a peptide antigen (A) coupled to a particle (P) or hydrophobic molecule (H). Peptide antigen conjugate refers to a compound that is formed by the adhesion, for example, through a covalent or other connection, of a peptide antigen (A) to a particle (P) or hydrophobic molecule (H). The hydrophobic molecule (H) or particle (P) causes the peptide antigen conjugate to aggregate into particles, resulting in a surprising improvement in immune responses directed 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- and C-termini of the peptide antigen (A), respectively, which offers unexpected improvements in manufacturing and biological activity; an optional charged molecule (C) that offers unexpected improvements in the stability of particles formed by peptide antigen conjugates, leading to improved manufacturing and improved biological activity; and an optional linker (L), which results from the reaction of the linker precursor X1 linked to the peptide antigen (A) and the linker precursor X2 proposed on a hydrophobic molecule (H) or particle (P), resulting in the connection of the peptide antigen (A) and hydrophobic molecule (H), and particle (P) in eff

- 23 046161 efficient process that leads to unexpected improvements in the efficiency of making peptide antigen conjugates. The peptide antigen conjugate-containing components may be coupled by any suitable mechanism and are described in more detail throughout.

In some embodiments of the present invention, the peptide antigen (A) is linked directly to a hydrophobic molecule (H) or particle (P) to form a peptide antigen conjugate of the formula A-H or A-P. In other embodiments of the present invention, a peptide antigen (A) is linked to a hydrophobic molecule (H) or particle (P) via a linker (L) to form a peptide antigen conjugate of the formula A-L-H or A-L-P. In other embodiments of the present invention, the peptide antigen (A) is linked to an extension (B1 or B2) that is linked either directly or via a linker (L) to a hydrophobic molecule (H) or particle (P) to form a peptide antigen conjugate of either from the formulas, A-B2-H, A-B2-L-H, H-B1A, H-L-B1-A, A-B2-P, A-B2-L-P, P-B1-A or P-L-B1-P. In some embodiments of the present invention, a peptide antigen (A) is linked directly or by extension (B1 and B2) to a linker precursor X1 to form a peptide antigen fragment of the formula A-X1, A-B2X1, Χι-A, or X1-B1-A, which reacts with the X2 linker precursor on a hydrophobic molecule (H) or Particle (P), i.e., X2-H or X2-P, to form a linker (L) that attaches the peptide antigen (A) to the hydrophobic molecule (H) or particle (P), resulting in a peptide antigen conjugate of any of the formulas, i.e., A-L-H, A-L-P, A-B2-L-H, A-B2-L-P, H-L-A, P-L-A, HB1-A, or P-B1- A. The present invention in such embodiments shows that the particles are formed under aqueous conditions that are shown to be suitable for inducing an immune response in a subject.

In some embodiments of the present invention, the peptide antigen (A) is linked to both extensions (B1 and B2). Such embodiments of the present invention include peptide antigen conjugates of the formula B1-A-B2-H, B1-A-B2-L-H, B1-A-B2-P, B1-A-B2-L-P, H-B1-AB2, H-L- B1-A-B2, P-B1-A-B2 or P-L-B1-A-B2. The present invention in such embodiments shows that the particles are formed under aqueous conditions that have been demonstrated to be suitable for inducing an immune response in a subject.

In some embodiments of the present invention, molecules containing functional groups that impart an electrostatic charge, that is, charged molecules (C), are linked directly or indirectly through optional extensions (B1 and/or B2), an optional linker (L), or a hydrophobic molecule (H), or particles (P), with peptide antigen (A). The charge imparted to the peptide antigen conjugate by the charged molecule stabilizes the supramolecular structures formed under aqueous conditions. Non-limiting examples of peptide antigen conjugates containing charged molecules (C) include C-A-H, C-B1-A-H, C-A-B2-H, CB1-A-B2-H, A-H(C), A-B2 -Н(С), В1-А-Н(С), В1-А-В2-Н(С), С1-А-Н(С2), С1-А-В2-Н(С2), С1-В1- A-H(C2), C1-B1-A-B2-H(C2), N-A-C, H-B1-A-C, H-A-B2-C, H-B1-A-B2-C, H( C)-A, N(C)-B1-A, N(C)-A-B2, N(C)B1-A-B2, N(C1)-A-C2, N(C1)-B1-A -C2, H(C1)-A-B2-C2, H(C1)-B1-A-B2-C2, C-A-L-H, C-B1-A-L-H, C-AB2-L-H, C-B1-A-B2-L-H , A-L-H(C), A-B2-L-H(C), B1-A-L-H(C), B1-A-B2-L-H(C), C1-A-L-H(C2), C1-AB2-L-H(C2), C1 -B1-A-L-H(C2), C1-B1-A-B2-L-H(C2), H-L-A-C, H-L-B1-A-C, H-L-A-B2-C, H-L-B1-A-B2C, H(C)-L-A, H(C)-L-B1-A, H(C)-L-A-B2, H(C)-L-B1-A-B2, H(C1)-L-A-C2, H(C1)-L-B1 -A-C2, H(C1)-L-AB2-C2, H(C1)-L-B1-A-B2-C2, C-A-P, C-B1-A-P, C-A-B2-P, C-B1-A -B2-P, A-P(C), A-B2-P(C), B1-A-P(C), B1-A-B2-P(C), C1-A-P(C2), C1-A-B2- P(C2), C1-B1-A-P(C2), C1-B1-A-B2-P(C2), P-A-C, P-B1-A-C, P-AB2-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)-AB2-C2, P(C1)-B1-A-B2-C2, C-A-L-P, C-B1-A-L-P, C-A-B2-L-P, C-B1-A-B2 -L-P, A-L-P(C), A-B2-L-P(C), B1-A-L-P(C), B1-A-B2-L-P(C), C1-A-L-P(C2), C1-A-B2-L-P( C2), C1-B1-A-L-P(C2), C1-B1-A-B2-L-P(C2), P-L-A-C, P-L-B1-A-C, P-L-A-B2-C, P-L-B1-A-B2-C, P (C)-L-A, P(C)-L-B1-A, P(C)-L-A-B2, P(C)-L-B1-AB2, P(C1)-L-A-C2, P(C1) -L-B1-A-C2, P(C1)-L-A-B2-C2 or P(C1)-L-B1-A-B2-C2.

The charged molecule (C) stabilizes the particles formed by peptide antigen conjugates. The charged molecule (C) can be linked directly to the peptide antigen conjugate. In an alternative embodiment of the present invention, the charged molecule (C) can be provided on a separate molecule that associates with particles formed by peptide antigen conjugates. In some embodiments of the present invention, a 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 (wherein A' is a conserved antigen) which is mixed with the peptide an antigenic conjugate of the formula [C]-[B1]-A-[B2]-[L]-H, where [ ] denotes that the group is optional, under aqueous conditions, and the resulting particles contain C-H or C-A '-H and peptide antigen conjugate.

The hydrophobic molecule (H) may comprise any suitable molecule that induces the aggregation of the peptide antigen conjugate into particles under aqueous conditions. In some embodiments of the present invention, the hydrophobic molecule (H) containing the peptide antigen conjugate is a polymer with limited water solubility. In some embodiments of the present invention, the hydrophobic molecule (H) is a polymer that senses

- 24 046161 temperature or pH sensitive, which has limited solubility in water at certain temperatures or pH values. In other embodiments of the present invention, the hydrophobic molecule (H) is a lipid, fatty acid, or cholesterol. Many hydrophobic molecules (H) are suitable for the present invention and are described in more detail throughout.

Particles formed by the peptide antigen conjugates disclosed herein are suitable for inducing an immune response in a subject. In some embodiments of the present invention, particles containing peptide antigen conjugates that contain 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 of the present invention, particles containing peptide antigen conjugates that contain 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 an infectious disease. In some embodiments of the present invention, particles containing peptide antigen conjugates that contain a self-antigen are provided to a subject to induce a tolerogenic or T-cell response to treat an autoimmune disease.

A peptide antigen conjugate contains a peptide antigen (A), optional N- 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(s). (WITH). Each of these components is described below and in more detail throughout.

Peptide antigen (A)

The peptide antigen (A) can be any antigen that is suitable for inducing an immune response in a subject. Peptide antigen (A) can be used to induce either a proinflammatory or tolerogenic immune response, depending on the nature of the immune response required for use. In some embodiments, the peptide antigen (A) is a tumor-associated antigen, such as a self-antigen, a neoantigen, or a tumor-associated viral antigen (eg, HPV E6/E7). In other embodiments of the present invention, the peptide antigen (A) is an infectious disease antigen, such as a peptide derived from a protein isolated from a virus, bacterium, fungus, or protozoan pathogen. In other embodiments of the present invention, the peptide antigen (A) is a peptide derived from an allergen or autoantigen that is known or suspected to cause allergic reactions or autoimmune diseases.

A peptide antigen (A) consists of a sequence of amino acids or a peptide mimetic that can induce an immune response, such as a T cell response or a B cell response, in a subject. In some embodiments of the present invention, the peptide antigen (A) comprises a post-translationally modified amino acid or amino acids, unnatural amino acids, or peptide mimetic. A peptide antigen can be any sequence of natural, non-natural or post-translationally modified amino acids, a peptide mimetic, or any combination thereof that has an antigen or predicted antigen, that is, an antigen with a T cell or B cell epitope.

The immunogenic compositions may contain at least one other peptide antigen conjugate, each having a different peptide antigen (A) composition. In some embodiments of the present invention, the immunogenic compositions contain particles containing up to 50 different peptide antigen conjugates, each of which has a unique peptide antigen (A) composition. In some embodiments of the present invention, the immunogenic compositions comprise mosaic particles that contain 20 different peptide antigen conjugates. In other embodiments of the present invention, the immunogenic compositions comprise mosaic particles that contain 5 different peptide antigen conjugates. In some embodiments of the present invention, the immunogenic compositions contain 20 different particle compositions, each of which is composed of a unique peptide antigen conjugate (ie, each particle contains a single peptide antigen conjugate composition). In some embodiments of the present invention, the immunogenic compositions contain 5 different particle compositions, each of which is aggregated from a unique peptide antigen conjugate (ie, each particle contains a single peptide antigen conjugate composition). In other embodiments of the present invention, the immunogenic compositions comprise a single particle composition consisting of a single peptide antigen conjugate composition.

The length of the peptide antigen (A) depends on the specific application and is typically from about 5 to about 50 amino acids. In preferred embodiments of the present invention, the peptide antigen (A) contains from about 7 to 35 amino acids, for example, 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.

- 25 046161

In other embodiments of the present invention, the peptide antigen is a fragment of a polypeptide. In other cases, the peptide antigen is a full-length polypeptide, such as a protein antigen, which can be recombinantly expressed. Peptide antigens (A) based on tumor-associated antigens, infectious disease antigens, allergens or autoantigens can be delivered as a full-length sequence, however, in the preferred embodiment of the present invention, no more than 50 amino acids in length. In preferred embodiments of the present invention, the peptide antigen (A) contains from 7 to 35 amino acids, typically about 25. Thus, for a tumor-associated antigen, infectious disease antigen, allergen or autoantigen greater than 25 amino acids in length, for example, an antigen of 100 amino acids, the antigen can be divided into 7-35 amino acids, for example, peptide antigens (A) of 25 amino acids, with each peptide antigen (A) containing a unique composition of amino acids; or, peptide antigens (A) may be overlapping peptide pools, wherein the antigen is divided into a predetermined number of amino acids: from 7 to 35, for example, peptide antigens (A) of 25 amino acids that have overlapping sequences. For example, an overlapping peptide pool containing an antigen of 100 amino acids can be divided into eight peptide antigens (A) of 25 amino acids, each offset by 12 amino acids (that is, each subsequent peptide of 25 amino acids containing a peptide sequence of 100 amino acids , begins at the 13th amino acid position from the previous peptide). Those skilled in the art will appreciate that there are many permutations for generating a peptide pool from an antigen.

In some embodiments of the present invention, the peptide antigen (A) is a minimal CD8 or CD4 T cell epitope that contains tumor-associated antigen, infectious disease antigen, allergen, or self-antigen regions that are predicted in silico (or measured empirically) for binding of MHC molecules -I or MHC-II. For tumor-associated antigens, the peptide antigen (A), which is the minimal CD8 or CD4 T cell epitope that is predicted in silico (or measured empirically) for binding of MHC-I or MHC-II molecules, must also be an amino acid sequence that is unique to a tumor cell. Algorithms for predicting MHC-I or MHC-II binding are widely available (see Landegaard et al., Nucleic Acids Res., 36:W509-W512, 2008 and http://www.cbs.dtu.dk/services/NetMHC/ ). In some embodiments of individualized therapy of the present invention for a particular subject (eg, a patient), the peptide antigen (A) containing peptide antigen conjugate may contain a minimal CD8 T cell epitope from a tumor-associated antigen, an infectious disease antigen, an allergen, or a self-antigen that is typically a 7-13 amino acid peptide that is predicted to have a binding affinity of <1000 nm for the particular MHCI allele that is expressed by that subject. In some embodiments of individualized therapy of the present invention for a particular subject (eg, a patient), the peptide antigen (A) may comprise a minimal CD4 T cell epitope from a tumor-associated antigen, an infectious disease antigen, an allergen, or a self-antigen, which is a 10- 16 amino acids that is predicted to have a binding affinity of <1000 nm for the specific MHC-II allele that is expressed by this subject. In a preferred embodiment of the present invention, 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 -cellular epitopes, peptide antigen (A) may contain from 16 to 35 amino acids, may contain up to 50 amino acids, for example up to 35 amino acids, up to 25 amino acids or up to 20 amino acids, or up to 16 amino acids, so it can contain all possible CD8 or CD4 T cell epitopes.

In some embodiments of the present invention, the peptide antigen (A) is derived from tumor-associated antigens. Tumor-associated antigens can be either self-antigens, which are present on healthy cells but are predominantly expressed by tumor cells, or neoantigens, which are aberrant proteins specific to tumor cells and unique to individual patients. Suitable self-antigens include antigens that are predominantly 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 affecting overall survival, prostatic acid phosphatase, NY-ESO-1 and MAGEA3. Neoantigens arise from the inherent genetic instability of cancers, which can lead to DNA mutations, RNA splice variants, and changes in post-translational modification, all of which potentially lead to de novo protein products collectively referred to as neoantigens or sometimes predicted neoantigens. DNA mutations include changes in DNA, including nonsynonymous missense mutations, nonsense mutations, insertions, deletions, chromosomal inversions and chromosomal translocations, all of which potentially lead to the emergence of new gene products and therefore neoantigens. RNA splice site changes can lead to new protein products, and missense mutations can introduce amino acids that allow post-translational modifications (eg, phosphorylation) that can be antigenic. In addition, tumor cell instability can lead to epigenetic changes and activation of certain transcription factors, which can lead to selective expression of certain antigens by tumor cells that are not expressed by healthy, non-cancerous cells.

Peptide antigen conjugates used in personalized cancer vaccines must include peptide antigens (A) that contain tumor-associated antigen regions that are unique to tumor cells. Peptide antigens (A) containing neoantigens resulting from a missense mutation must cover the change in the amino acid encoded by at least one nucleotide polymorphism. Peptide antigens (A) containing neoantigens resulting from frameshift mutations, splice site variants, insertions, inversions and deletions must cover new peptide sequences and compounds of new peptide sequences. Peptide antigens (A) containing neoantigens with novel post-translational modifications must include amino acids bearing post-translational modification(s), such as phosphate or glycan. In preferred embodiments of the present invention, the peptide antigen (A) contains 0-25 amino acids on both sides flanking the amino acid change or new compound that arises due to the mutation. In one embodiment of the present invention, the peptide antigen (A) is a neoantigen sequence that contains 12 amino acids on either side flanking an amino acid change that results from a single nucleotide polymorphism, for example, a 25 amino acid peptide, wherein the 13th amino acid represents is an amino acid residue obtained as a result of single nucleotide polymorphism. In some embodiments of the present invention, the peptide antigen (A) is a neoantigen sequence that contains 12 amino acids on either side flanking an amino acid with a novel post-translational modification, e.g., a 25 amino acid peptide, wherein the 13th amino acid is an amino acid residue , resulting from a new post-translational modification site. In other embodiments of the present invention, the peptide antigen (A) is a neoantigen sequence that contains 0-12 amino acids on both sides flanking the new junction created by the insertion, deletion or inversion. In some cases, the peptide antigen (A) containing neoantigens derived from new sequences may cover the entire new sequence, including 0-25 amino acids on either side of the new compounds that may also arise.

Tumor-associated antigens suitable as peptide antigens (A) for the immunogenic compositions of the present invention can be identified by various methods that are known to one skilled in the art. Tumor-associated antigens can be identified by assessing protein expression in tumor cells compared to healthy cells, that is, non-cancerous cells obtained from the subject. Suitable methods for assessing protein expression include, but are not limited to, immunohistochemistry, immunofluorescence, Western blotting, chromatography (ie, size exclusion chromatography), ELISA, flow cytometry and mass spectrometry. Proteins predominantly expressed by tumor cells rather than by healthy cells or a limited number of healthy cells (eg, CD20) are suitable tumor-associated antigens. DNA and RNA sequencing of patient tumor biopsies followed by bioinformatics to identify mutations in protein-coding DNA that are expressed as RNA and produce peptides predicted to bind to MHC-I or MHC-II alleles on the patient's antigen presenting cells (APCs) , can also be used to identify tumor-associated antigens that are suitable as peptide antigens (A) for the immunogenic compositions of the present invention.

In preferred embodiments of the present invention, tumor-associated antigens suitable as peptide antigens (A) for immunogenic compositions are identified using mass spectrometry. Suitable peptide antigens (A) are peptides identified by mass spectrometry after elution from MHC molecules from tumor biopsies of the patient, and not from healthy tissues of the same subject (that is, peptide antigens are present only in tumor cells and not in healthy cells of the same subject same subject). Mass spectrometry can be used alone or in combination with other methods to identify tumor-associated antigens. Those skilled in the art will appreciate that there are many methods for identifying tumor-associated antigens, such as neoantigens (see Yadav et al., Nature, 515:572-576, 2014), which are suitable as peptide antigens (A) for practical use. application

- 27 046161 development of the present invention.

In preferred embodiments of the present invention, the tumor-associated antigens used as peptide antigens (A) are clonal or nearly clonal within a population of nonplastic cells that may otherwise be considered heterogeneous.

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

Individualized cancer vaccines based on immunogenic compositions may contain at least one other peptide antigen conjugate, each of which has a unique peptide antigen (A) composition. In some embodiments of the present invention, individualized cancer vaccines may contain 10-50 different peptide antigen conjugate compositions, each having a unique peptide antigen (A). In preferred embodiments of the present invention, the immunogenic compositions contain 20 different peptide antigen conjugates, each of which contains peptide antigens (A) ranging from 7 to 35 amino acids in length, for example, 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, usually no more than 50. In a non-limiting example, immunogenic compositions consisting of particles formed from 20 different peptide antigen conjugates consisting of peptide antigens (A) of 8 amino acids in length are used as a personalized cancer vaccine. In another non-limiting example, immunogenic compositions consisting of particles formed from 20 different peptide antigen conjugates consisting of peptide antigens (A) of 25 amino acids in length are used as a personalized cancer vaccine.

For patients with highly mutated tumors that have more than 50 tumor-associated neoantigens, a pruning selection process can be applied to select peptide antigens (A) for use in customized cancer vaccines consisting of peptide antigen conjugates. In some embodiments of the present invention, a pruning selection process is used to select peptide antigens (A) containing epitopes that are predicted to have the highest binding affinity to MHC and RNA expression levels in tumor cells. Additional criteria may be used to select tumor-associated self-antigens or neoantigens. For example, predicted immunogenicity or the predicted ability of a peptide antigen (A) to result in T cells reacting with other self-antigens that may lead to autoimmune diseases are additional criteria considered. For example, peptide antigens (A) that contain tumor-associated antigens and have high predicted immunogenicity, but also low potential to lead to autoimmune diseases, are criteria used to select potential peptide antigens (A) for use in personalized cancer vaccines. In some embodiments of the present invention, neoantigens that are expected to result in T cell responses or antibody responses that react with self antigens found on healthy cells are not selected for use as peptide antigens (A). For patients with less than, for example, 20-50 predicted neoantigens, the pruning selection process may not be critical and therefore all 20-50 predicted neoantigens can be used as peptide antigens (A) in a customized cancer vaccine.

Cancer vaccines may include peptide antigens (A) that contain tumor-associated antigens that are specific to patients and/or tumor-associated agents that are common to patients. For example, the tumor-associated antigen may be a conserved self-antigen, such as NY-ESO-1 (testicular cancer) or gp100 (melanoma), or the antigen may be a critical epitope, such as Na17 (melanoma), that is not normally expressed by healthy cells , but is conservative for patients. The immunogenic compositions of the present invention may include peptide antigens (A) that arise from so-called hot spot mutations, which are frequent mutations in certain genes or regions of genes that occur more frequently than predicted by chance. Non-limiting examples of hot spot mutations include the V600E mutation in the BRAF protein, which is common to melanoma, papillary thyroid carcinoma and colorectal carcinoma, or KRAS G12 mutations, which are among the most common mutations, such as KRAS G12C. A range of suitable self-antigens as well as neoan

- 28 046161 tigens that arise from hot spot 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.

In some embodiments of the present invention, the peptide antigen (A) can be obtained from a hematological tumor. Non-limiting examples of hematologic tumors include leukemias, including acute leukemias (such as 11c.|2.3-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemia goats (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenic leukemia and chronic lymphocytic leukemia), true polycythemia, lymphoma, Hodgkin's disease, non -podkhkinsky lymphoma (unexpressed and highly differentiated forms), multiple myeloma, macroglobulinemia of Waldenstrem, severe macroglobulinemia, disease of severe chains , hairy cell leukemia and myelodysplasia.

In some embodiments of the present invention, the peptide antigen (A) can be obtained from a solid formation. Non-limiting examples of solid tumors such as types of sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other types of sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphocytic leukemia, pancreatic cancer, cancer breast (including basal carcinoma of the breast, ductal carcinoma and lobular carcinoma of the breast), types of lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary carcinoma thyroid, pheochromocytoma sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma and tumors CNS (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangiomas, melanoma, neuroblastoma and retinoblastoma). In some examples, the tumor is melanoma, lung cancer, breast lymphoma, or colon cancer.

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 of the present invention, the peptide antigen (A) is a tumor-associated antigen from prostate cancer. In some embodiments of the present invention, the peptide antigen (A) is a tumor-associated antigen from a skin cancer, such as basal 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, bronchiolavolar carcinoma, large cell carcinoma, or small cell carcinoma. In some embodiments, the peptide antigen (A) is a tumor-associated antigen from a 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 of the present invention, the peptide antigen (A) is a tumor-associated antigen from liver cancer, such as hepatocellular carcinoma. In some embodiments of the present invention, the peptide antigen (A) is a tumor-associated antigen from pancreatic cancer. In some embodiments of the present invention, the peptide antigen (A) is a tumor-associated antigen from kidney cancer, such as renal cell carcinoma. In some embodiments of the present invention, the peptide antigen (A) is a tumor-associated antigen from testicular cancer.

In some embodiments, the peptide antigen (A) is a tumor-associated antigen derived from precancerous lesions, such as carcinoma in situ variants or vulvar intraepithelial neoplasia, cervical intraepithelial neoplasia, or vaginal intraepithelial neoplasia.

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

In some embodiments of the present invention, the peptide antigen (A) is a self-antigen. The antimutagen may be identified and selected based on screening the subject's own T cells for autoreactivity against autoantigens presented in the context of the patient's own MHC-I molecules. In an alternative embodiment of the present invention

- 29 046161 tenia peptide antigens can be selected using in silico methods to predict potential autoantigens that (i) have a predicted high affinity for binding the subject's own MHC-I molecules and (ii) are expressed and/or are known to are associated with pathology that takes into account the subject's autoimmune syndrome. In other embodiments of the present invention, the peptide antigen is a CD4 epitope derived from an allergen and is selected based on a peptide antigen that has high binding affinity for the patient's own MHC-II molecules.

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

Extensions (B1 and B2):

Additional N- and C-terminal extensions (B1 and B2) designate molecules linked to the N- and C-terminus of the peptide antigen (A), respectively. The N- and C-terminal extensions B1 and B2 may consist of at least one of the following:

amino acids, including unnatural amino acids; hydrophilic ethylene oxide monomers (eg PEG); hydrophobic alkane chains or the like; or combinations thereof. The N- and C-terminal extensions B1 and B2 are linked to the peptide antigen (A) by any suitable mechanisms, for example through stable amide bonds.

In some embodiments of the present invention, the extensions (B1 and B2) function to control the rate of degradation of the peptide antigen (A), but they may perform any at least one additional function. In some embodiments, the N- and C-terminal extension (B1 or B2) may be free (one end of the N- or C-terminal extension is linked to the peptide antigen (A) and the other end is not linked to another molecule) and serve to slow down the degradation of the peptide antigen; for example, a B1 peptide-based extension can be linked to the N-terminus of the peptide antigen via an amide bond to retard degradation. In other embodiments of the present invention, the N- and C-terminal extensions (B1 and/or B2) can be linked to a heterologous molecule and can function as a linker and also modulate the degradation of the peptide antigen (A). N- and/or C-terminal extensions offering linker function can link the peptide antigen either directly or indirectly, via a Linker (L), to a particle (P) or to a hydrophobic molecule (H), and/or to a charged molecule ( WITH).

In some embodiments of the present invention, the extensions (B1 and/or B2) function to provide distance, that is, space between any two heterologous molecules. In other embodiments of the present invention, the extensions (B1 and/or B2) function to impart hydrophobic or hydrophilic properties to the peptide antigen conjugate. In other embodiments of the present invention, the composition of the extensions (B1 and/or B2) may be selected to impart rigidity or flexibility. In other embodiments of the present invention, N- or C-terminal extensions (B1 and/or B2) may help stabilize the particles formed by the peptide antigen conjugate.

In some embodiments of the present invention, the extensions (B1 and/or B2) consist of charged functional groups, such as charged amino acid residues (eg, arginine, lysine), which impart an electrostatic charge at physiological pH. The number of charged residues present in the extension can be used to modulate the net charge of the peptide antigen conjugate. Disclosed herein is an algorithm that describes a systematic process for selecting peptide-based extensions (B1 and/or B2) that are recognized by proteases and impart a specific electrostatic charge that functions to stabilize the particles formed by peptide antigen conjugates.

Additionally, in some embodiments of the present invention, C-terminal extensions (B2) added to peptide antigens (A) are selected to facilitate the manufacture of peptides containing [C]-[B1]-A-B2-[X1], wherein ,[ ] denotes that the group is optional by including amino acid sequences in B2 that disrupt β-sheet formation and prevent sequence truncation during solid-phase peptide synthesis. In a non-limiting example, the C-terminal dipeptide linker (B2), Gly-Ser, is included during solid-phase peptide synthesis as a pseudoproline dipeptide (eg, Gly-Ser(Psi(Me,Me)pro)). In additional embodiments of the present invention, the proline is included in a sequence with a cathepsin-cleavable C-terminal extension (B2), for example, Ser-Pro-Leu-Arg (SEQ ID NO: 21); however, proline is included both to facilitate production and to stimulate elongation processing by endosomal proteases.

In some embodiments of the present invention, the peptide antigen (A) is linked at the C-terminus to the B2 extension, which is linked either directly or indirectly by

- 30 046161 linker (L), with a hydrophobic molecule (H) or with a particle (P). In some embodiments of the present invention, 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), with either B1 or B2 linked either directly or via a linker (L) to the particle (P) or a hydrophobic molecule (H). In some embodiments of the present invention, the peptide antigen (A) is linked at the N-terminus to a B2 extension that is linked either directly or via a linker (L) to a particle (P) or hydrophobic molecule (H). In some embodiments of the present invention, the charged molecule (C) is linked to a B1 or B2 extension that is linked to the N- or C-terminus of the peptide antigen (A), respectively, wherein the extension that is not linked to the charged molecule (C) , linked either directly or via a linker (L) to a hydrophobic molecule (H) or particle (P). In additional embodiments of the present invention, charged molecules (C) are linked to both the B1 extension and the B2 extension, which are linked to both the N- and C-termini of the peptide antigen (A), respectively. In additional embodiments of the present invention, the charged molecules (C) are linked to the B1 extension linked to the N-terminus of the peptide antigen (A), but not to the B2 extension linked to the C-terminus of the peptide antigen (A), which can be linked either directly , either through a linker (L), with a hydrophobic molecule (H) or with a particle (P). The X1 linker precursor or linker (L) can be linked to either extension (B1 or B2) by any suitable mechanisms, such as an amide bond. In preferred embodiments of the present invention, the extensions (B1 and B2) are peptide sequences selected for recognition and hydrolysis by enzymes such as proteases. Extensions (B1 and B2) in preferred embodiments of the present invention are cleavable peptides, including amino acids recognized by any or all of the following: endosomal proteases or the immunoproteasome.

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

In some embodiments of the present invention, the N-terminal extension (B1) is a peptide sequence of about 1 to 8 amino acids in length, such as 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length, typically no more than 10 amino acids that is linked to the peptide antigen (A) through, for example, an amide bond formed between the carboxyl group of the extension (B1) and the alpha-amine residue of the N-terminus of the peptide antigen (A). The amide bond between B1 and peptide antigen (A) can be cleaved by enzymes. It is understood that amino acid positions are usually numbered in order, from proximal to distal positions from the cleavage site, with C-terminal amino acid positions relative to the cleavage site being designated by a simple symbol (eg, n'). For example, in relation to the extension of a tetrapeptide (PN4-PN3-PN2-PN1) linked to the N-terminus of a peptide antigen (A), which is an octapeptide (PA1'-PA2'-PA3'-PA4'PA5'-PA6'-PA7 '-PA8'), for example PN4-PN3-PN2-PN1-PA1'-PA2'-PA3'-PA4'-PA5'-PA6'-PA7'-PA8', the amide bond between PN1-PA1' is recognized and hydrolyzed by the enzyme .

In some embodiments of the present invention, the N-terminal extension (B1) is an enzyme-degradable tetrapeptide that is recognized by endosomal proteases, wherein the PN1 position of the tetrapeptide extension (e.g., PN4-PN3-PN2-PN1) is selected in a preferred embodiment of the present invention from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine, for example PN4-PN3-PN2-Arg; 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 of the present invention, the N-terminal extension (B1) is an enzyme-degradable tripeptide that is recognized by endosomal proteases, wherein the PN1 position of the tripeptide extension (e.g., PN3-PN2-PN1), in a preferred embodiment of the present invention, is 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 of the present invention, the N-terminal extension (B1) is an enzyme-degradable dipeptide that is recognized by endosomal proteases, wherein the PN1 position of the dipeptide extension (e.g., PN2-PN1), in a preferred embodiment of the present invention is selected from arginine, lysine , citrulline, glutamine, threonine, leucine, norleucine or methionine; and PN2 is selected from glycine, valine, leucine or isoleucine. In other further embodiments of the present invention, the N-terminal extension (B1) is an amino acid that is recognized by endosomal proteases, wherein the PN1 position, in a preferred embodiment of the present invention, is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine, or methionine.

In other embodiments of the present invention, the N-terminal extension (B1) is an enzyme-degradable peptide that is recognized by the immunoproteasome, wherein

- 31 046161 P1 extension tetrapeptide (PN4-PN3-PN2-PN1), in a preferred embodiment of the present invention selected from isoleucine, leucine, norleucine or valine, for example PN4-PN3PN2-Leu.

In additional embodiments of the present invention, the N-terminal extension (B1) is an enzyme-degradable peptide that is recognized by both endosomal proteases and the immunoproteasome, wherein the PN5 and PN1 positions of the octapeptide extension (PN8-PN7PN6-PN5-PN4-PN3 -PN2-PN1) selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine for the PN5 position recognized by cathepsins, and from isoleucine, leucine, norleucine or valine for the PN1 position recognized by the immunoproteasome; for example, PN8PN7-PN6-Arg-PN4-PN3-PN2-Leu. A non-limiting example of an N-terminal extension (B1) recognized by cathepsins and the immunoproteasome is Lys-Pro-Leu-Arg-Tyr-Leu-Leu-Leu (SEQ ID NO: 3).

Non-limiting examples of tetrapeptide N-terminal extensions (B1) that are recognized by the immunoproteasome include: Ser-Leu-Val-Cit, Ser-Leu-Val-Leu (SEQ ID NO: 4), Ser-Pro-Val-Cit, GluLeu-Val-Arg (SEQ ID NO: 5), Ser-Pro-Val-Arg (SEQ ID NO: 6), Ser-Leu-Val-Arg (SEQ ID NO: 7), Lys-ProLeu-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, GlyVal- Ser. Non-limiting examples of dipeptide N-terminal extensions (B1) include: Val-Cit, ValLeu, Val-Arg, Leu-Arg. Non-limiting examples of N-terminal single amino acid extensions (B1) include: Cit, Arg, Leu or Lys. In the examples above, Arg can be replaced with Lys; Lys can be replaced by Arg; Glu can be replaced with Asp; a Asp can be replaced by Glu. It should be noted that Cit = citrulline.

In some embodiments of the present invention, the extension (B2) is a degradable peptide linked to the C-terminal residue of the peptide antigen (A) and consists of amino acid sequences that are recognized and hydrolyzed by certain proteases. In some embodiments of the present invention, the C-terminal extension (B2) is a peptide sequence of about 1 to 8 amino acids in length, such as 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length, typically no more than 10 amino acids. In preferred embodiments of the present invention, the C-terminal extension (B2) is linked to the peptide antigen (A) via an amide bond formed between the C-terminal carboxyl group of the peptide antigen (A) and the alpha-amine residue of the N-terminal extension (B2). The amide bond between B2 and peptide antigen (A) can be cleaved by enzymes. It should be noted that amino acid positions are usually numbered in order from proximal to distal positions from the cleavage site, with terminal amino acid positions relative to the cleavage site being indicated by a simple symbol (eg, Pn'). For example, in relation to the extension of a tetrapeptide (PC1'-PC2'-PC3'-PC4') linked to the C-terminus of an octapeptide antigen (PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1), for example, PA8-PA7 -PA6PA5-PA4-PA3-PA2-PA1-PC1'-PC2'-PC3'-PC4', the amide bond between PA1-PC1' is recognized and hydrolyzed by the enzyme.

In preferred embodiments of the present invention, the C-terminal extensions (B2) are amino acid sequences that are selected to promote immunoproteasome recognition and degradation, and optional recognition by endosomal proteases. Since peptide antigens (A) typically contain a C-terminal residue, such as leucine, which stimulates hydrolysis by the immunoproteasome, for example, in the amide bond proximal to the C-terminal residue of the peptide antigen (A), it is necessary to select extensions linked to C-terminus of the peptide antigen (A), to stimulate recognition by the immunoproteasome and cleavage at the amide bond proximal to the C-terminus of the peptide antigen (A). The immunoproteasome has a preference for small, uncharged amino acids at position PC1', which is adjacent to the C-terminal amino acid, PA1, of the peptide antigen (A), for example, the amide bond between PA1-PC1'. However, endosomal proteases prefer bulky hydrophobic amino acids (eg, leucine, norleucine, methionine, or glutamine) and basic amino acids (eg, arginine and lysine). Therefore, C-terminal extensions can be selected to promote recognition by either or both classes of proteases.

In some embodiments of the present invention, a peptide antigen (A) with the sequence PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1 is linked to a C-terminal peptide extension (B2) with the sequence PC1'...PCn', wherein n represents an integer from 1 to 8, for example, PA8PA7-PA6-PA4-RAZ-PA2-PA1-RS1'...RSp'. The composition of the C-terminal extension (B2) depends on the length of the extension sequence used. In some embodiments of the present invention, the C-terminal extension, B2, is a single amino acid PC1' selected from Gly, Ala, Ser, Arg, Lys, Cit, Gln, Thr, Leu, Me, or Met. In further embodiments of the present invention, the C-terminal extension, B2, is a dipeptide PC1'-PC2', wherein PC1' is selected from Gly, Ala or Ser; a PC2' is selected from Gly, Ala, Ser, Pro, Arg, Lys, Cit, Gln, Thr, Leu, Me or Met. In additional embodiments of the present invention, the C-terminal extension of B2 is

- 32 046161 is a tripeptide PC1'-PC2'-PC3', wherein P1' is selected from Gly, Ala or Ser; PC2' selected from Gly, Ala, Ser or Pro; a PC3' is selected from Gly, Ser, Arg, Lys, Cit, Gln, Thr, Leu, Me or Met.

In additional embodiments of the present invention, 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 additional embodiments of the present invention, the C-terminal extension, B2, is a pentapeptide, PC1'-PC2'-PC3'PC4'-PC5', wherein 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 additional embodiments of the present invention, the C-terminal extension, B2, is a hexapeptide, PC1'-PC2'-PC3'-PC4'-PC5'-PC6', wherein 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.

Non-limiting examples of hexapeptide C-terminal extensions (B2) include Gly-Gly-LysLeu-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), LysPro-Leu-Arg (SEQ ID NO: 8), Glu-Leu-Val-Cit, Glu-Leu-Val-Leu (SEQ ID NO: 10), Glu-Pro-Val-Cit, Glu-GlyVal-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 C-terminal single amino acid (B2) extensions include Gly, Ser, Ala, Arg, Lys, Cit, Val, Leu, Met, Thr, Gln or Nle. In the examples above, Arg can be replaced with Lys; Lys can be replaced by Arg; Glu can be replaced with Asp; a Asp can be replaced with Glu.

The C-terminal linker (B2) linked to the C-terminus of the peptide antigen (A) can be selected for recognition (ie, hydrolysis) by both the immunoproteasome and endosomal proteases. In a non-limiting example, a peptide antigen (A) with the sequence PA8-PA7-PA6-PA5-PA4-PA3PA2-PA1 is C-terminally linked to a C-terminal tetrapeptide extension (B2) with the sequence PC1'PC2'-PC3'-PC4' wherein PC1' is selected from glycine, alanine or serine, and PC4' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine or methionine, for example, Ser-P3-P2-Arg. In some embodiments of the present invention, an antigen with the sequence PA8-PA7PA6-PA5-PA4-PA3-PA2-PA1 is C-terminally linked to a C-terminal hexapeptide extension (B2) to 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, for example, Gly- Gly-PC3'-PC4'-PC5'-Arg. A limiting example of a C-terminal extension (B2) that stimulates processing by both the immunoproteasome and cathepsins that is linked to the C-terminus of a peptide antigen (A) is Gly-Gly-Lys-Pro-Leu-Arg (SEQ ID NO : 12). An additional non-limiting example of a C-terminal extension (B2) that is linked at the C-terminus of a peptide antigen (A) that favors processing by the immunoproteasome and cathepsins is Gly-Gly-Ser-Leu-Val-Cit or Gly-Gly-Ser- Pro-Val-Cit.

Linkers (L) and linker precursors (X1 and X2)

The subset of linkers that perform the specific function of site-selective binding, that is, joining or linking a peptide antigen (A) to a hydrophobic molecule (H) or particle (P), are referred to as Linkers (L). The linker (L) is formed by the reaction between the linker precursor X1 and the linker precursor X2. For example, a linker precursor X1 that is linked directly or indirectly, via an extension (B1 or B2) or a charged molecule (C), to a peptide antigen (A) may react with a linker precursor X2 attached to a hydrophobic molecule (H) or to the particle (P) to form a linker (L), which links the peptide antigen (A) to a hydrophobic molecule (H) or particle (P). The X1 linker precursor provides site-selective coupling of a peptide antigen (A) to a particle (P) or hydrophobic molecule (H). In some embodiments of the present invention, the peptide anti

- 33 046161 gene (A), linked either directly or by extension (B 1 or B2), to the linker precursor X1, can be produced and isolated, and then separately added to the particle (P) or hydrophobic molecule (H) that contains a linker precursor X2 that selectively reacts to form a linker (L) connecting the peptide antigen (A) to a particle (P) or hydrophobic molecule (H).

The linker (L) or linker precursor X1 can be linked to the peptide antigen (A) either at the N- or C-terminus of the peptide antigen either directly or indirectly through an N-terminal extension (B1) or a C-terminal extension (B2), respectively. In preferred embodiments of the present invention, the linker (L) or linker precursor X1 is linked to the peptide antigen (A) or extension (B1 or B2) via an amide bond. It should be noted that a linker (L) or linker precursor X1 linked directly to the N- or C-terminus of a peptide antigen is not considered an extension.

Suitable X1 linker precursors are those that selectively react with X2 linker precursors on the particle (P) or hydrophobic molecule (H) without linkages occurring at any other site on the peptide antigen (A) or additional extensions (B1 and/or B2 ), or an additionally charged molecule (C). This selectivity is important to ensure the formation of adhesion between the peptide antigen (A) and the particle (P) or hydrophobic molecule (H) without modification of the peptide antigen (A). The linker (L) can link the peptide antigen (A), directly or indirectly by extension (B1 or B2), to a particle (P) or hydrophobic molecule (H) through covalent and/or high-affinity interactions. In preferred embodiments of the present invention, the linker precursor X1 forms a covalent bond with the linker precursor X2 on the particle or hydrophobic molecule (H).

In preferred embodiments of the present invention, the linker (L) is formed as a result of a bioorthogonal click chemical reaction between linker precursors X1 and X2. In some embodiments of the present invention, the click reaction is a catalyst-free click reaction, such as a strain-promoted azide-alkyne cycloaddition reaction, which does not require the use of copper or any catalyst. Non-limiting examples of X1 linker precursors that allow bioorthogonal reactions include molecules containing functional groups selected from azides, alkynes, tetrazines and trans-cyclooctenes. In some embodiments of the present invention, the azide-containing linker precursor X1 reacts with the linker precursor X2 to form a triazole linker. In other embodiments of the present invention, a tetrazine-containing linker precursor X1 reacts with a trans-cyclooctene (TCO)-containing linker precursor X2 to form a linker containing a reverse Diels-Alder ligation product. In preferred embodiments of the present invention, the linker precursor X1 is a non-natural amino acid bearing an azide functionality that reacts with a linker precursor X2 containing an alkyne present that undergoes a 1,3-dipolar cycloaddition to form a stable triazole ring. In preferred embodiments of the present invention, the X2 linker precursor linked to the particle (P) or hydrophobic molecule (H) contains an alkyne that undergoes strain-promoted cycloaddition, such as dibenzocyclooctine (DBCO). In additional embodiments of the present invention, the linker precursor X1 is an alkyne that reacts with a linker precursor X2 containing an azide that is present on the particle (P) or hydrophobic molecule (H).

In other embodiments of the present invention, X1 linker precursors that allow site-selective reactivity depending on the composition of the peptide antigen (A) may contain functional groups that include thiols, hydrazines, ketones and aldehydes. In some embodiments of the present invention, 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 of the present invention, a hydrazine-containing linker precursor X1 reacts with a ketone or aldehyde-containing linker precursor X2 to form a hydrazine Linker. In some embodiments of the present invention, the linker precursor X1 is a natural or unnatural amino acid residue with a thiol functionality, such as a cysteine, that reacts with a linker precursor X2 containing a thiol-reactive functionality, such as a maleimide or pyridyl disulfide .

In some embodiments of the present invention, the X1 linker precursor is a peptide sequence that is ligated to another peptide sequence containing an X2 linker precursor provided on a particle (P) or hydrophobic molecule (H). In other embodiments of the present invention, the linker precursor X1 binds to a complementary molecule containing the linker precursor X2 on the particle (P) or

- 34 046161 hydrophobic molecule (H), through high-affinity, non-covalent interactions, for example, through supercoil interactions or electrostatic interactions. In other embodiments of the present invention, the X1 linker precursor binds to a protein, such as biotin, that forms high affinity interactions with the protein, such as streptavidin.

Those skilled in the art will appreciate that suitable pairs of functional groups or complementary molecules selected for the linker precursors X1 and X2 may be transposable between X1 and X2. For example, a triazole linker may 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 resulting in the formation of a linker can be placed on either X1 or X2.

The particular linker precursors (X1 and X2) and linkers (L) provided in this invention offer 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 throughout.

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

In some embodiments of the present invention, the X1 linker precursor is an amino acid linked to the C-terminus of a peptide antigen (A) either directly or indirectly through a B2 extension or a charged molecule (C) and has the formula:

wherein the functional group (FG) is selected to react in a specific manner with the FG on the X2 o linker precursor

FG and it are usually selected from amine, azide, hydrazine or thiol;

R is typically selected from OH or NH ₂ and y1 is any integer such as 1, 2, 3, 4, 5, 6, 7 or 8; and the alpha amine of the amino acid is usually linked to the C-terminal amino acid of the B2 extension or the C-terminal amino acid of the peptide antigen (A) if there is no B2 extension.

In some embodiments of the present invention, the X1 linker precursor is linked to the N-terminus of the peptide antigen (A) either directly or indirectly through an extension (B1 or B2) or a charged molecule (C) and has the formula o

wherein the functional group (FG) is selected to react with the X2 linker precursor and is typically selected from an amine, azide, hydrazine or thiol; y2 is any integer, usually 1, 2, 3, 4, 5, 6, 7 or 8; and the carbonyl is usually linked to the alpha amine of the N-terminal amino acid of the B1 extension or peptide antigen (A) when B1 is absent.

The X2 linker precursor, provided on a hydrophobic molecule (H) or Particle (P), contains a functional group that is selected to selectively react with the X1 linker precursor to form a linker (L). In some embodiments of the present invention, functional groups containing X2 include carbonyls, such as activated esters/carboxylic acids or ketones, which react with the amines or hydrazines provided on the linker precursor X1 to form amide-containing linkers (L) or hydrazone. In other embodiments of the present invention, functional groups containing X2 include azides, which react with alkynes provided on the X1 linker precursor to form triazoles. In other embodiments of the present invention, the functional group containing X2 is selected from maleimides or disulfides, which react with the thiols provided on the linker precursor X1 to form linkers (L) containing thioesters or disulfides. The X2 linker precursor can be attached to a hydrophobic molecule (H) or particle (P) by any suitable mechanisms. In some embodiments of the present invention, the hydrophobic molecule (H) contains a peptide, and X2 is linked to the N-terminus of the hydrophobic molecule based on the peptide (H).

