LU102995B1

LU102995B1 - Immunization against coronavirus

Info

Publication number: LU102995B1
Application number: LU102995A
Authority: LU
Inventors: Wolfgang Schönharting; Mari Carmen Martos Contreras
Original assignee: PMCR GmbH
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2024-02-19

Abstract

The present invention relates to a pharmaceutical composition for use in the treatment or prophylaxis of a coronavirus infectious diseases, comprising at least one antigen polypeptide comprising at least one HLA peptide corresponding to MHC class I complexes, wherein the HLA antigen peptides are coronavirus exclusive and are directed against at least one MHC class I complex including combinations thereof; a pharmaceutical composition, a kit (or parts thereof), a method for determining a class I HLA peptide and/or antigen polypeptide, a method for preparing a formulation according to the invention, and the use of a formulation according to the invention for the preparation of a pharmaceutical composition for the treatment of a coronavirus infectious diseases.

Description

Immunization against coronavirus

Technical Field of the Invention

This disclosure relates to the field of amino acids to prevent or treat coronavirus infection. In particular, the present invention relates to a pharmaceutical composition/formulation, particularly to a combination agent/composition comprising of at least one, more particularly at least two and most particularly up to 12 different synthetic peptide complexes (antigen polypeptides) corresponding to HLA antigen peptides matching viral epitopes, for immunization against a coronavirus while avoiding infection-enhancing antibodies, a combination preparation (or parts thereof), a method for determining/identifying at least one

HLA antigen peptide corresponding to the MHC class | complexes and/or antigen polypeptide for use in the pharmaceutical composition and a method for preparing a pharmaceutical composition comprising at least one antigen polypeptide according to the invention.

State of the Art / Background

Viruses are infectious organic structures which, as virions (virion = single virus particle), spread outside cells (extracellularly) by transmission, but as viruses can only replicate within a suitable host cell (intracellularly). Viruses do not exhibit independent replication or metabolism, so they rely on the metabolism of a host cell. Viruses infect cells of eukaryotes (plants, fungi, and animals including humans) as well as prokaryotes (bacteria and archaea). Virions are particles that contain nucleic acids and usually have an enclosing protein capsule (capsid). Some virions additionally possess an envelope by a bio membrane whose lipid bilayer is interspersed with viral membrane proteins, which is called the viral envelope. The replication cycle of a virus begins when a virion attaches to a surface protein on a host cell that is used by the virus as a receptor. After ingestion, a virion must first be freed from its envelopes before replication. The genetic material of the virus, its nucleic acid, is then replicated in the host cell, and the envelope proteins and possibly other components of the virions are also synthesized by the host cell using the genes of the viral genome. In this way, new viruses can be formed in the cell and released as virions, either by dissolving the cell membrane or by being shed, taking parts of the host cell membrane with them as part of the viral envelope. Human pathogenic viruses are transmitted, for example, through the air by droplet infection or through contaminated surfaces

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung by smear infection. Coronaviruses (Orthocoronavirinae) are a group of related RNA viruses which cause respiratory tract infections.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a strain of the subgenus sarbecovirus, of the genus of betacoronaviruses and was responsible for the global pandemic outbreak in 2019 and closely related to SARS-CoV-1 virus which was responsible for the 2002-2004 SARS outbreak. These outbreaks lead to immense loss of human life and economical damage.

The virus still poses a drastic challenge to healthcare worldwide, as new and more contagious variants emerge due to mutation of the original Wuhan wild type (Hu-1). Some particularly worrisome strains are tracked by the World Health Organization (WHO) as variants of concern (VOC) such as the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P. 1), Delta (B.1.617.2) and

Omicron Variant (B.1.1.529). These variants commonly feature mutations especially on the spike (S) glycoprotein, also known as spike protein, which is targeted by many vaccines developed against the wild type and thus impeding immunization efforts worldwide.

The immune system continuously monitors the body for the presence of viral infections and degenerate or infected cells by checking whether cells present endogenous or foreign proteins, and for the presence of the MHC-(I-1ll) major histocompatibility complexes. MHC protein complexes are endogenous proteins that bind endogenous or exogenous peptides that are approximately 8-10 (MHC |) or 13-18 amino acids (MHC Il) in length. Antigens on the surface of body cells serve to identify the body's own cells. This is the origin of the name HLA system (Human Leucocyte Antigen) for the regulatory system of human immune defense, whose most important component is the MHC. The HLA system in other species is known as the major histocompatibility complex (MHC) and is found in all vertebrates.

There are two types of MHCs, class | MHC molecules (herein also "class | MHC complexes” or"class | HLA complexes") and class II MHC molecules (herein also "class II MHC complexes" or "class II HLA complexes"). Both show up on the cell surface of all nucleated cells in the bodies of gnathostomata. Class | MHC molecules, with (viral) antigens (peptides) presented on them, are expressed on the cell surface of all nucleated cells and are recognized by CD8+

T cells (also called killer T cells or cytotoxic T cells). The non-presenting HLA class | molecule (MHC-I) is recognized by NK cells. A missing MHC-I complex on the surface of an (e.g. infected) cell causes activation of the NK cell and killing of the (infected) cell. The immune system continuously monitors the body for the presence of viral infections and degenerate (infected) cells by checking whether cells present endogenous or foreign proteins and for the

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung presence of MHC-I protein complexes. The peptides presented reflect the proteins synthesized in the cells. Cytotoxic T lymphocytes (CD8+ T cells) are selected so that they generally do not bind with their T cell receptor (TCR) to cells presenting a peptide derived from an endogenous protein. MHC class II (MHC-Il) molecules are mainly exposed on the cell surface of antigen- presenting cells (including macrophages) and recognized by CD4+ T cells (also known as T helper cells), which function as a co-receptor of the T cell receptor (TCR) and play an important role in the antigen detection by the latter

With the identification of the genomes of pathogens such as SARS-CoV-2, (in this case a single-stranded RNA), their protein repertoire can be determined by DNA/RNA sequencing.

This involves the use of bioinformatics processes that recognize regions for epitopes in these proteins. Since T and B cells identify these regions, vaccines can be developed on this basis.

All existing and approved vaccines against SARS-CoV-2 target only one protein complex of this virus: the S1 part of the spike structural protein, which contains the receptor binding domain (RBD) with which the virus docks to the ACE2 receptor of the host cell. The formation of antibodies against this protein complex is intended to block this attachment site and thus prevent the virion from entering the host cell.

Vaccine developments deviating from this focus on further points of attack in the virus such as the nucleocapsid protein. For example, in 2020, Herst et al. demonstrated a nine amino acid epitope located in the conserved region of the nucleocapsid protein, which is a target for vaccine development against Ebola virus. Immunity is achieved in the mouse model through epitope-mediated stimulation of cytotoxic T leukocytes (CTL), which is why the vaccine is called a CTL peptide vaccine. The authors suggest that a similar approach may be possible for the development of SARS-CoV-2 immunity.