The linker (L) may contain a triazole formed between the linker precursors X1 and X2 containing an azide and an alkyne, respectively. In some embodiments of the present invention, the triazole-containing linker is produced by the reaction of an azide-containing precursor

- 35 046161 linker X1 and an alkyne (for example, cyclooctine) containing a precursor of linker X2. In some embodiments of the present invention, the azide-containing linker precursor X1 is a non-natural amino acid bearing an azide functionality that is linked to the N-terminus of a peptide antigen (A) or extension (B1) or to the C-terminus of a peptide antigen (A) or extension (B2 ). The azide functionality offers a reactive handle that is linked either directly to the peptide antigen (A) or indirectly, through an N-terminal extension (B1) or a C-terminal extension (B2), and selectively reacts with an alkyne (e.g., cyclooctine) containing a precursor of the X2 linker, which is linked to a hydrophobic molecule (H) or particle (P), thereby forming a triazole linker (L), which connects the peptide antigen (A) to the hydrophobic molecule (H) or particle (P).

In some embodiments of the present invention, the X1 linker precursor is an azidoamino acid linked to the C-terminus of a peptide antigen (A) either directly or indirectly through a B2 extension or a charged molecule (C), and has the formula

in this case, the alpha-amine of the amino acid is linked to the C-terminal amino acid of B2 or peptide antigen (A), if there is no B2 extension; R ¹ is selected from OH or NH ₂ , an is any integer such as 1, 2, 3, 4, 5, 6, 7, 8. Non-limiting examples of azide-containing linker X1 precursors include 5-azido-2-aminopentanoic acid, 4-azido-2-aminobutanoic acid and 3-azido-2-aminopropanoic acid.

In other embodiments of the present invention, when the azide-containing X1 linker precursor is linked to an N-terminal extension (B1) or directly to the N-terminus of a peptide antigen (A) when B1 is absent, the X1 linker precursor has the formula

wherein the carbonyl is usually linked to the alpha-amine of the N-terminal amino acid of the B1 extension or peptide antigen (A) when 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-azidohexanoic acid, 5-azidopentanoic acid, 4-azidobutanoic acid and 3-azidopropanoic acid .

In some embodiments of the present invention, wherein the linker precursor X1 contains an azide, the linker precursor X2 contains an alkyne moiety. Non-limiting examples of alkynes include aliphatic alkynes, cyclooctynes such as dibenzylcyclooctine (DBCO or DIBO), difluorooctine (DIFO), and biarylazacyclooctynone (BARAC). In some particular embodiments of the present invention, the alkyne-containing linker precursor X2 includes a DBCO molecule.

In some embodiments of the present invention, the C-terminal extension (B2) is linked to the peptide antigen (A) via an amide bond at the C-terminus of the peptide antigen (A), which in turn is linked via a Linker (L) to a hydrophobic block (H ). An example of such a C-terminal linked peptide antigen conjugate is (A) _7-35 -B2-LH, where (A) _7-35 is a peptide antigen containing from 7 to 35 amino acids. In a non-limiting example, a peptide antigen (A) with the octapeptide sequence PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1 is C-terminally linked to a tetrapeptide extension (B2), for example Ser-Leu-Val-Arg (SEQ ID NO : 7), which is linked to the azidolysine of the azido-containing linker precursor (X1) (6-azido 2-aminohexanoic acid, Lys(N3)), which in turn reacts with the dibenzocyclooctyne (DBCO) moiety of the cyclooctyne-containing linker precursor (X2) , which is linked to a hydrophobic molecule (h) to produce PA8-PA7-PA6-PA5-PA4-PA3-PA2-PA1-Ser-Leu-Val-Arg-Lys(N3-DBCOH).

In some embodiments of the present invention, the peptide antigen (A) is linked to an N-terminal extension (B1) at the N-terminus of the peptide antigen (A), which in turn is linked via a linker (L) to a hydrophobic block (H). In a non-limiting example, a peptide antigen with the sequence PA1'-PA2'-PA3'-PA4'-PA5'-PA6'-PA7'-PA8' is linked at the 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 dibenzocyclooctine (DBCO) moiety of the cyclooctine-containing linker precursor (X2), which is linked to the hydrophobic molecule (H): H-DBCO-Azp-Ser-Leu-Val-Arg-PA1'-PA2'-PA3'-PA4'-PA5'-PA6'-PA7'-PA8'.

- 36 046161

Other linkers and couplings

Linkers generally refer to any molecules that connect any at least two heterologous peptide antigen conjugate molecules and may additionally perform any of at least one of the following functions: i) increase or decrease water solubility; ii) increase the distance between any two components, that is, heterologous molecules of the peptide antigen conjugate; III) impart rigidity or flexibility; or IV) control/modulate the rate of degradation/hydrolysis of the cohesion between any at least two heterologous molecules. Specific types of linkers used herein are named. Linkers (L) are a specific type of linker molecule used to site-specifically link a peptide antigen (A) to a particle (P) or hydrophobic molecule (H), either directly or indirectly through extension (B1 or B2). It should be noted that N- and C-terminal extensions, B1 and B2, placed at the N- and C-terminus of the peptide antigen (A), respectively, can be linked to a heterologous molecule, such as a charged molecule (C), Linker (L), Particle (P) or hydrophobic molecule (H), or any heterologous molecule, and therefore function as a linker. For clarity, although any molecule placed at the N- or C-terminus of a peptide antigen (A) represents an N- or C-terminal extension (B1 or B2), the extension can also serve as a linker when it is linked to another molecule , for example, with a charged molecule (C). Thus, as defined herein, the linker at the N- or C-terminus of an antigen is always an extension (B1 or B2), but the extension is not always a linker.

Linkers used to connect any two components of a peptide antigen conjugate, for example, a peptide antigen (A) linked to a hydrophobic molecule (H), indirectly by extension (B1 or B2), can be produced by any suitable mechanisms. The linker can use covalent or non-covalent mechanisms to connect any at least two components, that is, heterologous molecules, for example, a peptide antigen (A) and a hydrophobic molecule (H).

In preferred embodiments of the present invention, the linker can connect, that is, link, any two components of the peptide antigen conjugate through a covalent bond. Covalent bonds are the preferred linkages used to connect any two components, that is, heterologous molecules of a peptide antigen conjugate and ensure that neither component is able to immediately disperse from the other components, for example, the peptide antigen and the hydrophobic molecule (H) after administration subject. In addition, covalent linkages generally offer greater stability than non-covalent linkages and help ensure that each component of the peptide antigen conjugate is co-delivered to the antigen presenting cells in the proportions of each component introduced or close to those proportions.

In a non-limiting example of covalent linkage, a click chemistry reaction can produce a triazole that links, that is, joins, any two components of a peptide antigen conjugate. In some embodiments of the present invention, the click reaction is a strain-promoted [3+2] azide-alkyne cycloaddition reaction. An alkyne group and an azide group can be offered on the corresponding molecules containing the peptide antigen conjugate, which must be linked by click chemistry. In some embodiments of the present invention, a ligand, such as a TLR agonist bearing an azide functionality, is linked to a hydrophobic molecule (H) having a corresponding reactive group, such as an alkyne, such as dibenzylcyclooctine (DBCO). In additional embodiments of the present invention, the X1 linker precursor bearing a thiol functionality is linked either directly or through an N- or C-terminal extension (B1 or B2) to a peptide antigen (A). The thiol can be linked to a hydrophobic molecule (H) having a corresponding reactive group, such as an alkyne, alkene, maleimide, resulting in a thioether linkage, or the thiol can react with a pyridyl disulfide, for example, resulting in a disulfide linkage. In some embodiments of the present invention, the amine is provided on one molecule and can be linked to another molecule by reacting the amine with any suitable electrophilic group, such as carboxylic acids, acid chlorides, or activated esters (e.g., N-hydroxysuccinimide (NHS) ester), which leads to the formation of an amide bond, or alkenes (via Michael addition), aldehydes and ketones (via a Schiff base) can react with the amine. In preferred embodiments of the present invention, the linker precursor X1, additional N- and C-terminal extensions (B1 and B2), an additional charged molecule (C) and a peptide antigen (A) are linked to each other via amide bonds as a single peptide sequence produced by solid-phase synthesis peptides. There are a number of suitable reactions available that result in a covalent bond, that is, a bond that will be known to one skilled in the art.

Type of linker used to connect any at least two heterologous molecules

- 37 046161 cool, that is, different components of the peptide antigen conjugate, can be selected to perform specific functions and meet specific application requirements. Typically, the linker is capable of forming covalent bonds between at least two heterologous molecules of a peptide antigen conjugate, the components being a peptide antigen (A), a particle (P) or a hydrophobic molecule (H), additional N- and C- terminal extensions (B1 and B2), additional linker (L); additional charged molecule (C); an additional second polymer (which is linked to a hydrophobic molecule (H)) and an additional ligand such as a PRR agonist.

There are many suitable linkers that are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, rigid aromatic linkers, flexible ethylene oxide linkers, peptide linkers, or combinations thereof. In some embodiments of the present invention, the carbon linker may include a C1-C18 alkane linker, such as a C4 lower alkyl; alkane linkers can serve to increase the space between at least two heterologous molecules, while longer chain alkane linkers can be used to impart hydrophobic characteristics. In an alternative embodiment of the present invention, hydrophilic linkers, such as ethylene oxide linkers, can be used instead of alkane linkers to increase the space between at least any heterologous molecules and increase water solubility. In other embodiments of the present invention, the linker may be an aromatic compound or a poly(aromatic) compound that imparts rigidity. The linker molecule may contain a hydrophilic or hydrophobic linker. In some embodiments of the present invention, the linker includes a degradable peptide sequence that is cleaved by an intracellular enzyme (such as cathepsin or immunoproteasome).

In some embodiments of the present invention, the linker may consist 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, for example, to reduce steric hindrance, or, in the case of a hydrophilic PEG linker, can be used to improve water solubility. The linker, such as PEG, can be a short linker that can be at least 2 monomers in length. The linker, such as PEG, can be from about 4 to about 24 monomers in length, for example, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 in length , 18, 19, 20, 21, 22, 23, 24 monomers. In some embodiments of the present invention, the ligand is linked to a hydrophobic molecule (H) via a PEG linker.

In some embodiments of the present invention, where the linker contains a carbon chain, the linker may contain a chain of about 1 or 2 and about 18 carbon atoms, such as a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 carbon atoms. In some embodiments of the present invention, where the linker contains a carbon chain, the linker may contain a chain of from about 12 to about 20 carbon atoms. In some embodiments of the present invention, where the linker contains a carbon chain, the linker may contain a chain containing no more than 18 carbon atoms. In some embodiments of the present invention, a ligand, such as a PRR agonist, is linked to a hydrophobic molecule (H) via a lower alkyl.

In some embodiments of the present invention, the linker is cleaved under intracellular conditions, whereby cleavage of the linker results in the release of any component linked to the linker, such as a peptide antigen.

For example, the linker can be cleaved by enzymes located in intracellular vesicles (eg, lysosome, endosome, or caveolae), or by enzymes in the cytosol, such as the proteasome, or the immunoproteasome. The linker may be, for example, a peptide linker that is cleaved by protease enzymes, including, but not limited to, proteases that are localized in intracellular vesicles such as cathepsins in the lysosomal or endosomal compartment. The peptide linker typically has a length of 1-10 amino acids, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (e.g., up to 20) amino acids, such as at least 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20 amino acids. Certain dipeptides are known to be hydrolyzed by proteases, which include cathepsins such as cathepsins B and D, and plasmin (see, eg, Dubovchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker that is cleaved by the thiol-dependent protease cathepsin-B (eg, a Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 23)) may be used. Other examples of such linkers are described, for example, in US Pat. No. 6,214,345, incorporated herein by reference. In a specific embodiment of the present invention, the peptide linker cleavable by the intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, for example, US Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a Val-Cit linker).

Specific cleavable peptide sequences in the linker can be used to

- 38 046161 stimulation of processing by immune cells after intracellular uptake. For example, several embodiments of the immunogenic compositions disclosed herein form particles under aqueous conditions that are internalized by immune cells, such as antigen presenting cells (eg, dendritic cells). The cleavable peptide linker may be selected to promote processing (ie, hydrolysis) of the peptide linker following intracellular uptake by immune cells. The cleavable peptide linker sequence can be selected to stimulate processing by intracellular proteases, such as cathepsins in intracellular vesicles or the proteasome or immunoproteasome in the cytosolic space.

In some embodiments of the present invention, linkers consisting of peptide sequences of the formula Pn...P4-P3-P2-P1 are used to promote recognition by cathepsins, wherein 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. In a non-limiting example, a tetrapeptide linker of the formula P4P3-P2-P1 is amide linked to a heterologous molecule and has the sequence Lys-Pro-Leu-Arg (SEQ ID NO: 8). For clarity, amino acid residues (Pn) are numbered in order from proximal to distal from the cleavage site, which is the C-terminus of the P1 residue, for example, the amide bond between P1-P1' is hydrolyzed. Suitable peptide sequences that stimulate cleavage by endosomal and lysosomal proteases such as cathepsin are widely described in the literature (see: Choi, et al., J. Bid. Chem., 281: 12824-12832, 2006).

In several embodiments of the present invention, linkers consisting 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, wherein P1 is selected from basic residues and hydrophobic branched residues such as arginine, lysine, leucine, isoleucine and valine; 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, which is recognized by the proteasome, is linked via an amide bond in P1 to a heterologous molecule and has the sequence Tyr-Leu-Leu-Leu (SEQ ID NO: 24). Sequences that promote degradation by the proteasome or immunoproteasome can be used alone or in combination with cathepsin-cleavable linkers. In some embodiments of the present invention, amino acids that stimulate immunoproteasomal processing are linked to linkers that stimulate processing by endosomal proteases. A number of suitable sequences for promoting digestion by the immunoproteasome are widely described in the literature (see: Kletzel, 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).

In preferred embodiments of the present invention, the N- and C-terminal extensions (B1 and B2) that are linked to the N- and C-termini of the peptide antigen (A), respectively, are linked to additional molecules, for example a charged molecule (C) and a hydrophobic molecule (H), respectively, and consist of cleavable peptides. In some embodiments of the present invention, cleavable peptide extensions are placed on either or both N- and/or C-termini of the peptide antigen (A). Cleavable peptide extensions placed at the N-terminus of a peptide antigen (A) are N-terminal extensions (B1) but can additionally function as linkers when the extension is linked to another molecule, such as a charged molecule (C), in addition with peptide antigen. Extensions placed at the C-terminus of a peptide antigen (A) are C-terminal extensions (B2), but can additionally function as linkers when the extension is linked to another molecule, such as a hydrophobic molecule (H). The preferred sequences of cleavage peptides containing linkers at either the N- or C-terminal end (B1 and B2) are different and are described in more detail throughout.

In other embodiments of the present invention, any at least two components of the peptide antigen conjugates can be connected via a pH-sensitive linker that is sensitive to hydrolysis under acidic conditions. A variety of pH-sensitive couplings are known to those skilled in the art, which include, for example, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, or the like (see, for example, US Patent Nos. 5,122,368; 5824805; 5622929; Dubovchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661). In preferred embodiments of the present invention, the adhesion is stable at a physiological pH, for example, at a pH of about 7.4, but is hydrolyzed at a lysosomal pH, about pH 56.5. In some embodiments of the present invention, a ligand, such as a TLR-7/8 agonist, is linked to a hydrophobic molecule (H) via an FG that forms a pH-sensitive bond, such as the reaction between a ketone and hydrazine to form a pH-labile hydrazone bond . In other embodiments of the present invention, the peptide antigen (A) is linked or unlinked

- 39 046161 indirectly, or indirectly, by extension (B1 or B2), with a linker precursor X1, which carries a ketone group and linked to a linker precursor X2 containing hydrazine on a hydrophobic molecule (H). The advantage of a pH-sensitive coupling such as hydrazone is that the coupling is stable at physiological pH, approximately pH 7.4, but hydrolyzes at lower pH values, such as the pH of intracellular vesicles.

In other embodiments of the present invention, the linker contains a linkage that is cleaved under reducing conditions, such as a reducible disulfide bond. Many different linkers 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., Immunoconjugates: Antibody Conjugates in Radio Imaging and Therapy cancer (S.V. Vogel et al., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008). See also, US Patent No. 4880935.). In other embodiments of the present invention, the peptide antigen (A) is linked either directly or indirectly by extension (B1 or B2) to a linker precursor X1 that carries a thiol functionality that forms a disulfide bond with a linker precursor X2 containing a pyridyl disulfide, linked to a hydrophobic molecule (H).

In other further embodiments of the present invention, the linkage between any two components of a peptide antigen conjugate can be formed by an enzymatic reaction, such as ligation of an expressed protein, or chemo-enzymatic sortase reactions (see: Fierer, et al., Proc. Natl. Acad. Sci., 111:W1176-1181, 2014 and Taile et al., Nat. Protoc, 8: 1800-1807, 2013. (Smith et al., Bioconjug. Chem., 25: 788-795, 2014), or non-covalent high-affinity interactions, such as, for example, biotin-avidin interactions and supercoil interactions (Pechar et al., Biotechnol. Adv., 31:90-96, 2013), or by any suitable means that are 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).

Particle (P) or hydrophobic molecule (H):

The immunogenic compositions of the present invention contain peptide antigen conjugates, which further contain a preformed particle (P) or hydrophobic molecule (H). Peptide antigen conjugates containing particle (P) occur as particles in aqueous conditions. In some embodiments of the present invention, peptide antigen conjugates containing hydrophobic molecules (H) may occur as single soluble molecules in organic solvents (eg, DMSO) but aggregate into particles under aqueous conditions. In other embodiments of the present invention, peptide antigen conjugates containing hydrophobic molecules (H) may occur as single soluble molecules in organic solvents (eg, DMSO) and aqueous conditions under certain temperature and pH ranges, but aggregate into particles under aqueous conditions at certain temperature and pH ranges, for example, at physiological temperature and pH.

The purpose of the particle (P) or hydrophobic molecule (H) is to shape the peptide antigen conjugate into a particle form as a means to modulate pharmacokinetics and promote uptake by antigen presenting cells. The particles formed by peptide antigen conjugates should have a size of from about 10 to 10,000 nm in diameter. In preferred embodiments of the present invention, the particles are nanoparticles that are of a size that can be introduced into the endosomal system of cells (such as immune cells). Nanoparticles can have an average size range of from about 10 to about 500 nm in diameter. Thus, in some embodiments of the present invention, the nanoparticles may have an average size of 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 of the present invention, the nanoparticles may have an average size of about 10-50 nm, or about 10-100 nm, or about 10-200 nm, or about 10-500 nm in diameter. In preferred embodiments of the present invention, the particle size is from 20 to 200 nm in diameter. The particles in the composition may vary in size, but generally fall within the size ranges specified 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%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the particles in the composition will fall within the size ranges specified herein. In some embodiments of the present invention, the peptide antigen (A) may be linked to an extension (B1 or B2) that is linked either directly or via a linker (L) to particles (P) or hydrophobic molecules (H) that aggregate into particles that are too large for absorption by immune cells (eg, particles larger than 5000 nm) and form a depot at the injection site.

In some embodiments of the present invention, the peptide antigen (A) is linked to a preformed particle (P). The particle (P) may consist of hydrophobic materials such as polymers or lipids, cross-linked hydrophilic polymers such as hydrogels, or cross-linked hydrophobic polymers such as cross-linked polystyrene, which retain

- 40 046161 structure in aquatic conditions. Non-limiting examples of particles (P) include polymer particles such as poly(lactic-co-glycolic acid) (PLGA), polymersomes or polexmers; 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 (ie, Alum). In some embodiments of the present invention, particle (P) is a liposomal nanoparticle. In other embodiments of the present invention, the particle (P) is an iron particle. In other embodiments of the present invention, the particle (P) is a polymer particle.

The efficiency of coupling of the peptide antigen (A) to preformed particles (P) depends on the nature of the peptide antigen and the type of coupling used. For example, peptide antigens (A) can be adsorbed or incorporated into particles (P), and the efficiency of this process can be determined empirically for each peptide antigen (A) since the nature of the peptide antigen (A) can influence adsorption and incorporation. Peptide antigens (A) can be linked to a particle (P) through high-affinity interactions (e.g., electrostatic) or covalent bonds, and the particle (P) has a given number of reactive sites that will determine the number of peptide antigens (A) that can be associated with particle (P). For immunogenic compositions containing multiple different peptide antigens (A), for example 20 different peptide antigens (A), multiple copies of each type of peptide antigen (A) may 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).

A limitation of preformed particles (P) is that the ratio of antigen to particle (P) cannot be easily controlled. In alternative preferred embodiments of the present invention, the peptide antigen (A) is linked, either directly or via a linker (L), to a hydrophobic molecule (H) that promotes particle aggregation in aqueous conditions. A peptide antigen conjugate consisting of a peptide antigen (A), optionally linked by an extension (B1 or B2), which is linked either directly or via a linker (L), to a hydrophobic molecule (H), is a molecularly defined entity level, and the ratio of peptide antigens (A) to hydrophobic molecule (H) can be precisely controlled. In preferred embodiments of the present invention, the ratio of peptide antigen (A) to hydrophobic molecule (H) is 1:1. In additional non-limiting examples, the ratio of peptide antigens (A) to hydrophobic molecules (H) can be from 1:3 to 3:1.

Unlike preformed particles, a peptide antigen conjugate can be formed by linking a peptide antigen (A), directly or indirectly, through an extension (B 1 or B2) and/or a linker (L), with a hydrophobic block (H), producing a single chemically defined molecule. A hydrophobic molecule (H) is a molecule having substantially limited solubility in water or having amphiphilic properties and capable of assembling into supramolecular structures, such as micellar, nano- or microparticles under aqueous conditions. In preferred embodiments of the present invention, the hydrophobic molecule (H) is insoluble or forms micelles under aqueous conditions to a concentration of about 0.1 mg/ml or to a concentration of about 0.01 mg/ml.

The hydrophobic molecule (H) can be selected from any molecules containing higher alkanes, cyclic aromatic compounds, fatty acids, compounds derived from terpenes/isoprenes or polymers that have limited water solubility and/or amphiphilic characteristics, resulting in molecular aggregation into particles under aqueous conditions. Typical higher alkanes include, but are not limited to, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane and octadecane. Typical cyclic aromatic compounds include, but are not limited to, benzene and fused benzene ring structures or heterocyclic aromatic molecules. Typical saturated and unsaturated fatty acids include, but are not limited to, myristic acid, palmitic acid, stearic acid or oleic acid. In some embodiments of the present invention, the hydrophobic molecule (H) is a fatty acid, such as myristic acid. In other embodiments of the present invention, 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 of the present invention, the fatty acid or lipid-based hydrophobic molecule (H) may further comprise PEG. Typical compounds derived from terpenes/isoprene include sterol derivatives such as cholesterol and squalene. In some embodiments of the present invention, the hydrophobic molecule (H) comprises cholesterol.

In preferred embodiments of the present invention, the hydrophobic molecule (H) is a polymer with limited solubility in water or is amphiphilic and can

- 41 046161 can accumulate into particles, for example micelles, under aqueous conditions. Typical polymers include, but are not limited to, PLGA, hydrophobic poly(amino acids), poly(benzyl glutamate), polystyrene, ethylene oxide, propylene oxide and temperature sensitive polymers such as poly(K,N'-diethyl acrylamide), poly(N-n-propylacrylamide), poly(N-isopropylacrylamide), poly[di(ethylene glycol) methacrylate methyl ether] and certain PEGylated poly(amino acids) such as poly(2-methoxyethoxy)esteryl-E-glutamate). In some embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) consisting of hydrophobic amino acids. In preferred embodiments of the present invention, the poly(amino acid) containing hydrophobic molecule (H) is composed of amino acids that contain aromatic rings. In other embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) that is linked to Ligands, such as PRR agonists. In other embodiments of the present invention, the hydrophobic molecule (H) is an A-B diblock copolymer. In some embodiments of the present invention, the diblock copolymer is temperature sensitive and capable of aggregating into particles in response to a temperature shift. In some embodiments of the present invention, the hydrophobic molecule (H) containing an A-B diblock copolymer further contains ligands, 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.

Polymers

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 consist of at least one other type of monomer unit. The polymer may be a random copolymer or an alternating copolymer. The polymer may be a block copolymer, such as type A-B, or the polymer may consist of a graft copolymer, whereby the two polymers are linked through a polymer-like reaction.

The hydrophobic molecule (H) or particle (P) may contain polymers containing monomers of natural and/or non-natural origin and combinations thereof.

Natural biopolymers may include peptides composed of amino acids (sometimes referred to as poly(amino acids)); a specific example is poly(tryptophan). Natural biopolymer can be chemically modified. For example, biopolymers consisting of glutamic acid or lysine residues can be modified with a gamma-carboxyl or epsilon-amine group, respectively, for ligand attachment. Biopolymers may be polysaccharides, which may include, but are not limited to, glycogen, cellulose and dextran. Additional examples include polysaccharides that occur naturally, including alginate and chitosan. Polymers may also be composed of naturally occurring small molecules such as lactic acid or glycolic acid, or may be a copolymer of the two (i.e., PLGA).

In some embodiments of the present invention, the hydrophobic molecule (H) or particle (P) consists of an anionic (eg, poly(acid)) polymer or a cationic (eg, poly(basic)) polymer, or combinations of anionic and cationic polymers. Cationic polymers can bind to negatively charged peptides through electrostatic interactions or may be suitable for complexation with negatively charged nucleic acids such as DNA and RNA. In some embodiments of the present invention, the polymer is a water-insoluble zwitterion at pH 7.4, but it carries a net positive charge at pH less than about 6 and is water soluble. In some embodiments of the present invention, the hydrophobic molecule (H) containing the first polymer carries a positive or negative charge that is complementary to the negative or positive charge, respectively, on the second polymer, and the first and second polymers form an electrostatic complex by neutralizing the charge, such that makes the complex insoluble. In some embodiments of the present invention, the cationic polymer may be a naturally occurring or synthetic poly(amine), such as poly(lysine) or poly(ethylenimine) (PEI). In additional embodiments of the present invention, the cationic polymer may be a poly(amidoamine) (PAA) or poly(beta-amino ester) (PBAE) produced by the Michael addition reaction of amines to either bis(acrylamides) or bis(acrylic esters). ). Non-limiting examples of cationic polymers that can be used in the disclosed embodiments of the present invention include poly(ethylenimine), poly(allylanion hydrochloride; RAS), putrescine, cadaverine, poly(lysine) (PL), poly(arginine), poly(trimethyleneimine) , poly(tetramethyleneimine), poly(propyleneimine), aminoglycoside-polyamine, dideoxydiamino-b-cyclodextrin, spermine, spermidine, cadaverine, poly(2-dimethylamino)ethyl methacrylate, poly(histidine), cationized gelatin, dendrimers, chitosan, and any combination thereof . The cationic polymer may contain a quaternary ammonium group, such as the group present on the methylated

- 42 046161 chitosan. In an alternative embodiment of the present invention, the polymer may be an anionic polymer. In some non-limiting examples, the polyanionic polymer is poly(glutamic acid). In alternative embodiments of the present invention, the polyanionic polymer is poly(aspartic acid). The polymer may be a polyphosphoester polymer. The polymer may contain natural anionic polysaccharides, including, for example, alginic acid, consisting of 1-4)-linked β-D-mannuronate and guluronic acid. The polymer may contain nucleotides. Other polyanionic polymers may also be suitable.

In some embodiments of the present invention, the hydrophobic molecule (H) is a water-soluble cationic polymer at certain pH ranges, but is uncharged and insoluble in water at pH ranges close to the physiological pH of 7.4. In some embodiments of the present invention, the hydrophobic molecule (H) is a polymer that contains aromatic amines, wherein the pKa of the conjugate acid base of the aromatic amine is less than 7.5. At a pH below the pKa of aromatic amines, the aromatic amine is protonated and therefore imparts a positive charge to the polymer. A non-limiting example of a hydrophobic molecule (H) consisting of a polymer containing aromatic amines is poly(phenylalanine amine). In some embodiments of the present invention, the hydrophobic molecule (H) is a polymer that contains nitrogen heterocycles such that the pKa of the nitrogen atom containing the heterocycle is less than 7.5. At a pH value below the pKa of the nitrogen atom containing the heterocycle, the nitrogen is protonated and imparts a positive charge to the polymer. A non-limiting example of a hydrophobic molecule (H) consisting of a polymer containing a heterocycle with protonable (ie, basic) nitrogen atoms is poly(histidine). Herein, we report the unexpected discovery that hydrophobic molecules composed of polymers that contain protonatable nitrogen (e.g., an aromatic amine) offer unexpected improvements in fabrication, particle stability, and biological activity.

In some embodiments of the present invention, the hydrophobic molecule (H) may be a poly(diethylene glycol methyl ether methacrylate)-(DEGMA) polymer. In further embodiments of the present invention, the hydrophobic molecule (H) is a polymer, which may include monomers of (meth)acrylates, (meth)acrylamides, styryl and vinyl moieties. Specific examples of (meth)acrylates, (meth)acrylamides, and styryl- and vinyl-based monomers include 2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA), styrene, and vinylpyrrolidone (PVP), respectively. The polymer may be a thermoset polymer consisting of N-isopropylacrylamide (NIPAAm) monomers; Nisopropyl methacrylamide (NIPMAm); C^-diethylacrylamide (DEAAm); Na(C)-(1-hydroxymethyl)propyl methacrylamide (HMPMAm); C^-dimethylethyl methacrylate (DMEMA), 2-(2methoxyethoxy)ethyl methacrylate (DEGMA). In some embodiments of the present invention, the hydrophobic polymer is a polymer containing HPMA or HPMA DEGMA monomers. In some embodiments of the present invention, the polymer comprising HPMA and DEGMA monomers is an A-B diblock polymer. An unexpected finding reported here is that peptide antigens (A) linked to A-B diblock copolymers containing a hydrophilic HPMA block accumulate into nanoparticle micelles of the same size, regardless of the composition of the peptide antigen (A ).

The hydrophobic molecule (H) may also contain polymers based on cyclic monomers, which include cyclic urethanes, cyclic ethers, cyclic amides, cyclic esters, cyclic anhydrides, cyclic sulfides and cyclic amines.

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; specific examples may include, but are not limited to, polycaprolactone and poly(ethylenimine) (PEI). Suitable polymers can also be produced by condensation reactions and include polyamides, polyacetals and polyesters.

In some embodiments of the present invention, the hydrophobic molecule (H) is a polymer that may include from 3 to 10,000 monomer units. In preferred embodiments of the present invention, the polymer includes from about 3 to 300 monomer units, such as from 3 to 10, such as 3, 4, 5, 6, 7, 8, 9 to 10 monomer units; or from about 10 to 100 monomer units, for example 10, 20, 30, 40, 50, 60, 70, 80, 90, 100; or from about 100 to 200 monomer units; or from about 200 to 300 monomer units, usually not more than 1000 monomer units. In some embodiments of the present invention, the polymer may contain up to 1000-10000 monomer units. Typically, at least five monomers are required to form a hydrophobic molecule (H) of sufficient size to stimulate the formation of peptide antigen conjugate particles, however, unexpectedly, hydrophobic molecules (H) consisting of polymers with only 3 monomers, which include aromatic rings, it was

- 43 046161 is sufficient to control the accumulation of peptide antigen conjugate particles. Increasing the polymer length from 3 to 5 and from 5 to 10 monomers increases the strength of the forces stimulating particle formation, resulting in more stable and larger particles formed by peptide antigen conjugates. In preferred embodiments of the present invention, the hydrophobic molecule (H) containing the peptide antigen conjugate is a polymer consisting of 5-100 monomers, resulting in particles with a diameter of approximately 10-300 nm in aqueous conditions. In additional embodiments of the present invention, the polymer containing the hydrophobic molecule (H) consists of about 300 monomers and results in peptide antigen conjugates that aggregate into particles ranging in size from about 20 to 500 nm or about 100-500 nm.

In some embodiments of the present invention, the average molecular weight of the polymer containing the hydrophobic molecule (H) may be from about 1000 to 1000000 g/mol. In preferred embodiments of the present invention, the average molecular weight of the polymer is from about 1000 to 60,000 g/mol. In some embodiments of the present invention, the molecular weight of the polymer is from about 1000 to 5000, or from about 5000 to 10000, or from about 10000 to 20000, or from about 20000 to 30000, or from about 25000 to 60000. In some embodiments of the present invention, the hydrophobic molecule (H) is an A-B type diblock polymer with an average molecular weight of from about 10,000 g/mol to about 60,000 g/mol, such as about 10,000 g/mol, 20,000 g/mol, 30,000 g/mol, 40,000 g /mol, 50000 g/mol or 60000 g/mol. In some embodiments of the present invention, the polymer is an A-B diblock polymer wherein the molecular weight ratio of the A block to the B blocks is from about 1:5 to about 5:1. In non-limiting examples, the A-B diblock polymer is an average molecular weight of approximately 60,000 g/mol consists of block A with an average molecular weight of approximately 10,000 g/mol and block B with an average molecular weight of approximately 50,000 g/mol; block A with an average molecular weight of about 20,000 g/mol and block B with an average molecular weight of about 40,000 g/mol; block A with an average molecular weight of about 30,000 g/mol and block B with an average molecular weight of about 30,000 g/mol; block A with an average molecular weight of about 40,000 g/mol and block B with an average molecular weight of about 20,000 g/mol; block A with an average molecular weight of approximately 50,000 g/mol and block B with an average molecular weight of approximately 10,000 g/mol.

The polydispersity, Mw/Mn, of the polymer can be from about 1.0 to about 5.0. Polymers can be formed by various polymerization methods. Peptide and nucleotide based polymers can be produced by solid phase synthesis and will have a polydispersity of 1.0 because the polymers are molecularly defined. Polymers formed by chain growth polymerization will have a polydispersity >1.0. Polymers can be synthesized by living polymerization or solution-free radial polymerization methods. In preferred embodiments of the present invention, peptide-based biopolymers are synthesized via solid phase peptide synthesis. Peptide-based polymers (or poly(amino acids)) containing amino acids with aromatic rings such as tryptophan, although hydrophobic under aqueous conditions, provide unexpected improvements in fabrication via solid-phase synthesis compared to peptides (or poly(amino acids)). ) without aromatic rings. Thus, in preferred embodiments of the present invention, the hydrophobic molecules (H) based on peptides produced by solid phase synthesis include amino acids containing aromatic rings. In additional embodiments of the present invention, acrylamide- and acrylate-based polymers are synthesized by reversible addition and fragmentation polymerization (RAFT). In additional embodiments of the present invention, poly(amino acids) and poly(phosphoesters) are synthesized by ring opening polymerization.

In some embodiments of the present invention, the hydrophobic molecule (H) may comprise a polymer that further contains a ligand or ligands, such as PRR agonists. In some embodiments of the present invention, polymer-based hydrophobic molecules (H) may include monomers that contain at least one functional group that can be linked to a ligand or a linker that can be linked to a ligand. In other embodiments of the present invention, the polymer-based hydrophobic molecule (H) may contain ligands that are linked to the ends and/or side chains of the polymer.

In preferred embodiments of the immunogenic compositions of the present invention for the treatment or prevention of cancer and infectious diseases, the hydrophobic molecule (H) is a polymer that is linked to ligands. In some embodiments of the present invention, the ligand that is linked to the hydrophobic molecule (H) on the polymer contains an aromatic ring structure. In some embodiments of the present invention, the ligand bound to the polymer is hydrophobic and promotes increased stability of the particles formed by the peptide antigen conjugate under aqueous conditions. In other variants

- 44 046161 In the implementation of the present invention, the ligand that is linked to the hydrophobic molecule (H) based on the polymer contains a heterocyclic aromatic ring. In some embodiments of the present invention, the ligand that is linked to the hydrophobic molecule (H) on the polymer contains an aromatic ring further comprising an arylamine. In some embodiments of the present invention, the ligand attached to the hydrophobic molecule (H) is a hydrophobic ligand containing a heterocyclic aromatic ring, optionally the hydrophobic ligand further comprising an aromatic amine (ie, Ar-NH2). In embodiments of the present invention, while the hydrophobic molecule (H) contains a ligand that contains an aromatic group, optionally containing a heterocycle and/or arylamine, we report the unexpected discovery that such a hydrophobic molecule (H) is highly soluble in pharmaceutically acceptable organic solvents such as DMSO and ethanol, but insoluble in aqueous buffer solutions.

In some embodiments, the ligand coupled to the hydrophobic molecule (H) on the polymer is a pattern recognition receptor agonist (PRRa), such as a STING, NOD, or TLR agonist that has adjuvant properties. The adjuvant ligand coupled to the polymer may be or be derived from any suitable adjuvant compound, such as a PRR agonist. Suitable ligands with adjuvant properties include compounds that include small organic molecules, that is, molecules having a molecular weight of less than about 3000 daltons, although, in some embodiments of the present invention, the adjuvant may have a molecular weight of less than about 700 daltons. and in some cases, the adjuvant may have a molecular weight of from about 200 daltons to about 700 daltons.

The hydrophobic molecule (H), in preferred embodiments of the immunogenic compositions of the present invention, used for the treatment or prevention of cancer and infectious diseases, is a polymer linked to ligands having adjuvant properties. Ligands with adjuvant properties, such as PRR agonists, can be linked to the side chains or terminal groups of the polymer via any suitable linker. In some embodiments of the present invention, monomers containing a polymer-based hydrophobic molecule (H) contain a side chain containing at least one functional group that can be linked to a ligand with adjuvant properties, or to a linker that can be linked to the ligand with adjuvant properties. In some embodiments of the present invention, the polymer-based hydrophobic molecule (H) contains a ligand with adjuvant properties, all of the polymer monomers are linked to the ligand with adjuvant properties. In other embodiments of the present invention, while the hydrophobic molecule (H) contains a ligand with adjuvant properties, not all monomers in the polymer are linked to the adjuvant.

In some embodiments of the present invention, wherein the hydrophobic molecule (H) comprises a polymer linked to a ligand with adjuvant properties through monomer units distributed along the polymer backbone, increasing the density of the ligand on the polymer results in a surprising improvement in immune responses to the peptide antigen (H).

In certain embodiments of the present invention, the molar ratio of ligands with adjuvant properties, such as PRR agonists, to polymer monomers can be selected from the following: from about 1:100 to 1:1 mol/mol (or from about 1 to about 100 moles). %), for example from 1: 2.5 to 1:1 mol/mol.

The density of adjuvant ligand, such as PRR agonists, bound to the polymer can be varied as needed for specific applications. Ligand with adjuvant properties, such as PRR agonists, can be coupled to the polymer from 1 to 100 mol.%, for example, from 1 to 10 mol.% or from 50-100 mol.%. Mol% refers to the percentage of polymer-containing monomers that are bound to a ligand with adjuvant properties, such as PRR agonists. For example, 10 mol% ligand (eg, PRR agonists) equals 10 ligand-linked monomer units out of a total of 100 monomer units. The remaining 90 may be monomeric units forming macromolecules that are not linked to a ligand.

The density of ligands, such as adjuvant ligands coupled to a polymer-based hydrophobic molecule (H), should be selected to ensure that the peptide antigen conjugate (i) is soluble in pharmaceutically acceptable organic solvents such as DMSO; (ii) could form stable nanoparticles under aqueous conditions at physiological temperature and pH; and/or (iii) was capable of inducing an immune response, in particular a T-cell response, in the subject.

The optimal density of ligands, such as PRR agonists, bound to a polymer depends on the polymer composition, the length of the polymer, as well as the composition of the ligand. When the ligand is a hydrophobic/amphiphilic molecule with low water solubility, such as an imidazoquinoline-based Toll-like receptor-7 and -8 (TLR-7/8a) agonist, and one polymer is water-soluble (i.e., a non-linked polymer with a ligand, is soluble in water), the ligand, like

- 45 046161 typically adheres to the polymer at a density of approximately 20-100 mol%, when the polymer consists of approximately 5-30 monomer units; 10-50 mol.%, when the polymer consists of 30-100 monomer units; or at a density of 5-20 mol.%, when the polymer consists of 100-300 monomer units. Typically, the mol % of ligand with adjuvant properties is higher for shorter polymers and lower for longer polymers.

Optimal ligand densities with adjuvant properties, such as PRRa, attached to hydrophilic or temperature-sensitive polymers that exceed 10,000 g/mol and are based on comonomers selected from 2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA) ), styrene, vinylpyrrolidone (PVP), N-isopropylacrylamide (NIPAAm); Nisopropyl methacrylamide (NIPMAm); ^^-diethylacrylamide (DEAAm); N-(L)-(1hydroxymethyl)propyl methacrylamide (HMPMAm); ^^-dimethylethyl methacrylate (DMEMA), 2-(2methoxyethoxy)ethyl methacrylate (DEGMA) or substituted poly(phosphoesters) is from 1 to 25%, for example, the density of the adjuvant attached to the polymer can be about 1%, about 2%, approximately, 3%, approximately, 4%, approximately, 5%, approximately, 6%, approximately, 7%, approximately, 8%, approximately, 9%, approximately, 10%, approximately, 11%, approximately, 12%, approximately, 13%, approximately, 14%, approximately, 15%, approximately, 16%, approximately, 17%, approximately, 18%, approximately, 19%, approximately, 20%, approximately, 21%, approximately, 22%, approximately, 23%, approximately 24% or approximately 25%.

In some embodiments of the present invention, the hydrophobic molecule (H) is an amphiphilic A-B diblock copolymer, wherein one block is hydrophobic and the other block is hydrophilic. In some embodiments of the present invention, wherein the hydrophobic molecule (H) is an amphiphilic A-B diblock copolymer and the ligand is hydrophobic, such as a TLR-7/8 imidazoquinoline agonist, the ligand, in a preferred embodiment of the present invention, is linked with a hydrophobic block at a density of from about 1 to 50 mol.%, typically from about 1 to 20 mol.%. In some embodiments of the present invention, where the hydrophobic molecule (H) is an amphiphilic diblock copolymer of type A-B and the Ligand is hydrophilic, the Ligand, in a preferred embodiment of the present invention, is linked to the hydrophilic block at a density of from about 1 to 20 mol .%, or at the hydrophilic end of the hydrophilic block and, therefore, the A-B type diblock copolymer is semi-telechelic with respect to the Ligand. In a non-limiting example, the hydrophobic molecule (H) comprises a temperature-sensitive A-B diblock copolymer containing an HPMA block and a DEGMA block, and the imidazoquinoline agonist TLR-7/8 is coupled to the DEGMA block at a density of from about 1 to 5 mol .%. In a further non-limiting example, the hydrophobic molecule (H) comprises an A-B diblock polymer containing a hydrophobic HPMA copolymer block and a hydrophilic HPMA homopolymer block, and the imidazoquinoline agonist TLR-7/8 is linked to the hydrophobic HPMA copolymer block at a density of approximately 20 mol % . In some embodiments of the present invention, the hydrophobic molecule is a triblock copolymer, such as A-B-A compositions or other multiblock copolymers.

An unexpected discovery reported here is that peptide antigens (A) linked to A-B diblock copolymers containing a hydrophilic HPMA block coupled to ligands with adjuvant properties accumulate into uniformly sized nanoparticle micelles , independent of the composition of the peptide antigen (A), and this increased reliability of nanoparticle micelle formation was associated with increased intensity of T-cell immunity. In a non-limiting example, the peptide antigen (A) is linked to a diblock copolymer consisting of a hydrophobic HPMA copolymer block (i.e., pKHPMAGcoDMA-b-Al2B)] and a hydrophilic HPMA homopolymer block (i.e., p(HPMA)), via a triazole linker ( Lys(N3)-DBCO), with the formation of the peptide antigenic conjugate pHHPMAGcoDMA-b-AP2B)]-b-p(HPMA)}-DBCO-(Lys(N3))-A, while 2B is TLR-7/8a, also referred to as Compound 1. In further embodiments of the present invention, the peptide antigen (A) is linked to a diblock copolymer consisting of a hydrophobic DEGMA copolymer block (i.e., p[(DEGMA)-co-(MA-b-Ala-2B) ]) and the hydrophilic block of the HPMA homopolymer (i.e., p(HPMA)), via a triazole linker (Lys(N3)-DBCO), to form the peptide antigen conjugate p{[(DEGMA)-co-(MA-b-Ala- 2B)]-b-p(HPMA)}-DBCO-(Lys(N3))-A, wherein 2B is TLR7/8a, also referred to as Compound 1. In other embodiments of the present invention, the peptide antigen (A) is linked to a diblock copolymer consisting of a DEGMA homopolymer linked to a ligand with adjuvant properties (i.e., 2BXy-p[(DEGMA)) and a hydrophilic HPMA homopolymer block (i.e., p(HPMA)), via a triazole linker (Lys(N3) -DBCO), with the formation of the peptide antigenic conjugate 2ВХу-р{[(DEGMA)-co-(MA-b-Ala-2B)]-b-p(HPMA)}-DBCO(Lys(N3))-A, while peptide antigen (A) and 2BXy (TLR-7/8a, also called Compound 2) are linked at one site at opposite ends of the polymer, making the polymer heterotelechelic.

In some embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) polymer that is composed of glutamic acid comonomers

- 46 046161 acid or aspartic acid, and aromatic and/or hydrophobic amino acids, such as phenylalanine, amino phenylalanine amine (or phenylalanineamine), tryptophan, tyrosine, benzyl glutamate, histidine, leucine, isoleucine, norleucine and valine, and at least one a ligand with adjuvant properties, such as PRRa, is attached to the polymer via the gamma-carboxylic acid of glutamic acid or the beta-carboxylic acid of aspartic acid. In preferred embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) polymer consisting of comonomers of glutamic acid and tryptophan, wherein at least one ligand with adjuvant properties, for example PRRa, is linked to the glutamic acid residues by gamma carboxylic acid. In additional embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) polymer consisting of comonomers of lysine and aromatic and/or hydrophobic amino acids, such as phenylalanine, amino phenylalanine, histidine, tryptophan, tyrosine, benzyl glutamate , leucine, isoleucine, norleucine and valine, wherein at least one ligand with adjuvant properties, for example PRRa, is attached to the polymer via the epsilon-amine of lysine. In preferred embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) polymer consisting of lysine and tryptophan comonomers, with at least one ligand with adjuvant properties, such as PRRa, linked to lysine through an epsilon-amine. In preferred embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) copolymer linked to at least one Ligand with adjuvant properties, for example PRRa, the polymer is 530 amino acids in length, and the adjuvant is attached at a density of 20 to 100 mol%, such as 30%, 50%, 60%, 80% and 100 mol%. In additional embodiments of the present invention, the ligand is attached to only one end of the poly(amino acid) polymer, that is, to the semi-telechelate polymer. Herein, we report the unexpected discovery that hydrophobic molecules (H) composed of poly(amino acid)-based copolymers that additionally contain aromatic groups such as aromatic amino acids (e.g., phenylalanine, amino phenylalanine, histidine, tryptophan, tyrosine, benzyl glutamate), or aromatic Ligands (eg, imidazoquinolines) coupled to the polymer, lead to unexpected improvements in processability due to improved solubility in an organic solvent, and improved particle stability, and biological activity of peptide antigen conjugates, compared with poly(amino acids) predominantly consisting of aliphatic amino acids or aliphatic ligands. Thus, in preferred embodiments of the present invention, hydrophobic molecules (H) consisting of poly(amino acids) or other classes of polymers include at least one aromatic amino acid and/or a ligand that contains an aromatic group.