Slathia et al. (2020) used immunoinformatic methods to predict T and B cell epitopes present in the proteins of SARS-CoV-2, where it is stated that the T-cell epitopes could be used in vaccine design and the B-cell epitopes could be used in the development diagnostic kits and vaccine design. They reported a population coverage analysis of the distribution of HLA alleles among different population groups with the goal to provide immunity to populations worldwide.

Ahmed et al. (2020) identified B-cell and T-cell epitopes of SARS-CoV that are identical between SARS-CoV and SARS-CoV-2 using experimentally determined B-cell and T-cell epitopes with bioinformatics methods, providing a potential basis for therapy development.

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A study by Chen et al. (2020) identified potential peptide epitopes for vaccine development.

Here, surface accessibility, antigenicity, and other features were characterized for immunogen potential assessment. Potential B-cell epitopes on the spike protein and T-cell epitopes within the nucleocapsid protein were identified that could potentially elicit a CD4+ as well as a CD8+- dependent immune response and could be used for vaccine development.

In order to effectively repel pathogens, a vaccine is needed that simultaneously prevents the virion from entering the host cell and also prepares the immune system for intracellular defense mechanisms. However, during vaccine development, infection-enhancing antibodies (antibody-dependent enhancement, ADE) may occur that bind to the surface of viruses but do not neutralize them. This leads to enhanced uptake of the virus into a host cell and thus favors the spread and multiplication of the viruses within the host. The result is a more difficult course of disease in the event of a secondary infection.

Current coronavirus vaccines, especially those for SARS-CoV-2, focus on rapid development of immunization platforms and do not consider the risk of ADE. For example, this problem was already encountered in vaccine development against the coronaviruses FIPV2,3, MERS4,5 and SARS, which is why no vaccines against these viruses have been developed and licensed to date. For SARS-CoV-1, for example, an aggravated disease course was demonstrated after immunization of macaques, with non-neutralizing antibodies involving Fc receptors (FcRs) leading to viral infection of monocytes, macrophages and B cells.

In vaccine development, the problem of ADE can be overcome by a combined and balanced activation of T and B cells leading to a specific immune response throughout the viral life cycle.

Thus, high technologies in bioinformatics, mathematical modeling, and artificial intelligence (Al) can predict different peptide epitopes that enable specific antibody properties or lead to balanced T-cell activation.

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Objection of the Invention / Technical Problem

Based on the state of the art, existing pharmaceutics for immunization against coronavirus are faced with the problem that their subject range is not broad enough (one fits all approach) therefore, an objection of the present invention is to provide a pharmaceutical active 5 combination preparation that reaches as broad a subject group as possible and at the same time is very specific response in the individual subject by means discussed further below in detail.

Based on the state of the art, single HLA antigen-based pharmaceutics face the problem that not every HLA allele that should/could be expressed according to the genotype is exposed on the cells, thus an objection of the present invention is to provide a pharmaceutical active combination which can target different HLA alleles simultaneously by means discussed further below in detail.

In addition, known HLA antigen-based pharmaceuticals against coronaviruses are faced with the problem that HLA antigen peptides are not stable for extended periods in vivo, therefore an objection of the present invention is to provide a pharmaceutical active combination preparation comprising at least one HLA antigen peptide as discussed further below in detail, which is stable over an extended period of time in the subject, and means of administration of said preparation.

Based on the state of the art, current immunization agents struggle to combat ever evolving and mutating viral strains of coronaviruses, especially SARS-CoV-2, which can overcome vaccines with only one point of attack more easily, therefor it is an objection of the present invention to provide a pharmaceutical composition for a combination drug which targets at least two points of attack.

In some embodiments of the invention, it may provide pharmacologically active agents, as well as pharmaceutical compositions comprising such active agents, which can be used for the prophylaxis and/or treatment of coronaviruses, preferably of betacoronaviruses, particularly preferably sarbecoviruses, and most preferably of SARS-CoV-2; and to provide methods for the prevention and/or treatment of such SARS-CoV-2 infectious diseases, which include the administration and/or use of such active agents and compositions.

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Solution

To overcome the technical problems of the prior art, a preferred embodiment of the present invention comprises of pharmacologically active agents (so called “antigen polypeptide” or “antigen polypeptide sequences”), pharmaceutical compositions and/or methods which have advantages over the active agents, compositions and/or methods currently in use and/or known in the prior art. These advantages result from the following further description.

A more preferred embodiment of the present invention is to provide therapeutically active HLA antigen peptides that may be used as pharmacologically active HLA antigen peptides and pharmacologically active agents, respectively, and to provide pharmaceutical compositions containing same, for the prevention and/or treatment of coronaviruses and other infections and disorders, such as SARS-CoV-2; and to provide methods for the prevention and/or therapeutic treatment of such infections and disorders involving the administration and/or use of such therapeutically active HLA antigen peptides and compositions.

In a particular preferred embodiment of the invention, such HLA antigen peptides are provided which are especially suitable for prophylactic and/or therapeutic use in warm-blooded animals, more particularly in mammals and most particularly in humans.

According to the invention, these objections are solved by a pharmaceutical composition for use in the therapeutic and/or prophylactic treatment of coronavirus infections, comprising a pharmacologically effective amount of at least one, preferably at least 2, more preferably 3, such as at least 4, at least 5, at least 6, at least 7, in particular at least 8, more particularly at least 10 and most particularly up to 12 antigen polypeptides comprising, inter alia, HLA antigen peptides, characterized in that exclusive targets are chosen, such as preferably a coronavirus, more preferably a betacoronavirus, most preferably a sarbecovirus, such as a SARS-CoV-2 spike glycoprotein (also called spike protein or S protein), nucleocapsid phosphoprotein (also called nucleocapsid protein or N protein), envelope protein (also called E protein), surface glycoprotein and ORF1ab polyprotein, wherein said antigen polypeptides particularly comprise or consist of sequences represented in the sequences SEQ ID NO: 38 — 58 (as defined in more detail below).