In additional embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) polymer consisting entirely of glutamic acid, aspartic acid, or unnatural amino acid residues bearing a carboxylic acid, wherein the ligand with adjuvant properties is linked to all of the following : glutamic acid, aspartic acid or unnatural amino acid residues, that is, a ligand with adjuvant properties is attached at a density of 100 mol.%. In additional embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) polymer consisting entirely of lysine or unnatural amino acids bearing a free amine, and the Ligand with adjuvant properties is linked to all of the following: lysine or naturally occurring amino acid residues, that is, the adjuvant is attached at a density of 100 mol.%. In additional embodiments of the present invention, PEGylated comonomers, such as γ-(2-methoxyethoxy)esteryl-Lglutamate), are included to impart temperature-sensing properties to the copolymer. In other embodiments of the present invention, temperature-sensitive polymers can be grafted onto pendant poly(amino acid) side chains to form a graft copolymer. In additional embodiments of the present invention, the temperature-sensitive polymer can be end-linked to poly(amino acid) polymers to form a temperature-sensitive diblock polymer. In other further embodiments of the present invention, a second polymer that is hydrophobic can be linked to a poly(amino acid) polymer that is linked to ligands with adjuvant properties, either through pendant side groups to form a graft copolymer, or to the end of the poly(amino acid) with formation of a diblock copolymer.

In some embodiments of the present invention, the hydrophobic molecule (H) is a poly(amino acid) polymer coupled to a ligand, such as a hydrophobic ligand with adjuvant properties, and has the formula

- 47 046161

Formula I

In Formula I, R ² is typically selected from one of hydrogen, hydroxyl or amine. In some embodiments of the present invention, R ² is linked to a ligand or other polymer via any suitable linker molecule. The number of methylene units, γ3, is typically from 1 to 6, for example 1, 2, 3, 4, 5 or 6. The N-terminal amine of a Formula I poly(amino acid) is usually linked to a precursor linker X2 or may be linked to a peptide antigen ( A) either directly or by extension (B1 or B2). The number of monomer repeats is denoted k and typically ranges from 3 to 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 of the present invention, linker X may be linked to a second polymer that is linked to ligands. In some embodiments of the present invention, the monomers may be linked to at least two different ligands.

In some embodiments of the present invention, the poly(amino acid) polymers of Formula I are

wherein y4 is any integer such as 0-6, such as 0, 1, 2, 3, 4, 5 or 6. The ligand may be linked to functional groups (FG) by any suitable means, and FG is any suitable a functional group, including an amine, carboxylic acid, thiol, hydroxyl, azide, alkyne, hydrazine, aldehyde or ketone, for attachment, i.e. coupling, to a ligand, either directly or via a linker.

When y3 is equal to 1, poly(amino acid) polymers of formula I are

The N-terminus of a poly(amino acid) of Formula I may be linked via a linker (L) to a peptide antigen (A) by any suitable means. In some embodiments of the present invention, the N-terminus of the Formula I poly(amino acid) 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 of the present invention, the N-terminus of a Formula I poly(amino acid) is linked to a linker precursor (X2) that reacts with a linker precursor (X1) that is linked, directly or by extension (B1 or B2), to a peptide antigen (A). In some embodiments of the present invention, a linker precursor (X2) containing a cyclooctyne (eg, DBCO) is attached to the N-terminus of a Formula I poly(amino acid) and reacts with an azide-containing linker precursor (X1) to form a triazole bond.

In some embodiments of the present invention, the hydrophobic poly(amino acid) molecule (H) may comprise hydrophobic amino acids (eg, aromatic amino acids) or hydrophobic amino acids (eg, aromatic amino acids) and ligand-linked amino acids, as well as additional amino acids such as charged or hydrophilic amino acids that are suitable for compensating or modulating the physical and chemical characteristics of a hydrophobic molecule (H) to produce a material that, in a preferred embodiment of the present invention, is soluble in organic solvents during manufacture but is capable of forming particles under aqueous conditions. Thus, in some embodiments of the present invention, the hydrophobic molecule (H) is

- 48 046161 polymer based on pol (amino acids), which has the formula oooo

Ligand

Formula II

The poly(amino acid) polymer of Formula II typically contains monomer 1 and additional monomers m, n and o. R ³ is typically selected from one of the following: hydrogen, hydroxyl or amine. In some embodiments of the present invention, R ³ is a ligand, such as a ligand with adjuvant properties, or other polymer that is linked to a hydrophobic molecule (H) through any suitable linker molecule. The number of methylene units, designated y5, y6, y7 and y8, is usually from 1 to 6, for example 1, 2, 3, 4, 5 or 6. The N-terminal amine of the poly(amino acid) of Formula I is usually linked to a precursor linker X2 or may be linked to a peptide antigen (A) either directly or by extension (B1 or B2). In exemplary embodiments of the present invention, the poly(amino acid) polymer of Formula II contains monomers 1 that are selected from any natural or unnatural amino acid, wherein R ⁴ is selected from lower alkyl or aromatic groups, and impart hydrophobic properties to the polymer backbone. In some embodiments of the present invention, the R ⁴ included in Formula II may be selected from

- 49 046161

wherein X of R ⁴ is any suitable linker.

In some embodiments of the present invention, the poly(amino acid) polymer of Formula II contains additional comonomers, m, that are selected from any natural or non-natural amino acid, such as a PEG amino acid spacer (for example, m from Formula II is -NH-(CH ₂ -CH ₂ -O) _y9 -(CH2) _y10 -(CO)-, wherein y9 is an integer, usually from 1 to 24 and y10 is an integer, usually from 1 to 3) or an amino acid with a small substituent, with here, for example, R ⁵ is selected from hydrogen, lower alkyl, or lower alkyl containing hydroxyl, and is proposed to increase the spacing or flexibility of the polymer backbone. In some embodiments of the present invention, the R ⁵ included in Formula II may be selected from

-pp ' ' b where b = 1 to 6 where b = 1-6.

In some embodiments of the present invention, the poly(amino acid) polymer of Formula II contains additional comonomers, n, that are selected from any natural or unnatural amino acid, wherein R ⁶ is selected from any group containing a functional group that carries a charge either permanently or at a certain pH. In some embodiments of the present invention, the R ⁶ included in Formula II may be selected from

- 50 046161

NH / H ₂ \ / H ₂ \ / ^H 2 \ n II —0С ή-ΝΗ ₂ “0 ^e -y-COOH —0 ^e J“ ^N -- ^s -- ^νη 2 where d = 1 to 6 , where d = 1 to 6 , where d = 1 to 6 ,

R/ _H2 \/ ^H2 \I

-(— C -)-St —f—C -)- N ⁺ --R where d = 1 to 6 where R = lower alkyl where d = 1 to 6 _R where R - lower alkyl

ABOUT

where d = 1-6, where d = 1-6, where d = 1-6, where d = 1-6, where R = lower alkyl, where d = 1-6, where R = lower alkyl, where d = 1-6, d = 1-6.

In some embodiments of the present invention, the poly(amino acid) polymer of Formula II contains additional comonomers, o, which are selected from any natural or unnatural amino acid, wherein the Ligand is linked, through any suitable linker, X, to the monomer, o. The ligand may be a ligand with adjuvant properties. The ligand linked to the poly(amino acids) of Formula II may be hydrophobic, hydrophilic, amphiphilic, charged or neutral in properties. The Formula II poly(amino acid) polymer containing o monomer may further contain l, m and n monomer units which compensate for the properties of the ligand attached to the o monomer.

In Formula II poly(amino acid) polymers, the number of monomer repeats is denoted as l, m, n and o, wherein the sum of l, m, n and o is typically any integer from 3 to 300. Each of the different types monomers, l, m, n or o, may be the same or different. The monomers designated as 1 impart hydrophobic properties to the poly(amino acid) polymer of Formula II, that is, they convert the polymer into a water-insoluble hydrophobic molecule (H). Hydrophobic monomers, 1, may be the same or different and usually contain an aromatic ring. In preferred embodiments of the present invention, the hydrophobic monomers, 1, contain a heterocyclic and/or amine-substituted aromatic ring. Additional comonomers, designated m, can be used to increase the flexibility or distance between different monomers containing the polymer backbone. Additional comonomers, designated n, contain charged functional groups. An additional comonomer, designated o, is used to attach a ligand, such as a PRR agonist. In some embodiments of the present invention, the ligand linked to the monomer, via any suitable linker, is a PRR agonist. In some embodiments of the present invention, the monomer, o, is linked to a ligand that carries a positive or negative charge, and is adjacent to the monomer, m, of the opposite charge. In some embodiments of the present invention, a charged comonomer, n, is located adjacent to a comonomer, o, containing a functional group of the opposite charge of the functional group containing comonomer n, and the opposite charges result in a zero net charge, such that monomer n functions to neutralize the charge, carried by a ligand attached to the o monomer.

The percentage of monomers, l, m, n and o, containing the poly(amino acid) polymer of Formula II depends on the specific application. In some embodiments of the present invention, the Formula II poly(amino acid) polymer consists entirely of l monomer. In other embodiments of the present invention, the poly(amino acid) polymer of Formula II consists of comonomers, l and o, such as about 5-95 mol% l monomer and about 95-5 mol% o monomer and about 95-5 mol% o monomer. In some embodiments of the present invention, the poly(amino acid) polymer of Formula II contains comonomers, l and m, wherein m provides space, that is, distance between hydrophobic monomers, l, and can reduce the stiffness of the polymer. In other embodiments of the present invention, the poly(amino acid) polymer of Formula II contains monomers l, m, and o, wherein the monomers m provide space between the bulky substituents containing the monomers l and o. In other embodiments of the present invention, the poly(amino acid) polymer of Formula II contains monomers l and o, and additional monomers m and n, wherein monomer n is used to modulate the charge of the polymer backbone. In certain embodiments of the present invention, the poly(amino acid) polymer of Formula II consists entirely of m and o monomers. In other embodiments,

- 51 046161 of the present invention, the poly(amino acid) polymer of Formula II consists entirely of m, n and o monomers or only n and o. In other embodiments of the present invention, the poly(amino acid) polymer of Formula II contains l, m, n, and o monomers.

If the poly(amino acid) polymer of Formula II contains an adjuvant, the percentage of monomers containing the polymer represented by monomer o, which is linked to the adjuvant by any suitable linker, is typically from 10 to 60%, for example from 2 to 12 amino acids of the polymer, the length of which is 20 amino acids, which is a monomer o. In some embodiments of the Formula II poly(amino acid) polymers of the present invention, l is the predominant monomer unit. In further embodiments of the present invention, the poly(amino acid) polymer of Formula II consists entirely of l monomer, that is, all monomers are l monomer, optionally with an adjuvant attached to the end of the poly(amino acid), either directly or indirectly, through a second polymer or through any suitable linker molecule.

If the Formula II polymer is a copolymer containing o monomers, then the ligand may be linked to pendant functional groups (FGs) distributed along the polymer backbone, as shown below:

oooh

FG

Ligand where y11 denotes the number of methylene units and is any integer such as 0-6, such as 0, 1, 2, 3, 4, 5 or 6. Functional group (FG) is any suitable functional group, including amine , carboxylic acid, thiol, hydroxyl, azide, alkyne, hydrazine, aldehyde or ketone, which provides attachment, that is, cohesion of the ligand, either directly or through a linker.

In some embodiments of the present invention, y5, y6, y7, and y8 are equal to 1, and the Formula II polymer is oooo

FG

I

Ligand

Ligands with adjuvant properties can be linked to any of the hydrophobic molecules (H) of the present invention. In certain embodiments of the present invention, ligands with adjuvant properties are coupled to polymers of Formula II. Ligands with adjuvant properties can be linked to poly(amino acid) polymers of Formula II via pendant functional groups (FG) on the monomers; at the ends of the polymer or indirectly, through another molecule or polymer that is grafted onto pendant functional groups (FGs) or at the ends of the polymer. In preferred embodiments of the present invention, the functional group (FG) of the o monomers links ligands with adjuvant properties, or other ligand molecules, to the poly(amino acid) backbone through a covalent bond. In some embodiments of the present invention, FG containing a Formula II o monomer may be linked to a second polymer. The FG included in Formula II may be selected from a carboxylic acid, aldehyde, ketone, amine, hydrazine, thiol, azide or alkyne, or any suitable functional group that can be used to link a ligand or other polymer to the polymer backbone.

In preferred embodiments of the present invention, the N-terminus of the poly(amino acid) of Formula II is linked via a linker (L) to a peptide antigen (A), either directly or by extension (B1 or B2), through the reaction of linker precursors X1 and X2. In some embodiments of the present invention, the N-terminus of the Formula II poly(amino acid) is linked directly (ie, there is no linker (L)) 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 of the present invention, the N-terminus of a Formula II poly(amino acid) is linked to a cyclooctine (DBCO)-containing linker precursor (X2), which reacts with an azide-containing linker precursor (X1), which is linked, either directly or by extension (B1 or B2), with peptide antigen (A).

- 52 046161

A poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) that can be linked either through an N-terminal amine, a C-terminal carboxylic acid, or through optional side chains, such as comonomer functional groups, directly or indirectly, through a linker (L) or extension (B1 or B2), with a peptide antigen (A). In some embodiments of the present invention, the poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) that is linked to a peptide antigen (A), resulting in a peptide antigen conjugate of the formula [C][B1]-A-[B2 ]-[L]-H, or [B1]-A-[B2]-[L]-H(C), with [ ] indicating that the group is optional.

In preferred embodiments of the present invention, the poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) that is linked at the N-terminus to a Linker (L) that is linked to a C-terminal extension (B2) that is linked to C - the end of the peptide antigen (A), which is linked at the N-terminus to the N-terminal extension (B1), which is linked to a charged molecule (C).

For example: S-B1-A-B2-T-N

In additional embodiments of the present invention, the charged molecule (C) may be linked directly to a hydrophobic molecule (H) consisting of a Formula I or Formula II poly(amino acid), or via a linker (L) that is linked to a peptide antigen (A) by elongation. As used herein, for A-B2-L(C)-H, the parenthetical notation is intended to indicate that L is concatenated with both C and H.

For example: A-B2-L(C)-H or A-B2-L-H(C)

In preferred embodiments of the present invention, the poly(amino acid) of Formula I or Formula II is a hydrophobic molecule (H) that is linked at the N-terminus to a Linker (L) that is linked to both an optionally charged (C) molecule and a C -terminal extension (B2), which is linked to the C-terminus of the peptide antigen (A).

For example: A-B2-(C-L)-H

In preferred embodiments of the present invention, the hydrophobic block comprises a poly(amino acid) with adjuvant ligand attached to pendant groups distributed along the poly(amino acid) backbone. A ligand with adjuvant properties can be either hydrophobic or hydrophilic, charged or uncharged in properties. In preferred embodiments of the present invention, the ligand with adjuvant properties is a PRR agonist.

In some embodiments of the present invention, the ligand with adjuvant properties may be a pattern recognition receptor (PRR) agonist. Non-limiting examples of pattern recognition receptor (PRR) agonists include the following: TLR-1/2/6 agonists (eg, lipopeptides and glycolipids, such as Pam2cys or Pam3cys lipopeptides); TLR3 agonists (eg, dsRNA such as PolyI:C, and nucleotide base analogs); TLR-4 agonists (eg, lipopolysaccharide (LPS) derivatives, eg monophosphoryl lipid A (MPL) and small molecule derivative or analogue of pyrimidoindole); TLR-5 agonists (eg, flagellin); TLR-7 and -8 agonists (eg, ssRNA and nucleotide base analogues, including derivatives of imidazoquinolines, hydroxyadenine, benzonaphthyridine and loxoribine); and TLR-9 agonists (eg, unmethylated CpGs); stimulator of interferon genes (STING) agonists (eg, cyclic dinucleotides such as cyclic diadenylate monophosphate); C-type lectin receptor (CLR) agonists (such as various mono-, di-, tri- and polymeric sugars, which may be linear or branched, for example mannose, Lewis-X trisaccharides, and so on); RIG-I-like receptor (RLR) agonists; and NOD-like receptor (NLR) agonists (such as peptidogylcanes and structural motifs from bacteria, such as mesodiaminopimelic acid and muramyl dipeptide); and their combinations. In some embodiments of the present invention, the pattern recognition receptor agonist may be a TLR agonist, such as an imidazoquinoline-based TLR-7/8 agonist. For example, a ligand with adjuvant properties may be imiquimod (R837) or resiquimod (R848), which are approved by the FDA for human use.

In some embodiments of the present invention, the ligand with adjuvant properties may be a TLR-7 agonist, a TLR-8 agonist, and/or a TLR-7/8 agonist. A variety of such agonists are known, including many different imidazoquinoline compounds.

Imidazoquinolines are used in the methods disclosed herein. Imidazoquinolines are synthetic immunomodulatory drugs that act by binding Toll-like receptors 7 and 8 (TLR-7/TLR-8) to antigen-presenting cells (eg, dendritic cells), structurally mimicking the natural ligand of these receptors, viral single-stranded RNA. Imidazoquinolines are heterocyclic compounds containing a fused quinoline-imidazole framework. Derivatives, salts (including hydrates, solvates and N-oxides) and prodrugs thereof are also contemplated by the present invention. Specific connections

- 53 046161 imidazoquinoline compounds are known in the art, see, for example, US Patent No. 6518265 and US Patent No. 4689338. In some non-limiting embodiments of the present invention, the imidazoquinoline compound is not imiquimod and/or is not resiquimod. In some embodiments of the present invention, ligand with adjuvant properties can be a small molecule, which has a 2-aminopyridine, merged with a five-membered nitrogen-containing heterocyclic ring, including, but this is not limited, imidazokhinolinamins and substituted imidazocynolinamins, such as, for example, amid-stained with imidasophinolinomins, sulfons, sulfons Dzema imidazokhinolinamins , urea-substituted imidazoquinoline amines, aryl ether-substituted imidazoquinoline amines, heterocycle ether-substituted imidazoquinoline amines, amide ester-substituted imidazoquinoline amines, sulfonamide ether-substituted imidazoquinoline amines, urea-substituted imidazoquinoline esters, thioether-substituted imidazoquinoline amines 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; tetrahydroimidazoquinoline amines, including, but not limited to, amide substituted tetrahydroimidazoquinoline amines, sulfonamide substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline amines, aryl ether substituted tetrahydroimidazoquinoline amines, heterocyclether substituted tetrahydroimidazoquinoline amines, amidoester-substituted tetrahydroimidazoquinoline amines, sulfonamide ester-substituted tetrahydroimidazoquinoline amines, urea-substituted tetrahydroimidazoquinoline ethers, thioether-substituted tetrahydroimidazoquinoline amines, hydroxylamine-substituted tetrahydroquinoline amines dazoquinoline amines , oxime-substituted tetrahydroimidazoquinoline amines and tetrahydroimidazoquinoline diamines;

imidazopyridine amines, including, but not limited to, amide-substituted imidazopyridine amines, sulfonamide-substituted imidazopyridine amines, urea-substituted imidazopyridine amines, arylester-substituted imidazopyridine amines, heterocycle ether-substituted imidazopyridine amines, amidoester-substituted imidazopyridine amines, sulfonamide ester-substituted imidazopyridine amines, urea-substituted imidazopyridine esters, and thioether-substituted imidazopyridine amines, 1,2 -bridged imidazoquinoline amines; 6,7-fused cycloalkylimidazopyridine amines; imidazonaphthyridine amines; tetrahydroimidazonaphthyridine amines; oxazolopyridine amines; thiazolopyridine amines; oxazolonaphthyridine amines; thiazolonaphthyridine amines; pyrazolopyridine amines; pyrazoloquinoline amines; tetrahydropyrazoloquinoline amines; pyrazolonaphthyridine amines; tetrahydropyrazolonaphthyridine amines; And

N-imidazo dimers fused with pyridine amines, quinoline amines, tetrahydroquinoline amines, naphthyridine amines or tetrahydronaphthyridine amines.

In some embodiments of the present invention, the ligand with adjuvant properties is an imidazoquinoline with the formula nh ₂

X / ^r7 U !·

Formula III

In Formula III, R ⁷ is selected from one of the following: hydrogen, optionally substituted lower alkyl, or optionally substituted lower ether; a R ⁸ is selected from one of the following: an optionally substituted arylamine or an optionally substituted lower alkylamine. R ⁸ may optionally be replaced by a linker that is combined with the polymer. An unexpected finding was that in some compounds where R ⁸ was selected from a lower alkylamine while the compound was less active than R ⁸ selected from an arylamine, the quality of the response improved. Thus, the moderately potent Formula III adjuvants resulted in better responses. Note: Formula III adjuvants are a type of ligand and may be referred to as Formula III adjuvants or ligands with adjuvant properties.

In some embodiments of the present invention, R ⁷ included in Formula III, , , 4c ^g )CH, Hg ", can be selected from hydrogen, ³ , or ³

In some embodiments of the present invention, R ⁸ may be selected from —(° ² )ξ ^ΝΗ 2 “CJ ---c ² --F \-^-C ² -^-NH ₂ '''-----' or

H ₂ / ⁼ \H ₂

---с $---с —nh ₂

- 54 046161 where e denotes the number of methylene units, which is an integer from 1 to

4.

In some embodiments of the present invention, R ⁸ may be n ₂ /=\ n ₂ -c -4 L -c -nh ₂

In some embodiments of the present invention, R ⁸ may be

-Vc/nh ₂

4.

In some embodiments of the present invention, R ⁷ may be m

L ^s / ^s z

3, _2 ² -/ ⁼ \-c ² - _nh and R ⁸ can represent.

Non-limiting examples of hydrophobic molecules (H) consisting of poly(amino acids) of Formula I coupled with adjuvants of Formula III include

in this case k is 3-300. For example, when k = 5, the peptide consists of 5 amino acids coupled to Formula III adjuvants. In some embodiments of the present invention, hydrophobic molecules (H) consisting of poly(amino acids) of Formula I linked to adjuvants of Formula III can be linked, either directly or indirectly, through a linker (L) and/or extension (either B1, or B2) with a peptide antigen (A) that is optionally linked to a charged molecule (C) by any suitable means to form a peptide antigen conjugate. In some embodiments of the present invention, the N-terminus of the Formula I poly(amino acid) linked to Formula III adjuvants 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 of the present invention, the N-terminus of a Formula I poly(amino acid) linked to Formula III adjuvants is linked to a click chemistry-capable linker precursor (X2), such as an alkyne or DBCO, or to a linker precursor X2 that is reactive with a thiol, for example a maleimide, which reacts with a precursor linker X2 that is linked directly or by extension (B1 or B2) to the peptide antigen (A). In preferred embodiments of the present invention, a DBCO linker precursor X1 is attached to the N-terminus of a Formula I poly(amino acid) linked to Formula III adjuvants and is used to react with a linker precursor X2 bearing an azide that is linked, either directly or via extension (B1 or B2), with peptide antigen (A).

A non-limiting example of a hydrophobic molecule (H) consisting of poly(amino acids) of Formula II coupled with adjuvants of Formula III includes

- 55 046161

For example, comonomer 1 is typically an integer from 3 to 300, and the optional comonomer o is typically an integer from 3 to 300 amino acid residues, with the sum of 1 and o typically being from 3 to 300. In an alternative embodiment of the present invention o is 0 and the polymer consists entirely of 1, that is, the polymer is a poly(tryptophan) polymer that is not bound to adjuvants. In some embodiments of the present invention, the N-terminus of a Formula II poly(amino acid) linked to Formula III adjuvants is linked, either directly or indirectly, via a Linker (L) and/or extension (B1 or B2), to a peptide antigen (A ) through any appropriate means. In some embodiments of the present invention, the N-terminus of the Formula II poly(amino acid) linked to Formula III adjuvants 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 of the present invention, the N-terminus of a Formula II poly(amino acid) linked to Formula III adjuvants is linked to a click chemistry-capable linker X2 precursor, such as DBCO, or to a linker X2 precursor that is reactive with a thiol, for example a maleimide, which reacts with a precursor linker X1 that is linked, either directly or by extension (B1 or B2), to the peptide antigen (A). In preferred embodiments of the present invention, the X2 DBCO linker precursor is attached to the N-terminus of a Formula II poly(amino acid) coupled to Formula III adjuvants and is used to react with an X1 linker precursor bearing an azide functionality.

An unexpected discovery disclosed herein is that the length, that is, the number of monomer units, of poly(amino acids) of either Formula I or Formula II coupled to Formula III adjuvants containing a hydrophobic molecule (H) of peptide antigen conjugates is a major determinant that influences the intensity of T cell responses generated against peptide antigen (A). Accordingly, peptide antigen conjugates consisting of hydrophobic molecules (H) consisting of Formula I or Formula II poly(amino acids) with at least 5 monomer units have been found to stimulate higher quality and intensity of T cell responses, compared to poly(amino acids) of the same formula, which are less than 5 amino acids in length. An additional discovery was that the amount of Formula III adjuvants conjugated to Formula I or Formula II poly(amino acids) also influenced the intensity of the immune response generated, with peptide antigen conjugates containing hydrophobic molecules (H) of Formula I or II containing at least 3 Formula III adjuvants result in higher intensity T cell responses compared to peptide antigen conjugates containing hydrophobic molecules (H) of Formula I or II containing less than 3 Formula III adjuvants. A non-limiting explanation for these findings is that the increased length of the hydrophobic molecule (H) of Formula I or Formula II coupled to Formula III adjuvants allows for the formation of micelles or other supramolecular structures upon coupling

- 56 046161 even with hydrophilic peptide antigens, and that such particle formation leads to improved immune responses, possibly due to improved pharmacokinetics and cellular uptake, which is characteristic of particles but not of soluble materials. An additional non-limiting explanation is that increasing the lengths of the hydrophobic molecule (H) consisting of Formula I or Formula II poly(amino acids) coupled to Formula III adjuvants results in improved stability, e.g., kinetic stability, of particles formed by peptide antigen conjugates in an aqueous buffer solution; Improved particle stability ensures that the particle remains intact, thereby delaying the clearance (either renal or hepatic) of peptide antigen conjugates containing the particles and promoting uptake by antigen presenting cells.

An additional unexpected finding disclosed herein is that the activity of a Formula III adjuvant coupled to Formula I or Formula II poly(amino acids) containing hydrophobic molecules (H) peptide antigen conjugates was found to be inversely proportional to the intensity and breadth of T cell responses generated against peptide antigens after multiple immunizations. Accordingly, we show, as disclosed herein, that Formula I poly(amino acids) are coupled to Formula III adjuvants, wherein

referred to as Compound 1, lead to higher intensity and breadth of T-cell responses compared to Formula I poly(amino acids) coupled with Formula III adjuvants, while

referred to as Compound 2.

A non-limiting explanation is the fact that the lower activity of Formula III adjuvants, while

leads to less inflammation than Formula III adjuvants, while

and the fact that moderate inflammation induced by lower potency agonists leads to less exhaustion of T cell responses, suggesting a higher intensity and breadth of T cell responses overall. Based on these findings, a preferred embodiment of the present invention for hydrophobic molecules (H) consisting of a poly(amino acid) of Formula I coupled with adjuvants of Formula III is:

Herein, we report the unexpected discovery that hydrophobic molecules (H) based on Formula II poly(amino acids) coupled with Formula III adjuvants lead to a significant increase in the intensity and quality of T cell responses when the poly(amino acid) polymer consists of from 5 to 10 amino acids, with 60 to 100% of the copolymer consisting of comonomer o linked to Formula III adjuvants, while

Connection 1.

In a preferred embodiment of the present invention, comonomer 0 contains from 20

- 57 046161 up to 60% of monomer units of poly(amino acids) of Formula II, for example 60%. For example:

□ o o o o

H _Z NH ₂ NH ₂ N

Surprising findings disclosed herein indicate that the length of the polymer containing the hydrophobic molecule (H), as well as the amount and potency of attached ligands with adjuvant properties (i.e., TLR-7/8 Formula III agonists) are major determinants of intensity and the quality of immune responses generated against peptide antigens (A) delivered as peptide antigen conjugates. These data suggest that hydrophobic molecules (H) composed of poly(amino acids), which consist of hydrophobic amino acids and/or amino acids linked to ligands, must be long enough to allow particle formation upon binding to any peptide antigen (A). , including highly hydrophilic peptide antigens (A), which will counteract the tendency of the poly(amino acid)-based hydrophobic molecule (H) to drive particle assembly. Poly(amino acids) of insufficient length, for example, less than 3 amino acids in length, may not offer sufficient hydrophobic surface area to promote particle formation under aqueous conditions when coupled to certain peptide antigens, particularly hydrophilic peptide antigens with high charge densities. Therefore, as disclosed herein, poly(amino acids) of Formula I or Formula II should be greater than 3 amino acids in length, in a preferred embodiment of the present invention from 5 to 30 amino acids, for example 5, 6, 7, 8, 9, 10, 20 or 30 amino acids in length. Longer poly(amino acids), such as poly(amino acids) containing more than 30 amino acids, such as about 30, 40 or 50 amino acids in length, can be generated, for example, by solid phase peptide synthesis. As an alternative to solid-phase peptide synthesis, solution polymerization reactions can be used to generate long-chain poly(amino acids) consisting of hydrophobic monomers.

Herein, we disclose an additional unexpected finding that Compound 1, which has some in vitro activity against TLR-7 activity (i.e., EC50) of 108 nM, leads to improved T cell responses after repeated immunizations, compared to the more active agonist, Compound 2, which has some in vitro activity against TLR-7 activity (ie, EC50) of 18.7 nM. These data suggest that the activity of adjuvants included in an immunogenic composition can be modulated to optimize immune responses, and that the natural immune activation of the adjuvant can be moderated so as to offer the appropriate level of stimulation needed to optimize T-cell immunity. The level of natural immune stimulation can be made moderate by delivering several, e.g., at least 3, moderately active agonists (i.e., agonists with EC50 > 100 nM) on peptide antigen conjugates, or by delivering less than 3 highly active agonists (i.e. , agonists with EC50 < 100 nM).

The activity of various TLR-7, TLR-8 and combined TLR-7 and -8 agonists can be easily determined from the literature. TLR-7 and TLR-7/8 agonists based on imidazoquinoline and adenine have been described (see: Shukla, et al., J. Med. Chem., 53:4450-4465, 2010 and Gerster, et al., J. Med. Chem., 2005, US Pat. No. 6,069,149, and Hirota, et al., J. Med. Chem., 45:5419-5422, 2002, which are incorporated herein by reference), and these works indicate that the linkers aromatic compounds, such as benzyl and xylyl linkers, selected for the R ² of the Formula III adjuvants, lead to compounds with increased activity against TLR-7, compared to a comparison compound where the R ² of the Formula III adjuvants is selected from lower alkyl.

Based on the surprising discoveries described herein, preferred embodiments of the hydrophobic molecules (H) of the present invention consisting of Formula I or Formula II poly(amino acids) coupled with Formula III adjuvants typically range from 5 to 30 amino acids in length, with In this case, the density of comonomers linked to the adjuvants of Formula III ranges from 20 to 100 mol.%. Optionally, when the adjuvant of Formula III includes R ⁸ selected from a lower alkyl, for example, Compound 1, the density of amino acids linked to Compound 1 should be from 40 to 100%, for example 60%. Optionally, when the Formula III adjuvant includes R ⁸ selected from an aromatic compound, eg Compound 2, the density of amino acids bound to Compound 2 should be less than 20% or 1-2 molecules of Compound 2 delivered on each polymer.

In preferred embodiments of the present invention, the density of Formula III adjuvants coupled to polymers with a molecular weight of less than about 10,000 g/mol and based on comonomers selected from the following: #2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA) , styrene, vinylpyrrolidone (PVP), N-isopropylacrylamide (NIPAAm); Nisopropyl methacrylamide (NIPMAm); HH-diethylacrylamide (DEAAm); N-(I/)-(1-1'hydroxymethyl)propyl methacrylamide (HMPMAm); Ν,Ν'-dimethylethyl methacrylate (DMEMA), 2-(2-methoxyethoxy)ethyl methacrylate (DEGMA) or substituted poly(phosphoesters), is from about 5 to 100 mol.%, for example the density of the adjuvant attached to the polymer can be about 5 -6%, approximately 6-7%, approximately 8-9%, approximately 9-10%, approximately 10-11%, approximately 11-12%, approximately 1213%, approximately 13-14%, approximately 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%.

In preferred embodiments of the present invention, the density of Formula III adjuvants is coupled to polymers with a molecular weight greater than about 10,000 g/mol and based on comonomers selected from the following:

#2-hydroxypropyl(methacrylamide) (HPMA), hydroxyethyl(methacrylate) (HEMA), styrene, vinylpyrrolidone (PVP), N-isopropylacrylamide (NIPAAm); N-isopropyl methacrylamide (NIPMAm); N,N'diethylacrylamide (DEAAm); Na^)-(1-hydroxymethyl)propyl methacrylamide (HMPMAm); N,N'dimethylethyl methacrylate (DMEMA), 2-(2-methoxyethoxy)ethyl methacrylate (DEGMA) or substituted poly(phosphoesters) is from about 1 to 25 mol.%, for example the density of the adjuvant attached to the polymer can be about 1- 2%, approximately 2-3%, approximately 3-4%, approximately 5-6%, approximately 6-7%, approximately 7-8%, approximately 8-9%, approximately 9-10%, approximately 10-11% , approximately 11-12%, approximately 13-14%, approximately 14-15%, approximately 16-17%, approximately 17-18%, approximately 19-20%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, about 24% or about 25%.

In additional embodiments of the present invention, the second polymer, which consists of hydrophobic and/or temperature-sensitive monomers, is linked to the terminal or pendant groups of Formula I or Formula II poly(amino acids) linked to Formula III adjuvants.

A non-limiting example of a hydrophobic molecule (H) consisting of a temperature-sensitive DEGMA-based polymer grafted to a Formula I poly(amino acid) coupled with Formula III adjuvants is provided herein for clarity:

wherein comonomer k is typically an integer from 3 to 300, and comonomer k' is typically an integer from 3 to 300 amino acid residues, with the sum of k and k' typically being from 3 to 300. In preferred embodiments of the present invention k is about 3-10 and k is about 1-10. Linker X can be any suitable linker molecule and m is typically about 10-300 monomer units. The N-terminus of the poly(amino acid) is linked, either directly or indirectly via a linker (L) and/or an extension (B1 or B2) , with a peptide antigen (A) by any suitable means to form a peptide antigen conjugate, which may further contain a charged molecule (C).

A non-limiting example of a hydrophobic molecule (H) consisting of a temperature-sensitive DEGMA-based polymer linked to the end of a Formula I poly(amino acid) linked to Formula III adjuvants is provided herein for clarity

- 59 046161

wherein comonomer k is usually an integer from 3 to 100 monomer units. The linker, X, may be any suitable linker molecule. In preferred embodiments of the present invention, k' is about 3-10 monomer units. The N-terminus of the poly(amino acid) is linked, either directly or indirectly, via a Linker (L) and/or extension (B1 or B2), to a peptide antigen (A) by any suitable means to form a peptide antigen conjugate, which may further comprise charged molecule (C).

A non-limiting example of a hydrophobic molecule (H) consisting of a polyphosphoester polymer linked to the poly(amino acid) end of Formula I linked to Formula III adjuvants is provided herein for clarity

wherein comonomer k is usually an integer from 3 to 100 monomer units. The linker, X, may be any suitable linker molecule. In preferred embodiments of the present invention, k is from 5 to 10 monomer units, and m is from 10 to 300 monomer units. The N-terminus of the poly(amino acid) is linked, either directly or indirectly, via a Linker (L) and/or extension (B1 or B2), to a peptide antigen (A) by any suitable means to form a peptide antigen conjugate, which may further comprise charged molecule (C).

A non-limiting example of a hydrophobic molecule (H) consisting of a poly(benzyl glutamate) polymer linked to the poly(amino acid) end of Formula I linked to Formula III adjuvants is provided herein for clarity

- 60 046161

wherein comonomer k is typically an integer of 3 to 100 monomer units, and m is typically an integer of 50 to 300 amino acid residues. Linker X may be any suitable linker molecule. In preferred embodiments of the present invention, x is from 5 to 10 monomer units and m is from 10 to 300 monomer units. The N-terminus of the poly(amino acid) is linked, either directly or indirectly, via a linker (L) and/or extension (B1 or B2), to a peptide antigen (A) by any suitable means to form a peptide antigen conjugate, which may further contain a charged molecule (C).

In a non-limiting example, a peptide antigen (A) is linked to a particle (P) or a hydrophobic molecule (H) and may further comprise optional extensions (B1 and/or B2) and an optional Linker (L) to obtain a peptide antigen conjugate of Formula IV, where This [ ] indicates that the group is optional:

[B1]-A-[B2]-[L]-P, [B1]-A-[B2]-[L]-H, P-[L]-[B1]-A-[B2] or H- [b]-[B1]-A-[B2]

Formula IV

Peptide antigen (A) of Formula IV consists of an integer number of amino acids, n, with n typically being from 7 to 35, for example 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, coupled with an adjuvant of Formula III.

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

Optional charged molecule (C)

Peptide antigen conjugates disclosed herein consist of peptide antigens (A) linked to a particle (P) or hydrophobic molecule (H), and may further contain optional extensions (B1 and/or B2), an optional Linker (L), and an optional charged

- 61 046161 molecule (C), wherein C = denotes a charged molecule bearing functional groups that impart an electrostatic charge. Immunogenic compositions disclosed herein containing peptide antigens bound to a particle (P) or a hydrophobic molecule (H) that aggregate into particles under aqueous conditions may flocculate without sufficient surface charge to stabilize the particles. Thus, charged molecules (C) may optionally be linked to peptide antigen conjugates as a means to stabilize particles and prevent flocculation; or, in an alternative embodiment of the present invention, charged molecules (C) can be included in particles containing peptide antigen conjugates as a means for stabilized particles. Thus, the purpose of the charged molecule (C) is to control the net charge of the particles formed by peptide antigen conjugates as a means to promote the stability of these particles.

Charged molecule (C) refers to any molecule that has at least one functional group that is positively or negatively charged in aqueous buffer solutions at a pH of about 7.4. The functional groups containing the charged molecule (C) may be partial or full integer charge values. A charged molecule (C) may be a molecule with a single charged functional group or with multiple charged functional groups. The net charge of a charged molecule (C) can be positive, negative or neutral. The charge of functional groups containing a charged molecule (C) may or may not depend on the pH of the solution in which the charged molecule (C) is dispersed, as is the case for tertiary amines and quaternary ammonium compounds, which are pH-dependent and pH-dependent. independent, respectively. The charge of a molecule can be easily estimated based on the Lewis molecular structure and conventional methods known to those skilled in the art. Charge can be caused by inductive effects, for example atoms bonded together having differences in electron affinity can result in a polar covalent bond resulting in an atom with a partial negative charge and an atom with a partial positive charge. For example, nitrogen bonded to hydrogen results in a partial negative charge on the nitrogen and a partial positive charge on the hydrogen atom. In an alternative embodiment of the present invention, an atom in a molecule may be considered to have a full integer charge value when the number of electrons inherent to that atom is less than or equal to the atomic number of that atom. The charge of a molecule is determined by summing the charge of each atom contained in that molecule. Those skilled in the art are familiar with the process of estimating the charge of a molecule by summing the formal charge of each atom in the molecule.

The charged molecule (C) may carry either a net negative, net positive, or neutral charge and depends on the net charge of the peptide antigen conjugate required for the particular use of the present invention disclosed herein. For example, it is known that most cell surfaces carry a net negative charge. Thus, particles with a net positive charge can interact with all cell surfaces without a high degree of specificity. Conversely, particles with a net negative charge will be electrostatically repelled from most cell surfaces, but have been shown to stimulate selective uptake by certain antigen-presenting cell populations. For example, positively charged particles delivered intravenously into the bloodstream have been found to accumulate in the liver and lungs, as well as in antigen-presenting cells in the spleen, and negatively charged particles have been found to preferentially accumulate in antigen-presenting cells in the spleen after intravenous administration. Thus, the net charge of the charged molecule (C) can be adjusted according to the specific application requirements.

In some embodiments of the present invention, the charged molecule (C) has a net negative charge and is composed of functional groups that carry a negative charge at physiological pH, at a pH of about 7.4. Suitable charged molecules (C) that carry a net negative charge include molecules bearing functional groups (eg, functional groups with a pKa of less than about 6.5) that occur as a conjugate acid base at physiological pH, at pH approximately 7.4. These include, but are not limited to, molecules bearing carboxylates, sulfates, phosphates, phosphoramidates and phosphonates. The charged molecule (C) bearing the caboxylate may be, but is not limited to, glutamic acid, aspartic acid, pyruvic acid, lactic acid, glycolic acid, glucuronic acid, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, and oxaloacetate and its derivatives. In preferred embodiments of the present invention, the negatively charged molecule (C) consists of a molecule with negatively charged functional groups, in an amount from 1 to 20, 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 functional groups, although typically with no more than 16 negatively charged functional groups. In some embodiments of the present invention, the charged molecule ( C) is a peptide

- 62 046161 li (glutamic acid) with a length of 2 to 6 amino acids. A poly(glutamic acid) sequence consisting of 1, 2, 3, 4, 5 or 6 amino acids is expected to have a negative charge of -1, -2, -3, -4, -5 and -6 at pH 7. 4 respectively. In additional embodiments of the present invention, the charged molecule (C) is phosphoserine or sulfoserine.

In some embodiments of the present invention, the charged molecule (C) has a net negative charge and consists of at least 1 negatively charged amino acid. In preferred embodiments of the present invention, a charged molecule (C) with a net negative charge consists of 1 to 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. In a non-limiting example, the charged molecule (C) consists of 16 aspartic acid monomers, for example Asp-Asp-Asp-AspAsp-Asp-Asp-Asp-Asp-Asp -Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 25) is used to obtain a charged molecule (C) with a net negative charge of -16; a charged molecule (C) consisting of 15 aspartic acid monomers, e.g. Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-AspAsp-Asp-Asp (SEQ ID NO: 26) is used to obtain a charged molecule (C) with a total negative charge of -15; a charged molecule (C) consisting of 14 aspartic acid monomers, e.g. Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 27) is used to obtain a charged molecule (C) with a total negative charge of -14; a charged molecule (C) consisting of 13 aspartic acid monomers, e.g. Asp-Asp-Asp-Asp-Asp-Asp-AspAsp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 28) is used to obtain a charged molecule (C) with a total negative charge of -13; a charged molecule (C) consisting of 12 aspartic acid monomers, e.g. Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 29) is used to produce a charged molecule (C) with a total negative charge of -12; a charged molecule (C) consisting of 11 aspartic acid monomers, for example, Asp-Asp-Asp-Asp-Asp-AspAsp-Asp-Asp-Asp-Asp (SEQ ID NO: 30) is used to obtain a charged molecule (C) with total negative charge -11; a charged molecule (C) consisting of 10 aspartic acid monomers, for example, Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 31) is used to obtain a charged molecule (C) with total negative charge -10; a charged molecule (C) consisting of 9 aspartic acid monomers, for example, Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 32) is used to obtain a charged molecule (C) with a net negative charge -9; a charged molecule (C) consisting of 8 aspartic acid monomers, for example, AspAsp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 33) is used to obtain a charged molecule (C) with a net negative charge of -8; a charged molecule (C) consisting of 7 aspartic acid monomers, for example, Asp-Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 34) is used to obtain a charged molecule (C) with a net negative charge of -7; a charged molecule (C) consisting of 6 aspartic acid monomers, for example, Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 35) is used to obtain a charged molecule (C) with a net negative charge of -6; a charged molecule (C) consisting of 5 aspartic acid monomers, for example, Asp-Asp-Asp-Asp-Asp (SEQ ID NO: 36) is used to obtain a charged molecule (C) with a net negative charge of -5; a charged molecule (C) consisting of 4 aspartic acid monomers, for example, Asp-Asp-Asp-Asp (SEQ ID NO: 37), is used to obtain a charged molecule (C) with a net negative charge of -4; a charged molecule (C) consisting of 3 aspartic acid monomers, for example, AspAsp-Asp, is used to obtain a charged molecule (C) with a total negative charge of -3; a charged molecule (C) consisting of 2 aspartic acid monomers, for example Asp-Asp, is used to obtain a charged molecule (C) with a net negative charge of -2; a charged molecule (C) consisting of 1 aspartic acid monomer, such as Asp, is used to obtain a charged molecule (C) with a net negative charge of -1. In the above examples, aspartic acid (Asp) may be replaced by any suitable negatively charged amino acid, including, but not limited to, glutamic acid, sulfoserine or phosphoserine, and the negatively charged amino acids may be the same or different.

In some embodiments of the present invention, the charged molecule (C) has a net positive charge and is composed of positively charged groups. Suitable positively charged molecules (C) include molecules with functional groups that carry a positive charge at physiological pH, at a pH of about 7.4, such as weak base conjugate acid, wherein the pKa of the base conjugate acid is greater than about 8.5. Suitable positively charged molecules (C) include, but are not limited to, molecules bearing primary, secondary and tertiary amines, as well as quaternary ammonium, guanidinium, phosphonium and sulfonium functional groups.

Suitable molecules bearing ammonium functional groups include, for example, imidazolium and tetraalkylammonium compounds. In some embodiments of the present invention, the charged molecule (C) consists of quaternary ammonium compounds that carry a constant positive charge that is independent of pH.

Non-limiting examples of positively charged functional groups that have

- 63 046161 charge, independent of pH, include

where f = 1-6, where R = lower alkyl, where f = 1-6, where R = lower alkyl, and X ^- represents any suitable counteranion.