Furthermore, these objections are solved by a pharmaceutical composition for use as a medicament, in particular for use in the therapeutic or prophylactic treatment of a coronavirus infectious disease, in particular in a subject or group of subjects suffering from or at risk of suffering from a coronavirus infectious disease, comprising of a pharmacologically effective

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung amount consisting of at least 1, preferably at least 2, more preferably 3, such as at least 4, at least 5, at least 6, at least 7, in particular at least 8, more particularly at least 10 and most particularly up to 12 antigen polypeptide sequence(s) comprising at least one HLA-A and/or

HLA-B antigen peptide, wherein the HLA antigen peptide corresponds, preferably is identical to at least one amino acid sequence in the coronavirus, such as spike S1 domain, S2 domain, nucleocapsid protein, envelope protein and/or ORF1ab polyprotein, characterized in that the antigen polypeptide sequence in accordance with the present invention comprises or consists of the following scaffold amino acid sequence: (a) a long peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which comprises one “HLA-A antigen peptide” or “HLA-B antigen peptide” as defined herein, preferably one native “HLA-A antigen peptide” or “HLA-B antigen peptide”, wherein the amino acid sequence comprises, in addition to the "HLA-A antigen peptide" or "HLA-B antigen peptide", up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or (b) a similarity peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), wherein the “HLA-A antigen peptide” or “HLA-B antigen peptide” as defined herein having less than 100% sequence identity or similarity to the native HLA antigen peptide, such as having at least 85%, more preferably at least 90%, sequence identity; and/or (c) a substitution peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), wherein the “HLA-A antigen peptide” or “HLA-B antigen peptide” as defined herein having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence of the native HLA antigen peptide; and/or (d) a tandem peptide, i.e. a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-B antigen peptide sequences, both as defined in items (a) to (c), in which the HLA antigen peptides are optionally connected to each other by suitable linkers, more preferably by a suitable amino acid linker (so-called oligopeptides); and/or (e) an overlapping tandem peptide, i.e. a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-

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B antigen peptide sequences, both as defined in items (a) to (c), in which the at least two HLA antigen peptides overlap in their amino acid sequence.

The combination of different peptide epitopes, in accordance with the present invention, enables comprehensive targeting of a virus throughout the infection chain by allowing multiple points of attack. Thus, the virus can be repelled, its entry into the host cell prevented by antibodies, and its replication in the host cell prevented by activated Th1 defense cells. This is made possible by the combination of HLA antigens targeting different points of attack of SARS-

CoV 2, such as the spike protein, the nucleocapsid protein, the envelope protein and the replicase polyprotein. The HLA antigen peptides used in the combination preparation are coronavirus exclusive, in particular wherein the HLA antigen peptides comprise sequences represented in the sequences SEQ ID NO: 1 — 37, 59 — 79 (as defined in more detail below).

Two or more HLA antigen peptides can also be present in an antigen polypeptide sequence in the form of a tandem peptide. Two or more HLA antigen peptides can also be present in the form of an overlapping tandem peptide. Some examples (but not limited to) for particularly preferred antigen polypeptides are listed in the sequences SEQ ID NOs.: 38 — 58.

Further advantageous embodiments are given in the dependent claims.

Description

Advantages and Description of the pharmaceutical combination

In one embodiment, the pharmaceutical use comprises inducing an immune response against coronavirus in a subject. In one embodiment, the pharmaceutical use comprises a therapeutic or prophylactic treatment of a coronavirus infection. In one embodiment, the pharmaceutical composition or medical preparation described herein is for administration to a human.

In one embodiment, the coronavirus is a betacoronavirus. In one embodiment, the coronavirus is sarbecovirus. In one embodiment, the coronavirus is SARS-CoV-2, especially a wild-type (WT) or mutant SARS-CoV-2, such as the Alpha (B.1.1.7), or Beta (B.1.351), or Gamma (P. 1), or Delta (B.1.617.2) and/or Omicron variant (B.1.1.529).

The present invention relates to a pharmaceutical composition, in particular a combination agent, to effect prophylactic protection against infection with a coronavirus, such as SARS-

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CoV-2, especially a WT or mutant SARS-CoV-2 and to prevent severe disease progression in the event that infection has occurred. In addition to B-cell-mediated antibody generation, the main focus is on the T-cell response. Preferably, administration of the combination agent induces more than 80%, more preferably 85% and most preferably 90% neutralizing antibodies against SARS-CoV-2, with the proportion of non-neutralizing antibodies preferably being less than 10%, particularly preferably less than 5%, and most preferably less than 2%.

Advantageously, antibody dependent enhancement (ADE) is thus reduced. As described herein, but without limitation to any explanation, mechanism of action or hypothesis in the present invention, 12 different targets of amino acid sequences of the invention have been determined based on their ability and interaction with specific SARS-CoV-2 structures. These structures of amino acid sequences of the invention are (as described below):

HLA antigen peptides in accordance to the present invention are corresponding to MHC class complexes (see particularly preferred examples in Tables 1-6).

Advantageously, the use/application of the pharmaceutical composition according to the invention or the specific combination of antigen peptides contained therein corresponding to the MHC class | complexes with at least one identical HLA allele is not merely a passive immunization (as in the case of treatment with antibodies, e.g. Herceptin) but an active immunization (i.e. specific activation of the T cells or B helper cells via information carriers).

For binding to a T cell receptor, an HLA antigen peptide of the invention typically has in its amino acid sequence one or more amino acid residues or one or more segments of amino acid residues (i.e., with each "segment" comprising two or more amino acid residues located adjacent to or in close proximity to each other, viz. in the primary or tertiary structure of the amino acid sequence) through which the amino acid sequence of the invention can bind to a

T cell receptor (particularly a binding pocket thereof), the amino acid residues or portions of the amino acid residues thus forming the "anchor" for binding to a T cell receptor (also referred to herein as "anchor amino acids"). Determination of this "anchor" can be determined, for example, by in silico methods (e.g., the artificial neural network NNAlign used in the publicly available NetMHC-4.0 server).

The HLA antigen peptides provided by the present invention are preferably present substantially as a mixture of at least one HLA antigen peptide of the invention having a sequence described herein (as defined herein) or form part of a protein or polypeptide which may comprise or consist essentially of one or more HLA antigen peptides of the invention, and which may optionally comprise one or more pharmaceutically active HLA antigen peptides

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(wherein the HLA antigen peptides in a so-called oligopeptide are all optionally connected via one or more linkers).

For example, and without limitation, the antigen peptides of the invention may be used as a binding moiety in such a protein or antigen polypeptide, which may optionally include one or more additional amino acid sequences that may serve as a binding moiety (i.e., against one or more targets other than a T cell receptor) to provide a monovalent, multivalent, or multispecific antigen polypeptide of the invention as described herein, respectively. Such a protein or antigen polypeptide may also be present in substantially as a mixture of at least one protein or polypeptide having at least one sequence described herein (as defined herein).

It has been shown that a pharmaceutical composition comprising a specific combination of antigen polypeptides comprising one or more HLA antigen peptides corresponding to MHC class | complexes and MHC class Il complexes disclosed herein is particularly advantageous in that it is capable of specifically activating T cells as well as specifically activating B cells.