In additional embodiments of the present invention, the charged molecule (C) consists of functional groups that occur as a conjugate acid base at physiological pH, such as, for example, primary, secondary and tertiary amines. In preferred embodiments of the present invention, the positively charged molecule (C) consists of 1 to 20 positively charged functional groups, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 positively charged functional groups, although typically no more than 16 positively charged functional groups. In some embodiments of the present invention, the charged molecule (C) is a poly(lysine) peptide with a length of 1 to 6 amino acids. A poly(lysine) sequence consisting of 1, 2, 3, 4, 5 or 6 amino acids is expected to have a positive charge of +1, +2, +3, +4, +5 or +6, respectively, at pH 7.4. In some embodiments of the present invention, the charged molecule (C) is a poly(arginine) peptide of 2 to 6 amino acids in length.

In certain embodiments of the present invention, the charged molecule (C) has a net positive charge and consists of at least 1 positively charged amino acid. In preferred embodiments of the present invention, the charged molecule (C) with a net positive charge consists of 1 to 20 positively 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. In a non-limiting example, the charged molecule (C) consists of 16 lysine monomers, for example Lys-Lys-Lys-Lys-Lys-Lys-LysLys-Lys-Lys- Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 38), used to obtain a charged molecule (C) with a total positive charge of +16; a charged molecule (C) consisting of 15 lysine monomers, e.g. Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 39) , is used to obtain a charged molecule (C) with a total positive charge of +15; a charged molecule (C) consisting of 14 lysine monomers, e.g. Lys-Lys-Lys-Lys-Lys-Lys-LysLys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 40), is used to produce charged molecule (C) with a total positive charge of +14; a charged molecule (C) consisting of 13 lysine monomers, e.g. Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 41), is used to produce charged molecule (C) with a total positive charge of +13; a charged molecule (C) consisting of 12 lysine monomers, e.g. Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-LysLys (SEQ ID NO: 42), is used to produce a charged molecule (C ) with a total positive charge of +12; a charged molecule (C) consisting of 11 lysine monomers, for example Lys-LysLys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 43), is used to obtain a charged molecule (C) with a total positive charge +11; a charged molecule (C) consisting of 10 lysine monomers, e.g. Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 44), is used to obtain a charged molecule (C) with total positive charge +10; a charged molecule (C) consisting of 9 lysine monomers, for example Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 45), is used to obtain a charged molecule (C) with a net positive charge +9; a charged molecule (C) consisting of 8 lysine monomers, for example Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 46), is used to obtain a charged molecule (C) with a net positive charge of +8 ; a charged molecule (C) consisting of 7 lysine monomers, for example Lys-Lys-Lys-LysLys-Lys-Lys (SEQ ID NO: 47), is used to obtain a charged molecule (C) with a net positive charge of +7; a charged molecule (C) consisting of 6 lysine monomers, for example LysLys-Lys-Lys-Lys-Lys (SEQ ID NO: 48), is used to obtain a charged molecule (C) with a net positive charge of +6; a charged molecule (C) consisting of 5 lysine monomers, for example Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 49), is used to obtain a charged molecule (C) with a net positive charge of +5; a charged molecule (C) consisting of 4 lysine monomers, for example, Lys-Lys-Lys-Lys (SEQ ID NO: 50), is used to obtain a charged molecule (C) with a net negative charge of +4; a charged molecule (C) consisting of 3 lysine monomers, for example Lys-Lys-Lys, is used to obtain a charged molecule (C) with a net positive charge of +3; a charged molecule (C) consisting of 2 lysine monomers, for example Lys-Lys, is used to obtain a charged molecule (C) with a net positive charge of +2; a charged molecule (C) consisting of 1 lysine, for example Lys, is used to obtain a charged molecule (C) with a net positive charge of +1. In the above examples, lysine (Lys) may be replaced by any suitable positively charged amino acid, including, but not limited to, trimethyllysine or arginine, and the positively charged amino acids may

- 64 046161 be the same or different.

Charged molecules (C) may further contain small uncharged, hydrophilic amino acids or hydrophilic linkers, such as ethylene oxide, which function to: i) improve water solubility and ii) increase the distance between charged functional groups to prevent incomplete ionization. For example, the ionization of one functional group on a polymer can affect the pKa of neighboring functional groups through local effects. For example, protonation of an amine in close proximity to a second amine can lower the pKa of the conjugate acid of the second amine. To reduce the influence of local effects on the ionization potential of adjacent functional groups, a linker molecule can be used to increase the distance between charged functional groups containing a charged molecule. The linker molecule may contain from 1 to 5 small uncharged hydrophilic amino acids, for example, 1, 2, 3, 4 and 5 amino acids. In an alternative embodiment of the present invention, the linker may comprise an ethylene oxide (ie, PEG) linker with a length of 1 to 4 monomer units, for example, 1, 2, 3 or 4 ethylene oxide monomers in length. In preferred embodiments of the present invention, 1-2 small uncharged hydrophilic amino acids are placed between adjacent charged amino acids containing a charged molecule (C), with the amino acids linked through amide bonds. In some embodiments of the present invention, serine is placed between each charged amino acid containing a charged molecule (C) with a net positive charge. In preferred embodiments of the present invention, the charged molecule (C) consists of repeating dipeptides of lysine and serine, that is, (Lys-Ser)n, wherein n is typically any integer from 1 to 20, for example 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. As other examples, serine is placed between each charged amino acid of a tripeptide charged molecule (C) with total charge +2, for example, Lys-Ser-Lys; serine is placed between each charged amino acid of a charged molecule (C) of 5 amino acids with a net charge of +3, for example, Lys-Ser-Lys-Ser-Lys (SEQ ID NO: 51); serine is placed between each charged amino acid of a charged molecule (C) of 7 amino acids with a net charge of +4, for example, Lys-Ser-Lys-Ser-Lys-Ser-Lys (SEQ ID NO: 52). In the above examples, lysine (Lys) may be replaced by any suitable positively charged amino acid, including, but not limited to, trimethyllysine or arginine, and the positively charged amino acids may be the same or different.

In some embodiments of the present invention, serine is placed between each charged amino acid containing a charged molecule (C) with a net negative charge. In preferred embodiments of the present invention, the charged molecule consists of repeating dipeptides of aspartic acid and serine, that is, (Asp-Ser)n, wherein n is typically any integer from 1 to 20, for example, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. As other examples, serine is placed between each charged amino acid of a tripeptide charged molecule ( C) with a total charge of -2, for example, Asp-Ser-Asp; serine is placed between each charged amino acid of a charged molecule (C) of 5 amino acids with a net charge of -3, for example, Asp-Ser-Asp-Ser-Asp (SEQ ID NO: 53); serine is placed between each charged amino acid of a charged molecule (C) of 7 amino acids with a net charge of -4, for example, Asp-Ser-Asp-Ser-Asp-Ser-Asp (SEQ ID NO: 54). In the above examples, aspartic acid (Asp) may be replaced by any suitable negatively charged amino acid, including, but not limited to, glutamic acid, sulfoserine or phosphoserine, and the negatively charged amino acids may be the same or different.

In additional embodiments of the present invention, the charged molecule (C) is composed of both negatively and positively charged amino acids. Dipeptides composed of amino acids with opposite charges, such as Lys-Asp, are called zwitterionic dipeptides because they are predicted to have a net neutral, 0, charge at pH 7.4. At least one zwitterionic dipeptide may be included in a charged molecule (C) as a means to i) improve water solubility and ii) offer a predominant charge (eg, net negative or net positive) in certain pH ranges. For example, a zwitterionic dipeptide can be used to increase the hydrophilicity of a peptide sequence without increasing or decreasing the charge of the peptide sequence at pH 7.4. However, a zwitterion can be used to impart a net charge at a specific pH. For example, excluding the contributions of the N-terminal amine and C-terminal carboxylic acid in this example, the zwitterionic dipeptide, LysAsp, has a net charge of 0 at pH 7.4, but a net charge of +1 at pH < 4 and a net charge of -1 at pH > 10. At least one zwitterionic dipeptide may be added to the sequence of charged molecules (C); for example, one dipeptide, Lys-Asp; two Lys-Asp-Lys-Asp dipeptides (SEQ ID NO: 55); three dipeptides, Lys-Asp-Lys-Asp-Lys-Asp (SEQ ID NO: 56) and so on. In the above examples, lysine (Lys) can be replaced by any suitable positively charged amino acid, including, but not limited to, trimethyllysine or arginine, and aspartic acid (Asp) can

- 65 046161 may be replaced by any suitable negatively charged amino acid, including, but not limited to, glutamic acid, sulfoserine or phosphoserine, and the positively or negatively charged amino acids may be the same or different.

The charged molecule composition (C) is selected to offer the net charge required for the peptide antigen conjugate for a particular application. In some embodiments of the present invention disclosed herein, the charged molecule (C) is a positively charged poly(amino acid) consisting of lysines or arginines or lysines or arginines and uncharged amino acids. In some embodiments of the present invention, the charged moiety consists of sulfonium or quaternary ammonium functional groups that carry a pH-independent positive charge. In some embodiments of the present invention disclosed herein, the charged molecule (C) is a negatively charged poly(amino acid) consisting of glutamic acid or aspartic acid, or glutamic acid or aspartic acid and uncharged amino acids. In some embodiments of the present invention, the charged moiety contains phosphate or sulfate groups, such as sulfoserine or phosphoserine. In additional embodiments of the present invention, the charged molecule consists of lysines or arginines and glutamic acid or aspartic acid, or lysines or arginines and glutamic acid or aspartic acid, as well as uncharged amino acids. Both positive and negatively charged functional groups can be included in the same charged molecule (C). The charged molecule (C) may be positive, negative or neutral, but the net charge of the peptide antigen conjugate must be non-zero, for example net charges greater than +3 or less than -3 are preferred and depend on the particular application.

An additional factor to consider regarding charged molecules (C) is the counterion chosen. Non-limiting examples of charged molecules (C) bearing functional groups with a positive charge include, but are not limited to, halides, including chloride, bromide and iodide anions, and conjugate acid bases, including phosphate, sulfate, sulfite and carboxylate anions, including formate, succinate, acetate and trifluoroacetate. Suitable counterions for charged molecules (C) bearing functional groups with a negative charge include, but are not limited to, hydrogen and alkali and alkaline earth metals, including, for example, sodium, potassium, magnesium and calcium or conjugate acids of weak bases such as compounds ammonium

The charged molecule (C) can be linked directly to the peptide antigen (A), either directly or indirectly through an extension (B1 or B2), Linker (L), Linker and extension (B1 or B2), or Particle (P) , or a hydrophobic molecule (H) that is linked, directly or indirectly, through a Linker (L) and/or an extension (B1 or B2), to a peptide antigen.

In some embodiments of the present invention, the charged molecule (C) is linked to an optional N- or C-terminal extension (B1 or B2) that is linked to either the N- or C-terminus, respectively, of the peptide antigen (A) that is linked to either at the C- or N-terminus, respectively, with an optional extension (B2), which is linked either directly or via a Linker (L), to a Particle (P) or a hydrophobic molecule (H) to yield a peptide antigen conjugate of Formula V, however, [ ] indicates that the group is optional:

C-[B1]-A-[B2]-[L]-P, C-[B1]-A-[B2]-[L]-H, P-[L]-[B1]-A-[B2 ]-C, or H-[L]-[B1]-A-[B2]-C

Formula V

In some embodiments of the present invention, a charged molecule (C) is placed at the N-terminus of a Formula V peptide antigen conjugate, wherein the charged molecule (C) is linked to an N-terminal extension (B1) consisting of a cathepsin-cleavable tetrapeptide extension (B1 = PN4- PN3-PN2-PN1), which is linked to the N-terminus of the peptide antigen (A), which is linked at the C-terminus to a C-terminal extension (B2), consisting of a combined immuno-proteasomal and cathepsin-cleavable hexapeptide extension (B2 = PC1' -PC2'-PC3'-PC4'-PC5'-PC6'), which is linked to a Linker (L), which is linked to a hydrophobic molecule (H) or Particle (P). The peptide antigen (A) of a peptide antigen conjugate of Formula V consists of an integer number of amino acids, n, with n typically ranging from 7 to 35 amino acids, and the hydrophobic molecule (H) typically being a poly(amino acid) of Formula I or II linked to Formula III adjuvant.

A non-limiting example of a Formula V peptide antigen conjugate consisting of a charged molecule (C = Lys-Lys) linked to a cathepsin-cleavable tetrapeptide extension (B1 = Lys-Pro-Leu-Arg SEQ ID NO: 8) at the N-terminus of the peptide antigen (A ), which is linked at the C-terminus to a cathepsin-cleavable hexapeptide extension (B2 = Gly-Gly-Ser-Leu-Val-Arg SEQ ID NO: 13), which is linked to a triazole linker (L), which is linked to a hydrophobic molecule (H ) consisting of a poly(amino acid) of Formula I, which is linked to an adjuvant of Formula I, is provided herein:

- 66 046161

In additional embodiments of the present invention, a charged molecule (C; or C1 and C2 when two charged molecules are present) may be linked directly to a hydrophobic molecule (H) or to a linker (L) that is linked to a C-terminal extension (B2), which is linked to the C-terminus of the peptide antigen (A), which is optionally linked at the N-terminus to an N-terminal extension (B1), which is optionally linked to an additional optional charged fragment (C1); or a charged molecule (C; or C1 and C2 when two charged molecules are present) can be linked directly to a hydrophobic molecule (H) or to a Linker (L) that is linked to an N-terminal extension (B1) that is linked to an N- end of a peptide antigen (A) which is optionally linked at the C-terminus to a C-terminal extension (B2) which is optionally linked to an additional optionally charged moiety (C2) to yield a peptide antigen conjugate of Formula VI, wherein [ ] denotes that group is optional:

[B1]-A-[B2]-L(C)-H, [B1]-A-[B2]-L-H(C), [C1]-[B1]-A-[B2]-L(C2) -H, [C1]-[B1]-A-[B2]-L-H(C2), H-L(C)[B1]-A-[B2], H(C)-[B1]-A-[B2] , H-L(C1)-[B1]-A-[B2]-C2 or H(C1)-[B1]-A-[B2]-C2

Formula IV

In some embodiments of the present invention, a charged molecule (C) is placed at the C-terminus of a peptide antigen conjugate of Formula VI, wherein the charged molecule (C) is linked to a linker (L), which is optionally linked to a C-terminal extension (B2) consisting of immunoproteasomal, cathepsin, or combined immunoproteasomal and cathepsin-cleavable extension, typically 1 to 6 amino acids long (B2 = PC1', PC1'-PC2', PC1'-PC2'-PC3', PC1'-PC2'PC3'-PC4', PC1'-PC2'-PC3'-PC4'-PC5' or PC1'-PC2'-PC3'-PC4'-PC5'-PC6'), which is linked to the C-terminus of the peptide antigen (A), which is optionally linked to N-terminal with a cathepsin-cleavable extension, usually 1 to 4 amino acids long (B1 = PN1, PN2-PN1, PN3-PN2-PN1 or PN4-PN3-PN2PN1), with the linker (L) additionally linked to a hydrophobic molecule (H ) as shown below:

B1 A B2 C

-------------------------------------------------- ------------------------I PN4-PN3-PN2-PN1-(Antlgen)-PC1 ^, -PC2'-PC3'-PC4'-PC5 '—PC6 ¹ —L—H

- 7-35 amino acids

The peptide antigen (A) of a peptide antigen conjugate of Formula VI consists of an integer number of amino acids, n, wherein n is typically from 7 to 35 amino acids or up to 50 amino acids, and the hydrophobic molecule is typically a Formula I or II poly(amino acid) linked with Formula III adjuvant.

A non-limiting example of a peptide antigen conjugate of Formula VI is provided, consisting of a charged molecule (e.g., C=Lys-Lys) linked via an amide bond to the C-terminus of a linker (L), which is linked to a combined immunoproteasomal and cathepsin-cleavable C-terminal hexapeptide extension ( e.g., B2 = Gly-Gly-Ser-Leu-Val-Arg SEQ ID NO: 13), which is linked to the C-terminus of a peptide antigen (A), which is linked at the N-terminus to a cathepsin-cleavable N-terminal tetrapeptide extension (e.g. , B1 = Lys-Pro-Leu-Arg SEQ ID NO: 8), wherein the linker (L) is additionally linked to a hydrophobic molecule (H), which consists of a poly(amino acid) of Formula I, which is linked to an adjuvant of Formula III:

- 67 046161

A further non-limiting example of a peptide antigen conjugate of Formula VI, B1-A-B2-L-H(C), is a charged moiety (C = Lys-Lys-Lys-Lys-Lys SEQ ID NO: 49) linked via a linker to a hydrophobic molecule (H ), which consists of a poly(amino acid) of Formula I, which is linked to an adjuvant of Formula III, which is linked to a linker (L), which is linked to a combined immunoproteasomal and cathepsin-cleavable C-terminal hexapeptide extension (B2 = Gly-Gly-Ser-Leu -Val-Arg SEQ ID NO: 13), which is linked to the C-terminus of the peptide antigen (A), and the N-terminus of the peptide antigen (A) is linked to the cathepsin-cleavable N-terminal tetrapeptide extension (B1 = Lys-Pro-Leu- Arg SEQ ID NO: 8):

In a further non-limiting example of a peptide antigen conjugate of Formula VI, B1-(A) _7-35 -B2-C-C)-H, peptide antigen (A) with the sequence Ala-Lys-Phe-Val-Ala-Ala-Trp-ThrLeu -Lys-Ala-Ala-Ala (SEQ ID NO: 22) is linked to the N-terminal extension (B1) with the sequence Ser-Leu-Val-Arg (SEQ ID NO: 7) and to the C-terminal extension (B2) with sequence Ser-Leu-ValArg (SEQ ID NO: 7), which is linked to a precursor linker X1, for example, Lys(N3), which is linked to both a charged fragment (C), consisting of a dipeptide with the sequence Glu-Lys, and to linker precursor X2 containing a DBCO molecule that is linked to a hydrophobic molecule (H), for example: Ser-Leu-Val-Arg-Ala-Lys-Phe-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala -Ala-Ser-LeuVal-Arg-Lys(N3-DBCO-H)-Glu-Lys, wherein the Glu-Lys sequence is linked to the C-terminus of the Linker (L) (Lys(N3-DBCO), resulting in peptide antigen conjugate with a predicted net charge of +4 at pH 7.4. In this case, the hydrophobic molecule (H) is considered to make a negligible contribution to the charge of the peptide antigen conjugate. It should be noted that the composition of the charged moiety (C) and extension sequences (B1 and B2) can be selected to offer a specific number of charged residues that offer the desired net charge and

- 68 046161 hydropathy of a peptide sequence containing a peptide antigen conjugate, as described in more detail below. In preferred embodiments of the present invention, the number of charged functional groups containing the charged fragment (C) is modulated such that the net charge of the peptide antigen conjugate containing the charged fragment (C), the peptide antigen (A), optional extensions (B 1 and/ or B2), linker (L) and hydrophobic molecule (H), are in the range from -3 to -10 or from +3 to +10.

Formula VI peptide antigen conjugates, where a charged moiety (C) is linked to a hydrophobic molecule (H), may be useful for the rapid production of personalized therapies, such as personalized cancer vaccines. A hydrophobic molecule (H) that is linked to a charged molecule (C) and an X2 linker precursor (e.g., an X2 containing cyclooctyne) can be produced as angro and then readily combined with any peptide antigen (A) bearing an X1 linker precursor (e.g., X1 containing an azide) to form a peptide antigen conjugate of Formula VI, [C1]-[B1]-A-[B2]-L-H(C2), or H(C)-L-[B1]-A[B2 ]-[C2], with [ ] indicating that the group is optional.

The function of the charged moiety (C) is to stabilize nanoparticles formed by peptide antigen conjugates under aqueous conditions. While the hydrophobic molecule (H) induces the formation of peptide antigen conjugate particles, the optional charged molecule (C) offers a counter force that prevents flocculation and, in some embodiments of the present invention, causes the peptide antigen conjugates to assemble into nanoparticle micelles with the surface charge suggested by the charged fragment (C).

In some embodiments of the present invention, the peptide antigen conjugate does not contain charged molecules, such as [B1]-A-[B2]-[L]-H, with [ ] indicating that the group is optional. Non-limiting examples include A-H, A-L-H, A-B2-H, A-B2-L-H, B1-A-B2L-H. Peptide antigen conjugates that do not contain a charged molecule (C) may undergo aggregation under aqueous conditions. To improve the stability of particles formed by peptide antigen conjugates that do not contain a charged moiety (C), a charged or amphiphilic molecule can be added. In some embodiments of the present invention, a first peptide antigen conjugate that does not contain a charged moiety (C) (i.e., [B1]-A[B2]-[L]-H) is mixed with a second peptide antigen conjugate containing a charged moiety (e.g. , C-[B1]-A-[B2]-[L]-H) in DMSO solution and then resuspended under aqueous conditions to form stable nanoparticles. In other embodiments of the present invention, a peptide antigen conjugate that does not contain a charged molecule (C) (i.e., [B1]-A-[B2]-[L]H) is mixed with a hydrophobic molecule (H) linked to the charged molecule (C), such as C-H, in DMSO solution and then resuspended under aqueous conditions to form stable nanoparticles.

In some embodiments of the present invention, a peptide antigen conjugate that does not contain a charged molecule (C), such as, [B1]-A-[B2]-[L]-H, wherein [ ] denotes that the group is optional, combined with an amphiphilic support, C-[B1]-[A']-[B2]-[L]-H, wherein [ ] denotes that the group is optional and the optional A' represents a conserved antigen (i.e. not intended for a specific patient). In some embodiments of the present invention, a peptide antigen conjugate containing a charged molecule (C) is combined with an amphiphilic carrier. The amphiphilic carrier serves to stabilize nanoparticles, such as nanoparticle micelles formed by peptide antigen conjugates.

Choice of charged molecule and extensions (B1 and B2)

The composition of the optional extensions, B1 and B2, linked at the N- and C-terminus of the peptide antigen (A), respectively, together with the optional charged molecule(s) (C), should be selected to: i) achieve an appropriate net charge and hydrophilic balance and hydrophobic characteristics, which are necessary to stabilize particles formed by peptide antigen conjugates in aqueous buffer solutions at physiological temperature and pH approximately 7.4; and ii) providing that the peptide antigens (A) delivered in the context of a peptide antigen conjugate can be processed to release the minimal CD4 and/or CD8 T cell epitopes proposed on the peptide antigen (A), whereby the epitopes can be presented in the context of MHC class I and class II molecules. The following description discloses specific compositions of B1 and B2 extensions with charged molecules (C) that result in unexpected improvements in control over the size and stability of particles formed by peptide antigen conjugates and/or improvements in the processing and presentation of CD4 and/or CD8 T cell epitopes , proposed as peptide antigens (A) on those particles that lead to significant enhancement of T-cell responses.

An unexpected discovery disclosed herein is that peptide antigens (A) containing minimal CD8 T cell epitopes, delivered as peptide antigen conjugates, for example, peptide antigen conjugates of Formulas IV, V and VI, are most efficiently processed in vitro and lead to higher levels of CD8 T cell responses in vivo when an enzyme-degradable C-terminal extension (B2) is applied at the C-terminus between the peptide

- 69 046161 antigen (A) and a hydrophobic molecule (H), and/or when an enzyme-degradable N-terminal extension (B1) is applied between the N-terminus of the peptide antigen (A) and the charged molecule (C). In some embodiments of the present invention, comprising a B1 extension containing a cathepsin cleavable site between a charged molecule (C) and a peptide antigen (A) (e.g., CB1-A-[B2]-[C]-H, wherein [ ] denotes that this group is optional), this resulted in more efficient processing of the minimal T cell epitope from the peptide antigen conjugate, which was associated with a higher intensity of T cell response in vivo. Thus, in preferred embodiments of the present invention, Formulas V and VI must employ cathepsin cleavage sequences in the form of N-terminal (B1) and C-terminal (B2) extensions in order to allow efficient processing and presentation of peptide antigens (A) proposed in the form of particles containing peptide antigen conjugates.

An additional unexpected discovery relates to the identification of the net charge required to stabilize the particles formed by certain embodiments of the peptide antigen conjugates of the present invention. Highly hydrophobic peptide antigens (A) delivered as peptide antigen conjugates tend to aggregate under aqueous conditions. To counter this tendency, charged molecules (C) and certain extensions (B 1 or B 2) can be selected for attachment to peptide antigen conjugates, for example, see Formulas V and VI, or a conjugate containing a charged molecule (for example, C-[ A']-[L]-H, wherein A' represents a conserved antigen and [ ] indicates that the group is optional), can be added to particles containing peptide antigen conjugates. An unexpected discovery disclosed herein is that the amount of charge (ie, net charge) required to stabilize particles formed by certain variants of peptide antigen conjugates, for example, peptide antigen conjugates of Formulas V and VI containing a hydrophobic molecule ( H) poly(amino acid) based, depends on the hydropathy (ie, hydrophobic properties) of the peptide antigen fragment, which contains the peptide antigen (A), optional charged molecules (C) and optional extensions (B1 and B2). We report the unexpected discovery that the net charge useful for stabilizing particles formed by certain embodiments of peptide antigen conjugates can be determined based on the hydropathy of the peptide antigen (A) or peptide antigen fragment. Thus, in preferred embodiments of the present invention, the net charge of the peptide antigen conjugate is selected based on the calculated hydropathy of the peptide antigen (A) and is modulated by the charged molecule (C) and optional extensions (B1 and/or B2). In other embodiments of the present invention, the net charge of the peptide antigen conjugate is selected based on the calculated hydropathy of the peptide antigen fragment and modulated by a charged molecule (C). For clarity, linker precursors (X1 and X2), linker (L) and hydrophobic molecule (H) or particle (P) are not taken into account when determining the hydropathy value for peptide antigens (A) or peptide antigen fragments used to select the net charge of peptide antigens conjugates. Thus, hydropathy of a peptide antigen conjugate is equivalent to hydropathy of a peptide antigen fragment.

The hydropathy of a peptide antigen (A) or a fragment of a peptide antigen, including the peptide antigen (A), an optional charged molecule (C), and optional extensions (B1 and/or B2), can be calculated using any of a variety of methods available for determining hydrophobic/ hydrophilic characteristics of the peptide. As a non-limiting example, a method for determining the hydropathy of peptide antigens (A) or a peptide antigen fragment is to calculate the overall average hydropathy value (GRAVY) using the Kite and Doolittle method as described below. The following hydropathy values with estimates proposed for unnatural amino acids (i.e., citrulline, sulfoserine, phosphoserine, and trimethyllysine) are based on the method of Kite and Doolittle, and are used to calculate GRAVY values herein:

- 70 046161

Amino acid Code Values Hydropathies Charge Isoleucine (Not) I 4.5 0 Valin (Vai) V 4.2 0 Leucine (Leu) L 3.8 0 Phenylalanine (Phe) F 2.8 0 Cysteine (Cys) WITH 2.5 0 Methionine (Met) M 1.9 0 Alanine (Ala) A 1.8 0 Glycine (Gly) G -0.4 0 Threonine (Thr) T -0.7 0 Tryptophan (Tgr) W -0.9 0 Serin (Ser) S -0.8 0 Tyrosine (Tug) Y -1.3 0 Proline (Pro) R -1.6 0 Histidine (His) n -3.2 0.1 Glutamic acid (Glu) E -3.5 -1 Glutamine (Gin) Q -3.5 0 Aspartic acid (Asp) D -3.5 -1 Asparagine (Asn) N -3.5 0 Lysine (Lys) TO -3.9 1 Citrulline (Cit) Z -4 0 Arginine (Arg) R -4.5 1 Trimethyllysine K(M e)3 -4.5 1 Phosphoserine SPhospho -4.5 -1 Sulfoserine Sulfo -4.5 -1

The process of determining the GRAVY value of a peptide is to add up the GRAVY values of each individual amino acid containing the peptide and then divide by the total length of the peptide. For example, a 9 amino acid (A) peptide antigen with the sequence Val-Val-De-Ala-Ile-Phe-IleIle-Leu (SEQ ID NO: 57) has a GRAVY value of 3.86 (e.g., (4.2 + 4, 2 + 4.5 + 1.8 + 4.5 + 2.8 + 4.5 + 4.5 + 3.8)/9 amino acids). As a non-limiting example, the same peptide antigen (A), i.e., Val-Val-Ile-Ala-Ile-Phe-Ile-Ile-Leu (SEQ ID NO: 57), obtained as a peptide antigen fragment by solid phase peptide synthesis , while the charged molecule (C = Lys-Ser-LysGly-Gly SEQ ID NO: 58) is linked to the N-terminal extension (B1 = Lys-Pro-Leu-Arg SEQ ID NO: 8) at the N terminus of the peptide antigen and to the C -terminal extension (B2 = Gly-Gly-Lys-Leu-Val-Arg SEQ ID NO: 11) at the C-terminus, which is linked to the precursor linker X1 (wherein X1 is Lys( _N3 ) _-NH2 ) : Ac-Lys-Ser-Lys-Gly-Gly-Lys-Pro-Leu-Arg-Val-Val-Ile-Ala-Ile-Phe-Ile-Ile-Leu-Gly-Gly-LysLeu-Val-Arg-Lys (N ₃ )-NH ₂ has a GRAVY value of 0.75 and a net charge of +6.

Determining the GRAVY value of a peptide antigen fragment or peptide (A) offers a description of the hydrophobic/hydrophilic characteristics of the peptide sequence. In general, GRAVY values less than zero generally indicate that the peptide is composed primarily of hydrophilic amino acids, whereas GRAVY values greater than zero indicate that the peptide is composed primarily of hydrophobic amino acid residues. We report herein the unexpected discovery that as the GRAVY value of a peptide antigen (A) or peptide antigen fragment increases, the intensity of the net charge (either positive or negative) required to stabilize particles formed by peptide antigen conjugates containing peptide antigen (A) (or fragment of peptide antigen) is also increased.

- 71 046161

Thus, peptide antigens (A) or fragments of peptide antigens with higher GRAVY values generally require a higher net charge to ensure the stability of the particles formed by peptide antigen conjugates.

The GRAVY value of a peptide antigen (A) or fragment of a peptide antigen, which is calculated based on the amino acid composition of the peptide antigen (A), optional charged molecule (C) and/or optional extensions (B 1 and/or B2) (and excludes the contribution of any amino acids that may contain linker precursors (X1 and X2), Linker (L) and hydrophobic molecule (H) or Particle (P)), can be calculated to determine the net charge required to ensure the formation of stable particles by some embodiments of the peptide antigen conjugates of the present invention. Note: GRAVY peptide antigen conjugate is considered equivalent to GRAVY peptide antigen fragment; thus, the GRAVY value of a peptide antigen fragment may also refer to the GRAVY value of a peptide antigen conjugate.

In some embodiments of the present invention, the GRAVY value of the peptide antigen (A) is used to determine the net charge required to stabilize the particles formed by the peptide antigen conjugates. As a non-limiting example, a peptide antigen conjugate must have a net charge > (greater than or equal to) +6 or < (less than or equal to) -6 when the GRAVY value for the peptide antigen (A) is greater than 0.75; the total charge must be > (greater than or equal to) +5 or < (less than or equal to) -5 when the GRAVY value is in the range of 0.25-0.75; the total charge must be > (greater than or equal to) +4 or < (less than or equal to) -4 when the GRAVY value is less than 0.25. In preferred embodiments of the present invention, the net charge is greater than or equal to +4 or less than or equal to -4, depending on whether the net charge is desired: positive or negative, respectively. In some embodiments of the present invention, peptide antigens (A) with a GRAVY value of less than 0.25 are synthesized as peptide antigen conjugates with a net positive charge of from about +4 to +15, such as +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14 or +15, however it is usually from about +4 to +12; or with a net negative charge of from about -4 to -15, such as -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, however, it is typically between about -4 and -12. In some embodiments of the present invention, peptide antigens (A) with a GRAVY value in the range of 0.25-0.75 are synthesized as peptide antigen conjugates with a net positive charge of about +5 to +15, such as +5, +6, +7 , +8, +9, +10, +11, +12, +13, +14, +15, however it is usually from about +5 to +12; or with a net negative charge of about -5 to -15, such as -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, but is usually ranges from about -5 to -15. In some embodiments of the present invention, peptide antigens (A) with a GRAVY value greater than 0.75 are synthesized as peptide antigen conjugates with a net positive charge of from about +6 to +15, for example, +6, +7, +8, + 9, +10, +11, +12, +13, +14, +15, but usually it ranges from about +6 to +12; or with a net negative charge of from about -6 to -15, such as -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, but is typically from about -6 to -12.

In one embodiment of the method of selecting a charged molecule (C) and optional extensions (B1 and/or B2) of the present invention to produce a peptide antigen conjugate with a desired net charge, the method may comprise the steps of (i) determining GRAVY and the charge of the peptide antigen ( A) and (ii) selection of the charged molecule (C) and optional extensions (B1 and/or B2) to achieve a sufficient number of charged functional groups to achieve the desired net charge. In a non-limiting example, the net charge required for a peptide antigen conjugate may vary depending on the hydropathy of the peptide antigen (A) according to the following criteria: a charge that is greater than or equal to +6 is required for peptide antigens (A) of 25-35 amino acids with a GRAVY value greater than 0.75; a charge greater than or equal to +5 is required for peptide antigens (A) of 25-35 amino acids with a GRAVY value of 0.25 to 0.75; and a charge greater than or equal to +4 is required for peptide antigens (A) of 25-35 amino acids with a GRAVY value of less than 0.25. Thus, in this non-limiting example, a 25 amino acid peptide antigen (A) with a GRAVY value of 2.0 and a charge of +2 would be combined with a charged molecule (C) and optional extensions (B1 and/or B2) containing the net charge, at least +4 to achieve a net charge of the peptide antigen conjugate of +6. In a further non-limiting example, the net charge required for a peptide antigen conjugate may vary depending on the hydropathy of the peptide antigen (A) according to the following criteria: a charge that is less than or equal to -6 is required for peptide antigens (A) of 25 -35 amino acids with a GRAVY value greater than 0.75; a charge that is less than or equal to -5 is required for peptide antigens (A) of 25-35 amino acids with a GRAVY value of 0.25 to 0.75; and a charge that is less than or equal to -4 is required for peptide antigens (A) of 25-35 amino acids with a GRAVY value of less than 0.25. Thus, in this non-limiting example,

- 72 046161 tide antigen (A) with a GRAVY value of 2.0 and a charge of +2 must be combined with a charged molecule (C) and optional extensions (B 1 and/or B2) containing a net charge of -8 to achieve a net charge of the peptide conjugate antigen -6.

In some embodiments of the present invention, determining the GRAVY value of a peptide antigen fragment, including an optional charged molecule (C), optional extensions (B1 and/or B2), and peptide antigen (A), is used to determine the net charge required to stabilize the particles , formed by peptide antigen conjugates. As a non-limiting example, a peptide antigen conjugate must have a net charge of > (greater than or equal to) +6 or < (less than or equal to) -6 when the GRAVY value of the peptide antigen fragment is greater than 0.75; the total charge must be > (greater than or equal to) +5 or < (less than or equal to) -5 when the GRAVY value is in the range of 0.25-0.75; the total charge must be > (greater than or equal to) +4 or < (less than or equal to) -4 when the GRAVY value is less than 0.25. In preferred embodiments of the present invention, the net charge is greater than or equal to +4 or less than or equal to -4, depending on whether the net charge is positive or negative, respectively. In some embodiments of the present invention, peptide antigen fragments with a GRAVY value of less than 0.25 have a net positive charge of about +4 to +15, such as +4, +5, +6, +7, +8, +9, +10 , +11, +12, +13, +14 or +15, however it is usually from about +4 to +12; or a net negative charge of about -4 to -15, such as -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, however, it is typically between about -4 and -12. In some embodiments of the present invention, peptide antigen fragments with a GRAVY value in the range of 0.25-0.75 have a net positive charge of about +5 to +15, such as +5, +6, +7, +8, + 9, +10, +11, +12, +13, +14, +15, however it is usually from about +5 to +12; or a net negative charge of about -5 to -15, such as -5, -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, but is typically from about -5 to -12. In some embodiments of the present invention, peptide antigen fragments with a GRAVY value greater than 0.75 have a net positive charge of from about +6 to +15, such as +6, +7, +8, +9, +10, +11 , +12. +13, +14, +15, however usually it is from about +, -6 to -15, for example -6, -7, -8, -9, -10, -11, -12, -13, -14 or -15, but is typically between about -6 and -12.

A further unexpected finding is that the intensity of the net charge required to stabilize particles formed by certain embodiments of peptide antigen conjugates, such as Formula V and Formula VI peptide antigen conjugates, generally increases with the length of the peptide antigen (A). In some embodiments of the present invention, peptide antigens (A) of 7-14 amino acids are synthesized as peptide antigen conjugates with a net charge that is greater than or equal to +6 or less than or equal to -6; peptide antigens (A) of 15-24 amino acids are synthesized in the form of peptide antigen conjugates with a total charge that is greater than or equal to +7 or less than or equal to -7; and peptide antigens (A) of 25-35 amino acids are synthesized as peptide antigen conjugates with a net charge that is greater than or equal to +8 or less than or equal to -8.

In some embodiments of the present invention, the immunogenic compositions comprise particles consisting of peptide antigen conjugates of the formula [B1]-A-[B2]-[L]-H and charged molecule (C) conjugates of the formula C-[A']-[L] -H, wherein [ ] denotes that the group is optional, and A' represents an optional conserved antigen (i.e., not patient specific), further wherein the molar ratio is [B1]-A-[B2]- [L]-H - C-[A']-[L]-H selected from the range 1:20 to 20:1, such as, 1:20, 1:10, 1:5, 1:2, 1 :1, 2:1, 5:1, 10:1 and 20:1, and the net charge of the formula C-[A']-[L]-H is negative and is chosen in the range from -2 to -20, such as , -2 , -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, - 18, -19 or -20, typically from about -4 to -12, or positive and selected in the range +2 to +20, such as +2, +3, +4, +5, +6, +7, +8 , +9, +10, +11, +12, +13, +15, +16, +17, +18, +19 or +20, usually +4 to +12. In a non-limiting example, the immunogenic composition contains particles containing 1 molar equivalent of the peptide antigen conjugate, Ala-Ser-Met-Thr-Asn-Met-Glu-Leu-Met-Ser-Ser-Lys(N3-DBCO-H)-NH ₂ . which carries a total charge of 0 (it should be noted that the peptide fragment has a C-terminal amide) and 1 molar equivalent of a conjugate of a charged molecule (C), consisting of (Lys) ₅ -Lys(N3-DBCO-NH)-NH ₂ , which carries total charge +6; the ratio of peptide antigen conjugate to charged molecule conjugate is 1:1, and the total charge of each set of these two molecules is +6. Thus, the net charge of the particles can be controlled by modulating the ratio of peptide antigen conjugate to charged molecule conjugate (C), or by modulating the net charge of the charged molecule conjugate. In preferred embodiments of the present invention, the ratio of peptide antigen conjugate to charged molecule is from about 1:4 to 4:1, and the net charge of the charged molecule is selected such that the average net charge of the molecules containing the particles is from about +4 to about +12 or from about -4 to about -12. For example, a particle containing a 1:1 molar ratio of peptide antigen conjugates to charged conjugates

- 73 046161 molecules (C), while the peptide antigen conjugate has a total charge of 0, the average total charge of the molecules is +4, when the total charge of the conjugate of a charged molecule (C) is +8.

Positively charged peptide antigen conjugates may be useful for certain applications. In some embodiments of the present invention, a peptide antigen conjugate may comprise a peptide antigen (A), a charged molecule (C) containing positively charged functional groups, a hydrophobic molecule (H) or particles (P), optional extensions (B1 and/or B2) and an optional linker (L). The charged molecule composition (C) and optional extensions (B1 and/or B2) are selected to maximize particle stability and biological activity of the peptide antigen conjugates. In some embodiments of the present invention, a Formula V peptide antigen conjugate consisting of a charged molecule (C) with a net positive charge is linked to an N-terminal peptide extension (B1) that is linked to a peptide antigen (A) that is linked to C- a terminal peptide extension (B2) that is linked, either directly or via a linker (L), to a hydrophobic molecule (H), and the resulting peptide antigen conjugate has a net positive charge. In this example, a hydrophobic molecule (H) linked through the C-terminus of the peptide antigen (A) functions to induce particle formation, and a charged molecule (C) linked through the N-terminus of the peptide antigen (A) functions to stabilize the particles through high density of positive charge on the surface of these particles. Thus, optional B2 extensions placed proximally to the hydrophobic molecule (H) of the peptide antigen conjugates with a net positive charge of Formula V, wherein the charged molecule (C) is linked via the N-terminus of the peptide antigen (A), in a preferred embodiment of the present invention consist of uncharged and hydrophobic amino acids that help stimulate particle formation and are usually selected from single amino acids such as glycine, serine, citrulline, leucine, norleucine or methionine; dipeptides such as Gly-Cit, Gly-Ser or Gly-Leu; tripeptides such as Gly-Ser-Cit, Gly-Pro-Cit or Gly-Pro-Gly; tetrapeptides such as Ser-Pro-Val-Cit or Gly-Pro-Gly-Cit; pentapeptides such as Gly-Ser-Val-Leu-Cit, Gly-Pro-Val-Leu-Cit; or hexapeptides such as Gly-Gly-Ser-Leu-Val-Cit or Gly-Gly-Ser-Pro-Val-Cit. Some embodiments of the present invention utilize a C-terminal extension (B2) that includes a single charged amino acid, and is typically selected from a single amino acid such as arginine or lysine; from dipeptides such as Gly-Arg or Gly-Lys; tripeptides such as Gly-Ser-Arg or Gly-SerLys; tetrapeptides such as Gly-Pro-Gly-Arg (SEQ ID NO: 59), Gly-Ser-Val-Arg (SEQ ID NO: 60), or Ser-Leu-Val-Arg (SEQ ID NO: 7) ( where Arg can be replaced by Lys); pentapeptides such as Gly-Ser-LeuVal-Arg (SEQ ID NO: 17) (where Arg can be replaced by Lys); and hexapeptides such as Gly-Gly-SerLeu-Val-Arg (SEQ ID NO: 13) or Gly-Gly-Ser-Pro-Val-Arg (SEQ ID NO: 61) (where Arg can be replaced by Lys). The B1 extensions placed proximal to the charged molecule (C) of positively charged peptide antigen conjugates of Formula V, wherein the charged molecule (C) is linked via the N-terminus of the peptide antigen (A), in a preferred embodiment of the present invention contain a charged amino acid such as Arg or Lys, and are usually selected from single amino acids selected from Arg or Lys; dipeptides selected from Val-Arg or Leu-Arg (where Arg may be replaced by Lys); tripeptides selected from Pro-Val-Arg or Gly-Val-Arg (where Arg may be replaced by Lys); or tetrapeptides selected from Lys-Leu-Val-Arg (SEQ ID NO: 62), Lys-ProVal-Arg (SEQ ID NO: 9), Lys-Pro-Leu-Arg (SEQ ID NO: 8), Ser- Leu-Val-Arg (SEQ ID NO: 7) and Ser-Pro-ValArg (SEQ ID NO: 6) (where Arg can be replaced by Lys). The various extensions, B1 and B2, can be combined with any charged molecule (C), peptide antigen (A), hydrophobic molecule (H) or particle (P) and linker (L) to achieve a global average hydropathy value (GRAVY) and total charge of the peptide antigen conjugate required for the intended use.

In some embodiments of the present invention, the peptide antigen conjugate with a net positive charge of Formula V contains a charged molecule (C), typically containing from 4 to 12 positively charged functional groups, for example, (Lys) ₄ - ₁₂ , which is linked to the N-terminal an extension (B1) of a dipeptide, for example Val-Arg, which is linked to a peptide antigen (A), which usually contains from 7 to 35 amino acids, which is linked to a C-terminal extension (B2), for example, Ser-Pro-Val-Cit, that is, linked to a linker (Lys(N3)-DBCO), which is linked to a hydrophobic molecule (H) consisting of poly(amino acids) of Formula I or Formula II linked to adjuvants of Formula III.

Certain applications require negatively charged peptide antigen conjugates. In some embodiments of the present invention, a peptide antigen conjugate may comprise a peptide antigen (A), a charged molecule (C) containing positively charged functional groups, a hydrophobic molecule (H) or particles (P), optional extensions (B1 and/or B2) and an optional linker (L). The charged molecule composition (C) and optional extensions (B1 and/or B2) are selected to maximize particle stability and biological activity of the peptide antigen conjugates. In some embodiments of the present invention, a Formula V peptide antigen conjugate consisting of a charged molecule

- 74 046161 ly (C) with a net negative charge, linked to the N-terminal peptide extension (B1), which is linked to the peptide antigen (A), which is linked to the C-terminal peptide extension (B2), which is linked, either directly, or via a linker (L), with a hydrophobic molecule (H), and the resulting peptide antigen conjugate has a net negative charge. Thus, the B2 extension placed proximally to the hydrophobic molecule (H) of the peptide antigen conjugates with a net negative charge of Formula V, wherein the charged molecule (C) is linked by the B1 extension at the N-terminus of the peptide antigen (A), in a preferred embodiment implementation of the present invention consists of uncharged and hydrophobic amino acids that help stimulate particle formation, and are usually selected from single amino acids such as glycine, serine, citrulline, leucine, norleucine or methionine; dipeptides such as GlySer, Gly-Cit or Gly-Leu; tripeptides such as Gly-Ser-Cit, Gly-Pro-Cit or Gly-Pro-Gly; tetrapeptides such as Ser-Pro-Val-Cit, Ser-Pro-Val-Cit or Gly-Pro-Gly-Cit; pentapeptides such as Gly-Ser-Val-LeuCit, Gly-Pro-Val-Leu-Cit; or hexapeptides such as Gly-Gly-Ser-Leu-Val-Cit or Gly-Gly-Ser-Pro-ValCit, or Gly-Gly-Ser-Pro-Leu-Cit. Some embodiments of the present invention employ a C-terminal extension (B2) that includes a single charged amino acid, and is typically selected from a single amino acid such as arginine or lysine; dipeptides such as Gly-Arg (where Arg can be replaced by Lys); tripeptides such as Gly-Ser-Arg (where Arg can be replaced by Lys); tetrapeptides such as Gly-Pro-Gly-Arg (SEQ ID NO: 59), Gly-Ser-Val-Arg (SEQ ID NO: 60), Ser-Leu-Val-Arg (SEQ ID NO: 7), Asp -Leu-Val-Cit or Asp-Leu-Val-Leu (SeQ ID NO: 63) (where Asp can be replaced by Glu and Arg can be replaced by Lys); pentapeptides such as Gly-Ser-Leu-Val-Arg (SEQ ID NO: 17) or Gly-Asp-Leu-Val-Leu (SEQ ID NO: 64) or Gly-Asp-Leu-Val-Arg (SEQ ID NO: 65); and hexapeptides such as Gly-Gly-Ser-Leu-Val-Arg (SEQ ID NO: 13) or Gly-Gly-Ser-Pro-Val-Arg (SEQ ID NO: 61) (where Asp can be replaced by Glu, a Arg can be replaced by Lys); or Gly-Ser-Glu-Leu-Val-Arg (SEQ ID NO: 66) or Gly-Gly-Asp-Pro-Val-Arg (SEQ ID NO: 67) (where Asp can be replaced by Glu and Arg can be replaced to Lys). The B1 extensions placed proximally to the charged molecule (C) of the peptide antigen conjugates with a net negative charge of Formula V, wherein the charged molecule (C) is linked through the N-terminus of the peptide antigen by the B1 extension, in a preferred embodiment of the present invention comprise a charged amino acid, such as Asp or Glu, and they are usually selected from single amino acids such as Cit, Leu, Arg or Lys; dipeptides selected from Val-Cit, Leu-Cit, Val-Arg or Leu-Arg (where Arg can be replaced by Lys); tripeptides selected from Pro-Val-Arg, Pro-Val-Cit, Gly-Val-Arg or Gly-Val-Cit (where Arg can be replaced by Lys); or tetrapeptides selected from Ser-Leu-Val-Arg (SEQ ID NO: 7), Ser-Pro-Val-Arg (SEQ ID NO: 6), SerPro-Leu-Cit, Ser-Leu-Val-Cit, Ser -Pro-Val-Cit, Asp-Leu-Val-Arg (SEQ ID NO: 68), Asp-Pro-Val-Arg (SEQ ID NO: 69), Asp-Leu-Val-Cit, Asp-Leu-Val -Leu (SeQ ID NO: 63), Ser-Gly-Val-Cit or Asp-Pro-Val-Cit (where Arg can be replaced by Lys and Asp can be replaced by Glu). The various extensions, B1 and B2, can be combined with any charged molecule (C), peptide antigen (A), hydrophobic molecule (H) or particle (P) to achieve a global average hydropathy value (GRAVY) and the required net charge of the peptide antigen conjugate .

In some embodiments of the present invention, the peptide antigen conjugate with a net negative charge of Formula V contains a charged molecule (C), typically containing from 6 to 14 negatively charged functional groups, for example, (Glu) _6-14 , which is linked to an N-terminal extension (B1) a dipeptide, e.g. Val-Cit, which is linked to a peptide antigen (A), which typically contains from 7 to 35 amino acids, which is linked to a C-terminal extension (B2), e.g. Ser-Pro-Val-Cit, that is, linked to a linker (Lys(N3)-DBCO), which is linked to a hydrophobic molecule (H) consisting of poly(amino acids) of Formula I or Formula II linked to adjuvants of Formula III.

In some embodiments of the present invention, the addition of a charged molecule (C) and/or extensions (B1 and B2) decreased the GRAVY value of the peptide antigen fragment containing peptide antigen (A) and increased the net charge of the sequence, which was associated with unexpected improvements in fragment production peptide antigen. In some embodiments of the present invention, the addition of a charged molecule (C) containing at least one amino acid residue of lysine (Lys) or arginine (Arg) resulted in the successful synthesis and HPLC purification of a peptide antigen fragment containing a peptide antigen (A) that could not otherwise be produced as a native peptide antigen (ie, a native peptide antigen without other modifications). In some embodiments of the present invention, a peptide antigen (A) linked to a C-terminal extension (B2) consisting of at least one amino acid proline, pseudoproline (Pro), arginine (Arg) or lysine (Lys) resulted in successful synthesis a peptide antigen fragment containing a peptide antigen (A) that would not otherwise be produced as a native peptide antigen (ie, a native peptide antigen without other modifications). In some embodiments of the present invention, adding at least one aromatic amine, such as phenylalanineamine, to a peptide antigen fragment or peptide antigen conjugate produced entirely by

- 75 046161 solid-phase peptide synthesis, has resulted in the successful synthesis of a peptide antigen fragment or peptide antigen conjugate containing a peptide antigen (A) that otherwise could not be produced as a native peptide antigen (that is, a native peptide antigen without other modifications) .

Selection of peptide antigens (A) for use in personalized cancer vaccines

In some embodiments of the present invention, the peptide antigen (A) comprising the peptide antigen conjugate is specific to an individual patient. A peptide antigen conjugate containing peptide antigens (A) that are specific to individual patients can be used for personalized therapies, for example, for use as a personalized cancer vaccine or a personalized vaccine to induce tolerance or suppression for the treatment of autoimmune diseases or allergic reactions.

The selection of peptide antigens (A) for inclusion in a personalized cancer vaccine is a multi-step process, and some steps may be optional.

The first step involves the identification of tumor-associated antigens that are specific to the tumor or, compared to normal tissue, overexpressed by the tumor. Accordingly, tumor tissue and healthy tissue are obtained. The tumor tissue and healthy tissue may be formalin-fixed and paraffin-embedded or may be freshly isolated tissue. Healthy tissue may be blood containing white blood cells. Tumor tissue and healthy tissue are processed to extract DNA. The DNA is then processed and sequenced to identify differences between tumor DNA and healthy tissue DNA. These DNA differences may be single nucleotide or dinucleotide or higher order nucleotide changes that result in a nonsynonymous mutation, insertions and deletions that lead to frameshift mutations, splice site mutations that lead to alternative splice variants, or stop splicing variants. codons that can be readthrough, resulting in single amino acid deletions. Further mutations may be possible through chromosomal translocations or inversions, or duplications. There are many ways to make changes at the DNA level that can give rise to aberrant peptide sequences and/or peptides with aberrant post-translational modifications, which are tumor specific and may be referred to as neoantigens or predicted neoantigens.

The second step involves determining whether the tumor-associated antigens identified in step 1 are actually expressed by the tumor. Tumor RNA is isolated, processed, and sequenced to determine whether the mutations identified in step 1 are expressed as RNA by tumor cells. Peptide antigens (A) containing mutations identified from DNA sequencing of tumor and healthy tissue can be selected for inclusion in a personalized cancer vaccine based on RNA expression levels. In addition, tumor-associated self-antigens that produce higher levels of RNA in tumors compared to non-malignant tissues can be selected as peptide antigens (A) for inclusion. Mutations where the RNA transcript has not been identified are not usually selected as the peptide antigen (A) for inclusion in the vaccine. Peptide antigens (A) containing mutations or tumor-associated self-antigens may be prioritized based on the RNA expression level of the mutant peptide (i.e., neoantigen) or tumor-associated self-antigens, e.g., mutations expressed at higher levels or tumor-associated self-antigens may be prioritized . Several criteria can be applied simultaneously when selecting peptide antigens (A) for inclusion in a personalized cancer vaccine. For example, the level of RNA expression and the predicted MHC binding affinity of epitopes contained in a mutant peptide (i.e., neoantigen) or tumor-associated self-antigen can be used together to select the optimal set of peptide antigens (A) for inclusion in a customized cancer vaccine. In such a scenario, a peptide antigen (A) containing a T cell epitope with moderate binding affinity that has very high expression may be prioritized over another peptide antigen (A) containing a T cell epitope that has more high binding affinity, but is expressed by the tumor at very low levels.

In addition, it should be taken into account that a clonal or conservative mutation or tumor-associated self-antigen occurs in different tumor cells that the tumor contains. The clonality of a mutation is assessed by comparing the frequency of the mutation with the frequency of the wild-type variant in DNA isolated from the tumor. Tumor-associated neoantigens or self-antigens can be selected for use as peptide antigens (A) for inclusion in personalized cancer vaccines consisting of peptide antigen conjugates based on the clonality or near-clonality of the mutation they contain. For example, peptide antigens (A) may be prioritized for inclusion if they are predicted to be present in >50%

- 76 046161 tumor cells, > 75% tumor cells, > 85% tumor cells, > 95% tumor cells or > 99% tumor cells.

In some embodiments of the present invention, peptide antigens (A) for inclusion in a customized cancer vaccine consisting of peptide antigen conjugates may also be prioritized based on the predicted binding of epitopes contained in the antigen for a given MHC class I and/or class II molecule. as determined by the in silico linking algorithm. In a non-limiting example, each subject's MHC type is first identified through sequencing. Each mutant peptide (i.e., neoantigen) or tumor-associated self-antigen, identified by any suitable means (e.g., DNA sequencing, RNA expression, or mass spectrometry), is then tested for predicted binding to each MHC molecule present in the subject that, e.g. , in human patients, may include up to 6 unique MHC Class I alleles. There are several publicly available algorithms that can be used to predict MHC binding, including the netMHC artificial neural network, the stabilized matrix method, and the Consensus Analytical Resources and Immune Epitope Database (IEDB) algorithm. . Internal algorithms can also be applied. Peptide antigens (A) that contain an epitope with a high predicted binding affinity are more likely to induce an immune response than peptide antigens (A) containing epitopes with a low predicted binding affinity. An unexpected finding disclosed herein is that more than 50% of peptide antigens (A), including predicted neoantigens, that have an epitope with a predicted binding affinity of less than 0.5 percentile by the IEDB Consensus algorithm are capable of generating T cell responses ( that is, CD8 T cell responses) when administered using immunogenic compositions containing peptide antigen conjugates described herein. Based on this unexpected discovery, peptide antigens (A), including peptide antigens (A), containing predicted neoantigens that contain an epitope with a binding affinity of less than 0.5 percentile according to the IEDB Consensus algorithm, can be selected for use in individualized cancer vaccines containing peptide antigen conjugates.

In addition, confirmation of antigen binding to MHC obtained by mass spectrometry or algorithms for binding antigens to MHC developed based on mass spectrometry can be used to select peptide antigens (A) for inclusion in a personalized cancer vaccine. In this scenario, tumor tissue is processed to identify peptides that are associated with MHC molecules. Peptides that are identified on the surface of tumor cells but not healthy cells, which may be mutant peptides (i.e., neoantigens), proteasomal splice variants, or tumor-associated self-antigens, may be prioritized for inclusion in a personalized cancer vaccine. An unexpected finding disclosed herein is that a high proportion (i.e., 7 out of 7) of predicted neoantigens that were selected for use in a personalized cancer vaccine based on mass spectrometrically confirmed MHC-I binding on tumor cells resulted in high intensity CD8 T cell responses when delivered as peptide antigen (A) in immunogenic compositions containing a peptide antigen conjugate, suggesting that mass spectrometry or mass spectrometry-based predictive algorithms may be reliable filters for selecting neoantigens for use as peptide antigens (A) in individualized cancer vaccines consisting of peptide antigen conjugates.

Peptide antigens (A) can also be selected for inclusion based on a T cell recognition assay. For example, an assay may be employed in which synthetic peptides (or expression systems that produce the peptide in situ) containing predicted neoantigens or tumor-associated self-antigens are added in vitro to a culture of T cells derived from the blood, tumor tissue, or other tissue of a subject. T cell recognition of a given peptide can be assessed, for example, by enzyme-linked immunospot (ELISpot) or flow cytometry. Antigens recognized in in vitro T cell assays may be prioritized for inclusion as peptide antigen (A) in a customized peptide antigen conjugate vaccine.

Finally, peptide antigens (A) can be selected based on any number of predictive algorithms. Peptide antigens (A) that are predicted to be immunogenic or effective based on predictive algorithms developed for large data sets can be used. In addition, predictive algorithms can be applied to select peptide neoantigens that are predicted to result in T cell responses that are specific for the mutant epitope but not for the wild type epitope, that is, for T cells that are not cross-reacting to self-antigens.

Any combination of the above methods can be used to identify and select

- 77 046161 peptide antigens (A) containing neoantigens or tumor-associated self-antigens, or the like, for use in a personalized cancer vaccine consisting of peptide antigen conjugates.

Peptide antigen length (A) and influence on induction of CD8 and CD4 T cell responses for personalized cancer vaccines

A single nucleotide polymorphism that results in a nonsynonymous amino acid substitution can occur at any position in a peptide epitope that binds MHC Class I, which is typically 8-13 amino acids in length. Therefore, to cover all possible epitopes that a given MHC Class I may bind, the peptide antigen (A) containing neoantigen for use in a customized cancer vaccine may include 12 amino acids on either side of the nonsynonymous mutation, forming a 25 amino acid long peptide with the mutant amino acid as the middle (13th) remainder. In an alternative embodiment of the present invention, the peptide antigen (A) may include only the minimal epitope (8-13 amino acids in length) that is predicted to bind to MHC Class I. In an alternative embodiment of the present invention, a customized cancer vaccine may contain: a peptide antigen (A) containing a neoantigen of 25 amino acids, and a peptide antigen (A) containing only the predicted minimal epitope of the neoantigen.

The MHC Class II peptide binding pocket typically binds peptides of 12-16 amino acids, and some have at least 20 amino acids. Therefore, a personalized cancer vaccine that consists of peptide antigens (A) that contain only predicted Class I binding minimal epitopes (which are typically 8-13 amino acids in length) is unlikely to induce CD4 T cell responses. However, a cancer vaccine that contains 25 amino acid (or 25-mer) peptide antigens (A) can, but does not always, induce CD4 T cell responses targeting the mutation.

A 25 amino acid (or 25-mer) peptide antigen (A) in a cancer vaccine may induce lower levels of CD8 T cell responses compared to a peptide antigen (A) containing a precise minimal epitope consisting of ~7- 12 amino acids, possibly due to differences in the efficiency of processing and presentation of peptide antigens of different lengths in the soluble organic fraction. Thus, 25 amino acid peptide antigens (A) included in a cancer vaccine may result in lower intensity CD8 T cell responses compared to the intensity of responses induced by peptide antigens (A) containing minimal epitopes. Accordingly, in some embodiments of the present invention, a 25 amino acid peptide antigen (A) included in a cancer vaccine consisting of peptide antigen conjugates does not result in detectable CD8 T cell responses, whereas a 7 amino acid peptide antigen (A) -12 amino acids containing the exact minimal epitope results in detectable CD8 T cell responses. In additional embodiments of the present invention, a 25 amino acid peptide antigen (A) included in a cancer vaccine consisting of peptide antigen conjugates results in detectable CD4 T cell responses, whereas a 7-amino acid peptide antigen (A) The 12 amino acids containing the exact minimal epitope do not result in detectable CD4 T cell responses. Based on these unexpected findings, in some embodiments of the present invention, two lengths of peptide antigens (A) containing the same epitope, both the minimal CD8 T cell epitope (referred to as Min or minimal epitope (ME)) and a peptide containing 25 amino acids (referred to as synthetic long peptide (SLP) or long peptide), can be included as peptide antigens (A) in personalized cancer vaccines consisting of peptide antigen conjugates. In some embodiments of the present invention, peptide antigens (A) that consist of minimal CD4 and CD8 T cell epitopes are included in a personalized cancer vaccine consisting of peptide antigen conjugates. In additional embodiments of the present invention, peptide antigens (A) consisting of minimal CD8 T cell and peptide antigens (A) containing a universal CD4 T cell epitope and optional peptide antigens (A) consisting of minimal CD4 T cell epitopes, included in individualized cancer vaccines consisting of peptide antigen conjugates. In preferred embodiments of the present invention, the peptide antigens (A) included in the personalized cancer vaccine consisting of peptide antigen conjugates contain from 7 to 35 amino acids, usually from 15 to 35 amino acids, for example 25 amino acids.

Combination immunotherapy

The peptide antigen conjugates disclosed herein can be used in immunogenic compositions for the treatment of tumors or infectious diseases. Peptide antigen conjugates can be used alone or in combination with other therapies. In the treatment of cancer, immunogenic compositions consisting of peptide antigen conjugates can be used before, during or after any form of treatment such as surgery, radiation therapy or

- 78 046161 chemotherapy. In preferred embodiments of the present invention, immunogenic compositions containing peptide antigen conjugates are used in combination with immunomodulators such as cytokines (eg, IL-2), antitumor antibodies, checkpoint inhibitor antibodies (eg, anti-PD1) or other small molecules or biologics that reverse immunosuppression, directly kill tumor cells, or enhance the immune response against the tumor. In preferred embodiments of the present invention, a peptide antigen conjugate cancer vaccine is offered to a tumor-bearing subject in combination with a checkpoint inhibitor (eg, αntu-PD1, anti-PDLL·anti-CTLA4). In preferred embodiments of the present invention, a cancer vaccine, such as a personalized cancer vaccine, is offered to the subject prior to administration of the checkpoint inhibitor. The peptide antigen conjugates disclosed herein can also be used in heterologous prime-boost immunization, such as a prime or boost with a peptide antigen conjugate and a prime or boost with a heterologous vaccine, such as a viral vector.

Composition of the composition

Immunogenic compositions consisting of the peptide antigen conjugates disclosed herein can be formulated into pharmaceutical compositions prepared for administration to a subject and which include a therapeutically effective amount of at least one immunogen as described herein. A therapeutically effective amount of the disclosed compound will depend on the route of administration, the type of subject, and the physical characteristics of the subject being treated. Specific factors that may be taken into account include the severity and stage of the disease, weight, diet and types of concomitant drug therapy. The relationship of these factors to the determination of a therapeutically effective amount of the disclosed compounds will be apparent to those skilled in the art.

The immunogenic compositions for administration to a subject may be pharmaceutical compositions and may include at least one additional pharmaceutically acceptable additive, such as carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like, in addition to those selected molecule. Immunogenic compositions may also include at least one additional active ingredient, such as an antimicrobial agent, an anesthetic, and the like. Pharmaceutically acceptable carriers used for these compositions are standard. In Pharmaceutical Sciences of Remington, E.W. Martin, Mack Publishing Co., Easton, Pennsylvania, 19th edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compounds described herein.

In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a transporter. In addition to biologically neutral carriers, pharmaceutical compositions for administration may contain small amounts of non-toxic excipients such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

To formulate immunogenic compositions, the disclosed nanoparticle components or a solution containing the disclosed nanoparticle components can be combined with various pharmaceutically acceptable additives, as well as a carrier or carrier for dispersing the nanoparticles. Desirable additives include, but are not limited to, pH adjusting agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid and the like. In addition, local anesthetics (eg, benzyl alcohol), isotonic agents (eg, sodium chloride, mannitol, sorbitol), adsorption inhibitors (eg, Tween 80 or Migliol 812), solubility enhancing agents (eg, cyclodextrins and their derivatives), stabilizers (eg serum albumin) and reducing agents (eg glutathione). Adjuvants such as aluminum hydroxide (eg, Amphogel, Wyeth Laboratories, Madison, NJ), Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN), and IL-12 (Genetics Institute, Cambridge , Massachusetts), among many other suitable adjuvants well known in the art, may be included in the compositions. When the composition is a liquid, the tonicity of the composition, measured relative to the tonicity of 0.9% (vol%) physiological saline solution taken as a unit, is generally adjusted to a value that will not induce significant irreversible tissue damage at the site of administration. Typically, the tonicity of the solution is adjusted to about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7.

The immunogenic compositions of the present invention are generally sterile and stable under the conditions of manufacture, storage and use. Sterile solutions can be prepared by including the compound in the required amount in an appropriate solvent with one or

- 79 046161 combination of ingredients listed herein, as necessary, followed by filter sterilization. Typically, dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains the basic dispersion medium and other required ingredients among those listed herein. For sterile powders, methods of preparation include vacuum drying and freeze drying to yield a powder of the compound plus any additional desired ingredient from its solution, previously sterilized by filtration. Prevention of the action of microorganisms can be achieved through various antibacterial and antifungal agents, for example, through parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.

The present invention also includes kits, packages and multi-container units containing the immunogenic compositions, active ingredients and/or means for administering them described herein for use in the prevention and treatment of diseases and other health conditions in mammalian subjects. In one embodiment of the present invention, these kits include a container or composition that contains at least one immunogenic composition described herein. In one example, the immunogenic composition is formulated as a pharmaceutical preparation for delivery to a subject. In one example, the immunogenic composition is optionally contained in a dosage container in the form of an angro, or in a monoblock or multiblock packaging in the form of a dosage form. Additional dosing devices may be offered, for example, an applicator for inhalation or intranasal spraying. The packaging materials do not necessarily include a label or instructions indicating for what medicinal purposes and/or how the pharmaceutical agent packaged therein may be used.

Methods for inducing an immune response

Immunogenic compositions comprising peptide antigen (A) as described herein can be used to induce an immune response to peptide antigen (A) in a subject. Subjects that may benefit from the methods disclosed in the present invention include humans and veterinarians.

In some embodiments of the present invention, a subject who has developed or is at risk of developing an infection from an infectious agent that contains a peptide antigen is selected for treatment, for example, due to exposure or potential exposure to the infectious agent. Following administration of a therapeutically effective amount of the disclosed immunogenic composition, the subject may be monitored for the presence of infection, symptoms associated with infection, or both.

In some embodiments of the present invention, a subject who has developed or is at risk of developing cancer, such as a malignant tumor, is selected for treatment. After administration of a therapeutically effective amount of the disclosed immunogen, the subject may be monitored for the presence of cancer, reduction in tumor volume, any relevant symptom of cancer, or a combination thereof.

Typical subjects to be treated with the therapeutic agents and methods of the present invention include humans, as well as non-human primates and other animals. To identify subjects for prevention or treatment in accordance with the methods of the present invention, conventional screening methods are used to determine risk factors associated with a specific or suspected disease or health condition, or to determine the status of an existing disease or health condition in a subject. These screening methods include, for example, routine tests to determine environmental, family, occupational and other risk factors that may be associated with a specific or suspected disease or health condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods that are available and are widely known in the art for identifying and/or characterizing a disease or health condition. These and other routine methods allow the physician to select patients in need of therapy using the methods and pharmaceutical compositions of the present invention. In accordance with these methods and principles, the composition can be administered in accordance with the directions given herein or other standard methods known to one of ordinary skill in the art, as an independent prophylactic or treatment program, or as a medical follow-up, treatment regimen, complementing or coordinating other treatments.

Administration of a therapeutically effective amount of an immunogenic composition comprising a peptide antigen as described herein may be for prophylactic or therapeutic purposes. When used prophylactically, the immunogenic composition is offered before the onset of any symptoms, for example, before infection or tumor development. Prophylactic administration of an immunogenic composition serves to prevent or eliminate the subsequent development of a disease or health condition. Therefore, in some embodiments of the present invention, the methods include selecting a subject who is at risk of contracting an infectious disease.

- 80 046161 tion or tumor development, and administration of a therapeutically effective amount of a disclosed therapeutically effective amount of a disclosed immunogenic composition. Thus, an immunogenic composition may be offered prior to anticipated exposure to an infectious agent or tumor development to reduce the expected severity, duration or extent of the infection or tumor, and/or any associated disease symptoms.

For therapeutic use, the disclosed immunogenic composition may be provided during or after the onset of a symptom of a disease or health condition, such as after the development of a symptom of an infection or diagnosis of an infection, or the development of a symptom of a tumor or diagnosis of a tumor. Treatment of an infection or tumor may include delaying and/or reducing signs or symptoms of the infection or tumor in a subject. In some examples, treatment using methods disclosed herein prolongs the survival time of a subject.

The immunogenic composition can be used in coordinated immunization protocols or in combinatorial formulations.

In some embodiments of the present invention, a therapeutically effective amount of the disclosed immunogenic composition may be administered to a subject being treated or to inhibit an infectious agent in the subject. An infectious agent is an agent that can infect a subject, including, but not limited to, viruses, bacteria and fungi. A subject who is suspected or at risk of developing an infection with an infectious agent may be selected for treatment. In some embodiments of the present invention, the infectious agent is a virus, bacterium, or fungus as described above, and the peptide antigen includes an antigen from the particular virus, bacterium, or fungus.

In some embodiments of the present invention, a therapeutically effective amount of the disclosed immunogenic composition may be administered to a subject being treated or to inhibit a tumor and/or cancer in the subject. A subject who is suspected or at risk of developing a tumor and/or cancer may be selected for treatment. In some embodiments of the present invention, treating a tumor and/or cancer in a subject reduces tumor growth and/or proliferation. The tumor can be any tumor of interest and can be benign or malignant.

Tumor treatment usually begins after the tumor is diagnosed or after the onset of a pre-existing health condition (such as dysplasia or the development of a benign tumor). Treatment may be initiated in the early stages of the cancer, for example, may be initiated before the subject exhibits symptoms of a medical condition, such as at the time of diagnosis of stage I or at the time of diagnosis of dysplasia. However, treatment can be initiated during any stage of the disease, such as, but not limited to, stage I, stage II, stage III and stage IV cancer. In some examples, the treatment is administered to these subjects in the presence of a benign tumor that may develop into a malignant or even metastatic tumor.

Treatment initiated after the development of a health condition, such as cancer, may result in a reduction in the severity of symptoms of one of the health conditions or complete elimination of symptoms, or a reduction in metastasis, tumor volume, or number of tumors. In some examples, the tumor becomes undetectable after treatment. In one aspect of the present invention, tumor formation, such as metastasis, is delayed, prevented or reduced. In another aspect, the size of the primary tumor is reduced. In a further aspect, the tumor symptom is reduced. In yet another aspect, the volume of the tumor is reduced.

Subjects may be screened prior to initiation of the disclosed therapies, for example, to determine whether the subject has a tumor. The presence of a tumor can be determined by methods known in the art, and these usually include cytological and morphological evaluation. The tumor may be an advanced tumor. The cells may be in vivo or ex vivo, including cells obtained from a biopsy. The presence of a tumor indicates that the tumor can be treated using the methods proposed herein.

The therapeutically effective amount will depend on the severity of the disease and the general health of the patient. A therapeutically effective amount is that amount that offers either subjective relief of symptom(s) or objectively identifiable improvement, as noted by a physician or other qualified observer. In one embodiment of the present invention, a therapeutically effective amount is an amount necessary to inhibit tumor growth, or an amount that is effective to reduce a sign or symptom of a tumor. In another embodiment of the present invention, a therapeutically effective amount is an amount necessary to inhibit infection by an infectious agent, or an amount that is effective to reduce a sign or symptom of an infection. The therapeutically effective amount of agents administered may vary depending on the desired effects and the subject being treated. In some examples, therapeutic amounts are amounts that eliminate or reduce the tumor burden of a patient, or that prevent or reduce the proliferation of metastatic tumors.

- 81 046161 cells, or which reduce the infectious agent load in the subject.

The actual dosage of the immunogenic composition will vary depending on factors such as the disease symptom and the specific status of the subject (eg, the subject's age, size, physical condition, severity of symptoms, susceptibility factors, and the like), time and route of administration, other drugs, or treatments administered simultaneously, as well as the specific pharmacology of the compound to induce production of the desired activity or biological response in a subject. Dosage regimens can be adjusted to offer the optimal prophylactic or therapeutic response. A therapeutically effective amount is also an amount at which any toxic or detrimental side effects of the compound and/or other biologically active agent are outweighed in clinical terms by the therapeutically beneficial effects.

Dosage may be varied by the prescribing physician to maintain the desired concentration at the target site (eg, lungs or systemic circulation). Higher or lower concentrations may be selected based on the mode of delivery, for example, transepidermal, rectal, oral, inhalation, intraosseous or intranasal delivery, compared to intravenous or subcutaneous or intramuscular delivery. Dosing may also be adjusted based on the rate of release of the administered formulation, e.g., intrainhalation spray versus powder, orally administered sustained release formulation versus injectable dispersible or transdermal delivery formulations, and so on.

Any route of administration may be used for the disclosed therapeutic agents, including local and systemic administration. For example, topical, oral, intravascular, such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration can be used. The specific mode of administration and dosage regimen will be selected by the attending physician based on the characteristics of the case (eg, subject, disease, relevant stage of disease, and whether the treatment is prophylactic). In cases where more than one agent or more than one composition is administered, at least one route of administration may be used.

The disclosed therapeutic agents may be formulated in a single dosage dosage form suitable for individual administration of precise dosages. In addition, the disclosed therapeutic agents may be administered as a single dose or as a multiple dose regimen. A multiple-dose regimen is one in which the initial course of treatment may include more than one individual dose, for example 1-10 doses, followed by further doses at subsequent intervals as necessary to maintain or enhance the effects of the compositions. Treatment may involve daily or multi-day doses of the compound(s) over a period of days to months or even years. Thus, the dosage regimen will also be determined, at least in part, based on the specific needs of the subject being treated and will depend on the judgment of the practitioner.

Examples

Connection 1

nh ₂

Compound 1, 1-(4-ami11obutyl)-2-butyl-111-imidazo|4,5-c|chi11oli11-4-ami11, referred to as 2B, was synthesized according to the scheme shown in FIG. 3.

1st. Starting with 2,4-quinolindiol, intermediate 1-b was prepared as previously described (Lynn GM, et al., Nat Biotechnol 33(11): 1201-1210, 2015). To 21 g of 1-b (87.8 mmol, 1 eq.) in 210 ml of triethylamine (TEA) (10% by weight) was added 16.34 g (87.8 mmol, 1 eq.) Naboc-1.4 -butanediamine, shaking vigorously. The reaction mixture was heated to 70°C and monitored by HPLC, which confirmed that the reaction was complete after 2 hours. Triethylamine was removed in vacuo and the resulting oil was dissolved in 200 ml of dichloromethane and then washed 3 times with 100 ml of DI H ₂ O (deionized water ). The organic layer was dried with Na ₂ SO ₄ and then removed in vacuo and the resulting oil was triturated with a 1:1 (v:v) mixture of hexane and diethyl ether to yield 30.7 g of yellow crystals of intermediate 1-c. Mass spectrometry (atmospheric pressure chemical ionization) (MS (APCI)), calculated for C ₁₈ H ₂₃ ClN ₄ O ₄ , mass/charge 394.1, the result was 394.9.

1-d. 30.7 g (76.4 mmol) of intermediate 1-c were dissolved in 300 ml of ethyl acetate in a Parr reaction vessel, which was bubbled with argon, after which 3 g of 10% platinum on carbon was added. The reaction vessel was maintained under argon and then evacuated and pressurized with H ₂ (g) several times before increasing the pressure to 55 psi H ₂ (g), vigorously

- 82 046161 shaking. H2(g) was added continuously until the pressure stabilized at 55 psi, at which point the reaction was determined to be complete. The reaction mixture from the Parr Reactor was then filtered through a celite end, evaporated to dryness to yield a yellow oil, which was triturated with 1:1 hexane/ether to yield white crystals, which were collected by filtration to yield 27.4 g of spectroscopically pure white crystals 1-d. MS (APCI) was calculated for C ₁₈ H ₂₅ ClN ₄ O ₂ , mass/charge 364.2, the result was 365.2.

1st. To 10 g (27.4 mmol, 1 eq.) of 1-d in 50 ml of tetrahydrofuran (THF) was added 7.7 ml of triethylamine (54.8 mmol, 2 eq.), after which 3.6 g of valeroyl chloride was added dropwise (30.1 mmol, 1.1 eq.) in 30 ml THF, shaking vigorously while the reaction mixture was on ice. After 90 min, the ice bath was removed and THF was removed in vacuo to give a yellow oil, which was dissolved in 100 ml dichloromethane (DCM), which was washed 3 times with 50 ml 100 ml acetate buffer pH 5.5. The DCM was removed in vacuo into the oil, which was triturated with ethyl acetate to give 10.4 g of a white solid, which was dissolved in methanol with 1 g of CaO(s), which was heated at 100°C for 5 hours with vigorous shaking. The reaction mixture was filtered and dried to yield 10.2 g of an off-white solid, intermediate 1st. MS (electrospray ionization (ESI)) was calculated for C ₂₃ H ₃₁ ClN ₄ O ₂ , mass/charge 430.21, the result was 431.2.

1-f. To 10.2 g (23.7 mmol, 1 eq.) of 1st was added 30.4 g (284 mmol, 12 eq.) benzylamine liquid, which was heated to 110°C, shaking vigorously. The reaction was completed after 10 hours and the reaction mixture was added to 200 ml of ethyl acetate and washed 4 times with 100 ml of 1 M HCl solution. The organic layer was dried with Na ₂ SO ₄ and then removed in vacuo and the resulting oil was recrystallized from ethyl acetate to give 10.8 g of spectroscopically pure white crystals of intermediate, 1-f. MS (ESI) was calculated for C ₃₀ H ₃₉ N ₅ O ₂ , mass/charge 501.31, the result was 502.3.

Compound 1: 10.8 g (21.5 mmol) 1-f was dissolved in 54 ml concentrated (>98%) H2SO4 and the reaction mixture was shaken vigorously for 3 hours. After 3 hours, the viscous red reaction mixture was slowly added to 500 ml DI H2O, shaking vigorously. The reaction mixture was shaken for 30 minutes and then filtered through celite, after which a 10 M NaOH solution was added until the pH of the solution became pH 10. The aqueous layer was then extracted 6 times into 200 ml of DCM, and the resulting organic layer was dried with with Na2SO4 and reduced in vacuo to yield a spectroscopically pure white solid. ¹ H NMR (400 MHZ, DMSO-d ₆ ) δ 8.03 (d, J=8.1 HZ, 1H), 7.59 (d, J=8.1 Hz, 1H), 7.41 (t , J=7.41 Hz, 1H), 7.25 (t, J=7.4 Hz, 1H), 6.47 (s, 2H), 4.49 (t, J=7.4 Hz, 2H ), 2.91 (t, J=7.78 Hz, 2H), 2.57 (t, J=6.64 Hz, 1H), 1.80 (m, 4H), 1.46 (sep, J =7.75 Hz, 4H), 0.96 (t, J=7.4 Hz, 3H). MS (ESI) was calculated for C ₁₈ H ₂₅ N ₅ , mass/charge 311.21, the result was 312.3.

Connection 2.

NH;

Compound 2, 1-(4-(aminomethyl)benzyl)-2-butyl-III-imidazo[4,5-c]quinoline-4-amine, referred to as 2BXy, was previously described (see G. M. Lynn, et al., In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity. Nat Biotechnol 33(11): 1201-1210, 2015, and Shukla N.M., et al., Synthesis of fluorescent imidazoquinoline conjugates as Toll-like receptor 7 probes. Bioorg Med Chem Lett 20(22):6384-6386, 2010). 1H NMR (400 MHz, DMSO-d6) δ 7.77 (dd, J=8.4, 1.4 Hz, 1H), 7.55 (dd, J=8.4, 1.2 Hz, 1H) , 7.35-7.28 (m, 1H), 7.25 (d, J=7.9 Hz, 2H), 7.06-6.98 (m, 1H), 6.94 (d, J =7.9 Hz, 2H), 6.50 (s, 2H), 5.81 (s, 2H), 3.64 (s, 2H), 2.92-2.84 (m, 2H), 2 .15 (s, 2H), 1.71 (q, J=7.5Hz, 2H), 1.36 (q, J=7.4Hz, 2H), 0.85 (t, J=7.4 Hz , 3H). MS (APCI) was calculated for C ₂₂ H ₂₅ N ₅ , mass/charge 359.2, the result was 360.3.

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Connection 3.

Compound 3, referred to as DBCO-2BXy ₃ , 2BXy ₃ or DBCO-(Glu(2BXy) ₃ ), was synthesized starting from the Fmoc-(Glu) ₃ -NH ₂ precursor obtained by solid-phase peptide synthesis. 50 mg Fmoc(Glu) ₃ -NH ₂ (0.08 mmol, 1 eq.), 143 mg Compound 2 (0.40 mmol, 5 eq.), 84 mg 2-chloro-4,6-dimethoxy1,3, 5-triazine (CDMT) (0.48 mmol, 6 eq.) and 48.5 mg of 4-methylmorpholine (NMM) (0.48 mmol, 6 eq.) were added to 3.25 ml DMSO, shaking vigorously at room temperature in the surrounding air. The progress of the reaction was monitored by HPLC (Area under the pharmacokinetic curve (AUC) 254 nm). After 30 minutes, 1 additional equivalent of Compound 2 and 2 additional equivalents of both CDMT and NMM were added. After 2 hours, the reaction was completed and the reaction mixture was added to 50 ml of 1 M HCl to precipitate the Fmoc-protected intermediate, which was collected by centrifuging the solution at 3000 g at 4°C for 10 minutes. The HCl solution was discarded and the Fmoc protected intermediate was collected as a solid white pellet. The white solid was resuspended in 50 ml of 1 M HCl and centrifuged at 3000 g at 4°C for 5 min; The 1 M HCl solution was discarded and the product was collected as a solid pellet. This process was repeated and then the solid was collected and dried in vacuo to yield 156.1 mg of the Fmoc protected intermediate in quantitative yield. The Fmoc-protected product was then added to 1.5 ml of 20% piperidine in dimethylformamide (DMF) for 30 min at room temperature to yield the deprotected product, which was then precipitated from 50 ml of ether and centrifuged at 3000 g at 4°C for 30 min. The product was collected as a solid pellet and then washed twice with ether and then dried in vacuo to yield 126.4 mg of intermediate. 60 mg of the resulting intermediate, NH ₂ -(Glu-2BXy) ₃ -NH ₂ , (0.042 mmol, 1 eq.) was then reacted with 18.6 mg (0.046 mmol, 1.1 eq.) of DBCO-NHS ester (Scottsdale, AZ, USA) and 8.5 μL triethylamine (0.084 mmol, 2 equiv.) in 1 mL DMSO for 6 h at room temperature. The resulting product, Compound 3, was purified on a preparative HPLC system using a 30-70% acetonitrile/H ₂ O (0.05% trifluoroacetic acid (TFA)) gradient over 12 min on the column.

Agilent Prep-C18, 50x100 mm, 5 µm. The product was eluted for 7.0 min and the resulting fractions were collected, frozen and then lyophilized to yield 40.12 mg (55.7% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₀₀ H ₁₀₆ N ₂₀ O ₈ , mass/charge 1714.85, the result was 858.9 (M/2) ⁺

Connection 4.

Compound 4, referred to as DBCO-2BXy ₅ , 2BXy ₅ or DBCO-(Glu(2BXy) ₅ ), was synthesized using the same procedure as described for Compound 3, except for the fact that Fmoc-(Glu) ₅ -NH ₂ was used as a starting material for the conjugation of Compound 2. Compound 4 was purified in a preparative HPLC system using a gradient of 38-48% acetonitrile/H ₂ O (0.05%

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TFA) for 12 min on an Agilent Prep-C18 column, 50x100 mm, 5 µm. The product was eluted for 8.0 min and the resulting fractions were collected, frozen and then lyophilized to yield 45.9 mg (63.4% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₅₄ H ₁₆₆ N ₃₂ O ₁₂ , mass/charge 2655.34, the result was 886.6 (M/3) ⁺ .

Connection 5.

Compound 5, referred to as DBCO-2B5, 2B5 or DBCO-(Glu(2B) ₅ ), was synthesized using the same procedure as described for Compound 3, except that Fmoc-(Glu) ₅ NH2 was used as starting material for conjugation of Compound 1. Compound 5 was purified in a preparative HPLC system using a 33-45% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 50x100 mm, 5 µm column. The product was eluted for ~10.0 min and the resulting fractions were collected, frozen and then lyophilized to yield 25.2 mg (62.6% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₃₄ H ₁₆₆ N ₃₂ O ₁₂ , mass/charge 2415.34, the result was 1209.3 (M/2) ⁺ .

Connection 6.

Compound 6, referred to as DBCO-2B ₃ W ₂ , 2B ₃ W ₂ or DBCO-(Glu(2B) ₃ (Trp) ₂ ), was synthesized using the same procedure as described for Compound 3, except for the fact that Fmoc-Glu-Trp-Glu-Trp-Glu-NH ₂ was used as a starting material for the conjugation of Compound 1. Compound 6 was purified in a preparative HPLC system using a gradient of 33-47% acetonitrile/H ₂ O (0.05% TFA) for 12 min on an Agilent Prep-C18 column, 50x100 mm, 5 µm. The product was eluted over ~8 min and the resulting fractions were collected, frozen and then lyophilized to yield 197 mg (50.6% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₁₀ H ₁₂₆ N ₂₄ O ₁₀ , mass/charge 1943.01, the result was 973.0 (M/2) ⁺ .

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Connection 7.

Compound 7, referred to as DBCO-2B2W3, 2B ₂ W ₃ or DBCO-(Glu(2B) ₂ (Trp) ₃ ), was synthesized using the same procedure as described for Compound 3, except that Fmoc-Trp -Glu-Trp-Glu-Trp-NH ₂ was used as a starting material for the conjugation of Compound 1. Compound 7 was purified in a preparative HPLC system using a gradient of 35-65% acetonitrile/H ₂ O (0.05% TFA) over 12 min on an Agilent Prep-C18 column, 50x100 mm, 5 µm. The product was eluted over ~9 min and the resulting fractions were collected, frozen and then lyophilized to yield 11.6 mg (62.5% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₉₈ H ₁₀₆ N ₂₀ O ₉ , mass/charge 1706.85, the result was 854.9 (M/2) ⁺ .

Connection 8.

Compound 8, referred to as DBCO-2B ₂ W ₈ , 2B ₂ W ₈ or DBCO-(Glu(2B) ₂ (Trp) ₈ ), was synthesized using the same procedure as described for Compound 3, except for the fact that Pmoc-Trp-Trp-Glu-Trp-Trp-Trp-Trp-Glu-Trp-Trp-NH2 was used as a starting material for the conjugation of Compound 1. Compound 8 was purified in a preparative HPLC system using a gradient of 35-85% acetonitrile/ H ₂ O (0.05% TFA) for 12 min on an Agilent Prep-C18 column, 50x100 mm, 5 µm. The product was eluted for ~8.0 min and the resulting fractions were collected, frozen and then lyophilized to yield 3.3 mg (16.3% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₅₃ H ₁₅₆ N ₃₀ O ₁₄ , mass/charge 2637.24, the result was 1320.2 (M/2) ⁺ .

Connection 9.

Compound 9, referred to as DBCO-2B1W4, 2B1W4 or DBCO^GIu^B^Tip)^, was synthesized using the same procedure as described for Compound 3, except for the fact that Fmoc-Trp-Trp-Glu-Trp- Trp-NH ₂ was used as starting material for the conjugation of Compounds

- 86 046161

1. Compound 9 was purified on a preparative HPLC system using a 50-55% acetonitrile/ _H2O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 50x100 mm, 5 µm column. The product was eluted for 8.9 min and the resulting fractions were collected, frozen and then lyophilized to yield 9.7 mg (55.4% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₈₆ H ₈₆ N ₁₆ O ₈ , mass/charge 1470.68, the result was 736.6 (M/2) ⁺ .

Connection 10.

Compound 10, referred to as DBCO-W ₃ , W ₃ or DBCO-(Trp) ₃ , was synthesized by reacting with 50.2 mg (0.08 mmol, 1 eq.) of the tripeptide precursor NH ₂ -(Trp) ₃ - NH ₂ which was prepared by solid phase peptide synthesis with 35 mg DBCO-NHS (0.096 mmol, 1.1 eq.) and 44 mg triethylamine (0.44 mmol, 5 eq.) in 1.5 ml DMSO. Compound 10 was purified by preparative HPLC using a 30-95% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-d8, 50x100 mm, 5 µm column. The product was eluted over ~9 min and the resulting fractions were collected, frozen and then lyophilized to yield 31.2 mg (41.5% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder.

Connection 11.

Compound 11, referred to as DBCO-W ₅ , W ₅ or DBCO-(Trp) ₅ , was synthesized using the same procedure as described for Compound 10, except for the fact that NH ₂ -(Trp) ₅ -NH ₂ ( SEQ ID NO: 70) was used as a peptide precursor for conjugation with DBCO-NHS. Compound 11 was purified by preparative HPLC using a 30-95% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 50x100 mm, 5 µm column. The product was eluted over ~10 min and the resulting fractions were collected, frozen and then lyophilized to yield 28.7 mg (44.1% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₇₄ H ₆₆ N ₁₂ O ₇ , mass/charge 1234.52, the result was 1235.6 (M+H)+.

Connection 12.

Compound 12, referred to as DBCO-2BXy3W2, 2BXy3W2 or DBCO-(Glu(2BXy)3(Trp)2), was prepared using Fmoc-Glu-Trp-Glu-Trp-Glu-NH2 and Compound 2 as starting materials. 500 mg Fmoc-Glu-Trp-Glu-Trp-Glu-NH2 (0.5 mmol, 1 eq.), 595.6 mg thiazolin-2-thiol (TT) (5 mmol, 10 eq.),

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575.7 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (3 mmol, 6 eq.) was suspended in 26 ml of DCM. 18.3 mg of 4-(dimethylamino)pyridine (DMAP) (0.2 mmol, 0.3 eq.) was added and the reaction mixture was shaken at room temperature. The progress of the reaction was monitored using analytical HPLC. After 4 hours, another four equivalents of TT and two equivalents of EDC were added. After shaking overnight, two equivalents of TT and half an equivalent of EDC were added. After 6 hours the reaction was completed. The DCM was removed in vacuo and the solid was taken up in 6 ml dry DMSO. 539.3 Compound 2 (1.5 mmol, 3 eq.) was added and the reaction mixture was shaken for 2 hours at room temperature. The conjugated intermediate was then precipitated from 300 ml of 1 M HCl and centrifuged at 3000 g at 4°C for 10 min. The pellets were collected and washed again with 1 M HCl and once with DI water. The final collected pellet was frozen and vacuum dried. 809.06 mg of Fmoc-2BXy ₃ W2-NH2 (0.4 mmol, 1 eq.) was dissolved in 4 ml of 20% piperidine in DMF. The reaction mixture was shaken at room temperature for 1 hour. The deprotected intermediate was then precipitated from 100 ml of ether and centrifuged at 3000 g at 4°C for 10 minutes. The product was collected as a solid pellet and then washed twice with ether and then dried in vacuo to yield the intermediate. 729 mg of NH2-2BXy ₃ W ₂ -NH2 (0.4 mmol, 1 eq.) was dissolved in 6 ml of dry DMSO. 488.8 mg of DBCO-NHS (1.2 mmol, 3 eq.) was added and the reaction mixture was shaken at room temperature for 1 hour. The resulting product was purified on a preparative HPLC system using a 36-46% acetonitrile/H ₂ O gradient ( 0.05% TFA) for 12 min on an Agilent Prep-C18, 50x100 mm, 5 µm column. The resulting fractions were combined, frozen and lyophilized to yield 239 mg (38.1% yield) of a spectroscopically pure white powder. MS (ESI) was calculated for C ₁₂₂ H ₁₂₆ N ₂₄ O ₁₀ , mass/charge 2087.65, the result was 697 (M/3) ⁺ .