Classical methods for the treatment of infectious diseases of the invention are, for example, the injection of vaccines (also: vaccines). Comprehensively, a biologically or genetically engineered antigen consists mostly of protein or DNA or RNA segments or killed or attenuated pathogens. A vaccine can consist of an antigen from a single pathogen, as well as a mixture of several antigens from different pathogens or strains of pathogens and possibly other additives (adjuvants). In the context of a vaccination, the vaccine serves to specifically activate the immune system with regard to a specific pathogen or group of pathogens. For this purpose, the reaction of the immune system is exploited, which provokes an immune response in the presence of pathogens or their specific parts (e.g. surface proteins as antigens). This leads to the formation of antibodies and specialized T-helper cells, which then provide a long-lasting protection against the respective pathogen, which, depending on the pathogen, can last between a few years and lifelong. Classic known vaccine types include, for example, conjugated vaccines, DNA vaccines, RNA vaccines, T-cell vaccination (TCV).

Thus, it is a particular achievement of the inventors to have found that for effective treatment with the antigen polypeptides used in accordance with the invention, with different target sequences of the different peptides of SARS-CoV-2, "Antibody Dependent Enhancement” (ADE) can be avoided in the individual to be treated. ADE has occurred in the development of vaccines against the coronaviruses FIPV2,3, MERS4,5, and SARS. The objective of overcoming the ADE problem led to the development of the vaccine of the present invention, which causes combined and balanced T- and B-cell activation, leading to multiple specific

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung immune responses throughout the viral replication cycle. The development of high technologies in bioinformatics, mathematical modeling and artificial intelligence (Al) allows for the first time to specifically predict epitopes that elicit specific antibody properties or lead to balanced T- and B-cell activation. tis also an outstanding achievement of the inventors to have discovered that a pharmaceutical composition based on antigen polypeptides is particularly effective when both T lymphocytes (T cells for short) and B lymphocytes (B cells for short) are activated by them. T and B cells belong to the lymphocyte cell group and play an important role in the human immune system.

T cells recognize antigens via a specific receptor, the so-called T cell receptor (TCR). However, for this to occur, the antigen must be offered to the T cell by an antigen-presenting cell (APC) via a class | or class || HLA. Stable binding of the T cell to the APC requires the participation of so-called auxiliary proteins. These include CD4 and CD8 (CD = "Cluster of Differentiation"), with the presence of such a cluster denoted by a "+" (CD4+, CD8+).

T cell lymphocytes contain the CD4+ and the CD8+ positive T cells. T cells that carry the CD4 feature are also called CD4-positive T cells or T helper cells. In normal adult blood, CD4+ T cells account for 27-57% of lymphocytes, or approximately 310-1,570 cells/jul. The group of

CD8-positive (CD8+) T cells is also called cytotoxic or killer T cells. They play a special role in killing the body's own cells that are infected by pathogens, such as viruses.

In contrast, B cells are the only cells capable of producing antibodies and, together with T cells, make up the critical component of the adaptive immune system. While T cells are involved in the cell-mediated immune response, B cells are the carriers of the humoral immune response (and are responsible for the formation of antibodies). It has been shown in experiments that pharmaceutical compositions containing an absolute concentration per antigen polypeptide of preferably more than 100 ug and particularly preferably more than 500 ug are quite preferred, as this greatly intensifies the information and thus the activation and/or training of the immune system. In one embodiment, the concentration of a peptide is 600 ug. Accordingly, 600 ug x12 = 7,200 pg are contained in the pharmaceutical composition (per vial). It is highly advantageous if the pharmaceutical composition comprises the adjuvants already mentioned above, which, when the composition is applied to a patient, are capable of intensifying the body's immune response.

Comprehensive of a balanced Th1/Th2 based robust immune response, CpG (oligonucleotide of cytidine and guanosine) is a Toll-like receptor 9 (TLR-9) agonist and is, for example, a component of the market-approved prophylactic HBV vaccine Heplisav-B (Dynavax, USA).

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The letter C stands for the nucleotide cytosine, the p for phosphate and the G for the nucleotide guanine. The targeted activation of TLR9 by CpG oligonucleotides opens up a wide range of possibilities for using these compounds, for example, to improve the efficacy of vaccines. In the context of prophylactic HBV vaccination, the use of CpG leads to a shortening of the vaccination schedule from three (all other approved HBV vaccines) to two vaccinations. In addition, CpG as an adjuvant in Heplisav-B also results in a better response in type 2 diabetics, smokers, the obese, and the elderly, who generally show poorer response rates.

Furthermore, it is an outstanding achievement of the inventors to have discovered a pharmaceutical composition of up to 12 amino acid sequences used in accordance with the present invention which can take advantage of highly preserved parts of the virus, thus enabling a vaccination more effective against new mutations. In coronaviruses, the spike glycoprotein consists of trimers that form large structures, referred to as spikes or peplomers, which project from the surface of the virion. The spike protein is divided into two parts, the S1 and S2 regions. The S1 subunit is responsible for the interaction with the cells receptor proteins and docks to the angiotensin converting enzyme 2 (ACE2) membrane protein. The S2 region consist of the fusion peptide, which function is to destabilize the host cell membrane and is mostly made up of hydrophobic amino acids. The S2 region is more conserved in coronaviruses compared to the S1 region. In a preferred embodiment of the invention, at least one amino acid sequence is selected to target the S2 region of the coronavirus specifically, since for effective entry of a virion into a host cell, both functions are required: a) docking to the ACE2 receptor via S1 and b) fusion of the host cell and virion membranes primed by the serine 2 transmembrane protease TMPRSS2 which is able to cleave 51/52, therefore blocking the fusion protein S2 is equally effective as blocking the receptor docking mechanism (S1) as the virus requires booth S1 and S2 motives for effective virion penetration into the host cell.

In a preferred embodiment of the present invention at least one of the antigen polypeptide sequences of the pharmaceutical composition comprises epitopes of the SARS-CoV-2 spike glycoprotein, in a more preferred embodiment, at least one of the antigen polypeptide sequences of the pharmaceutical composition comprises epitopes of the of the S2 membrane fusion subunit of the spike protein specifically rather than the S1 receptor binding domain (RBD). In this respect, blocking the S2 fusion protein by antibodies is expected to advantageously show the same efficacy as blocking the S1-mediated receptor docking mechanism. In contrast to the S1 subunit, the S2 subunit is highly conserved and shows only a low mutation rate. Advantageously, the use of S2 specific epitopes results in improved protection against different SARS-CoV-2 strains.

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It is a severe shortcoming of existing vaccines that only small parts of the epitope of the virus, such as but not exclusively the spike protein, which is subject to strong mutations, can be targeted and thus easily bypassed by new virus variants, such as the Alpha (B.1.1.7), or Beta (B.1.351), or Gamma (P. 1), or Delta (B.1.617.2) and/or Omicron Variant (B.1.1.529).