Connection 13.

Compound 13, referred to as DBCO-2B6W4, 2B6W4 or DBCO-(Glu(2B) ₆ (Trp)4), was synthesized using the same procedure as described for Compound 3, except for the fact that Fmoc-(Glu-Trp -Glu-Trp-Glu) ₂ -NH ₂ was used as starting material for the conjugation of Compound 1. Compound 13 was purified on a preparative HPLC system using a 24-45% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18 column, 30x100 mm, 5 µm. Fractions were collected, frozen and then lyophilized to yield a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₂₀₁ H ₂₃₆ N ₄₆ O ₁₈ , mass/charge 3582.4, the result was 717.7 (M/5) ⁺ .

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Connection 14.

Compound 14, referred to as DBCO-2B4W6, 2B4W6 or DBCO/GIu^B^Ttp^), was synthesized using the same procedure as described for Compound 3, except for the fact that Fmoc-(Trp-Glu-Trp-Glu -Trp) ₂ -NH ₂ was used as starting material for the conjugation of Compound 1. Compound 14 was purified on a preparative HPLC system using a 24-45% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent column Prep-C18, 30x100 mm, 5 µm. Fractions were collected, frozen and then lyophilized to yield a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₇₇ H ₁₉₆ N ₃₈ O ₁₆ , mass/charge 3111.7, the result was 777.5 (M/4) ⁺ .

Connection 15.

Compound 15, referred to as DBCO-2BXy ₁ W4, 2BXy ₁ W4 or DBCO-(Glu(2BXy) ₁ (Trp)4), was prepared using the same procedure as described for Compound 3, except for the fact that Fmoc- Trp-Trp-Glu-Trp-Trp-NH2 was used as the starting material. Compound 15 was purified by preparative HPLC using a 40-70% acetonitrile/H ₂ O (0.05% TFA) gradient over 16 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to yield 3.4 mg (73.3% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₉₀ H ₈₅ N ₁₅ O ₉ , mass/charge 1519.67, the result was 760.5 (M/2) ⁺ .

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Connection 16.

Compound 16, referred to as DBCO-(GG2B)5, 2B5G ₁₀ or DBCO-(Glu(2B)5(Gly) ₁₀ ), was synthesized using the same procedure as described for Compound 3, except for the fact that Fmoc- (Gly-Gly-Glu)5-NH ₂ and Compound 1 were used as starting materials. Compound 16 was purified in a preparative HPLC system using a 22-42% acetonitrile/ _H2O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The product was eluted for 7 minutes and the resulting fractions were collected, frozen and then lyophilized to yield 22.8 mg (36.2% yield) of a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for Ci ₅₄ H ₁₉₆ N ₄₂ O ₂₂ , mass/charge 2985.51, the result was 598.5 (M/5) ⁺ .

Connection 17.

Compound 17, referred to as DBCO-(GG2BGGW)2GG2B, 2B ₃ W2G ₁₀ or DBCO(Glu(2B) ₃ (Trp)2(Gly) ₁₀ ), was synthesized from the precursor Fmoc-(Gly-Gly-Glu-Gly-Gly- Trp) ₂ -Gly ₂ -GluNH2 obtained by solid-phase peptide synthesis, and Compound 1. 235.4 mg Fmoc-(Gly-Gly-Glu-GlyGly-Trp) ₂ -Gly ₂ -Glu-NH ₂ (0.15 mmol, 1 equiv.) was dissolved in 2 ml of 20% piperidine in DMF. After 30 min, the reaction was completed and the product was precipitated from 100 ml of ether and centrifuged at 3000 g at 4°C for 10 min. The product was collected as a solid pellet and then washed twice with ether and then dried in vacuo to yield ~200 mg of deprotected intermediate. 200 mg (0.15 mmol, 1 eq.) NH ₂ -(Gly-Gly-Glu-Gly-Gly-Trp) ₂ -Gly ₂ -Glu-NH ₂ was dissolved in 2 ml of dry DMSO and 89.73 mg of DBCO was added -NHS (0.22 mmol, 1.5 eq.), after which TEA (0.22 mmol, 1.5 eq.) was added. The reaction mixture was shaken at room temperature for 1 hour. The resulting DBCO intermediate was purified on a preparative HPLC system using a 30-50% acetonitrile/ _H2O (0.05% TFA) gradient over 12 minutes on an Agilent Prep-C18 column. 50x100 mm, 5 microns. The resulting fractions were pooled, frozen and lyophilized to give the intermediate. 25 mg DBCO-(Gly-Gly-Glu-Gly-Gly-Trp) ₂ -Gly ₂ -Glu-NH ₂ (0.015 mmol, 1 eq.) and 17.11 mg of Compound 1 (0.055 mmol, 3.6 eq. ) was dissolved in 1.2 ml. dry DMSO. TEA (0.183 mmol, 12 eq.) was added and the reaction mixture was shaken at room temperature for 5 min. 19.17 mg HATU (0.05 mmol, 3.3 eq) was added and the reaction mixture was shaken at room temperature. The progress of the reaction was monitored using liquid chromatography with mass spectrometry (LC-MS). After 1 hour, 1.2 additional equivalents of Compound 1 and 1.1 equivalents of HATU were added. After 2 hours the reaction was completed. The resulting product was purified on a preparative HPLC system using a 3060% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were combined, frozen and lyophilized to obtain a spectroscopically pure white powder. MS (ESI) was calculated for C130H156N34O20, m/c 2515.96, resulting in 839 (m/3) ⁺ .

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Connection 18.

Compound 18, referred to as Bis(TT), was synthesized using suberic acid and 2thiazoline-2-thiol (TT) as starting materials. To put it briefly, 500 mg suberic acid (2.87 mmol, 1 eq.), 752.7 mg TT (6.31 mmol, 2.2 eq.) and 1.431 g EDC (7.46 mmol, 2.6 eq.) was dissolved in 17.5 ml of dry DMSO. 70.15 mg DMAP (0.57 mmol, 0.2 eq.) was added and the reaction mixture was shaken at room temperature for 1 hour. The reaction mixture was diluted with DCM and washed twice with 1 M HCl and once with DI water. The organic fractions were dried over sodium sulfate and evaporated under reduced pressure to give a yellow solid in quantitative yield.

Connection 19.

Compound 19, referred to as 2B-TT, was synthesized using Compound 18 and Compound as starting materials. Briefly, 50 mg (0.16 mmol, 1 eq.) of Compound 1 was dissolved in 0.6 ml of methanol and added dropwise to a vigorously shaken solution containing 301.1 mg of Compound 18 (0.8 mmol, 5 eq. .), in 1.93 ml DCM. After 30 min, the reaction mixture was injected directly onto the column and purified by flash chromatography using a 2-step gradient: 5% methanol in DCM at 5 column volumes (CV), then 5-50% methanol in DCM gradient at 20 CV. The fractions were combined and the solvent was removed in vacuo. MS (ESI) was calculated for C ₂₉ H ₄₀ N ₆ O ₂ S ₂ , mass/charge 568.27, the result was 569.3 (m +1) +

Connection 20.

Compound 20, referred to as

DBCO-(2BGWGWG) ₅ , 2B ₅ W ₁₀ G ₁₅ or DBCO-91 046161 (Glu(2B) ₅ (Trp) ₁₀ (Gly) ₁₅ ), was synthesized from the peptide precursor Fmoc-(Lys-Gly-Trp-Gly-TrpGly ) ₅ -NH ₂ , which was prepared by solid-phase peptide synthesis, and Compound 19. 49.8 mg (0.01 mmol, 1 eq.) Fmoc-(Lys-Gly-Trp-Gly-Trp-Gly) ₅ -NH ₂ was dissolved in 0.5 ml dry DMSO. To this solution was added 0.492 ml of Compound 19 (0.03 mmol, 2.5 eq.) as a 40 mg/ml stock solution in dry DMSO. TEA (0.01 mmol, 1 eq.) was added and the reaction mixture was shaken at room temperature for 4 hours. Analytical HPLC using a 45-65% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 minutes showed complete conversion to the penta-substituted intermediate. The reaction was quenched by the addition of amino-2-propanol (0.03 mmol, 2.5 eq) and then 0.5 ml of 20% piperidine in DMF was added and the reaction mixture was shaken at room temperature for 30 min. The reaction mixture was added to 50 ml of ether and centrifuged at 3000 g at 4°C for 10 min. The product was collected as a solid pellet and then washed twice with ether and then dried in vacuo to yield a deprotected intermediate. 73.4 mg of the deprotected intermediate (0.0131 mmol, 1 eq.) was dissolved in 0.5 ml of dry DMSO, followed by the addition of 0.066 ml (0.0196 mmol, 1.5 eq.) DBCO-NHS (40 mg/ml) and TEA (0.0131 mmol, 1 eq.). The reaction mixture was shaken for 1 h at room temperature and then quenched by the addition of amino2-propanol (0.0196 mmol, 1.5 eq.). Then the product was precipitated from 50 ml of 1 molar HCl solution and centrifuged at 3000 g at 4°C for 10 min. The product was collected as a solid pellet and then washed again with 1 M HCl and once with DI water. The final collected pellet was vacuum dried to yield 15.1 mg (26% yield) of final product. MS (ESI) was calculated for C ₃₁₉ H ₃₉₆ N ₇₂ O ₄₂ mass/charge 5909.1, the result was 1183 (m/5) ⁺ .

Connection 21.

Compound 21, referred to as Mal-2B ₃ W2 or Mal-(Glu(2B) ₃ (Trp)2), was synthesized starting with Fmoc-(Glu) ₃ (Trp) ₂ -NH ₂ and Compound 1. 100 mg Fmoc- (Glu)3(Trp) ₂ -NH ₂ (0.1 mmol, 1 eq.) and 112.1 mg of Compound 1 (0.36 mmol, 3.6 eq.) were dissolved in 8 ml of dry DMF. TEA (1.2 mmol, 12 eq.) was added and the solution was cooled in an ice bath with shaking for 5 min. HATU (0.33 mmol, 3.3 eq.) was added and the reaction mixture was shaken at 4°C for 1 hour. The reaction mixture was then added to 50 ml of 1 M HCl and centrifuged at 3000 g at 4°C within 10 min. The product was collected as a solid pellet and then washed again with 1 M HCl and again with DI water to obtain the conjugated intermediate. Then, 188 mg of Fmoc-2B ₃ W ₂ -NH ₂ (0.1 mmol, 1 eq.) was dissolved in 2 ml of 20% piperidine in DMF solution and shaken for 30 min at room temperature. The deprotected intermediate was precipitated from 100 ml of ether and centrifuged at 3000 g at 4°C for 30 min. The product was collected as a solid pellet and then washed twice with ether and then dried in vacuo to yield a deprotected intermediate. 30 mg of NH2-2B3W2-NH2 (0.018 mmol, 1 eq.) was dissolved in 0.3 ml of dry DMSO, after which 22 mg of succinimidyl 6-((betamaleimidopropionamido)hexanoate (SMPH) (0.058 mmol, 3.2 eq.) was added. The reaction mixture was shaken for 1 hour at room temperature and the resulting product was purified on a preparative HPLC system using a 30-50% acetonitrile/ _H2O (0.05% TFA) gradient over 12 minutes on an Agilent Prep-C18, 50x100 column mm, 5 µm. The product was eluted over 4.75 min and the resulting fractions were pooled, frozen and lyophilized to give 23 mg (66.7% yield) of a spectroscopically pure white powder. MS (ESI) was calculated for C1o ₄ H ₁₂₉ N ₂₅ O ₁₂ , mass/charge 1921.33, the result was 961 (m/2) ⁺ .

- 92 046161

Connection 22.

Compound 22, referred to as NHS-2B3W2, was synthesized by reacting 125 μg (0.0004 mmol, 1 eq.) of 3-azidopropionic acid sulfo-NHS ester (Az-NHS, click chemistry tools) with 0.8 mg (0. 0004 mmol, 1 eq.) Compound 6 in dry DMSO. HPLC showed complete conversion to the NHS-activated compound, which was used immediately to conjugate the peptides bearing the reactive amine. MS (ESI) was calculated for C ₁₁₇ H ₁₃₃ N ₂₈ O ₁₇ S, mass/charge 2234, the result was 1119 (m/2) ⁺ .

Connection 23.

Compound 23, referred to as DBCO-2BXy, was synthesized by reacting 5.0 mg (0.01 mmol, 1 eq.) of DBCO-NHS with 4.91 mg (0.011 mmol, 1.1 eq.) of Compound 2 in dry form. DMSO. The DMSO solution was dissolved in ethyl acetate and then washed with 1 M HCl solution. The organic layer was removed in vacuo to obtain ~3 mg of a spectroscopically pure white solid. MS (ESI) was calculated for C ₄₁ H ₄₀ N ₆ O ₂ , mass/charge 646.31, the result was 647.3 (m/2) ⁺ .

Connection 24.

Compound 24, referred to as DBCO-WWTTWW or W4TT, was prepared using NH ₂ -TrpTrp-Glu-Trp-Trp-NH ₂ obtained by solid phase peptide synthesis. 32.8 mg of NH ₂ -Trp-Trp-Glu-TrpTrp-NH ₂ (0.04 mmol, 1 eq.) was dissolved in 0.5 ml of dry DMSO and 15.5 mg (0.04 mmol, 1 eq.) was added. ) DBCO-NHS, after which TEA (0.04 mmol, 1 eq.) was added. The reaction mixture was shaken at room temperature for 1 hour. The resulting DBCO intermediate was purified on a preparative HPLC system using a 40-70% acetonitrile/ _H2O (0.05% TFA) gradient over 16 minutes on an Agilent Prep-C18 column. 30x100 mm, 5 microns. The resulting fractions were combined, frozen and lyophilized. For TT activation of DBCO peptide, 6.1 mg DBCO-Trp-Trp-Glu-Trp-Trp-NH ₂ (0.005 mmol, 1 eq.), 0.68 mg TT (0.006 mmol, 1.1 eq.) and 1.26 mg EDC (0.007 mmol, 1.3 eq.) was dissolved in 0.2 ml of a 1:1 solution of DMSO and DCM. 0.06 mg DMAP (0.001 mmol, 0.1 eq.) was added to the reaction mixture with vigorous shaking at room temperature. After 2 hours, an additional 3.3 eq was added. CT, 4.0 eq. EDC and 0.1 eq. DMAP and the reaction was completed after a total of 3 hours. DBCO-WWTTWW was used as an intermediate in subsequent steps.

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Connection 25.

Connection 25, designated DBCO-WWPIWW, WW(PI)WW, or PIW4. Primido-indole-Red ₄ NH2 (PI-NH2, a TLR-4 agonist) was prepared as previously described (Lynn, G.M., et al., In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity Nat Biotechnol 33(11): 1201-1210, 2015). To 3.6 mg of PINH2 (0.005 mmol, 2 eq.) was added 3.31 mg of Compound 33 (0.0026 mmol, 1 eq.) and TEA (0.003 mmol, 1.1 eq.) in DMSO, and shaken for overnight at room temperature. The resulting product was purified in a preparative HPLC system using a 45-75% acetonitrile/H ₂ O (0.05% TFA) gradient over 16 min on an Agilent PrepC18, 30x100 mm, 5 µm column. The resulting fractions were combined, frozen and lyophilized. MS (ESI) was calculated for C ₁₀₃ H ₁₀₇ N ₁₇ O ₁₅ S, mass/charge 1854.77, the result was 928.6 (m/2) ⁺ .

Connection 26.

Compound 26, referred to as Pam2Cys-TT or P2C-TT, which is a TLR-2/6 agonist, was synthesized using the same procedure as described for Compound 24, except that Fmoc-Pam2Cys-Acid was used as the starting material material, and the reaction was monitored using thin layer chromatography (TLC) (1% methanol in DCM). The procedure produced a yellow solid in quantitative yield, which was used as an intermediate in subsequent reaction steps.

Connection 27.

Compound 27, referred to as DBCO-WWPam2CysWW, WW(P2C)WW or P2C(W) ₄ , was prepared with

- 94 046161 using Compound 24, PEG2-guaMUHa and Compound 26 as starting materials. First, 1.15 mg of Peg2-diamine (0.008 mmol, 3 eq.) was added to the reaction mixture containing 3.31 mg of Compound 24 (0.0026 mmol, 1 eq.) and the reaction mixture was shaken for 1 h at room temperature .

The reaction mixture was added to 15 ml DI water and centrifuged. The pellet was collected and washed an additional three times with DI water and lyophilized. The intermediate (DBCOWWPeg2NH2WW) was taken up in 0.1 mL of dry DMSO and 2.58 mg of Compound 26 (0.0026 mmol, 1 eq.) was added as a 100 mg/mL stock solution in DCM. The reaction mixture was shaken for 1 hour at room temperature, and then the DCM was removed in vacuo, after which 0.2 ml of 20% piperidine in DMF was added. The reaction mixture was shaken for 30 min at room temperature, then diluted with DCM and washed three times with DI water. The organic layer was dried with sodium sulfate and evaporated. The resulting oil was dissolved in DMSO and purified on a preparative HPLC system using a 70-100% acetonitrile/ _H2O (0.05% TFA) gradient over 16 min on an Agilent Prep-C18, 9.4 x 100 mm, 5 µm column. The resulting fractions were combined and evaporated to obtain the product DBCO-WWPam2CysWW. The molecular weight of the product was confirmed based on MS (ESI+) for peptide antigen conjugates of Compound 27.

Connection 28.

Compound 28, referred to as NH ₂ -GK(DBCO)GW ₅ , was synthesized using Fmoc-Gly-LysGly-(Trp) ₅ -NH ₂ obtained by solid-phase peptide synthesis and DBCO-NHS as starting materials. 50 mg of Fmoc-Gly-Lys-Gly-(Trp) ₅ -NH ₂ (0.04 mmol, 1 eq.) was dissolved in 0.25 ml of dry DMSO. TEA (0.04 mmol, 1 eq.) was added and the solution was shaken at room temperature for 5 min. 14.24 mg of DBCO-NHS (0.04 mmol, 1 eq.) was added and the reaction mixture was shaken at room temperature for 1 h. The reaction was quenched with amino-2-propanol (0.04 mmol, 1 eq.) and 0 .5 ml 20% piperidine in DMF. The reaction mixture was shaken at room temperature for 30 hours. Then the product was precipitated from 50 ml of ether and centrifuged at 3000 g at 4°C for 10 minutes. The product was collected as a solid pellet and then washed twice with ether and then dried in vacuo. The product was purified on a preparative HPLC system using a 25-45% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The product was eluted for 9.4 minutes and the resulting fractions were combined, frozen and lyophilized to give a spectroscopically pure off-white solid. MS (ESI) was calculated for C ₈₄ H ₈₄ N ₁₆ O ₁₀ , mass/charge 1477.64, the result was 739.6 (m/2) ⁺ .

- 95 046161

Connection 29.

Compound 29, referred to as CL264-a3ug, hydroxyadenine azide or TLR-7-azide, which is a TLR-7 agonist, was synthesized using CL264 (Invivogen, San Diego, CA, USA) and azidopropylamine as starting materials. 5 mg CL264 (0.012 mmol, 1 eq.) and 6.1 mg azidopropylamine (0.061 mmol, 5 eq.) were dissolved in dry DMF. TEA (0.073 mmol, 6 equiv) was added and the solution was cooled to 0°C in an ice bath. 27.6 mg HATU (0.073 mmol, 6 eq.) was added and the reaction mixture was shaken at 0°C for 1 hour. Compound 29 was purified on a preparative HPLC system using a 20-40% acetonitrile/H ₂ O (0. 05% TFA) for 10 min on an Agilent Prep-C18 column, 30x100 mm, 5 µm. The product was eluted over 5 min and the resulting fractions were pooled, frozen and lyophilized. MS (ESI) was calculated for C ₂₂ H ₂₉ NnO ₃ , mass/charge 495.25, the result was 496.3 (m+H)^

Connection 30.

Compound 30, referred to as DBCO-GK(CL264)GW ₅ , C1264-(W) ₅ or DBCO-Gly-Lys(DBCOCL264)-Gly-(Trp) ₅ -NH ₂ , was synthesized using Compounds 28 and 29 as starting materials materials. 5 mg of Compound 28 (0.0034 mmol, 1 eq.) was dissolved in 0.25 ml of dry DMSO and 1.68 mg of Compound 29 (0.0034 mmol, 1 eq.) was added and the reaction mixture was shaken for 2 hours, after of which 1.5 mg of DBCO-NHS (0.0037 mmol, 1.1 eq.) and 0.5 eq. TEA. The reaction mixture was shaken at room temperature and the resulting product was then purified on a preparative HPLC system using a 25-55% acetonitrile/ _H2O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. . The product was eluted for 9.4 minutes and the resulting fractions were pooled, frozen and lyophilized to give a spectroscopically pure white solid. MS (ESI) was calculated for C ₁₂ 5H ₁₂₈ N ₂₈ O ₁ 5, mass/charge 2261.01, the result was 1132.2 (m/2) ⁺ .

- 96 046161

Compound 31.

CH;

Compound 31, referred to as DMXAA azide, which is a STING agonist, was synthesized using the same procedure as described for Compound 29, except that DMXAA (Invivogen) was used as a starting material. Compound 31 was purified by preparative HPLC using a 35-65% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The product was eluted over 6 minutes and the resulting fractions were pooled, frozen and lyophilized to give a spectroscopically pure white solid. MS (ESI) was calculated for C ₂₀ H ₂₂ N ₄ O ₃ , mass/charge 366.42, the result was 365.2.

Connection 32.

Compound 32, referred to as DBCO-GK(DMXAA)-G-W5, DMXAA-(W) ₃ , DMXAA-W5, or DBCO-Gly-Lys(DBCO-DMXAA)-Gly-(Trp) ₅ - _NH2 , was synthesized with using the same procedure as described for Compound 30, except that Compound 31 was used as a cargo agonist. Compound 32 was purified by preparative HPLC using a 45-85% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. MS (ESI) was calculated for C ₁₂₃ H ₁₂₁ N ₂₁ O ₁₅ , mass/charge 2131.94, the result was 1067.6 (m/2) ⁺ .

- 97 046161

Connection 33.

as NH2-GRK(DBCO)GW5, NH ₂ -Gly-Arg-Lys(DBCO)-Gly-(Trp) ₅ -NH

Compound 33 was synthesized using the same procedure as described for Compound 28, except for Fmoc-GRKGW ₅ -NH ₂ which was produced by solid phase peptide synthesis and used as starting material. Compound 33 was purified by preparative HPLC using a 25-55% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-d8, 30x100 mm, 5 µm column. The product was eluted over 7.5 min and the resulting fractions were pooled, frozen and lyophilized. MS (ESI) was calculated for C ₉₀ H ₉₆ N ₂₀ O11, mass/charge 1633.74, the result was 817.5 (m/2) ⁺ .

Connection 34.

Compound 34, referred to as Muramyl Azide, which is a NOD 2 agonist, was synthesized using Muramyl trilysine (M-TriLys, Invivogen) and sulfo NHS azidopropionic acid as starting materials. 5 mg of muramyltrilysine (0.008 mmol, 1 eq.) was dissolved in 0.5

- 98046161 ml dry DMSO with TEA (0.02 mmol, 2.5 eq.) and added 2.2 mg sulfo NHS azidopropionic acid (0.008 mmol, 1 eq.). The reaction mixture was shaken at room temperature for 2 hours. The reaction mixture was quenched with amino-2-propanol (0.008 mmol, 1 eq.) and the intermediate was used immediately for the reaction. Connection 35.

Compound 35, referred to as DBCO-GRK(Muramil)GW5, Muramyl-W ₅ or DBCO(Muramil)W ₅ , was synthesized using the same procedure as described for Compound 30, except for the fact that Compounds 33 and 34 were used in quality of the starting material. Compound 35 was purified by preparative HPLC using a 35-65% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 minutes on an Agilent Prep-C18, 30x100 mm, 5 µm column. The product was eluted over 4.6 min and the resulting fractions were pooled, frozen and lyophilized. MS (ESI) was calculated for C ₁₃ 7H ₁₅₈ N ₃₀ O ₂₆ , mass/charge 2639.2, the result was 1319.5 (m/2) ⁺ .

- 99 046161

Connection 36.

Compound 36, referred to as NH ₂ -GGRK(DBCO)RGGW ₅ , was synthesized using the same procedure as described for Compound 28, except that Fmoc-GGRKRGGW5NH2, which was prepared by solid-phase peptide synthesis, was used as starting material. Compound 36 was purified in a preparative HPLC system using a 25-45% acetonitrile/ _H2O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The product was eluted over 7.8 min and the resulting fractions were collected, frozen and then lyophilized to yield a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₀₀ H ₁₁₄ N ₂₆ O ₁₄ , mass/charge 1902.9, the result was 951.4 (m/2) ⁺ .

Connection 37.

Compound 37, referred to as DBCO-GGRK(AMP)RGW5 or

DBCO(AMP)W5, was synthesized using the same procedure as described for Compound 30, except for the fact that Compound 36 and adenosine monophosphate 8-(6-aminohexyl)aminoadenosine 5' monophosphate (Sigma-Aldrich) substituted with azidopropionic acid, were used as starting materials. Compound 37 was purified by preparative HPLC using a 30-60% acetonitrile/H2O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The product was eluted over 5.2 min and the resulting fractions were pooled, frozen and lyophilized to obtain spectroscopic

- 100 046161 scopically pure white solid. MS (ESI) was calculated for C ₁₃₈ H ₁₅₈ N ₃₇ O ₂₄ P, mass/charge 2748.2, the result was 917.5 (m/3) ⁺ .

Connection 38.

Compound 38, referred to as NH2-GRRK (DBCO)-RGW ₅ , was synthesized using the same procedure as described for Compound 28, except that Fmoc-GRRKRGW ₅ -NH ₂ was used as the starting material. Compound 38 was purified in a preparative HPLC system using a 25-55% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent PrepC18, 30x100 mm, 5 µm column. The product was eluted for 5.1 min and the resulting fractions were collected, frozen and then lyophilized to yield a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₀₂ H ₁₂₀ N ₂₈ O ₁₃ , mass/charge 1944.96, the result was 973.4 (m/2) ⁺ .

Connection 39.

Compound 39, referred to as NH ₂ -GK(DBCO)[GGK(DBCO)] ₂ GW ₅ , was synthesized using the same procedure as described for Compound 28, except for the fact that FmocGK(GGK) ₂ GW ₅ -NH ₂ was used as starting material, and three additional equivalents of TEA and DBCO-NHS were used. Compound 39 was purified by preparative HPLC using a 45-85% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to obtain the title compound as a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁₄₂ H ₁₄₆ N ₂₆ O ₂₀ , mass/charge 2535.12, the result was 1269 (m/2) ⁺ .

- 101 046161

Connection 40.

Compound 40, referred to as NH2-[GRK(DBCO)RG] ₃ -W5, was synthesized using the same procedure as described for Compound 39, except that NH2[GRK(DBCO)RG] ₃ -W5 was used in quality of the starting material. Compound 40 was purified by preparative HPLC using a 35-65% acetonitrile/H2O (0.05% TFA) gradient over 10 min on an Agilent Prep-d8, 30x100 mm, 5 µm column. Fractions were collected, frozen and then lyophilized to yield a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for C ₁ 78H ₂₁₈ N ₅₀ O ₂₆ , mass/charge 3471.31, the result was 695.6 (m/5) ⁺ .

Compound 41.

Compound 41, referred to as E ₁₀ -2B ₃ W2, was synthesized using Azido-(O1u) ₁₀ -KH ₂ and Compound 6 as starting materials. 5 mg of Azido-(O1u) ₁₀ -KH ₂ (0.0035 mmol, 1 eq.) was dissolved in dry DMSO and 6.77 mg of Compound 6 (0.0035 mmol, 1 eq.) was added as a 40 mg/ml solution in dry DMSO. The reaction mixture was shaken overnight at room temperature. Compound 41 was purified by preparative HPLC using a 25-45% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to yield 11.8 mg of spectroscopically pure (>95% AUC at 254 nm) white powder in quantitative yield. MS (ESI) was calculated for mass/charge 3377.31, the result was 1127 (M/3) ⁺ .

- 102 046161

Compound 42.

Compound 42, referred to as K ₁₀ -2B3W ₂ , was synthesized using the same procedure as described for Compound 41, except that azido-(Lys) ₁₀ -NH ₂ was used as starting materials. Compound 42 was purified by preparative HPLC using a 20-40% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to obtain a spectroscopically pure (>95% AUC at 254 nm) white powder in quantitative yield. MS (ESI) was calculated for mass/charge 3367.58, the result was 482 (M/7) ⁺ .

Connection 43.

Compound 43, referred to as DBCO-G ₅ -2B ₃ W ₂ -G5-D ₉ or DBCO-2B ₃ W ₂ (D) ₉ , was synthesized using NH ₂ -(Gly) ₅ -Lys(N ₃ )-(Gly ) ₅ -(Asp) ₉ -NH ₂ , Compound 6 and DBCO-NHS as starting materials. 5 mg NH ₂ -(Gly) ₅ -Lys(N ₃ )-(Gly) ₅ -(Asp) ₉ -NH ₂ (0.0028 mmol, 1 eq.) was dissolved in 0.1 ml of dry DMSO and 6 was added, 56 mg of Compound 6 (0.0034 mmol, 1.2 eq.). The reaction mixture was shaken overnight at room temperature. 3.55 mg of DBCO-NHS (0.0084 mmol, 3 eq.) was added, followed by TEA (0.0028 mmol, 1 eq.). The reaction mixture was shaken for 2 hours at room temperature. Compound 43 was purified by preparative HPLC using a 30-40% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to provide 3.8 mg (33.7% yield) of the title compound as spectroscopically pure (>95% AUC at 254 nm) white

- 103 046161 powder. MS (ESI) was calculated for mass/charge 4008.31, the result was 1003.3 (m/4) ⁺ .

Compound 44, referred to as DBCO-G5-2B3W2-G5-D8 or DBCO-2B3W2(D)8, was synthesized using the same procedure as described for Compound 43, except for the fact that NH ₂ -(Gly) ₅ - Lys(N ₃ )-(Gly)5-(Asp) ₈ -NH ₂ was used as the starting material. Compound 44 was purified by preparative HPLC using a 30-40% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to obtain the title compound as a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for mass/charge 3894.52, the result was 974.6 (m/4) ⁺ .

Compound 45, referred to as DBCO-G ₅ -2B ₃ W2-G ₅ -D ₇ or DBCO-2B ₃ W2(D) ₇ , was synthesized using the same procedure as described for Compound 43, except for the fact that NH ₂ -(Gly) ₅ -Lys(N ₃ )-(Gly) ₅ -(Asp) ₇ -NH ₂ was used as the starting material. Compound 45 was purified by preparative HPLC using a 29-39% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to obtain the title compound as a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for mass/charge 3779.43, the result was 945.8 (m/4) ⁺ .

Compound 46, referred to as DBCO-G5-2B3W2-G5-D6 or DBCO-2B ₃ W2(D) ₆ , was synthesized using the same procedure as described for Compound 43, except for the fact that NH ₂ -(Gly) ₅ -Lys(N ₃ )-(Gly) ₅ -(Asp) ₆ -NH ₂ was used as the starting material. Compound 46 was purified by preparative HPLC using a 29-39% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to obtain the title compound as a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for mass/charge 3664.35, the result was 917.1 (m/4) ⁺ .

Compound 47, referred to as DBCO-G5-2B3W2-G5-D5 or DBCO-2B ₃ W ₂ (D) ₅ , was synthesized using the same procedure as described for Compound 43, except for the fact that NH ₂ -(Gly ) ₅ -Lys(N ₃ )-(Gly) ₅ -(Asp) ₅ -NH ₂ was used as the starting material. Compound 47 was purified by preparative HPLC using a 29-41% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to obtain the title compound as a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for mass/charge 3549.26, the result was 1184.1 (m/3) ⁺ .

Connection 48.

Compound 48, referred to as DBCO-2B3W2-R6 or DBCO-2B ₃ W2(R)6, was synthesized using the same procedure as described for Compound 43, except for the fact that NH2Lys(N ₃ )-(Arg) ₆ -NH ₂ was used as the starting material. Compound 48 was purified by preparative HPLC using a 26-36% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-d8, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen, and then

- 104 046161 was filtered to obtain 17.8 mg (51.8% yield) of the title compound as a spectroscopically pure (>95% AUC at 254 nm) white powder. MS (ESI) was calculated for mass/charge 3338.19, the result was 478.1 (m/7) ⁺ .

Connection 49.

Compound 49, referred to as Myr-DBCO or C14-DBCO, was prepared from Myristoyl chloride and DBCO-Amin. 50 mg of DBCO-amine (0.18 mmol, 1 eq.) was dissolved in 0.5 ml of DCM. TEA (0.22 mmol, 1.2 eq.) was added and the solution was shaken for 5 min at room temperature. Myristoyl chloride (0.16 mmol, 0.9 eq.) was added and the reaction mixture was shaken for 1 hour at room temperature. TLC (2.5% methanol in DCM) showed a new spot with a retention factor (rf) of 0.5 for Myr-DBCO. The reaction mixture was injected onto a flash chromatography column and purified using a gradient of 0-3% methanol in DCM at 12 CV. Fractions were collected and dried to obtain 86 mg of MyrDBCO in quantitative yield. MS (ESI) was calculated for C ₃₂ H ₄₂ N ₂ O ₂ , mass/charge 486.32, the result was 487.3 (m+H)^

Connection 50.

Compound 50, referred to as Val-DBCO or C5-DBCO, was prepared using the same procedure as described for Compound 49, except that Valeroyl chloride was used as the starting material. Val-DBCO was purified on a preparative HPLC system using a 40-60% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. The resulting fractions were collected, frozen and then lyophilized to obtain 80 mg ValDBCO in quantitative yield. MS (ESI) was calculated for C ₂₃ H ₂₄ N ₂ O ₂ , mass/charge 360.18, the result was 361.2 (m+H)^

Connection 51.

Compound 51, referred to as Plm-DBCO or C16-DBCO, was prepared using the same procedure as described for Compound 49, except that Palmitoyl chloride was used as the starting material. Compound 51 was purified by flash chromatography using a gradient of 0-3% methanol in DCM at 12 CV. Fractions were collected and dried to obtain 80 mg of PlmDBCO in quantitative yield. MS (ESI) was calculated for C ₃₄ H ₄₆ N ₂ O ₂ , mass/charge 514.36, the result was 515.3 (m+H)^

Connection 52.

Compound 52, referred to as Myr-Pegn-DBCO or C14-PEG11-DBCO, was synthesized using Myristoyl chloride, Boc-Pegn-Amin and DBCO-NHS as starting materials. 116.06 mg Boc-Pegn-Amin (0.18 mmol, 1 eq.) was dissolved in 0.5 ml DCM. TEA (0.22 mmol, 1.2 eq.) was added and the reaction mixture was shaken for 5 min at room temperature. Myristoyl chloride was added and the reaction mixture was shaken at room temperature for 1 hour. The reaction mixture was diluted with DCM and washed twice with 1 M HCl and once with DI water. The organic layer was dried with sodium sulfate and evaporated. The intermediate was dissolved in 0.35 ml DCM and 0.15 ml TFA was added. The reaction mixture was shaken for 30 min at room temperature, then air-dried and further dried under high vacuum. 122.5 mg of oil (0.162 mmol, 1 eq.) was dissolved in 0.5 ml of DCM. TEA (0.49 mmol, 3 eq.) was added and the solution was shaken for 5 min at room temperature. 65.19 mg of DBCO-NHS (0.162 mmol, 1 eq.) was added and the reaction mixture was shaken for 1 hour at room temperature. The reaction mixture was injected onto a flash chromatography column and purified using a gradient of 0-15% methanol in DCM at 15

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CV. Fractions were collected and dried to obtain Myr-Pegn-DBCO. The molecular weight of the product was confirmed based on MS (ESI+) for peptide antigen conjugates of Compound 52.

Connection 53.

Compound 53, referred to as Plm-Pegn-DBCO or C16-PEG11-DBCO, was synthesized using the same procedure as described for Compound 52, except that palmitoyl chloride was used as the starting material.

Plm-Pegn-DBCO was purified by flash chromatography using a gradient of 0-15% methanol in DCM at 15 CV. Fractions were collected and dried to obtain Plm-Pegn-DBCO. The molecular weight of the product was confirmed based on MS (ESI+) for peptide antigen conjugates of Compound 53.

Connection 54.

□

Compound 54, referred to as NH ₂ -Pam2Cys-DBCO, was synthesized using Compound 26 and DBCO-Amin as starting materials. 30 mg of Compound 26 (0.03 mmol, 1 eq.) was prepared as a stock solution at a concentration of 100 mg/ml in DCM. To this stock solution was added 8.34 mg of DBCO-Amin (0.03 mmol, 1 eq.) also as a stock solution at a concentration of 100 mg/ml in DCM. The reaction mixture was shaken for 1 h at room temperature and then injected onto a flash chromatography column and purified. The gradient applied was a step gradient from 0-5% methanol in DCM (hold 5CV, 1CV to increase methanol concentration by 1%). Fractions were collected and dried to yield the DBCO intermediate. 26 mg of FmocPam2Cys-DBCO (0.023 mmol, 1 eq.) was dissolved in 1 ml of 20% Piperidine in DMF solution and shaken for 30 min at room temperature. The reaction mixture was diluted with DCM and washed three times with DI water. The organic layer was dried with sodium sulfate and evaporated. The solid was taken up in 0.5 ml DCM and injected onto a flash chromatography column and purified. The gradient used was a step gradient from 0-5% methanol in DCM. The fractions were collected and dried to obtain NH2-Pam2Cys-DBCO. The molecular weight of the product was confirmed based on MS (ESI+) for peptide antigen conjugates of Compound 54.

Connection 55.

Compound 55, referred to as NH ₂ -Pam2Cys-Peg ₁₁ -DBCO, was synthesized using Compound 26, Boc-Pegn-Amine and DBCO NHS as starting materials. 60 mg of Compound 26 (0.06 mmol, 1 eq.) was dissolved in 0.6 ml of DCM. 38.9 mg of Boc-Pegn-Amine (0.06 mmol, 1 eq.) was added and the reaction mixture was shaken at room temperature for 30 minutes. The DCM was removed in vacuo and the oil was placed in DMSO and added to DI water. The water was centrifuged at 3000 g for 5 min, and the pellets were collected and dried in vacuum. The PEGylated intermediate was dissolved in 0.35 ml DCM and 0.15 ml TFA was added. The reaction mixture was shaken at room temperature for 30 min and then air-dried and further dried under high vacuum. 71.76 mg of Boc deprotected oil (0.05 mmol, 1 eq.) was dissolved in 1 ml DMSO and TEA (0.1 mmol, 2 eq.) was added, followed by the addition of 20.3 mg DBCO-NHS (0 .05 mmol, 1 eq.). The reaction mixture was shaken for 1 hour at room temperature. 0.5 ml of 20% piperidine in DMF was added and the reaction mixture was shaken for 30 min at room temperature. The reaction mixture was diluted with DCM and washed three times with sodium bicarbonate pH 9.5. The organic layer was dried with sodium sulfate and evaporated. The solid was taken up in 0.5 ml DCM and injected onto a flash chromatography column and purified. The gradient used was 0-10% methanol in DCM at 30 CV. The result was a product with a total yield of 6.7%. The molecular weight of the product was confirmed based on MS (ESI+) for peptide antigen conjugates of Compound 55.

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Connection 56.

Compound 56, named Chol-TT, was prepared using cholesteryl hemisuccinate and TT as starting materials. 100 mg cholesteryl hemisuccinate (0.21 mmol, 1 eq.), 26.94 mg TT (0.23 mmol, 1.1 eq.) and 51.20 g EDC (0.27 mmol, 1.3 eq.) were dissolved in 1 ml DCM. 2.51 mg DMAP (0.02 mmol, 0.1 eq.) was added and the reaction mixture was shaken at room temperature. After 1 h, the bright yellow reaction mixture was diluted with DCM and washed twice with 1 M HCl and once with DI water. The organic layer was dried over sodium sulfate and evaporated to obtain 120 mg of a yellow solid in quantitative yield. The molecular weight of the product was confirmed based on MS (ESI+) for peptide antigen conjugates of Compound 56.

Connection 57.

Compound 57, called Chol-DBCO, was prepared from Compound 56 and DBCO-Amin. 106.28 mg of Compound 56 (0.18 mmol, 1 eq.) and 50 mg of DBCO-amine (0.18 mmol, 1 eq.) were dissolved in 0.5 ml of DCM and shaken at room temperature. Within an hour, the bright yellow color of the solution disappeared and TLC in DCM indicated the complete absence of Compound 56. The reaction mixture was injected onto a flash chromatography column and purified using a gradient of 0-3% methanol in DCM at 12 CV. Fractions were collected and dried to obtain 139 mg of Chol-DBCO in quantitative yield. The molecular weight of the product was confirmed based on MS (ESI+) for peptide antigen conjugates of Compound 57.

Connection 58.

Compound 58, called Chol-Peg ₁₁ -DBCO, was prepared from Compound 56, Boc-Peg11-amine and DBCO-NHS. 50 mg of Compound 56 (0.085 mmol, 1 eq.) was dissolved in 0.5 ml DCM. 60.38 mg of Boc-Pegn-amine (0.09 mmol, 1.1 eq.) was added and the reaction mixture was shaken at room temperature. Within an hour, the bright yellow color of the solution disappeared and TLC in DCM indicated the complete absence of Compound 56.

150 μL of TFA was added to the reaction mixture to obtain a 30% solution in DCM. The reaction mixture was shaken for 30 min at room temperature, then air-dried and further dried under high vacuum. 86.25 mg of oil (0.085 mmol, 1 eq.) was dissolved in 0.5 ml of DCM. TEA (0.26 mmol, 3 eq.) was added and the solution was shaken for 5 min at room temperature. 34.26 mg of DBCO-NHS (0.085 mmol, 1 eq) was added and the reaction mixture was shaken for 1 h at room temperature. The reaction mixture was injected onto a flash chromatography column and purified using a gradient of 0-15% methanol in DCM at 15 CV. Fractions were collected and dried to obtain Chol-Pegn-DBCO.

Connection 59.

Compound 59, referred to as LA-TT, was synthesized using Levulinic acid and TT in

- 107 046161 quality of starting materials. 500 mg Levulinic acid (4.3 mmol, 1 eq.), 564.7 mg TT (4.7 mmol, 1.1 eq.) and 1.032 g EDC (5.4 mmol, 1.3 eq.) were dissolved in 10 ml dry DMSO. 52.6 mg DMAP (0.4 mmol, 0.1 eq.) was added and the reaction mixture was shaken at room temperature for 2 hours. Compound 59 was treated by dilution in Ethyl acetate and washed twice with 1 M HCl and once with DI water. The organic layer was dried over sodium sulfate and removed in vacuo. The solid was recrystallized from Ethyl acetate. MS (ESI) was calculated for C ₈ H11NO ₂ S ₂ , mass/charge 217.02, the result was 218 (M+H)+.

Connection 60.

Compound 60, referred to as LA-2BXy, was synthesized using Compound 59 and Compound 2 as starting materials. 50 mg of Compound 59 (0.23 mmol, 1 eq.) and 82.62 mg of Compound 2 (0.23 mmol, 1 eq.) were dissolved in 1 ml of DMSO. The reaction mixture was shaken at room temperature for 1 hour. TLC (35% ethyl acetate in hexane) showed the disappearance of both starting materials. Compound 60 was purified by preparative HPLC using a 15-30% acetonitrile/ _H2O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. MS (ESI) was calculated for C ₂₇ H ₃₁ N ₅ O ₂ , mass/charge 457.25, the result was 458.3 (M+H)+.

Connection 61.

Compound 61, referred to as CTA-TT, was synthesized using the same procedure as described for Compound 59, except that 4-cyano-4-(thiobenzoylthio)pentanoic acid (CTA) was used as starting material and DCM was used as a solvent. Compound 61 was treated by dilution in DCM and washed twice with 1 M HC1 and once with DI water. The organic layer was dried over sodium sulfate and removed in vacuo. The solid was recrystallized from ethyl acetate. MS (ESI) was calculated for C ₁₆ H ₁₆ N ₂ OS ₄ , mass/charge 380.01, the result was 381.1 (M+H)+.

Connection 62.

Compound 62, referred to as CTA-DBCO, was synthesized from CTA-NHS (4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, (Sigma-Aldrich) and DBCO-amine (click chemistry tools). 210 mg CTA -NHS (0.56 mmol, 1 eq.) and 167.8 mg DBCO-amine (0.61 mmol, 1.1 eq.) were dissolved in 2 ml of dry DMSO and shaken at room temperature for 2 hours. The reaction mixture diluted with DCM and washed twice with 1 M HCl and once with DI water. The organic layer was dried with sodium sulfate and evaporated. Compound 62 was purified on a preparative HPLC system using a 50-70% acetonitrile/H ₂ O (0.05% TFA) gradient in for 10 min on an Agilent Prep-C18 column, 30x100 mm, 5 µm MS (ESI) calculated for C ₃₁ H ₂₇ N ₃ O ₂ S ₂ , mass/charge 537.7, the result was 538.2 (M+H) +.

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Connection 63.

Compound 63, referred to as CTA-2BXY, was synthesized using the same procedure as described for Compound 60 except that Compound 61 and Compound 2 were used as starting materials. Compound 63 was purified by preparative HPLC using a 40-50% acetonitrile/H ₂ O (0.05% TFA) gradient over 12 min on an Agilent PrepC18, 50x100 mm, 5 µm column. MS (ESI) was calculated for C ₃₅ H ₃₆ N ₆ OS ₂ , mass/charge 620.83, the result was 621.2 (M+H) ⁺ .

Connection 64.

Compound 66, referred to as ACVA-TT, was synthesized using the same procedure as described for Compound 59, except that 4,4'-azobis(4-cyanovaleric acid) was used as starting material and TT equivalents EDC and DMAP were doubled to reflect the doubling of active sites. Compound 66 was treated by dilution in ethyl acetate and washed twice with 1 M HCl and once with DI water. The organic layer was dried over sodium sulfate and removed in vacuo. The solid was recrystallized from DCM:Ether. MS (ESI) was calculated for C ₁₈ H ₂₂ N ₆ O ₂ S ₄ , mass/charge 482.65, the result was 483.1 (M+H) ⁺ .

Connection 65.

Compound 65, referred to as ACVA-2BXy, was synthesized using the same procedure as described for Compound 60, except that Compound 64 was used as starting material and Compound 2 equivalents were doubled to reflect the doubling of the active sites. Compound 65 was purified by preparative HPLC using a 28-48% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 50x100 mm, 5 µm column. MS (ESI) was calculated for C ₅₆ H ₆₂ N ₁₄ O ₂ , mass/charge 963.21, the result was 482.4 (M/2) ⁺ .

Connection 66.

Compound 66, referred to as ACVA-DBCO, was synthesized using the same procedure as described for Compound 60, except that Compound 64 and DBCO-Amin (click chemistry tools) were used as starting materials. Compound 66 was purified by flash chromatography using a gradient of 0-3% methanol in DCM at 15 CV. MS (ESI) was calculated for C48H44N8O4, m/c 796.93, resulting in 797.3 (M+H)+.

- 109 046161

Connection 67.

Compound 67, referred to as Azp-2BXY, was synthesized from 5-Azidopentanoic acid (Azp) and Compound 2. 16.2 mg Azp (0.114 mmol, 1.1 eq.), 12.3 mg TT (0.103 mmol, 1 eq. ) and 27.2 mg EDC (0.142 mmol, 1.3 eq.) were dissolved in 0.5 ml dry DMSO. 1.4 DMAP was added and the reaction mixture was shaken for 2 hours at room temperature. 37.09 of Compound 2 (0.103 mmol, 1 eq.) was added and the reaction mixture was shaken for 1 hour at room temperature. Compound 67 was purified by preparative HPLC using a 22-42% acetonitrile/H ₂ O (0.05% TFA) gradient over 10 min on an Agilent Prep-C18, 30x100 mm, 5 µm column. MS (ESI) was calculated for C ₂₇ H ₃₂ N ₈ O, mass/charge 484.27, the result was 485.3 (M+H)+.

Connection 68.

Compound 68, also referred to as (HPMA-2B)-b-(HPMA)-DBCO or p{[(HPMA)-co-(MA-b-Ala2B)]-b-p(HPMA)}-DBCO. A micelle-forming diblock copolymer (type A-B) was produced by polymerizing RAFT in two synthetic steps using HPMA and MA-b-Ala-TT precursors prepared as previously described (see G. M. Lynn, et al. Nat Biotechnol 33 (11): 1201-1210, 2015), as well as 2-cyano-2propylbenzodithioate (CTA-ABIN, Sigma-Aldrich), azobiisobutyronitrile (AIBN, Sigma-Aldrich), Compound 1 and Compound 66. Hydrophobic block A was obtained by copolymerizing HPMA with MA-b-AlaTT using CTA-AIBN as chain transfer agent and AIBN as initiator at 70°C for 16 h in tert-butyl alcohol/DMSO. Hydrophobic block B was subsequently subjected to chain extension polymerization via the RAFT mechanism by adding HPMA in the presence of AIBN at 70°C for an additional 16 hours. Diblock copolymer A-B, p{[HPMA)-co-(MA-b-TT )]-b-p(HPMA)}-DTB was isolated by precipitation to yield a green solid. The DTB group at one end of the copolymer was replaced by reacting Compound 66 at 80°C for 2 hours in DMSO, after which Compound 1 was added. The product was precipitated and then purified by LH-20 to yield Compound 68, which was then used for the reaction with peptide antigens yielding various types of peptide antigen conjugates, for example p{[(HPMA)-co-(MA-b-Ala-2B)]-b-p(HPMA)}-DBCO-(peptide antigen), which are fully described below.

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Connection 69.

N.H.

I

Compound 69, also referred to as (HPMA-NHNH-2BXy)-b-(HPMA)-DBCO or p{[(HPMA)-co(MA-Acap-NHN=CH-2BXy)]-b-p(HPMA)}-DBCO .

A micelle-forming diblock copolymer (type A-B) was produced by polymerizing RAFT in two synthetic steps using HPMA and MA-Acap-NHNH-Boc precursors prepared as described above (see G. M. Lynn, et al. Nat Biotechnol 33 (11): 1201-1210, 2015), as well as 2-cyano-2propylbenzodithioate (CTA-ABIN, Sigma-Aldrich), azobiisobutyronitrile (AIBN, Sigma-Aldrich), Compound 60 and Compound 66.

Hydrophobic block A was prepared by copolymerizing HPMA with MA-Acap-NHNH-Boc using CTA-AIBN as chain transfer agent and AIBN as initiator at 70°C for 16 hours in tert-butyl alcohol/DMSO . Hydrophobic block B was subsequently subjected to chain extension polymerization via the RAFT mechanism by adding HPMA in the presence of AIBN at 70°C for an additional 16 hours. Diblock copolymer A-B, p p{[(HPMA)co-(MA-Acap- NHN=CH-2BXy)]-b-p(HPMA)}-DTB was isolated by precipitation and then deprotected with Boc in 30% trifluoroacetic acid TFA/DCM. TFA/DCM was removed in vacuo and then the DTB group at one end of the copolymer was replaced by reacting Compound 66 at 80°C for 2 hours in DMSO, after which Compound 60 was added. The product was precipitated and then purified using LH-20 with yield of Compound 69, which was then used to react with peptide antigens to yield various types of peptide antigen conjugates, for example, p^HPMA/co/MA-b-A^2B)]-b-p(HPMA)}-DBCO-(peptide antigen), which are described in full below.

Connection 70.

h ₂ n

Compound 70, also designated (DEGMA-2B)-b-(HPMA)-DBCO or p{[(DEGMA)-co-(MAb-Ala-2B)]-b-p(HPMA)}-DBCO. A temperature-sensitive micelle-forming diblock copolymer (type A-B) was produced by polymerizing RAFT in two synthetic steps using the DEGMA precursors, HPMA and MA-L-AL-TT, prepared as described above (see Lynn, G.M., and al. Nat Biotechnol 33(11): 1201-1210, 2015), as well as 2-cyano-2-propylbenzodithioate (CTA-ABIN, Sigma-Aldrich), Azobiisobutyronitrile (AIBN, Sigma-Aldrich), Compound 1 and Compound 66.

Temperature-sensitive hydrophobic block A was prepared by copolymerizing DEGMA with MA-bAla-TT using CTA-AIBN as chain transfer agent and AIBN as initiator at 70°C for 16 h in tert-butyl alcohol/DMSO. Hydrophobic block B was subsequently subjected to chain extension polymerization via the RAFT mechanism by adding HPMA in the presence of AIBN at 70°C for an additional 16 hours. Diblock copolymer A-B, p{[(DEGMA)-co-(MA-b- Ala-2B)]-b-p(HPMA)}-DTB was isolated by precipitation to yield a green solid. The DTB group at one end of the copolymer was replaced by reacting Compound 66 at 80°C for 2 hours in DMSO, after which Compound 1 was added.

- 111 046161

The product was precipitated and then purified by LH-20 to yield Compound 70, which was then used to react with peptide antigens to yield various types of peptide antigen conjugates, for example p{[(DEGMA)-co-(MA-b-A1a-2B) ]-b-p(HPMA)}-DBCO-(Peptide antigen), which are fully described below.

Connection 71.

h ₂ n

Compound 71, also referred to as (DEGMA-2BXy)-b-(HPMA)-DBCO or p{[(DEGMA)-co-(MAb-Ala-2BXy)]-b-p(HPMA)}-DBCO, was prepared using this The same procedure as described for Compound 70, except for the fact that Compound 2 was used as the ligand. The product was precipitated and then purified by LH-20 to yield Compound 68, which was then used to react with peptide antigens to yield various types of peptide antigen conjugates, for example p{[(DEGMA)-co-(MA-b-Ala-2BXy) ]-b-p(HPMA)}-DBCO-(Peptide antigen), which are fully described below.

Connection 72.

SN _E

Compound 72, also referred to as 2BXy-DEGMA-b-HPMA-DBCO. A micelle-forming diblock copolymer (type A-B) was produced by polymerizing RAFT in two synthetic steps using the precursors DEGMA, HPMA, Compound 63, Compound 65 and Compound 66. Hydrophobic block A was produced by copolymerizing DEGMA using Compound 63 as a chain transfer agent and Compound 65 as an initiator at 70°C for 16 hours in a mixture of tert-butyl alcohol/DMSO. Hydrophobic block B was subsequently subjected to chain extension polymerization via the RAFT mechanism by adding HPMA in the presence of AIBN at 70°C for an additional 16 hours. Diblock copolymer AB, 2BXy-[p(DEGMA)-b-p(HPMA)]- DTB was isolated by precipitation and the DTB group at one end of the copolymer was replaced by reacting Compound 66 at 80°C for 2 hours in DMSO. The product was precipitated and then purified by LH-20 to yield Compound 72, which was then used to react with peptide antigens to yield various types of peptide antigen conjugates, for example, 2BXy-p[(DEGMA)-bp(HPMA)]-DBCO-(Peptide antigen), which are described in full below.

Example 2. Reaction of a fragment of a peptide antigen and a hydrophobic molecule (H) to produce a peptide antigen conjugate.

A peptide antigen fragment consisting of a peptide antigen (A), an optional charged molecule (C), optional extensions (B1 and/or B2) and a precursor linker X1, e.g. [C][B1]-A-[B2]-X1, where [ ] denotes that the group is optional in this example, can react with X2 linked to a hydrophobic molecule (H), such as X2-H, to produce a peptide antigen conjugate, such as [C]-[B1]-A-[B2 ]-L-H. In this case, the peptide antigen fragment contains a linker precursor X1 containing an azide, for example, azidolysine (Lys(N3)), sometimes referred to as K', the peptide antigen fragment can react with a linker precursor X2 containing an alkyne, such as DBCO, which linked to a hydrophobic molecule (H) to form a triazole linker (L). By way of non-limiting examples, Compounds 3-5 are examples of hydrophobic Formula I molecules (H) bearing an X2 linker precursor with DBCO that is further linked to Formula III adjuvants (ie, TLR-7/8a); Compounds 6-11 represent

- 112 046161 are examples of hydrophobic molecules (H) of Formula I carrying a precursor linker X2 with DBCO, which is further linked to adjuvants of Formula III (ie, TLR-7/8a); and Compounds 10-11 are examples of hydrophobic molecules (H) of Formula II bearing the X2 linker precursor with DBCO, but not containing a Ligand with adjuvant properties.

A non-limiting example shown in FIG. 2 is the reaction of a hydrophobic molecule (H) linked to a DBCO-containing X2 linker precursor which reacts in a 1.1:1 molar ratio with an azide-containing peptide antigen moiety carrying the X1 linker precursor. As indicated in the HPLC chromatogram, the DBCO on the hydrophobic molecule (H) reacts with the azide-containing linker precursor X1, resulting in the formation of a triazole linker (L), which is linked to the B2 extension, which is linked to the peptide antigen (A), which linked to the B1 extension, which is linked to a charged molecule (C), thereby connecting the peptide antigen (A) to a hydrophobic molecule (H) to produce a peptide antigen conjugate. Hundreds of examples of various peptide antigen conjugates formed by the reaction between a peptide antigen fragment bearing an azide and a hydrophobic molecule (H) bearing DBCO have been described throughout. Because the azide-DBCO reaction is reliable and consistent for all peptide antigen conjugates produced, a complete description of the reaction used to form the conjugates and their characteristics are not proposed. Note: The molecular weight of peptide antigen conjugates resulting from cycloaddition is the sum of the weights of the starting materials.

In this case, the peptide antigen fragment contains a linker precursor X1 containing a thiol, for example, cysteine (Cys), sometimes referred to as C, the peptide antigen fragment can react with a hydrophobic molecule (H) carrying a linker precursor X2 containing a maleimide, for example, Compound 21, resulting in the formation of a thioester linker (L). In this case, the peptide antigen fragment contains an X1 linker precursor containing an amine, for example lysine (Lys), sometimes referred to as K, the peptide antigen fragment can react with a hydrophobic molecule (H) carrying an X2 linker precursor containing an activated ester, such as NHS, for example, Compound 22, resulting in the formation of an amide bond.

Example 3/ Synthesis and biological characterization of peptide antigen conjugates of Formula IV

Previous studies have reported the delivery of T cell epitopes as ~20–40 amino acid synthetic long peptides (SLP or LP) in combination with various vaccine adjuvants as a means to induce CD8 T cell responses. However, the influence of peptide length and hydrodynamic behavior, as well as co-delivery of the peptide with immunostimulants such as TLRa, on immunogenicity is not well understood. To better understand how different parameters of peptide-based antigens influence T-cell immunity, we systematically assessed how different properties of the Formula IV peptide antigen conjugates described herein affect CD4 and CD8 T-cell responses in vivo.

The influence of the hydrodynamic behavior of the peptide antigen, the length of the peptide antigen (A), and co-delivery of TLR-7/8a on CD8 T cell responses was not clear a priori, how the length of the peptide antigen (A) delivered in the form of peptide antigen conjugates of the present invention would be influence immunogenicity, in particular the intensity and quality of the generated CD8 and/or CD4 T-cell response.

To systematically investigate how the length of the peptide antigen (A) affects the immunogenicity of Formula IV peptide antigen conjugates, two models of neoantigenic CD8 T cell epitopes, Irgq and Cpne1, derived from a mouse tumor model, MC38, were produced as peptide antigen conjugates, while the peptide antigens (A) were either a synthetic long peptide of 26 amino acids (SLP or LP) or a minimal epitope of 10 amino acids (ME or Min) (Fig. 4). The SLP-based peptide antigens (A) were linked at the C-terminus to the X1 linker precursor, azidolysine (K'), which was linked to the X2 linker precursor, DBCO, which was linked to the N-terminus of a hydrophobic molecule (H); whereas peptide antigens (A) with minimal epitopes were linked to a C-terminal extension (B2), which was linked to the X2 linker precursor, DBCO, which was linked to the N-terminus of a hydrophobic molecule (H) to generate Formula IV peptide antigen conjugates , summarized in Fig. 4.

The general scheme for the synthesis of peptide antigen conjugates was the reaction of 1.0 equivalents of a peptide antigen fragment bearing a reactive azide with 1.2 equivalents of a hydrophobic molecule (H) bearing DBCO in DMSO at room temperature. The reaction was completed after 24 hours and no further purification was required.

We first assessed the hydrodynamic behavior of peptide antigens (A) and peptide antigen conjugates (Figure 4). It is noteworthy that peptide antigens (A) containing minimal epitopes (Min) and LPs from both Irgq and Cpne1 were found to be soluble in water up to a concentration of 0.1 mg/ml; however, the attachment of peptide antigens (A) to either two hydrophobic molecules (H), W ₃ or 2BXy ₃ , caused the resulting peptide antigen conjugates to undergo particle formation, with the exception of the peptide antigen conjugate delivering the peptide

- 113 046161 antigen (A) containing LP from Cpnel attached to W3. These results indicate that Formula IV peptide antigen conjugates induce the formation of water-soluble peptide particles, and that increasing the length of the hydrophobic molecule (H) or using more hydrophobic monomers (eg, Glu(2BXy), compared to Tryptophan) increases the reliability of particle formation .

Accordingly, it was unexpectedly found that most of the tested peptide antigen conjugates formed particles when the hydrophobic molecule (H) of Formula I consisted of at least 5 monomer units linked to adjuvants of Formula III, whereas the hydrophobic molecules (H) of Formula I or II with less than 5 monomer units, for example 1 or 3 monomer units, induced particle formation less reliably. Based on these data, in preferred embodiments of the present invention, the hydrophobic molecules (H) used in peptide antigen conjugates consist of at least 5 monomer units to provide formation of highly hydrophilic and/or charged peptide antigen (A) particles. Although in some embodiments of the present invention the hydrophobic molecule (H) may be at least 3 monomer units in length, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 monomer units, typically up to about 300 monomer units.

We further assessed how the hydrodynamic behavior of the peptide antigen conjugate, as well as the length of the peptide antigen (A) and co-delivery of a TLR-7/8a adjuvant containing the peptide antigen conjugate affected immunogenicity in vivo. Peptide antigen conjugates delivering peptide antigens (A) consisting of either long peptides (LP) of 26 amino acids or minimal epitopes (Min) of 10 amino acids, or were mixed with the small molecule TLR-7/8a, Compound 2, called like 2ВХу; linked to a hydrophobic molecule (H), W ₃ and mixed with 2ВХу; or the peptide antigens (A) were linked to a hydrophobic molecule (H), 2BXy3, which forms particles and co-deliveries the peptide antigen (A) with the TLR-7/8a adjuvant (see FIG. 46). Mice immunized with peptide antigen conjugates delivering peptide antigens (A) consisting of minimal epitopes linked to a hydrophobic molecule (H), 2BXy ₃ , offered CD8 T cell responses of the greatest intensity (> 1% IFNg ⁺ CD8 T cells of total numbers) after a single immunization, indicating that co-delivery of a minimal epitope with TLR-7/8a in a particulate format is an effective approach for generating CD8 T cell responses. Notably, soluble minimal epitope and soluble LP mixed with TLR-7/8a induced the lowest CD8 T cell responses, which were not statistically significant, compared to naïve groups, indicating that the soluble form of the peptide antigen (A) is the least immunogenic (Fig. 5 and 6). Finally, while peptide antigen conjugates delivering peptide antigens (A) consisting of minimal Irgq epitopes or LPs from Irgq linked to a hydrophobic molecule (H), 2BXy ₃ , offered comparable intensity of CD8 T cell responses, peptide antigen conjugates , delivering peptide antigens (A) consisting of a minimal epitope of Cpne1 induced a nearly 10-fold increase in CD8 T cell responses compared with peptide antigen conjugates delivering Cpne1 as an LP.

To further explore the role of peptide antigen (A) length in immunogenicity to stimulate CD4 and CD8 T cell immunity for Formula IV peptide antigen conjugates, 7 predicted CD8 T cell minimal epitopes (i.e., predicted neoantigens) derived from the B16 mouse tumor model , were delivered either as long peptides (LP) of 27 amino acids or as minimal epitopes of 10 amino acids (referred to as ME or Min) as peptide antigens (A) containing Formula IV peptide antigen conjugates (see FIG. 7) . The LP-based peptide antigens (A) were linked at the C-terminus to the X1 linker precursor, azido-lysine (K'), which was linked to the X2 linker precursor, DBCO, which was linked to the N-terminus of a hydrophobic molecule (H); whereas peptide antigens (A) with minimal epitopes were linked to a C-terminal extension (B2), which was linked to the X2 linker precursor, DBCO, which was linked to the N-terminus of a hydrophobic molecule (H) to generate Formula IV peptide antigen conjugates , summarized in Fig. 7. Peptide antigens (A) were linked to either 2BXy ₅ or 2B ₅ hydrophobic molecules (H) to form peptide antigen conjugates. Comparison of 2BXy ₅ with 2B5, which are structurally similar but differ in activity against TLR-7, allowed us to evaluate how inflammation affects immunogenicity. Mice were immunized with immunogenic compositions consisting of peptide antigen conjugates delivering peptide antigens (A) as minimal epitopes (Min), LP, or both minimal epitopes and LP. The intensity of CD4 and CD8 T-cell responses was assessed 2 weeks after the 2nd immunization (Fig. 8).

A striking and unexpected result was that mice that received peptide antigen conjugates delivering peptide antigens (A) containing minimal epitopes (Min) induced CD8 T cell responses of the highest intensity, whereas peptide antigen conjugates delivered peptide antigens (A ), containing LP, induced CD8 T

- 114 046161 lower level cellular responses. This trend was evident for both hydrophobic molecules (H), 2BXy5 and 2B5. Accordingly, for peptide antigen conjugates delivering a peptide antigen (A) containing LP linked to a hydrophobic molecule (H), 2BXy5, CD8 T cell responses were ~0.35% and CD4 T cell responses were ~0.25 % (Fig. 8). In contrast, for a group of mice that received peptide antigen conjugates delivering a peptide antigen (A) containing a minimal epitope linked to a hydrophobic molecule, 2BXy ₅ , CD8 T cell responses were ~1.9% and CD4 T cell responses were ~0.1%, which offered an approximately 6-fold increase in CD8 T cell responses compared to peptide antigen conjugates delivering LP but undetectable CD4 T cell responses. In a group of mice that received peptide antigen conjugates delivering peptide antigens (A) containing both the minimal epitope and LP (LP + min), CD8 T cell responses were ~0.6% and CD4 T cell responses were ~0.5%, which suggested a balance of both CD4 and CD8 T cells (Fig. 8).

A similar trend was observed for peptide antigen conjugates using the hydrophobic molecule (H) 2B5, which includes the less active TLR-7/8 agonist, 2B, compared to the hydrophobic molecule (H), 2BXy ₅ , which includes 2BXy. Accordingly, in the group that received peptide antigen conjugates that included the hydrophobic molecule (H) 2B5 and peptide antigens (A) containing LP, CD8 T cell responses were detected at ~1.0% and CD4 T cell responses at ~0. 75%, whereas in the group that received peptide antigen conjugates delivering peptide antigens (A) containing only Min, CD8 T cell responses at ~4.5% and CD4 T cell responses at ~0.1% were observed. There was an almost 4-fold increase in CD8 T cell responses, but CD4 T cell responses were undetectable. In the group that received the vaccine that contained peptide antigen conjugates delivering peptide antigens (A) containing a minimal epitope and LP linked to a hydrophobic molecule (H) 2B5, CD8 T cell responses were ~2.2%, and CD4 T cell responses responses were ~0.95% (Fig. 8)

In summary, the use of ME (or Min) as the peptide antigen (A) of the Formula IV peptide antigen conjugates resulted in an unexpected and significant increase in CD8 T cell response compared to the peptide antigen (A) containing LP. However, it also resulted in decreased CD4 T cell responses. Importantly, the inclusion of peptide antigen conjugates delivering LP and Min as peptide antigens (A) into immunogenic compositions administered to mice resulted in restored CD4 T cell responses and an unexpectedly higher CD8 T cell response compared to peptide antigen conjugates delivering peptide antigens (A) consisting only of LP. Another key finding was that the extent of inflammation had a marked effect on immunogenicity. The hydrophobic molecule (H), 2B5, contains TLR-7/8a, Compound 1, which is almost 10 times less active than Compound 2, 2BXy, delivered on the hydrophobic molecule (H), 2BXy5. Thus, the nearly 3- to 4-fold increase in CD4 and CD8 T cell responses to peptide antigen conjugates containing the hydrophobic molecule (H), 2B5, compared with peptide antigen conjugates that use 2BXy ₅ suggests that the reduction in the extent of inflammation due to modulation of adjuvant activity may be preferable to achieve optimal levels of immunogenicity with the peptide antigen conjugates of the present invention. Indeed, when screening the immunogenicity of 40 different neoantigens based on peptides obtained from the mouse tumor cell line MC38, individualized anticancer vaccines based on peptide antigen conjugates delivering peptide antigens (A) consisting of minimal epitopes linked to a hydrophobic molecule (H), 2B5 , resulted in a 6-fold improvement in the breadth of CD8 T cell responses achieved compared to peptide antigen conjugates consisting of minimal epitopes linked to a hydrophobic molecule (H), 2BX _y5 (Fig. 9).

An additional unexpected finding relates to studies aimed at identifying optimal mechanisms for dosing several different T cell epitopes as peptide antigen conjugates. According to previous studies, dosing multiple T cell epitopes at the same site that bind the same MHC molecule may compete with binding to MHC molecules on the APC, and this may also result in competition with presentation to T cells, which thereby reducing the intensity of T cell responses to epitopes delivered together, compared to epitopes delivered separately as a single epitope, to the immunization site. Although we observed that administration of immunogenic compositions, including peptide antigen conjugates containing a single epitope, to mice resulted in a higher intensity of T cell response to that epitope compared to co-administration of the same epitope with 3 different peptide antigen conjugates containing different epitopes, all of which bind the same MHC molecule at either 1 or 4 sites (Fig. 10), however, it was unexpectedly found that delivery of the same 4 epitopes to 4 separate sites (i.e., 1 unique epitope per site) the same animal resulted in lower response rates than delivery of all 4 epitopes together to 4 sites (Fig. 10). This was unexpected and paradoxical for the current paradigm, which assumes

- 115 046161 that it is better to dose multiple epitopes at different sites to avoid competition between epitopes than to deliver multiple epitopes together at multiple sites. These data suggest a new paradigm for delivering multiple different epitopes based on maximizing the number of sites where each epitope is introduced. Therefore, in preferred embodiments of the methods for inducing an immune response of the present invention, immunogenic compositions containing multiple different peptide antigens (A) are dosed at multiple sites to maximize the immune response.

Example 3 Peptide Antigen Conjugates of Formula V

The previous data demonstrate how the parameters of Formula IV peptide antigen conjugates influence immunogenicity. These data indicate that peptide antigen conjugates, which co-deliver peptide antigens (A) with TLR-7/8a in particles, are a preferred embodiment of the present invention for inducing CD8 T cell immunity. A limitation of Formula IV peptide antigen conjugates is that the particles formed according to this approach may have variable size and stability due to variability in the composition of peptide antigens (A) coupled to hydrophobic molecules (H). One possible mechanism for stabilizing particles formed by peptide antigen conjugates is the introduction of a surface charge, which prevents particle aggregation and stabilizes particles formed by peptide antigen conjugates in aqueous media.

Peptide antigen conjugates of Formula V allow the formation of stable particles of a certain size by attaching a charged molecule (C) to a peptide antigen (A) that is linked to a hydrophobic molecule (H) (Fig. 11). However, direct conjugation of a charged molecule (C) and/or a hydrophobic molecule (H) immediately adjacent to a peptide antigen (A) may interfere with the processing and presentation machinery of the APC, which allows the presentation of minimal epitopes in the context of MHC-I and MHC-II . Thus, in order to ensure efficient processing of peptide antigens (A) based on minimal epitopes (Min) or LPs delivered using Formula V peptide antigen conjugates, we evaluated the use of enzyme-degradable peptide antigen-coupling extensions (B1 and B2). A) with a charged molecule (C) and a hydrophobic molecule (H) at the N- and C-termini, respectively (Fig. 11).

To evaluate the effect of extensions (B1 and B2) on the processing of minimal CD8 T cell epitopes from Formula V peptide antigen conjugates, we synthesized a series of peptides in which a charged molecule (C) was linked to an optional extension (B1) at the N-terminus minimal epitope, Cpne1, which was linked to an optional extension (B2) at the C-terminus to the X1 linker precursor (Lys(N3)), and assessed how the charged molecule (C) and extensions (B1 and/or B2) affected activity peptide antigens (A) in vitro in relation to the activation of Cpne1-specific CD8 T cells. As shown in FIG. 12, peptide sequences were generated using cathepsin-cleavable extensions or combined cathepsin-cleavable and immunoproteasomal extensions at either one or both the N- and C-termini (B1 and/or B2) of the minimal epitopes. These peptide antigen (A) sequences linked to extensions (B1 and/or B2) were added to splenocyte cultures at various concentrations to evaluate the effectiveness of different constructs to promote uptake and presentation of the minimal epitope, Cpne1, by APC.

As expected, the minimal epitope (Fig. 12), which does not require processing and can be directly loaded in the context of MHC-I and presented to APC, was most effective and provided the highest activity (lowest EC50 level) in vitro. However, adding a charged molecule (C) (Glu-Lys-Glu-Lys-Glu-Lys SEQ ID NO: 71) directly to the N-terminus of the minimal epitope (♦) without using the degradable extension (B1) resulted in an almost 10,000-fold decrease in activity , compared to the minimal epitope applied alone. Notably, the activity of the minimal epitope was restored by simply placing an enzymatically degradable extended tetrapeptide (B1) (i.e., Ser-Leu-Val-Arg SEQ ID NO: 7) between the charged molecule (C) and the minimal epitope (Hexagons, Fig. 12). . Placing an additional cathepsin-cleavable extension (B2) at the C-terminus (i.e., Ser-Leu-Val-Arg SEQ ID NO: 7) or combining an immunoproteasome-degradable sequence with a cathepsin-cleavable extension at the N-terminus (B1) (SerLeu-Val- Arg-Tyr-Leu-Leu-Leu SEQ ID NO: 72) or at the C-terminus (B2) (Gly-Gly-Ser-Leu-Val-Arg SEQ ID NO: 13) had minimal additional effect on activity, compared using a single tetrapeptide extension (B1) between a charged molecule (C) and a peptide antigen (A) with a minimal epitope.

The following studies attempted to evaluate the effect of enzymatically degradable extensions (B1 and B2) on the efficiency of in vivo antigen cross-presentation using peptide antigen conjugates of Formula V, wherein the hydrophobic molecule (H) was 2BXy ₃ .

The general scheme for the synthesis of peptide antigen conjugates of Formula V was a reaction of 1.0 eq. peptide antigen carrying a chemically active azide, with 1.2 eq. hydrophobic molecule (H),

- 116 046161 carrier DBCO, in DMSO at room temperature. The reaction was completed after 24 h and no further purification was required.

As shown in FIG. 13, peptide antigen conjugates have been synthesized with various charged molecules (C) (e.g. Lys-Ser-Lys-Ser-Lys-Ser SEQ ID NO: 73, Glu-Ser-Glu-Ser-Glu-Ser SEQ ID NO: 74 and Glu-Lys-Glu-Lys-Glu-Lys SEQ ID NO: 71) using various combinations of enzyme-degradable extensions at the N- and C-termini (B1 and B2) of the minimal epitope, Cpne1. Mice were vaccinated with various immunogenic compositions presented in FIG. 13, on days 0 and 14, and T-cell responses were assessed relative to whole blood on days 10 (Fig. 14A) and 28 (Fig. 14B). Consistent with the in vitro data shown in FIG. 12, placement of any of the three charged molecules (C) at the N-terminus of the minimal epitope, without application of the B1 extension, resulted in a complete abrogation of CD8 T cell responses (~0.1% IFN-γ + total CD8 + T cells) after assessing responses relative to the whole blood of animals primed with a single immunization (Fig. 14). However, placing a cathepsin-cleavable extension (B1) between a charged molecule (C) and the N-terminus of a peptide antigen (A) consisting of a minimal epitope resulted in an almost 10-fold increase in CD8 T cell responses to peptide antigen conjugates using a positively charged molecule (C). (Lys-Ser-Lys-Ser-Lys-Ser SEQ ID NO: 73) and to a moderate improvement in CD8 T cell responses to peptide antigen conjugates with charged molecules (C) ES and EK (Fig. 14). Among groups vaccinated with peptide antigen conjugates containing a positively charged molecule (C) (Lys-Ser-Lys-Ser-Lys-Ser SEQ ID NO: 73), the addition of both an N-terminal and a C-terminal cathepsin-cleavable extension ( B1 and B2) resulted in CD8 T cell responses of approximately 3.5% after a single immunization, while the addition of immunoproteasomal sequences to B1 and/or B2, in addition to cathepsin-cleavable sequences, resulted in only modest further increasing the intensity of CD8 T cell responses (Fig. 14). The trends were consistent with data after 2 immunizations for peptide antigen conjugates using all 3 sets of charged molecules (C): including N- and C-terminal cathepsin-cleavable extensions (B1 and B2) between the charged molecule (C) and hydrophobic molecules (H) , respectively, which generally resulted in the highest intensity responses, with little or no improvement in responses by the addition of an immunoproteasomal linker.

These data illustrate the unexpected discovery that a cathepsin-cleavable peptide extension (B1), e.g., Ser-Leu-Val-Arg (SEQ ID NO: 7), located at the N-terminus of the peptide antigens of the Formula V peptide antigen conjugates, represents is a preferred embodiment of the present invention for promoting antigen presentation in vitro and cross-priming of CD8 T cells in vivo. Moderate additional improvements in CD8 T cell responses were observed when cathepsin-cleavable peptide extensions were placed at both the N- and C-termini, and when a cathepsin-cleavable extension (B2) at the C-terminus was combined with a sequence that additionally favored immunoproteasomal processing (for example, Gly-Gly dipeptide, of Gly-Gly-Ser-Leu-Val-Arg SEQ ID NO: 13). Thus, preferred embodiments of the peptide antigen conjugates of the present invention include a cathepsin-cleavable extension (B1) between the N-terminus of the peptide antigen (A) and any additional components, eg, charged molecules (C); and, optionally, a cathepsin-cleavable or combined cathepsin-cleavable and immunoproteasome-degradable extension (B2) at the C-terminus.

To extend these results, we synthesized peptide antigen conjugates with various combinations and compositions of cathepsin-degradable extensions at the N- and C-termini (B1 and B2) of the minimal epitope, Adpgk (Fig. 15). The various peptide compositions shown in FIG. 15 were added to splenocyte cultures at various concentrations to evaluate the effectiveness of various constructs to promote the uptake and presentation of a minimal epitope, Adpgk, by APC. The in vitro activity (EC50) for the Adpgk peptide antigen, with various combinations and compositions of cathepsin-degradable N- and C-terminal extensions (B1 and B2), is shown in FIG. 16. To confirm the in vitro results, a subset of sequences based on the formula C-B1-A-B2-X1 shown in FIG. 15 were linked to a hydrophobic molecule (H), 2B ₃ W ₂ , and then administered to mice. CD8 T cell responses were assessed at day 13 (Fig. 17) and were consistent with the results of the in vitro screen, confirming that cathepsin-degradable extensions linking the peptide antigen (A) to a charged molecule (C) and a hydrophobic molecule (H) to N- and C-termini, respectively, can lead to improved antigen presentation in vitro, as well as to increased efficiency of cross-presentation of antigens in vivo.

Effect of total charge and sequence extensions (B1 and B2) on particle size

The fact that the surface charge of particles affects their stability in solution is well known, but the influence of the charge density and total charge of peptide antigen conjugates on the size and stability of the supramolecular associates of these peptide antigen conjugates in aqueous solutions has not been well studied.

To better understand how charge density and net charge of amphiphilic macromolecules influence

- 117 046161 on the hydrodynamic behavior of self-assembling particles, we synthesized a series of peptide antigen conjugates of Formula V (Fig. 18) with different compositions of peptide antigens (A), charged molecules (C), extensions (B1 and B2) and hydrophobic molecules (H), and assessed how these parameters affect the size and stability of particles formed by peptide antigen conjugates suspended in aqueous solutions at a pH of approximately 7.4 (Fig. 18).

Our initial studies were aimed at examining how increasing net charge affects the particle size and stability of Formula V peptide antigen conjugates (FIGS. 18 and 19). Our results show an inverse relationship between charge intensity and particle size formed by Formula V peptide antigen conjugates delivering a wide range of different peptide antigens (A) (N=754), with approximately 90% of peptide antigens (A) delivered as peptide antigens Formula V conjugates with a total charge (absolute value) > 6 were assembled into nanoparticle micelles with a size of ~ 20-40 nm. Notably, the intensity of the net charge required to mediate nanoparticle micellization is largely dependent on the length and hydropathy of the peptide antigen (A), with most peptide antigen conjugates delivering L-based peptide antigens (A) forming micelles as peptide antigen conjugates with a net charge of +8 or higher, or -8 or lower, but with the most highly hydrophobic antigens, which require a net charge of up to +10 or higher, or -10 or lower, to ensure stable formation of nanoparticle micelles.

The main challenge for peptide-based PCVs is that they must ensure compositional homogeneity using any possible peptide antigen that may arise from the human genome. To assess the generalizability of SP-7/8a as a PCV, we first calculated the charge and hydropathy frequency distribution of all possible 25 amino acid peptides (11.3 g/mol 25-mer) resulting from the canonical transcripts, including all possible missense mutations (72.6 g/mol 24-mer). Surprisingly, our results show that more than 98% of the 25 amino acid peptide-based neoantigens will have a charge between -6 and +6 (at pH 7.4) and a global average hydropathy value (GRAVY) between +2 and -2. These results indicate that the charge distribution and hydropathy of mouse neoantigens used herein represent the in silico characteristics of predicted human neoantigens (Figure 19 C and D). Notably, even a modest increase of about 2 to 4 whole number units of net charge results in markedly improved stability of particles formed by Formula V peptide antigen conjugates. Accordingly, while at least about 25% of LP neoantigens are delivered as peptide antigen conjugates Formula V with a net charge of +6 aggregated, the same LP neoantigens, are delivered as Formula V peptide antigen conjugates with a net charge greater than or equal to +8 aggregated into stable nanoparticle micelles (Figure 19 E). These results indicate that Min and LP peptide antigens, delivered as Formula V peptide antigen conjugates with net charges greater than or equal to +6 and +8 (or less than or equal to -6 and -8), respectively, reliably accumulate in nanoparticle micelles. Importantly, these unexpected results suggest that peptide antigen conjugates must be able to accommodate 25 amino acid peptide antigens (A) with a charge range of about -6 to about +6 to ensure that sufficient net charge is applied to 98% of possible neoantigens with 25 amino acids; therefore, it is necessary to have at least 13 unique combinations of charged molecules (C) and optional extensions (B1 and/or B2) to ensure that sufficient charge is added (e.g., carrying 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 charged functional groups) to the peptide antigen (A) to achieve the required net charge of the peptide antigen conjugate.

To more closely examine the trend between the net charge of peptide antigen conjugates and the size and stability of particles formed by peptide antigen conjugates in aqueous buffers, we examined the interaction between the overall average hydropathy value (GRAVY) of the peptide sequence containing the peptide antigen conjugates (e.g., peptide antigen fragment , including the charged peptide-based molecule (C), peptide extensions (B1 and B2), and peptide antigen (A)), as well as the size and stability of the resulting particles (Fig. 19F). There was a strong relationship between the GRAVY value, the net charge and the particle size. Accordingly, many of the peptide antigen conjugates with a net charge of +4 and a global average hydropathy value (GRAVY) less than 0 formed stable particles (Fig. 19F). In contrast, about 25% of peptide antigen conjugates with a net charge of +4 and a GRAVY value greater than 0 formed aggregates, while few peptide antigen conjugates with a net charge of +6 and a GRAVY value greater than 0 formed aggregates. These data indicate that in preferred embodiments of the present invention, it is necessary to select the net charge intensity to compensate for the GRAVY value of the delivered peptide antigen (A). In preferred embodiments of the present invention, the composition of the charged molecule (C) and extensions (B1 and B2) is selected to provide peptide antigen conjugates whose net charge is greater than or equal to +4, or

- 118 046161 less than or equal to -4, to stimulate the formation of stable particles formed by peptide antigen conjugates, the total charge of which is greater than +6 or less than -6, which is used to ensure stable formation of nanoparticles even of the most hydrophobic peptide antigens (A) (Fig. .19F). Indeed, highly hydrophobic LP-based antigens may require an even higher absolute value of net charge. As such, peptide antigen conjugates must have a net charge that is: greater than or equal to +8, or less than or equal to -8, for LP peptide antigens (A) with a GRAVY value of less than 0.25; greater than or equal to +9, or less than or equal to -9, for LP peptide antigens (A) with a GRAVY value of approximately 0.25-0.75; and greater than or equal to +10, or less than or equal to -10, for LP peptide antigens (A) with a GRAVY value greater than 0.75, wherein LP is used to denote a peptide antigen (A) that is longer than the minimum epitope, for example a peptide antigen containing more than 15 amino acids, usually at least 20, for example 25 amino acids.

It should be noted that our analysis included peptide antigens with compositional extremes in terms of charge and hydropathy. Accordingly, the 3 unique peptide antigens presenting to the greatest extent, i.e., 90th percentile or higher, hydrophobic (Val-Val-Ile-Ala-IlePhe-Ile-Ile-Leu (SEQ ID NO: 57)), positively charged (Lys-Asn-His-Arg-Asn-Arg-Qln-Val-Ile SEQ ID NO: 75) and negatively charged (Ser-Pro-Glu-Arg-Asn-Asp-Trp-Glu-Pro-Leu SEQ ID NO : 76) sequences of 1377 predicted neoantigens taken from 4 mouse tumor cell lines, as well as a confirmed CD8 T cell epitope (Ala-Ser-Met-Thr-Asn-Met-Glu-Leu-Met-Ser-Ser (SEQ ID NO: 2)) were selected for evaluation. Peptide antigen (A) containing a confirmed CD4 T cell epitope (PADRE = Ala-Lys-Phe-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala-Ala SEQ ID NO: 22) was also included in the analysis as a known universal CD4 T cell epitope (Fig. 18). Effect of Hydrophobic Molecule (H) Length and Composition on Particle Size Given that the net charge of peptide antigen conjugates appears to be critical for promoting the stability of the formed particles, we next examined additional factors that influence particle size. Previous studies have shown that the length of macromolecules containing micelles, liposomes, and polymersomes can be modulated to change the size of the resulting particles. Thus, using the CD8 T cell epitope model as a peptide antigen (A) delivered as a Formula V peptide antigen conjugate, we assessed how the length and composition of the hydrophobic molecule (H) affects particle size (Figure 20). Consistent with previous data, we found that the size and stability of particles formed by peptide antigen conjugates were highly dependent on the net charge of the peptide antigen conjugate across a range of different hydrophobic molecules (H) used (Figure 20). When applying a net charge of +4 or greater, the smallest hydrophobic molecule (H), W ₅ , consisting of 5 tryptophan units, resulted in the smallest particles with an average diameter of ~10 nm, while the largest hydrophobic molecule (H) used in In this example, 2B2W8 resulted in particles with diameters ranging from 30 to 100 nm (Figure 20). Notably, the intermediate size hydrophobic molecules (H), 2B ₅ and 2BXy ₅ , resulted in the formation of intermediate size particles, with particle diameters of ~10-30 nm when the net charge was greater than or equal to +4.

Overall, these results led to unexpected discoveries establishing how the precisely defined chemical parameters of a charged molecule (C) and a hydrophobic molecule (H) can be used to regulate the size and stability of particles formed by peptide antigen conjugates.

Effect of net charge on particle size and stability of peptide antigen conjugates delivering highly hydrophobic peptide antigens (A)

To demonstrate the tolerability of Formula V peptide antigen conjugates designed to deliver peptide antigens (A) that are highly hydrophobic, we selected the highly hydrophobic neoantigen LP from a library of 1377 peptide neoantigens from 4 mouse tumor lines and synthesized the peptide antigen as either LP or Min delivered as a Formula V peptide antigen conjugate that self-assembles into TLR-7/8a co-delivering nanoparticles (SNP-7/8a) (FIG. 21). While the hydrophobic Min (GRAVY = 3.96) delivered as SNP-7/8a required a net charge equal to or greater than 8 to stabilize the hydrophobic LP (GRAVY = 2.0) delivered in form of SNP-7/8a, a total charge equal to or greater than 10 was required (Fig. 21). An additional unexpected finding was that native LP and min were not prepared by solid-phase peptide synthesis; however, the addition of a charged molecule (C) and extensions (B1 and B2) to the peptide antigen (A) on the resin during solid-phase peptide synthesis improved fabrication by preventing sequence truncation and allowing HPLC purification due to improved solubility in aqueous and organic solvents.

Effect of a composition of a charged peptide antigen molecule (C) and a hydrophobic molecule (H) on immunogenicity

Our above results indicate that the net charge is critical for

- 119 046161 stabilization of micelles with nanoparticles formed by peptide antigen conjugates. We next examined the effect of compositions of a charged molecule (C) and a hydrophobic molecule (H) on the immunogenicity of peptide antigen conjugates delivering the minimal epitope, Adpgk (Fig. 22-24). While the Formula V peptide antigen conjugate without adjuvant did not induce CD8 T cell responses (FIG. 22), all other TLR-7/8a-linked peptide antigen conjugate compositions induced robust CD8 T cell responses (FIG. 22). It is noteworthy that while the Formula IV peptide antigen conjugate does not have a charged molecule (C) and the Formula V peptide antigen conjugates with either lysine (i.e., poly(K)) or arginine (i.e., charged molecules ( C) based on poly(L)). induced robust CD8 T cell responses, even at doses as low as 0.25 nmol, of the same neoantigen administered as a Formula V peptide antigen conjugate with aspartic acid (i.e., a charged molecule (C) based on poly(B) ), did not induce CD8 T cell responses at 0.25 or 1 nmol doses, but induced high intensity CD8 T cell responses at 4 and 16 nmol doses. Notably, the negatively charged peptide antigen conjugate (FIG. 22) induced CD8 T cell responses of the highest intensity after multiple immunizations (~30% IFNg+ of total CD8 T cells), approximately 1.5-2 times CD8 T-cell responses. cellular responses induced by either Formula IV peptide antigen conjugates or positively charged Formula V peptide antigen conjugates. These results indicate that, while peptide antigen conjugates that self-assemble into microparticles (Fig. 22) or positively charged nanoparticles (Fig. 22) are more active in inducing CD8 T cell responses compared to negatively charged peptide antigen conjugates; negatively charged peptide antigen conjugates that self-assemble into TLR-7/8a co-delivering nanoparticles (SNP-7/8a) can induce CD8 T cell responses that are more capable of a secondary immune response, offering higher intensity responses after multiple immunizations.

To extend these findings, we assessed how the charged molecule (C) composition of peptide antigen conjugates affected CD8 T cell responses following the subcutaneous route of administration (FIG. 23). Notably, Formula V peptide antigen conjugates with either negatively or positively charged poly(amino acid), poly(K), or poly^ molecules, respectively, induced CD8 T cell responses of higher intensity. than the Formula V neutral extension B1 peptide antigen conjugates based on PEG (FIG. 23).

Importantly, Formula V peptide antigen conjugates, which self-assemble into nanoparticles co-delivering TLR-7/8a (TLR-7/8a), have been found to be highly immunogenic for eliciting CD8 T cell responses following common vaccination routes. (intramuscular (IM), subcutaneous (SC)), as well as intravenous routes of administration (Fig. 24).

Effect of Peptide Antigen Conjugate Particle Size and Adjuvant Activity and Composition on Immunogenicity

It was previously unknown how particle size and the amount and activity of TLR agonists influence T cell responses after a single or multiple (secondary) immunization. The current state of the art suggests that increasing the amount or activity of the immunostimulant will result in higher intensity, or at least will not reduce the intensity of CD8 T cell responses; however, the effects of immunostimulant, such as TLR agonist, amount and potency on the priming and secondary immunization of CD8 T cell responses in vivo have not been well studied. Therefore, in our next studies, we attempted to evaluate how particle size and the amount and potency of the agonist delivered on Formula V peptide antigen conjugates affect immunogenicity.