Therefore, it is an outstanding achievement of the inventors, that in a preferred embodiment of the invention at least two amino acid sequences are selected in such a manner, that the virus can be targeted in all reproduction steps, not only the spike glycoprotein required for membrane fusion, nucleocapsid phosphoprotein, envelope protein, surface glycoprotein and

ORF1ab polyprotein, particularly wherein said peptides particularly comprise sequences represented in the sequences SEQ ID NOs: 38-58.

It is a further outstanding achievement of the inventors to have discovered that a combination preparation containing at least two antigen polypeptides, preferably selected from SEQ ID

NOs: 38-58, preferably match a total of at least 20, more preferably at least 25, in a particular embodiment at least 30 HLA alleles, advantageously allowing more than 95% of the world's population to be immunized with the pharmaceutical composition of the invention. This allows a worldwide use of the vaccination without need for adaptation for different populations. In an alternative embodiment, if the combination preparation in accordance with the present invention contains the 12 antigen polypeptide sequences SEQ ID NOs.: 38-49, a total of 37

HLA alleles are matched, allowing more than 95% of the world population to be immunized.

In an alternate embodiment of the invention, where at least two amino acid sequences, preferably at least two HLA antigen peptides are selected for the pharmaceutical composition in accordance with the present invention, these two sequences are preferably selected to target at least two different parts of the viral epitope, chosen for example from the spike glycoprotein, nucleocapsid phosphoprotein, envelope protein, surface glycoprotein and

ORF1ab polyprotein of the SARS-CoV-2 virus. In a particularly preferred embodiment, at least three sequences are selected to match at least three different regions, in a most preferred embodiment at least four regions corresponding to different parts of the virus are chosen. The benefit of this embodiment is to target different steps in reproductive cycle of the virus, thus reducing the possibility of mutation caused resistance.

In a preferred embodiment of the invention the pharmaceutical composition contains at least two antigen polypeptides which are selected to match viral epitopes of the coronavirus, particularly SARS-CoV-2, particularly preferably selected from SEQ ID NOs: 38-58 in such manner to match enough HLA alleles to archive prophylactic protection for at least 95% of the

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung world population and to match different viral proteins, which are highly conserved and thus be more resistant against mutations.

Bis-(3',5')-cyclic-adenosine monophosphate is a bacterial messenger involved in the control of cell wall metabolism, osmotic stress responses and sporulation. C-di-AMP is a STING agonist that also induces a balanced Th1/Th2 immune response. STING (STimulator of INterferon

Genes) is a protein expressed by various cell types (including T cells, macrophages, and dendritic cells) that acts as a sensor of cytosolic, pathogen DNA and induces type | interferon production as part of the innate immune response.

Aluminum salts (usually a mixture of aluminum sulfate, potassium or sodium hydroxide, and phosphate) have been used as adjuvants for more than 100 years and are adjuvant components of a variety of market-approved vaccines. Aluminum-containing adjuvants adsorb antigens most commonly due to electrostatic interactions. The exact mechanism of how the immune response is stimulated is still the subject of research. A depot mechanism is assumed, that the adjuvant gradually releases the adsorbed antigen at the site of injection. This mechanism is supported by the observation that antigens that bind more strongly to the adjuvant cause a stronger immune response. In particular, alum-dependent T cell differentiation to CD4+ Th2 cells is an important effect in prophylactic vaccines for targeting an antibody-based immune response. Alum can further promote antigen uptake in antigen- presenting cells (APCs) and enhance MHC-II gene cluster expression.

The lipopeptide Pam3Cys-GDPKHPKSF is a TLR-2/TLR-1 ligand with the precise designation

N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteinyl-GDPKHPKSF. This water- soluble adjuvant induces potent responses from CD8 and CD4 positive T cells.

MF59 and AS03 are squalene-based oil in water emulsion adjuvants which were successfully used in a variety of market approved human vaccines. They can lead to an improved and more balanced immunity, possibly by stimulating the CD4+ and CD8+ T-cell immune responses. MF59 is currently used as an adjuvant in the United States Canada and Europe.

Poly-IC is a TLR3/MDA5 agonist. Poly I:C binds to and activates TLR3 in animals, subsequently activating the innate immune response. TLR3 is produced by B cells, macrophages and dendritic cells. Poly I:C has been studied as an adjuvant in vaccines and for the induction of apoptosis in cancer cells.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

The physiological pH-value of the blood of a healthy adult human is between 7,35-7,45, thus for better physiological compatibility the pH of the pharmaceutical composition is considered, the total charge of the amino acids in the pharmaceutical composition should be mostly basic or only slightly acidic. In a preferred embodiment of the present invention single peptides have a maximum excess of acidic amino acids of 5, more preferred of 4, and most preferred of 3.

The preferred excess of basic amino acids is up to 10 amino acids, more preferred is an excess of 8, and most preferred is an excess of 6 basic amino acids. Amino acid excess is hereby defined as absolute value of the difference of basic and acid amino acids of the sequence.

The S2 region of the corona spike protein contains the fusion peptide, a stretch of mainly hydrophobic amino acids, thus in a preferred embodiment of the invention, the amino acid sequence (as set forth in the Examples SEQ ID NOs: 38-58) is mainly compromised of hydrophobic amino acids, preferably the share of hydrophobic amino acids is between 20-90% of the sequence, more preferably between 35%-80% of the sequence, while having a preferred share of hydrophilic amino acids between 0%-60% of the total amino acids of the sequence bearing hydrophobic amino groups, more preferred a share of hydrophilic amino acids between 10%-50% of the total amino acids of the sequence.

In a preferred embodiment of the invention, the antigen polypeptides used in the pharmaceutical composition have an amino acid length between 9 and 40, more preferred between 9 and 35 and most preferred between 9 and 30 amino acids. In an alternative embodiment of the invention the amino acid chain is linear (not cyclic).

In an alternative embodiment of the invention, the antigen polypeptides can be further modified at the C or N-terminus. In a preferred embodiment the modification of the C-terminus can reduce excess positive charge to align to the physiological pH-value of the subjects, while the modification of the N terminus can reduce the basic character of the peptide. The means to modify peptides in this way are known to the person skilled in the art, for example using the

Merrifield peptide synthesis.