To determine how particle size and agonist composition affected immunogenicity, mice were vaccinated with immunogenic compositions containing two peptide antigen conjugates, one delivering a peptide antigen (A) containing a minimal CD8 T cell epitope, and the other delivering a peptide antigen (A) , containing a minimal CD4 T cell epitope (Fig. 25). Formula V peptide antigen conjugates have been differentiated by whether they form small particles (nanoparticles (NP) ~20-100 nm in diameter) or large particles (microparticles (MP) ~1000 nm in diameter) delivering TLR-7/8 agonists in various quantities and with different activities. In each set of small or large particles, the peptide antigen conjugates contained a hydrophobic molecule (H) that contained 1, 2, 3 or 5 molecules of Compound 1, 2B or 5 molecules of the higher activity agonist, Compound 2, 2BXy. Mice were vaccinated twice with this immunogenic composition, and T cell responses were measured in the blood 1 week after each vaccination.

A striking and unexpected finding was that for both small (20-100 nm) and large particles (~1000 nm), increasing the amount of 2B agonist coupled to a hydrophobic molecule (H) resulted in an increase in CD4 and CD8 T- cellular responses, while peptide antigenic conjugate

- 120 046161 you linked to hydrophobic molecules (H) contained only one 2B molecule, resulting in the lowest levels of CD8 and CD4 T cell responses. The level of both CD4 and CD8 T cell responses increased after the addition of two 2B molecules, and further increased modestly after the addition of 3 or 5 2B molecules to hydrophobic molecules (H). Consistent with our previous and unexpected results (Figs. 6 and 7) showing that peptide antigen conjugates containing hydrophobic molecules (H) delivering at least 3 molecules of the more active TLR-7/8a agonist, 2BXy, resulted in more low levels of CD8 T cell responses after multiple immunizations, compared to peptide antigen conjugates containing hydrophobic molecules (H) delivering at least 3 molecules of the less active 2B agonist, leading to the results shown in FIG. 25 show that 2BXy ₅ results in lower levels of CD8 and CD4 T cell responses compared to the use of a hydrophobic molecule (H) delivering the moderately potent agonist, 2B5. This unexpected finding—that an agonist with higher potency results in lower levels of immune responses—was not predicted based on previous findings in the literature.

Thus, increasing the amount of the agonist, Compound 1, 2B, to peptide antigen conjugates generally resulted in higher levels of CD8 T cell responses, but these responses were of lesser intensity when the natural stimulant was changed to an agonist with higher activity. Therefore, in preferred embodiments of the present invention, either high densities of moderately active TLR-7/8a agonists or low densities of highly active TLR-7/8a agonists are used to induce optimal CD4 and CD8 T cell responses using immunogenic compositions, containing peptide antigen conjugates.

Particle size

Another striking and unexpected finding was that immunogenic compositions containing peptide antigen conjugates that formed small particles, particles with a diameter of ~10-200 nm, resulted in higher intensity CD4 and CD8 T cell responses compared to peptide antigen conjugates , which formed larger particles (>1000 nm in diameter). For example, in the group that received immunogenic compositions containing peptide antigen conjugates linked to the hydrophobic molecule (H)2B5 and which formed small particles, CD8 T cell responses were approximately ~2.5%, compared to ~0.5 % in the group that received immunogenic compositions containing peptide antigen conjugates linked to a hydrophobic molecule (H) 2B5, which formed large particles (Fig. 25). Similarly, the level of CD8 T cell responses in the group that received immunogenic compositions containing peptide antigen conjugates linked to the hydrophobic molecule (H) 2B ₃ W2 in the form of a small particle was approximately 10 times higher than in the group that received immunogenic compositions containing peptide antigen conjugates linked to a hydrophobic molecule (H) 2B3W2 in the form of a large particle.

Thus, these data indicate that the particle size formed by the peptide antigen conjugates used in immunogenic compositions had a large and unexpected impact on the CD8 T cell response. Therefore, peptide antigen conjugates that form particles with a size of 20-100 nm are a preferred embodiment of the present invention for use in immunogenic compositions to generate optimal T-cell immunity.

Overall, these studies elucidate the precise physical and chemical parameters that are optimal for maximizing CD4 and CD8 T cell responses using the peptide antigen conjugates of the present invention.

An additional unexpected finding was that the hydrodynamic behavior and stability of the particles formed by peptide antigen conjugates depended on both the net charge and the overall average hydropathy value (GRAVY), as determined using the methods of Kite and Doolittle (see: Kight J, Doolittle RF, A simple method for displaying the hydropathic character of a protein, Biol, 157: 105-32, 1983), a peptide sequence containing a peptide antigen conjugate. Note: The determination of the GRAVY value and charge excludes contributions from the Linker (L) and the hydrophobic molecule (H) or particle (P).

Accordingly, peptide antigen conjugates delivering highly hydrophobic peptide antigens (A) with GRAVY values >0.75 reliably formed stable particles in aqueous buffers at pH approximately 7.4 when the net charge of the peptide antigen conjugate was > (greater than or equal to ) up to +6 or < (less than or equal to) up to -6; Moderately hydrophobic peptide antigens (A) with GRAVY values of 0.25-0.75 reliably formed stable particles in aqueous buffers at pH approximately 7.4 when the net charge of the peptide antigen conjugate was > (greater than or equal to) +5 or < (less than or equal to) -5; and peptide antigens (A) with GRAVY values less than 0.25 reliably formed stable particles in aqueous buffers at pH approximately 7.4 when the net charge was > (greater than or equal to) +4 or < (less than or equal to) -4 . Thus, based on our unexpected results, it is possible to select the appropriate composition of charged molecules (C) and extensions along

- 121 046161 sequences (B1 and B2) for charge modulation as a mechanism to stimulate the formation of stable particles of peptide antigen conjugates delivering any composition of peptide antigen (A).

Influence of hydrodynamic behavior of peptide neoantigen on immunogenicity

We then assessed whether more uniform formulations using Formula V peptide antigen conjugates that self-assemble into TLR7/8a co-delivering nanoparticles (SNP-7/8a) (Figure 26) would result in improved immunogenicity of CTL responses compared to coupling of LP neoantigens to TLR-7/8 hydrophobic polymer without charge stabilization, resulting in the formation of microparticles/aggregates (MP-7/8a), or with the more standard approach of mixing LP neoantigens with a particle adjuvant (LP + poly-ICLC ) (Fig. 27). We selected three LP neoantigens for evaluation: Adpgk, Cpne1, and Irgq. While Adpgk aggregates into particles as native LP, both Cpne1 LP and Irgq LP are water soluble (Fig. 27 A).

Consistent with our earlier data, only neoantigen LPs delivered as particulates induced CD8 T cell responses in vivo (Fig. 27 B and C). Accordingly, soluble Cpne1 and Irgq LPs mixed with pICLC did not induce CD8 T cell responses, while the same neoantigens delivered as either MP-7/8a (microparticle/aggregates) or SNP-7/8a ( nanoparticle) resulted in the generation of high intensity CD8 T-cell immunity (Fig. 27 B-E). Although soluble LPs mixed with pICLC were inactive as described previously, the particle LP, Adpgk, induced a significant increase in CD8 T cell responses compared to background, indicating that the physical form of the peptide neoantigen, but not the adjuvant, is, pICLC likely accounted for weak T cell responses from Cpne1 and Irgq LPs mixed with pICLC.

Among formulations delivering neoantigens in particulate format, nanoparticle micelle (SNP-7/8a) based on Formula V peptide antigen conjugates offered responses with the highest intensity, followed by microparticle formulations (MP-7/8a) and in last place only LP mixed with adjuvant (Fig. 27 B-E). Notably, the rate of responses from nanoparticle micelles (SNP7/8a) after a single immunization was significantly (~5-10 times) higher than the rate of responses from microparticle formulations, while responses after two or three immunizations were comparable, which indicates that the T cell induction kinetics of smaller particles, nanoparticle micelles, are faster compared to the T cell induction kinetics of microparticles.

Mechanistic studies to account for the observed differences in immunogenicity showed that peptide antigen conjugates, which form stable micelles with nanoparticles, resulted in the uptake of the greatest amount of neoantigens into the draining lymph nodes with a peak before the main component in concentrations and uptake into the lymph node APCs on day 1, at while the microparticle showed slower uptake into the lymph nodes, with concentrations peaking at day 7, which may indicate slower kinetics of CD8 T cell responses resulting from the microparticles (Figure 28). In contrast, soluble neoantigen mixed with either a soluble adjuvant or a particle adjuvant showed limited neoantigen uptake into the draining lymph nodes. Although all adjuvant particle formulations induced comparable intensity and kinetics of natural immune activation in lymph nodes, only the antigen particle formulations resulted in CD8 T cell responses. Limited accumulation of soluble neoantigen in lymph nodes likely explains its failure to induce CD8 T cell responses.

Effect of peptide length on immunogenicity

Peptide length is considered to be the main factor that influences the immunogenicity of peptide antigen (A). We therefore examined how the length of peptide neoantigens delivered as Formula V peptide antigen conjugates that self-assemble into TLR-7/8a co-delivering nanoparticles (TLR-7/8a) influences the intensity of CD4 and CD8 T cell responses. , generated in vivo (Fig. 29). Seven peptide neoantigens derived from the MC38 tumor cell line were formulated as either minimal epitope (min) or LP-based peptide antigens (A) delivered as Formula V peptide antigen conjugates, herein referred to as SNP-7/ 8a and then administered to mice. Our results show that both min and LP forms of neoantigens delivered as SNP-7/8a generate comparable intensities of CD8 T cell responses that are approximately 10- to 100-fold above background, including against 4 neoantigens , which were previously reported to be non-immunogenic. (Fig. 29A). However, as expected, LPs elicited significantly higher levels of CD4 T cell responses compared to min, likely because min were too short to encode a CD4 epitope (FIG. 29B). Importantly, these results indicate that peptide length has minimal impact on CD8 T cell responses and indicate that short peptides (7-14 amino acids) containing minimal CD4 or CD8 epitopes are more effective for manufactured when properly formulated, may be preferable for vaccine use over LP (>20 amino acids). In this regard, it can be noted that improvements in MHC-II prediction algorithms should make it possible to use minimal epitopes to generate CD4 and CD8 T-cell responses, thereby avoiding the need for LP.

Comparative analysis of the immunogenicity of peptide neoantigens delivered as peptide antigen conjugates compared with standard approaches

We then comparatively analyzed the activity of our Formula V peptide antigen conjugates, which self-assemble into TLR-7/8a co-delivering nanoparticles (SNP7/8a), with the standard peptide vaccine approach of mixing native LPs with a particle adjuvant poly-ICLC (Fig. 30). According to Yadav M, et al. Nature 515(7528):572-576 (2014), only 2 of 7 mass spectrometrically confirmed predicted neoantigens (Reps1 and Adpgk, both of which are particles) were immunogenic when combined with pICLC, however, all 7 LP neoantigens generated high intensity CD8 T cell responses as SNP-7/8a, that is, as peptide antigens (A) delivered as Formula V peptide antigen conjugates (FIG. 30). Notably, all 7 neoantigens shown in FIG. 30 bind tumor MHC-I through mass spectrometry, which offers the surprising discovery that the mass spectrum is a reliable filter for predicting the immunogenicity of peptide antigens (A), such as neoantigens.

Relationship between predicted minimal epitope binding affinity and immunogenicity

Our results indicate that peptide-based neoantigens delivered as Formula V peptide antigen conjugates that self-assemble into TLR-7/8a co-delivering nanoparticles (SNP-7/8a) are a highly effective approach for generating CD4 and CD8 T cell responses. Due to the increased efficacy of the vaccine platform, algorithms for predicting immunogenicity can be improved. Indeed, we observed a strong correlation between predicted MHC-I binding and immunogenicity after screening 192 unique CD8 T cell epitopes in vivo with SNP-7/8a, with nearly 50% of the epitopes having high affinity (IEDB Consensus Score < 0.5 percentile) lead to CD8 T cell responses, which increases the efficiency of priming CD8 T cell responses by almost 5 times compared to published responses (Fig. 31). These results indicate that in silico predicted binding affinity, particularly the IEDB consensus score, is highly predictive of the immunogenicity of peptide antigen (A).

Peptide antigen conjugates induce robust, tumor-associated (self-antigen, neoantigen, and viral antigen)-specific CD8 and CD4 T cell responses that mediate tumor clearance in vivo

We investigated whether CD8 T cell responses to non-immunogenic epitopes, as previously reported, could lead to improved clearance of established tumors (Figs. 32-33). Our results show that several neoantigens from two tumor models, MC38 and B16.F10, previously reported to be non-immunogenic, resulted in clearance of established tumors when delivered as Formula V peptide antigen conjugates that self-assemble into TLR-delivering nanoparticles. 7/8a (SNP-7/8a). Notably, although Kreiter et al previously reported that neoantigen-specific CD4 T cell responses predominantly mediated clearance of established B16.F10 tumors, we observed that increased vaccine efficacy could mobilize both neoantigen-specific CD4 and CD8 T cell responses to mediate clearance of B16.F10 (Figs. 32 and 34). It is important to note that these results go beyond the use of neoantigens, since both viral antigens (HPV E6 and E7, Fig. 35) and self-antigens (Trp1, Fig. 32), delivered as 8N-7/8a, produced High intensity CD8 T cell responses that were associated with improved tumor clearance.

Particles containing several different peptide antigen conjugates are stable and exhibit uniform particle size.

PCV approaches will likely require administration of several different epitopes to the patient to increase the breadth of T cell responses. We therefore assessed the tolerance of Formula V peptide antigen conjugates that self-assemble into TLR7/8a co-delivering nanoparticles (SNP-7/8a) for use in multi-antigen vaccines based on a particle containing several different peptide antigen conjugates (Figure 36). The particle containing several different peptide antigen conjugates offered a consistent particle size of 20-40 nm, regardless of neoantigen LP composition (Figure 36).

Formula V peptide antigen conjugates delivering tumor-associated self-antigens or infectious disease antigens

To extend our findings described above, we assessed the generalizability of using peptide antigen conjugates as a universal vaccine platform for inducing T-cell immunity. As shown in FIG. 37, Formula V peptide antigen conjugates delivering peptide antigens (A) containing an oncogene (Kras), a virus-derived peptide (HIV), and a foreign antigen (i.e., ovalbumin) all formed stable nanoparticles and induced T cell responses with high intensity in vivo.

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Particle size and biological activity of peptide antigen conjugates of Formula V, consisting of various charged molecules (C) or various hydrophobic molecules (H) based on poly(amino acids) of Formula II, coupled with adjuvants of Formula III

As demonstrated above, peptide antigen conjugate vaccines are a modular platform that can offer a wide range of different compositions of each of the components, for example, charged molecules (C), extensions (B1 and B2), peptide antigens (A), Linkers ( L) and hydrophobic molecules (H). In some embodiments of the present invention, it may be necessary to include a charged molecule (C) that contains functional groups that are independent of pH over most physiologically relevant pH ranges, for example, from pH 4.5 to about 8.5. Thus, we synthesized peptide antigen conjugates with either a net negative or a net positive charge using charged molecules (C) that contain the amino acids phosphoserine or trimethyllysine (pH-independent quaternary ammonium), respectively (Fig. 38). Our results show that peptide antigen conjugates, including charged molecules (C) based on phosphoserine or trimethyllysine, form stable nanoparticles and induce T cell responses with high intensity, whereas peptide antigen conjugates without a charged molecule (C) form aggregates.

To extend these results, we produced peptide antigen conjugates of varying lengths, 30 amino acids (Compound 20, referred to as 2B ₅ W ₁₀ G ₁₀ ) and 15 amino acids (Compound 16, referred to as 2B5G10) DBCO-bearing hydrophobic molecules (H) based on poly( amino acids) of Formula II, coupled with an adjuvant of Formula III. An unexpected finding was that increasing the content of amine and aromatic groups in the hydrophobic blocks resulted in improved solubility in the organic solvent and therefore improved fabrication, compared to hydrophobic blocks with fewer aromatic groups or amines. Accordingly, despite the longer variation, 2B ₅ W ₁₀ G ₁₀ and its peptide precursors were more efficient in production than 2B ₅ G ₁₀ and its precursors.

Two different hydrophobic molecules (H), 2B5W ₁₀ G ₁₀ and 2B5G10, were linked to peptide antigen fragments containing neoantigens based on long peptides as peptide antigen (A), via a triazole Linker (L), to form peptide antigen conjugates of Formula V Consistent with our previous results, the formation of stable nanoparticles depended on the net charge of peptide antigen conjugates containing different hydrophobic molecules (H) (Fig. 38). Surprisingly, it was found that despite the high density of hydrophobic groups on the hydrophobic molecules (H), both peptide antigen conjugates formed stable micelles with the nanoparticles and induced T cell responses with high intensity in vivo (Fig. 38). In some embodiments of the present invention, a longer hydrophobic molecule (H) is used to improve the kinetic stability of peptide antigen conjugates, which may be required for vaccines delivered by the intravenous route.

Particle size and biological activity of Formula V peptide antigen conjugates consisting of various hydrophobic molecules (H) based on Formula II poly(amino acids) linked to various PRR agonists

Although the above examples of immunogenic compositions predominantly used peptide antigen conjugates consisting of hydrophobic molecules (H) coupled to TLR-7/8a (e.g., Formula III adjuvants) or not coupled to a Ligand, hydrophobic molecules (H) can be coupled to any ligand. In some, a hydrophobic molecule (H), while in other embodiments of the present invention, a Ligand that does not have adjuvant properties is included in the hydrophobic molecule. To demonstrate the generalizability of peptide antigen conjugates delivering various ligands with adjuvant properties, we synthesized Formula V peptide antigen conjugates consisting of hydrophobic Formula II molecules (H) coupled to various ligands, including a TLR-4 agonist (i.e., WW(PI) WW), TLR-2/6 agonist (i.e., WW(PI)WW), TLR-7 agonist (i.e., CL264-W5), NOD agonist (Muramil-\U5), and STING agonist (i.e., DMXAA- W5). Consistent with our previous results, the formation of stable nanoparticles depended on the net charge of peptide antigen conjugates containing various hydrophobic molecules (Fig. 39), with all different immunogenic compositions based on hydrophobic molecules (H) containing various ligands with adjuvant properties inducing measurable T-cell immunity. Notably, the TLR-7 agonist induced responses with the highest intensity, and therefore, TLR-7 or TLR-7/8 agonists are used in preferred embodiments of the peptide antigen conjugates of the present invention to induce T cell responses for either prophylaxis or treatment of infectious diseases or cancer.

Attachment of a peptide antigen (A) to a hydrophobic molecule (H) using various linker chemicals (amide and thioester)

Because in preferred embodiments of the peptide antigen conjugates

- 124 046161 The present invention uses click chemistry to form a linker (L) from a linker precursor X1 and a linker precursor X2, and other types of linker chemistries are also suitable. Thus, Formula V peptide antigen conjugates were linked to hydrophobic molecules (H) using either amide or thioester Linkers (L), with the linker (L) linked through either the N- or C-terminus of the peptide antigen. (A). Our results indicate that peptide antigens (A) linked via either the N- or C-terminal extension of the peptide antigen (via either B1 or B2) with a linker (L) containing either an amide or a thioester resulted in peptide antigen conjugates , which formed stable nanoparticles and induced T cell responses with high intensity in vivo (Fig. 40), although peptide antigens (A) linked via an amide linker (L) to a hydrophobic molecule (B) induced responses with higher intensity, compared to peptide antigens linked using a thioether linker (L). Thus, in preferred embodiments of the present invention, stable amide or triazole linkers (L) are used to link peptide antigens (A) to hydrophobic molecules (H), either directly or through extensions (B1 or B2).

Particle size and biological activity of Formula V peptide antigen conjugates consisting of various hydrophobic molecules (H) based on fatty acids, lipids and cholesterol

Since preferred embodiments of the peptide antigen conjugates of the present invention employ hydrophobic molecules (H) composed of polymers, such as poly(amino acids) or A-B diblock copolymers, the hydrophobic molecules (H) can be based on a variety of other hydrophobic molecules such as fatty acids, lipids and cholesterol. Thus, peptide antigenic conjugates of Formula V were linked to hydrophobic molecules (H) based on myristic acid (Myr or C14, Compounds 49 and 52), palmitic acid (Palm or C16, Compounds 51 and 53), cholesterol (Chol, Compounds 57 and 58) and diacyl lipid (Pam2Cys, Compounds 54 and 55). Consistent with our previous results, stable nanoparticle formation was dependent on the net charge of peptide antigen conjugates containing various hydrophobic molecules (Figure 41), with a net charge greater than or equal to +6, resulting in the formation of stable nanoparticles. Importantly, immunogenic compositions of fatty acid, cholesterol, and lipid-based hydrophobic molecules (H) peptide antigen conjugates induced T cell responses with high intensity in vivo (FIG. 41). Although fatty acid, cholesterol, and lipid-based particles exhibited suitable characteristics in terms of particle formation and biological activity as peptide antigen conjugates, one limitation is that fatty acid, cholesterol, and lipid-based hydrophobic molecules (H) are more difficult to prepare from -due to the limited solubility of these materials in many solvents (e.g., poor solubility in DMSO, DMF and many alcohols), which requires biologically harsher chlorinated solvents (e.g., dichloromethane and chloroform), making characterization and purification difficult, especially with regard to strategies individualized vaccines, and increases safety risks compared, for example, with hydrophobic molecules (H), consisting of polymers containing aromatic groups that are soluble in most organic solvents (for example, DMSO, methanol, ethanol, acetonitrile, etc.).

Particle size and biological activity of peptide antigen conjugates of Formula V, consisting of various hydrophobic molecules (H) based on diblock copolymers of type A-B

Peptide antigen conjugates of Formula IV and Formula V were prepared by attaching peptide antigens (A) to HPMA-based diblock copolymers (see Compounds 68-72). Surprisingly, the micelle-forming diblock copolymers formed highly stable nanoparticle micelles regardless of the net charge of the peptide antigen fragment (Figure 42). Thus, in some embodiments of the present invention, wherein the hydrophobic molecule (H) is a diblock copolymer consisting of HPMA monomers, Formula IV peptide antigen conjugates may be preferred. Diblock may form particles under aqueous conditions regardless of temperature, or particle formation may be temperature dependent. In addition, the ligand can be included in either of the two blocks, either at the ends or in the side groups of the diblock copolymer. Our results show that an A-B diblock polymer consisting of HPMA monomers provides stable nanoparticle formation and leads to high-intensity T cell responses when included as a hydrophobic molecule (H) in immunogenic compositions containing peptide antigen conjugates (Fig. 42).

Particle size and stability of Formula V peptide antigen conjugates based on peptide antigens (A) coupled to particles

In preferred embodiments of the present invention, the peptide antigen conjugate contains a hydrophobic molecule (H); however, in some embodiments of the present invention, the peptide antigen conjugate contains a preformed particle (P). Dependence of the charge of a peptide antigen fragment on the stability of peptide-based particles

- 125 046161 antigen conjugates of Formula V containing particle (P) were evaluated by attaching peptide antigen fragments (C-B1-A-B2-) with different net charges to preformed particles (P) based on liposomes, polystyrene, silicon particles and iron oxide. Consistent with our previous results, the formation of stable nanoparticles depended on the net charge of peptide antigen conjugates containing different hydrophobic molecules (Fig. 43).

Particle size and biological activity of peptide antigen conjugates delivering autoantigens used to induce tolerance

Peptide antigen conjugates can also be used to induce tolerance or suppressive regulatory T cells. In preferred embodiments of the present invention, a ligand with adjuvant properties is generally not included to induce tolerance or immunosuppression. Instead, a peptide antigen conjugate must contain a particle containing a peptide antigen (A), a hydrophobic molecule (H) or particle (P) and an optional linker (L), a charged molecule (C) and extensions (B1 and/or B2), however, a ligand with adjuvant properties is not included. To evaluate the generalizability of the peptide antigen conjugates described herein for use in tolerance induction or suppression, models of autoantigens for inducing arthritis (i.e., Collagen II) and a CNS syndrome that resembles MS (i.e., myelin oligodendrocyte glycoprotein, MOG), as peptide antigens (A) on Formula V peptide antigen conjugates. Consistent with our earlier results, autoantigens delivered on Formula V peptide antigen conjugates aggregated into stable nanoparticles and induced low levels of regulatory T cell responses (Fig. 44).

Particle size and biological activity of particles consisting of A-H + C-H or A-H(C) (Formula VI)

In some embodiments of the present invention, the immunogenic composition contains a particle consisting of peptide antigen conjugates without charged molecules (for example, A[B2]-[L]-H) mixed with peptide antigen conjugates (for example, C-[A']-[ L]-H]). For example, a peptide antigen conjugate consisting of a neoantigen LP (Adpgk) was linked via a triazole linker (L) to a hydrophobic molecule 2B ₃ W2 to form a peptide antigen conjugate (ALH), which was mixed with a charged molecule (C-H) conjugate,

Compound 42 (K10-2B3W2) to form stable nanoparticles that induced T cell immunity with high intensity (Fig. 45). In another embodiment of the present invention, a peptide antigen conjugate consisting of a neoantigen LP (Adpgk) was linked via a triazole linker (L) to a hydrophobic molecule 2B3W2 to form a peptide antigen conjugate (A-L-H), which was mixed with a charged molecule conjugate further comprising conserved antigen A' (ie, C-A'-H), to form stable nanoparticles that induced T-cell immunity with high intensity (Fig. 45).

In an alternative embodiment of the present invention, a charged molecule (C) can be coupled to a hydrophobic molecule (H) that is coupled to a peptide antigen (A) to form a peptide antigen conjugate of Formula VI. In a non-limiting example, a peptide neoantigen (Adpgk) is delivered as a peptide antigen (A) on a peptide antigen conjugate of the formula A-[L]-H(C), and the resulting conjugate has been found to produce stable nanoparticles and high T-cell immunity intensity. Thus, in preferred embodiments of particulate immunogenic compositions, a charged molecule (C) may be provided to be linked, either directly or indirectly, to a peptide antigen (A), or it may be provided on a separate molecule that associates with particles formed by peptide antigen conjugates.

Importantly, the present invention describes new approaches to peptide-based vaccines, called peptide antigen conjugates, which lead to unexpected improvements in the intensity and breadth of T cells generated in response to peptide antigens (A), including tumor-associated antigens (self-antigens). , neoantigens and viral antigens), infectious disease antigens and foreign antigens. It has also been found that immunogenic compositions containing peptide antigen conjugates, which exclude adjuvants, induce low levels of tolerogenic/suppressive responses to autoantigens, which may be useful for the treatment of autoimmune diseases and/or allergic reactions.

In the present invention, we demonstrate the unexpected result that peptide antigens (A) containing a minimal epitope (ME) can lead to CD8 T cell responses with higher intensity than the same ME delivered as a synthetic long peptide (SLP), when they are both normalized pharmacokinetics by delivering them both in the form of nanoparticles consisting of peptide antigen conjugates. In addition, we provide examples where ME delivered to SLPs as a peptide antigen conjugate does not result in CD8 T cell responses after a single immunization, but ME delivered as a peptide antigen conjugate does, presumably due to more efficient processing , which results in increased presentation of cell surface MEs in the context of MHC I molecules via APC. Was

- 126 046161 found that such ME-based vaccines expand the breadth of T cell responses, which have been further shown to be functional in the context of reducing the growth of established tumors. Therefore, based on our unexpected results, preferred embodiments of the immunogenic compositions of the present invention employ peptide antigen conjugates delivering peptide antigens (A) based on minimal epitopes, or a combination of ME with SLP. Although in some embodiments of the present invention, the peptide antigen (A) is an SLP (or LP).

In addition, to provide improved control over the loading of materials and the size and stability of particles delivering peptide antigens (A) with a number of properties, we disclose herein a new approach to delivering peptide antigens (A) in the form of peptide antigen conjugates that accumulate in micellar and nano-sized supramolecular associates in aqueous buffers. Importantly, we show how the composition of peptide antigen conjugates, including an optional particle-stabilizing charged molecule (C), a hydrophobic molecule (H), an optional linker, and optional sequence extensions (B1 and B2), can be optimized to achieve stable particles specific sizes for any possible peptide antigen (A). We show that robust particle formation and improved stability can be achieved by adjusting the length and hydrophobic characteristics of the hydrophobic molecule (H), as well as by modulating the charge of the particles formed by peptide antigen conjugates.

As a mechanism to improve the processing of T cell epitopes containing a peptide antigen conjugate, we demonstrate the utility of using N- and C-terminal extensions (B1 and B2 or extensions) that are stable under aqueous conditions but, in a preferred embodiment of the present invention, are hydrolyzed by enzymes in the endosomes of antigen-presenting cells, thereby limiting the processing and presentation of antigen to such cells and stimulating the effective release of the minimal CD8 and/or CD4 T-cell epitope in the APC.

In conclusion, in the present discovery, we systematically evaluated the optimal composition, amount, and potency of PRR agonist-based immunostimulants required to stimulate T-cell immunity. We present evidence suggesting that progressive increases in the number and activity of TLR agonists that induce the production of IL-12 and type I IFN initially lead to increased intensity of T-cell responses, but in the context of a new and unexpected finding that that this results in an additional increase in the narrower breadth of the antigenic spectrum and lower intensity of T-cell responses. We therefore report the composition, amount and activity of immunostimulants (i.e., PRR agonists) attached to a hydrophobic molecule (H) that are preferred to offer optimal intensity, quality and breadth of T cell immunity.

In summary, the unexpected findings 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 sequence extensions, i.e. N- and/or C-terminal extensions (B1 and/or B2), which facilitate the manufacture of peptide-based vaccines, promote particle stability and facilitate processing to allow efficient presentation by ARS;

iii) the composition of charged molecules (C) and the net charge of peptide antigen conjugates necessary to induce and stabilize particles of optimal size to stimulate T-cell immunity;

iv) how hydrophobic molecule length (H) and composition affect the size and stability of particles formed by peptide antigen conjugates, as well as their immunogenicity in vivo:

v) how the activity and quality characteristics of the ligand (eg, PRR agonist) incorporated into hydrophobic molecules (H) influence the breadth, intensity and quality of T cell responses.

Throughout the specification of the present invention and in the claims set forth below, unless the context otherwise requires, the words contains and includes, and such variations as containing and including, are to be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other an integer or group of integers; or the inclusion of a specified composition or compositions, but not the exclusion of any other composition or compositions.

Reference to any prior art in this specification does not constitute, and should not be construed as, an endorsement of any form of suggestion that such prior art is part of the prior art.

Those skilled in the art will appreciate that the use of this ISO

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Claims (41)

shaving is not limited to the specific application described. The present invention is also not limited to its preferred embodiment in the context of the specific elements and/or features described or depicted herein. It should be understood that the present invention is not limited to the disclosed embodiment or embodiments, but is susceptible of numerous reconfigurations, modifications and substitutions without departing from the scope of the present invention as set forth and defined in the following claims. CLAIM 1. A peptide antigenic conjugate having the formula C-[B1]-A-[B2]-[L]-H or H-[L]-[B1]-A[B2]-C, wherein A is a peptide antigen, H is a hydrophobic molecule that is insoluble in water up to a concentration of 0.1 mg/ml at physiological pH 7.4, and molecule C is a charged molecule containing one or more functional groups at physiological pH , B1 is an N-terminal extension, B2 is a C-terminal extension, L is a linker, where [ ] denotes that the group is optional, - denotes a covalent bond; wherein the peptide antigen is a tumor-associated antigen, an infectious disease antigen, an allergen, or a self-antigen that contains a sequence of 5 to 50 amino acids in length; and wherein the peptide antigen conjugate has a net electrostatic charge that is greater than or equal to +6 or less than or equal to -6, in an aqueous buffer solution at pH 7.4. 2. The peptide antigen conjugate according to claim 1, characterized in that the B1 extension is present and contains an enzyme-degradable peptide sequence containing the amino acid PN1, characterized in that PN1 is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine and methionine . 3. The peptide antigen conjugate according to claim 2, characterized in that the enzyme-degradable peptide sequence containing the PN1 amino acid further contains at least one of the following: a) a PN2 amino acid which is selected from glycine, valine, leucine and isoleucine; b) amino acid PN3, which is selected from glycine, serine, proline and leucine; c) a PN4 amino acid which is selected from arginine, lysine, glutamic acid, aspartic acid, glycine and serine or d) any of the combinations a-c. 4. Peptide antigen conjugate either according to claim 2 or claim 3, characterized in that the enzyme-degradable peptide sequence contains a tetrapeptide selected from: 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 and n) Glu-Gly-Val-Cit, wherein Glu can be replaced by Asp and/or where Arg can be replaced by Lys. 5. Peptide antigen conjugate according to any one of claims 1 to 4, characterized in that the B2 extension is present and contains an enzyme-degradable peptide sequence containing: i) a single amino acid PC1' selected from glycine, serine, arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine and methionine; ii) a dipeptide PC1'-PC2', wherein PC1' is selected from glycine and serine and PC2' is selected from glycine, serine, proline, arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine and methionine; iii) tripeptide PC1'-PC2'-PC3', wherein PC1' is selected from glycine and serine; and PC2' is selected from glycine, serine and proline, and PC3' is selected from glycine, serine, arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine and methionine; iv) tetrapeptide PC1'-PC2'-PC3'-PC4', wherein PC1' is selected from glycine and serine; PC2' selected from - 128 046161 glycine, serine, proline and leucine; PC3' is selected from glycine, valine, leucine and isoleucine; and PC4' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine and methionine; v) pentapeptide PC1'-PC2'-PC3'-PC4'-PC5', wherein PC1' is selected from glycine and serine; PC2' is selected from glycine, serine, proline, arginine, lysine, glutamic acid and aspartic acid; PC3' is selected from glycine, serine, proline and leucine; PC4' is selected from glycine, valine, leucine and isoleucine; and PC5' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine and methionine; or vi) hexapeptide PC1'-PC2'-PC3'-PC4'-PC5'-PC6', wherein PC1' is selected from glycine and serine; PC2' is selected from glycine, serine and proline; PC3' is selected from glycine, serine, proline, arginine, lysine, glutamic acid and aspartic acid; PC4' is selected from proline and leucine; PC5' is selected from glycine, valine, leucine and isoleucine and PC6' is selected from arginine, lysine, citrulline, glutamine, threonine, leucine, norleucine and methionine. 6. Peptide antigen conjugate according to claim 5, characterized in that the B2 extension contains an enzyme-degradable peptide sequence containing: a) a hexapeptide selected from: 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 and wherein Glu may be replaced by Asp and/or wherein Arg may be replaced by Lys; or b) a pentapeptide selected from: 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) and wherein Glu may be replaced by Asp and/or wherein Arg may be replaced by Lys; or c) a tetrapeptide selected from: 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 and xii) Glu-Gly-Val-Cit and wherein Glu may be replaced by Asp and/or wherein Arg may be replaced by Lys; or d) a tripeptide selected from: 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; or e) a dipeptide selected from: i) Gly-Ser; ii) Gly-Pro; iii) Gly-Arg; - 129 046161 iv) Gly-Cit; or f) a single amino acid selected from the group consisting of Gly, Ser, Ala, Arg, Lys, Cit, Val, Leu, Met, Thr, Gln and Nle. 7. Peptide antigen conjugate according to any one of claims 1 to 6, characterized in that the hydrophobic molecule is a branched, long-chain or cyclic aliphatic molecule, or a derivative of a fatty acid, lipid or sterol. 8. Peptide antigenic conjugate according to any one of claims 1 to 6, characterized in that the hydrophobic molecule contains a polymer based on monomer units selected from acrylates, acrylamides, meth(acrylates), meth(acrylamides), styryl monomers, vinyl monomers, dioxophospholanes , cyclic esters, natural and non-natural amino acids and polymers resulting from the reactions of diols or diamines with di- or polyisocyanates, optionally, wherein the adjuvant is linked to at least one monomer containing the polymer. 9. Peptide antigenic conjugate according to claim 8, characterized in that the hydrophobic molecule is a poly(amino acid) of Formula I Formula I wherein R ² is selected from one of the following: hydrogen, hydroxyl and amine, k is an integer from 3 to 30; y3 is an integer from 1 to 8 and X is a linker that attaches a ligand to a peptide antigen conjugate, and the ligand is an agonist of Toll-like receptor (TLR)-7, TLR-8, or both TLR-7 and -8 (TLR -7/8). 10. Peptide antigen conjugate according to claim 8, characterized in that the hydrophobic molecule is a poly(amino acid) of Formula II Formula II in which l is an integer 3-30, and o is an integer 0-30; R ³ is selected from one of the following: hydrogen, hydroxyl and amine; R ⁴ is selected from one of the following: hydrogen, lower alkyl y11 is selected from an integer from 1 to 8, and FG represents a functional group selected from carboxylic acid, aldehyde, ketone, amine, thiol, azide and alkyne, which is linked to a ligand with adjuvant properties; and the N-terminus is linked either directly, through an amide bond, or indirectly, through a linker, either directly to the peptide antigen, or through a B1 extension of the linker and/or a charged molecule, where the ligand is a Toll-like receptor (TLR)-7 agonist, TLR-8 or both TLR-7 and -8 (TLR-7/8). 11. Peptide antigen conjugate according to claim 8, characterized in that the hydrophobic molecule (H) is a poly(amino acid) of Formula IIa - 130 046161 o o o o I igand Formula IIa where the sum of l, m, n and o is an integer from 3 to 30; R ³ represents hydrogen, hydroxyl or amino; each R ⁴ is independently selected from and their combinations; each R ⁵ independently represents -H, -CH ₃ or -(CH2) _b OH; each R ⁶ independently represents -(CH2 ₎ dNH2, -(CH2) _dCOOH , -(CH2) _dN (=NH)NH2, -(CH2)dS ⁺ (R)2, -(CH2)dN ⁺ ( R)3, -(CH2)dSO2OH or -(CH2)dOP(O)(OH)2; each a is independently an integer from 1 to 6; b is an integer from 1 to 6; each d is independently an integer from 1 to 6; each R independently represents a lower alkyl; the ligand is an agonist of Toll-like receptor (TLR)-7, TLR-8 or both TLR-7 and -8 (TLR-7/8) and each X is independently a linker; wherein 3 or more amino acids of the poly(amino acid) contain an aromatic group. 12. Peptide antigenic conjugate according to claim 8, characterized in that the hydrophobic molecule contains a homopolymer or copolymer consisting of poly(leucine), poly(tryptophan), poly(γ-glutamyl diethylene glycol monomethyl ether), poly(γ-benzyl glutamate), poly (^isopropylacrylamide) (NIPAM), poly[2(2-methoxyethoxy)ethyl methacrylate)] (DEGMA) or poly[I-(2-hydroxypropyl)methacrylamide) (HPMA). 13. Peptide antigenic conjugate according to claim 9, characterized in that the hydrophobic molecule, with - 131 046161 containing poly(amino acids) of Formula I, grafted or linked at the end with a second polymer based on poly(leucine), poly(tryptophan), poly(N-glutamyl diethylene glycol monomethyl ether), poly(N-benzyl glutamate), poly(N-isopropylacrylamide) ) (NIPAM), poly[2(2-methoxyethoxy)ethyl methacrylate)] (DEGMA) or poly[N-(2-hydroxypropyl)methacrylamide) (HPMA). 14. Peptide antigenic conjugate according to claim 10, characterized in that the hydrophobic molecule containing poly(amino acids) of Formula II is grafted or linked at the end with a second polymer based on poly(leucine), poly(tryptophan), poly^-glutamyl diethylene glycol monomethyl ether), polyN-benzylglutamate), poly(N-isopropylacrylamide) (NIPAM), poly[2(2-methoxyethoxy)ethyl methacrylate)] (DEGMA) or poly[N-(2-hydroxypropyl)methacrylamide) (HPMA). 15. Peptide antigenic conjugate according to claim 10, characterized in that the hydrophobic molecule containing poly(amino acids) of Formula IIa is grafted or linked at the end with a second polymer based on poly(leucine), poly(tryptophan), poly^-glutamyl diethylene glycol monomethyl ether), polyN-benzylglutamate), poly(N-isopropylacrylamide) (NIPAM), poly[2(2-methoxyethoxy)ethyl methacrylate)] (DEGMA) or poly[N-(2-hydroxypropyl)methacrylamide) (HPMA). 16. Peptide antigen conjugates according to any one of claims 13-15, characterized in that the second polymer contains a plurality of monomer units from 30 to 500 monomer units. 17. Peptide antigen conjugate according to any one of claims 8 to 16, characterized in that at least one adjuvant is linked to one end of the polymer (semi-telechelate). 18. Peptide antigen conjugate according to any one of claims 8-11, characterized in that the adjuvant/ligand is an imidazoquinoline of Formula III Formula III wherein R ⁷ is selected from one of the following: hydrogen, optionally substituted lower alkyl and optionally substituted lower ether; a R ⁸ is selected from one of the following: an optionally substituted arylamine and an optionally substituted lower alkylamine. 19. The peptide antigen conjugate according to claim 18, characterized in that at least three imidazoquinoline molecules of Formula III are linked to a polymer containing a hydrophobic molecule, and R ⁸ is selected from one optionally substituted lower alkylamine. 20. The peptide antigen conjugate according to claim 19, characterized in that three or less imidazoquinoline molecules of Formula III are linked to a polymer containing a hydrophobic molecule, and R ⁸ is selected from one optionally substituted lower arylamine. 21. Peptide antigen conjugate according to any one of claims 1 to 20, characterized in that the peptide antigen conjugate accumulates into particles consisting of micelles or other types of supramolecular associates of nano- or microsize containing at least 2 peptide antigen conjugates, and their size is greater than 5 nm in an aqueous buffer solution at pH 7.4. 22. The peptide antigen conjugate according to claim 21, characterized in that the peptide antigen conjugate accumulates into micelles with a diameter of from about 5 to 200 nm in an aqueous buffer solution at pH 7.4. 23. Peptide antigen conjugates according to claim 21, characterized in that the peptide antigen conjugate is an aggregate in an aqueous buffer solution at pH 7.4. 24. Peptide antigen conjugate according to any one of claims 1 to 23, characterized in that the linker (L) is formed as a result of the reaction between a linker precursor X1 and a linker precursor X2, wherein the linker precursor X1 is linked either directly or by extension B2 to the peptide antigen and the X2 linker precursor is associated with a hydrophobic molecule. 25. Peptide antigen conjugate according to claim 24, characterized in that the X1 linker precursor has the formula wherein the N-terminal amine of the X1 linker precursor is linked either directly or by extension B2 to the peptide antigen; R ¹ is selected from one of the following: hydrogen, hydroxyl and amine; y1 is an integer from 1 to 8; and FG is selected from one of the following: thiol, amine, azide, alkyne, or hydrazine, wherein the linker precursor X2 contains an alkyne group, an activated ester, a ketone, an azide, a maleimide, or a disulfide. 26. Peptide antigen conjugate according to claim 25, characterized in that FG is an azide, - 132 046161 a y1 is 4, additionally, wherein the azide is linked to an alkyne group, which is linked to a hydrophobic molecule. 27. Peptide antigen conjugate according to any one of claims 1 to 24, characterized in that the X1 linker precursor is linked to the B1 extension and has the formula o wherein the carbonyl is linked either directly or through a B1 extension to the N-terminus of the peptide antigen; y2 is an integer from 1 to 8 and FG is selected from one of the following: thiol, amine, azide and alkyne. 28. The peptide antigen conjugate according to any one of claims 1 to 27, characterized in that the peptide antigen fragment consists of a peptide antigen, an optional B1 extension, an optional B2 extension and a charged molecule, and/or a linker precursor X1, synthesized as a single peptide by solid phase peptide synthesis, and wherein the B2 extension contains proline (or pseudoproline during solid phase peptide synthesis while the sequence is attached to the resin used for peptide synthesis). 29. Peptide antigen conjugate according to any one of claims 1 to 28, characterized in that the charged molecule contains 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 and phosphonate. 30. Peptide antigen conjugate according to claim 29, characterized in that the charged molecule contains both negatively and positively charged functional groups. 31. The peptide antigen conjugate according to any one of claims 1 to 30, characterized in that the series of charged functional groups containing the charged molecule have certain characteristics adjusted as follows: a) the total electrostatic charge of the peptide antigen conjugate contains a positive value that is greater than or equal to +6 when the peptide antigen has an overall average hydropathy value that is greater than or equal to 0.75; b) the net electrostatic charge of the peptide antigen conjugate contains a negative value that is less than or equal to -6 when the peptide antigen has an overall average hydropathy value that is greater than or equal to 0.75; c) the net electrostatic charge of the peptide antigen conjugate contains a negative value that is less than or equal to -5 when the peptide antigen has an overall average hydropathy value that is greater than or equal to 0.25 and less than 0.75; or d) the net electrostatic charge of the peptide antigen conjugate contains a negative value that is less than or equal to -4 when the peptide antigen has an overall mean hydropathy value that is less than 0.25. 32. Peptide antigen conjugate according to claim 31, characterized in that the peptide antigen fragment consists of a peptide antigen, a B1 extension, a B2 extension, a charged molecule and a precursor linker X1, produced as a single peptide, which is soluble in an aqueous buffer solution to a concentration of 1 .0 mg/ml at pH 7.4. 33. A delivery particle containing peptide antigen conjugates according to any one of claims 1 to 32. 34. The particle according to claim 33, characterized in that the particle contains a plurality of different peptide antigen conjugates. 35. An immunogenic composition containing peptide antigen conjugates according to any of claims 132 or particles according to claims 33, 34. 36. Immunogenic composition according to claim 35, additionally containing a vaccine adjuvant. 37. The immunogenic composition of either claim 35 or claim 36, characterized in that the composition contains a plurality of different peptide antigen conjugates. 38. Peptide antigen conjugate according to any one of claims 1-32, a particle according to any one of claims 33, 34 or an immunogenic composition according to any one of claims 35-37, characterized in that the tumor-associated antigen is a neoantigen. 39. Peptide antigen conjugate according to any one of claims 1-32 and 38, a particle according to any of claims 33-34 and 38 or an immunogenic composition according to any one of claims 35-37, characterized in that the peptide antigen is a minimum CD4 or CD8 T cell epitope. 40. An immunogenic composition according to any one of claims 35 to 39, characterized in that the immunogenic composition contains from 10 to 50 different peptide antigen conjugates consisting of peptide antigens selected from minimal CD4 T-cell epitopes, CD8 T-cell epitopes, or both from minimal CD4 and CD8 T-cell epitopes. 41. An immunogenic composition according to any one of claims 35-38, characterized in that the immunogenic composition contains 10-50 different peptide antigen conjugates consisting of peptide antigens selected from both minimal CD4 T-cell epitopes and minimal CD8 T - 133 -