In one embodiment, an “antigen polypeptide”, preferably for use as a medicament, in particular for use in the therapeutic or prophylactic treatment of a coronavirus infectious disease, in particular in a subject or group of subjects suffering from or at risk of suffering from a coronavirus infectious disease, is preferred corresponding to at least one HLA antigen peptide corresponding to MHC class | comprising the following scaffold amino acid sequence:

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

(a) a long peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which comprises one “HLA-A antigen peptide” or “HLA-B antigen peptide” as defined herein, preferably one native “HLA-A antigen peptide” or “HLA-B antigen peptide”, most preferably one “HLA-A antigen peptide” or “HLA-B antigen peptide” selected from the group consisting of SEQ ID NOs: 1 — 37 and 59 — 79, wherein when the amino acid sequence comprises an aforementioned HLA antigen peptide, the amino acid sequence comprises, in addition to the "HLA-A antigen peptide" or "HLA-B antigen peptide", up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or (b) a similarity peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), wherein the “HLA-A antigen peptide” or “HLA-B antigen peptide” as defined in item (a) having less than 100% sequence identity or similarity to the native HLA antigen peptide, such as having at least 85%, more preferably at least 90%, sequence identity; and/or (c) a substitution peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), wherein the “HLA-A antigen peptide” or “HLA-B antigen peptide” as defined in item (a) having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence of the native HLA antigen peptide; and/or (d) a tandem peptide, i.e. a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-B antigen peptide sequences, both as defined in items (a) to (c), in which the HLA antigen peptides are optionally connected to each other by suitable linkers, more preferably by a suitable amino acid linker (so-called oligopeptides); and/or (e) an overlapping tandem peptide, i.e. a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-

According to one preferred embodiment, one of the aforementioned amino acid sequences of the antigen polypeptide, in particular as defined in items (a) to (e), may consist in total of up to (but not more than) 10 to 40, more preferably up to 10 to 30, most preferably up to 10 to 25

PMCR GmbH

PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung amino acids, which has the advantage that the stability of the incorporated HLA-A antigen peptides and/or HLA-B antigen peptides is increased thereby. In particular, the addition of amino acids at the C- and/or N-terminus increases the stability (depending on the method of administration e.g., in the bloodstream, in muscle tissue, in skin tissue) after administration to a subject, as they may be undergoing partial degradation from the C- and/or N-terminus after administration.

In a preferred embodiment, the C-terminus and/or N-terminus of the antigen polypeptide comprises so called “capping groups”. A preferred capping group at the N-terminus can be an acetyl group. A preferred capping group at the C-terminus can be an amide group. These capping groups not only increase metabolic stability but may also increase in vitro binding affinities.

According to one preferred embodiment, one of the aforementioned amino acid sequences of an antigen polypeptide, in particular as defined in items (a) to (e), that may consist of up to (but not more than) 10 to 40, more preferably up to 10 to 30, most preferably up to 10 to 25 amino acids, additionally comprises so called “anchor amino acids” or “anchor residues” that strengthen the linkage to an HLA allele. For examples the antigen polypeptide as defined herein comprises one or more HLA-A antigen peptides and/or HLA-B antigen peptides flanked by an anchor amino acid in C-terminal and/or N-terminal position. Examples for anchor amino acids are acidic amino acids like D and/or E at the C-terminus and/or N-Terminus of the long

HLA antigen peptide and/or hydrophobic amino acids like A, F, G, |, L, M, P, V and/or W in the center of the long HLA antigen peptide. Furthermore, those anchor residues include phosphorylated peptides, which can be recognized by T cells.

According to one preferred embodiment, the pharmaceutical composition comprises at least one tandem antigen polypeptide as defined in item (d) and/or one overlapping tandem antigen polypeptide as defined in item (e) that comprises at least two different HLA antigen peptides, such as (i) one HLA-A antigen peptide and at least one HLA-B antigen peptide or (ii) at least two HLA-A antigen peptide or (iii) at least two HLA-B antigen peptide. This has the advantage that multiple of targeting multiple HLA alleles. Thus, the inventors have found that although certain genotypes encode specific HLA alleles, these are not expressed and/or presented on the cells and are therefore not accessible. Furthermore, this allows different genotypes to be addressed by the administration of a single "antigen polypeptide" (as defined herein).

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

In addition, a tandem antigen polypeptide as defined in item (d) or an overlapping tandem antigen polypeptide as defined in item (e) can be tailored in such a way that one antigen polypeptide comprises at least two different HLA-A and/or HLA-B antigen peptides, wherein (i) each one of the HLA-A and/or HLA-B antigen peptide located within the tandem antigen polypeptide or the overlapping tandem antigen polypeptide corresponds to an amino acid sequence located within a region of a coronavirus, such as within the spike S1 domain, S2 domain, nucleocapsid protein, envelope protein and/or

ORF1ab polyprotein, wherein the amino acid sequence of the first HLA-A and/or

HLA-B antigen peptide differs from the amino acid sequence of the second or the other HLA-A and/or HLA-B antigen peptide(s) of the antigen polypeptide, for example corresponds to and thus “target” a different region within a specific virus sub-species (such SARS-CoV 1, SARS-CoV 2, MERS-CoV). As a result, the tandem antigen polypeptide or the overlapping tandem antigen polypeptide according to the invention can be used to “target” a specific/single region of a coronavirus, especially a specific/single region of a virus sub-species (such as only the spike S1 domain, S2 domain, nucleocapsid protein, envelope protein or

ORF1ab polyprotein), but moreover for the use in broad targeting, preferably for use in the “targeting” of multiple regions. and/or (ii) each one of the HLA-A and/or HLA-B antigen peptide located within the tandem antigen polypeptide or the overlapping tandem antigen polypeptide corresponds to an amino acid sequence located within a region of a different coronavirus, such as within the spike S1 domain, S2 domain, nucleocapsid protein, envelope protein and/or ORF1ab polyprotein, wherein the amino acid sequence of the first HLA-A and/or HLA-B antigen peptide differs from the amino acid sequence of the second or the other HLA-A and/or HLA-B antigen peptide(s) of the antigen polypeptide in this way that first HLA-A and/or HLA-B antigen peptide corresponds to and “target” a first region in a first coronavirus, especially first virus sub-species (such SARS-

CoV 1, SARS-CoV 2, MERS-CoV) and the second or the other HLA-A and/or HLA-

B antigen peptide(s) corresponds to and “target” a region in a second coronavirus, especially second virus sub-species. As a result, the tandem antigen polypeptide or the overlapping tandem antigen polypeptide according to the invention can be used not only for the treatment of a specific/single coronavirus, especially specific/single virus sub-species (such as SARS-CoV 1, SARS-CoV 2, MERS-

CoV), but moreover for the use in the broad-spectrum immunization, preferably for

PMCR GmbH

PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung use in the therapeutic or prophylactic treatment of a family-spanning, preferably at least subfamily-spanning, particularly preferably genus-spanning, especially subgenus-spanning coronavirus infectious disease.

In one embodiment, the “antigen polypeptide” corresponding to at least one HLA antigen peptide corresponding to MHC class | comprises or consists of an amino acid sequence according to the following sequences: (a) an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 — 58, more preferably of SEQ ID NO: 43, 44, 50, 51, 53, 54 and/or 58, most preferably an amino acid sequence of SEQ ID NO: 43, 44, 50, 54 and/or 58, and/or (b) an amino acid sequence having less than 100% sequence identity or similarity to the amino acid sequence according to item (a), such as at least 85%, more preferably at least 90%, sequence identity (as defined herein) to an amino acid sequence of SEQ ID

NOs: 38 — 58, more preferably of SEQ ID NO: 43, 44, 50, 51, 53, 54 and/or 58, most preferably to an amino acid sequence of SEQ ID NO: 43, 44, 50, 54 and/or 58; and/or (c) an amino acid sequence, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence(s) selected from the group consisting of SEQ ID

NOs: 38 — 58, more preferably of SEQ ID NO: 43, 44, 50, 51, 53, 54 and/or 58, most preferably an amino acid sequence of SEQ ID NO: 43, 44, 50, 54 and/or 58; and/or (d) a tandem peptide, i.e., a compound, construct, or polypeptide comprising or consisting of at least two identical or different peptide sequences of at least one sequence as defined in items (a) to (c) above, in which the amino acid sequences are optionally linked to each other by suitable linkers (so-called oligopeptides); and/or (e) an overlapping tandem peptide comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-B antigen peptide sequences (as defined herein), encoding for at least two, different HLA-A and/or HLA-

B antigen peptides (as defined herein), wherein the at least two HLA antigen peptides overlap in their amino acid sequence.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

In one preferred embodiment, the pharmaceutical composition comprises at least one amino acid sequence according to the following sequences: (a) an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 — 58; and/or (b) an amino acid sequence having less than 100% sequence identity or similarity to the amino acid sequence according to item (a), such as having at least 85%, more preferably at least 90%, sequence identity (as defined herein) to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 — 58; and/or (c) an amino acid sequence, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence(s) selected from the group consisting of SEQ ID

NOs: 38 — 58; and/or (d) a tandem peptide, i.e., a compound, construct, or polypeptide comprising or consisting of at least two identical or different peptide sequences of at least one sequence as defined in items (a) to (c) above, in which the amino acid sequences are optionally linked to each other by suitable linkers (so-called oligopeptides); and/or (e) an overlapping tandem peptide comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-B antigen peptide sequences (as defined herein), encoding for at least two, different HLA-A and/or HLA-

In one embodiment, the pharmaceutical composition comprises (a) an amino acid sequence of SEQ ID NO: 43, 44, 50, 51, 53, 54 and/or 58, more preferably an amino acid sequence of SEQ ID NO: 43, 44, 50, 54 and/or 58, or the amino acid sequence having at least 85%, more preferably at least 90%, sequence identity (as defined herein) to an amino acid sequence of SEQ ID NO: 43, 44, 50, 51, 53, 54 and/or 58, more preferably an amino acid sequence of SEQ ID NO: 43, 44, 50, 54 and/or 58; and/or

PMCR GmbH

PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

(b) an amino acid sequence having less than 100% sequence identity or similarity to the amino acid sequence according to item (a), such as at least 85%, more preferably at least 90%, sequence identity (as defined herein) to an amino acid sequence of SEQ ID

NO: 43, 44, 50, 51, 53, 54 and/or 58, more preferably to an amino acid sequence of

SEQ ID NO: 43, 44, 50, 54 and/or 58; and/or (c) an amino acid sequence, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence(s) selected from the group consisting of SEQ ID

NO: 43, 44, 50, 51, 53, 54 and/or 58, more preferably an amino acid sequence of SEQ

ID NO: 43, 44, 50, 54 and/or 58; and/or (d) a tandem peptide, i.e., a compound, construct, or polypeptide comprising or consisting of at least two identical or different peptide sequences of at least one sequence as defined in items (a) to (c) above, in which the amino acid sequences are optionally linked to each other by suitable linkers (so-called oligopeptides); and/or (e) an overlapping tandem peptide comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-B antigen peptide sequences (as defined herein), encoding for at least two, different HLA-A and/or HLA-

A preferred aspect of the present invention are HLA antigen peptides corresponding to MHC class | complexes, in particular for use in the treatment or prophylaxis of a coronavirus infectious disease such as SARS-CoV-2 in a person or group of persons who has contracted or is at risk of contracting a coronavirus such as SARS-CoV-2 and/or for a pharmaceutical composition according to the invention, wherein the HLA-A antigen peptides or HLA-B antigen peptides are preferably selected from the group consisting of the amino acid sequences shown in Table 1-6 or which have at least one mutation, preferably an amino acid substitution, with respect to one of these amino acid sequences.

Particularly preferred herein is the use of the foregoing HLA antigen peptides and/or antigen polypeptides for use in a method of treating SARS-CoV-2 in a patient or group of patients having at least one identical HLA allele, the method comprising administering/applying a treatment regimen to a subject or group of subjects such that a pharmacologically effective amount comprises at least one of the foregoing HLA antigen peptides.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

In one embodiment, a pharmaceutical composition as defined herein is for use in therapeutic or prophylactic treatment of a coronavirus infectious disease in a living subject, wherein the therapeutic or prophylactic treatment comprises administering an effective amount of the pharmaceutical composition to a subject and/or group of subjects.

In one embodiment, the HLA-A and/or HLA-B antigen peptide(s) corresponding to MHC class complexes of the at least one antigen polypeptides, preferably as used in the pharmaceutical composition, is/are an amino acid sequence selected from the group consisting of SEQ ID NO: 1-37, 59 - 79.

In one embodiment, the antigen polypeptide(s), preferably as used in the pharmaceutical composition, is/are an amino acid sequence selected from the group consisting of SEQ ID

NOs 38 — 58.

In one embodiment, the pharmaceutical composition further comprises at least one amino acid sequence comprising or consisting of an HLA-A or HLA-B antigen peptide, wherein the HLA peptide corresponds, preferably is identical to at least one amino acid sequence in the coronavirus, such as spike S1 and S2 domain, nucleocapsid protein, envelope protein and/or

ORF1ab polyprotein. Preferably, the HLA-A or HLA-B antigen peptide comprises or consisting of an amino acid sequence as defined herein, more preferably, the amino acid sequence is selected from the group consisting of SEQ ID NO: 1 — 37, 59 — 79.

An "HLA-A antigen peptide corresponding to MHC class | complexes" is defined herein as an "HLA-A antigen peptide of the invention” (also called “HLA-A antigen peptide”) or "amino acid sequence of the invention" (as defined herein) comprising: (a) an amino acid sequence preferably consisting of 6 to 12 amino acids, more preferably consisting of 7 to 10 amino acids, and most preferably consisting of 8 or 9 amino acids corresponding to MHC class | complexes which is similar or more preferably identical to a protein encoding RNA sequence in the genome of a coronavirus, such as SARS-

CoV-2, for example the spike glycoprotein, nucleocapsid phosphoprotein, envelope protein, surface glycoprotein and ORF1ab polyprotein. (b) preferably, the HLA-A antigen peptide is present in the pharmaceutical composition according to the invention at a concentration, as defined above, of at least 100 ug and particularly preferably at least 500 pg, alternatively preferably at an absolute

PMCR GmbH

PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung concentration of at least 600 ug relative to the volume of the pharmaceutical composition to be applied.

Preferably, the HLA-A antigen peptides are selected as defined above under point (b).

According to a particularly preferred embodiment, an "HLA-A antigen peptide", is preferred, comprising the following scaffold sequence: (a) an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-7, 9, 16, 18-19, 21-23, 27, 29-31, 33- 34, 36, wherein when the amino acid sequence comprises an aforementioned amino acid sequence, the amino acid sequence preferably further comprises up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or (b) an antigen peptide comprising or consisting of an amino acid sequence having less than 100% sequence identity or similarity to the native HLA-A antigen peptide, such as having at least 85%, more preferably at least 90%, sequence identity (as defined herein) to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-7, 9, 16, 18-19, 21-23, 27, 29-31, 33-34, 36, wherein when the amino acid sequence comprises an aforementioned amino acid sequence, the amino acid sequence preferably further comprises up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or (c) an antigen peptide having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence(s) selected from the group consisting of SEQ ID NOs: 1, 3-7, 9, 16, 18-19, 21-23, 27, 29-31, 33- 34, 36, wherein when the amino acid sequence comprises an aforementioned amino acid sequence, the amino acid sequence preferably further comprises up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or (d) a long peptide, i.e., a compound, construct, or polypeptide, the amino acid sequence of which comprises at least one antigen peptide as defined in items (a) to (c) above; and/or (e) a tandem peptide, i.e., a compound, construct, or polypeptide comprising or consisting of at least two identical or different peptide sequences of at least one HLA-A antigen peptide and one HLA-A and/or HLA-B antigen peptide, preferably both as defined in

PMCR GmbH

PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung items (a) to (c), in which the HLA antigen peptides are optionally linked to each other by suitable linkers (so-called oligopeptides); and/or

Very preferably, HLA-A antigen peptides are as defined in (b), (d) and (e), respectively. Also in the embodiment according to (e), the HLA-A antigen peptides are preferably defined as described under (a) or (d). In the case that the HLA-A antigen peptides as defined under (e) are linked to each other via a linker, suitable linkers are known to the skilled person from the prior art.

That one of the aforementioned amino acid sequences, in particular as defined in items (a) to (c), may further comprise up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids in addition to the HLA-A antigen peptide, has the advantage that the stability of the HLA-A antigen peptides is increased thereby. In particular, this increases the stability after administration to a subject, as they may be undergoing partial degradation from the C- and/or N-terminus after application.

In a particular embodiment of the present invention, one of the aforementioned amino acid sequences, in particular as defined in items (a) to (c), comprises of preferably up to (but not more than) 3 HLA-B antigen peptides, more preferably up to two HLA-B peptides, and further comprise up to (but not more than 1 to 20, more preferably 1 to 5 most preferably 1 to 10 amino acids. This has the particular advantage that more than one viral epitope can be targeted with a single amino acid sequence in accordance to the present invention, thus increasing a potential B/T cell response.

The use of compounds, constructs or polypeptides as defined under (e) according to the invention, which consist of at least two identical or different peptide sequences of HLA antigen peptides, has the further advantage that the longer amino acid sequences of the compounds, constructs, proteins or polypeptides result in a longer retention time in the tissue of the subject after application, whereby the compounds, constructs, proteins or polypeptides after application to the subject can, for example by endogenous enzymes into smaller fragments (pharmaceutically active form comprising at least 6 to 12 amino acids) which have a biologically desired function in the sense of the invention. That is, the individual fragments of the oligopeptide exhibit activity as HLA-A and/or HLA-B antigen peptides and thus contribute tothe activation of T cells.

In a particular embodiment, any HLA-A peptide sequence may be a humanized and/or sequence optimized sequence as further described herein.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

An "HLA-B antigen peptide corresponding to MHC class | complexes" is defined herein as an "HLA-B antigen peptide of the invention” (also called “HLA-B antigen peptide”) or "amino acid sequence of the invention" (as defined herein), which comprises the following: (a) an amino acid sequence preferably consisting of 6 to 12 amino acids, more preferably consisting of 7 to 10 amino acids, and most preferably consisting of 8 or 9 amino acids corresponding to MHC class | complexes which is similar or more preferably identical to a protein encoding RNA sequence in the genome of a coronavirus, such as SARS-

CoV-2, for example the spike glycoprotein, nucleocapsid phosphoprotein, envelope protein, surface glycoprotein and ORF1ab polyprotein. (b) preferably, the HLA-B antigen peptide is present in the pharmaceutical composition according to the invention at a concentration, as defined above for the HLA-A antigen peptide, of at least 100 ug, alternatively in absolute concentration of preferably at least 600 pg relative to the volume of the pharmaceutical composition to be applied.

Preferably, the pharmaceutical composition contains (per vial) 600 ug x12 = 7,200 pg of antigen peptides.

Preferably, the HLA-B antigen peptides are selected as defined above in point (b).

According to a particularly preferred embodiment, an "HLA-B antigen peptide", preferably comprising the following scaffold sequence: (a) an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 10-15, 17, 20, 24-26, 28, 31-32, 35, 37, wherein when the amino acid sequence comprises an aforementioned amino acid sequence, the amino acid sequence preferably further comprises up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or (b) an antigen peptide comprising or consisting of an amino acid sequence having less than 100% sequence identity or similarity to the native HLA-B antigen peptide, such as having at least 85%, more preferably at least 90%, sequence identity (as defined herein) to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 10-15, 17, 20, 24-26, 28, 31-32, 35, 37, wherein when the amino acid sequence comprises an aforementioned amino acid sequence, the amino acid sequence preferably further comprises up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

(c) an antigen peptide having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence(s) selected from the group consisting of SEQ ID NO: 2, 8, 10-15, 17, 20, 24-26, 28, 31-32, 35, 37, wherein when the amino acid sequence comprises an aforementioned amino acid sequence, the amino acid sequence preferably further comprises up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids; and/or (d) along peptide, i.e., a compound, construct, or polypeptide whose amino acid sequence comprises at least one antigen peptide as defined in items (a) through (c) above; and/or (e) a tandem peptide, i.e. a compound, construct, or polypeptide comprising or consisting of at least two identical or different peptide sequences of at least one HLA-B antigen peptide and one HLA-A and/or HLA-B, preferably both as defined in items (a) to (c), in which the HLA antigen peptides are optionally connected to each other by suitable linkers (so-called oligopeptides).

Very preferably, HLA-B antigen peptides are as defined in (a), (b), (c) and (e), respectively.

Also in the embodiment according to (e), the HLA-B antigen peptides are preferably defined as described in items (a) through (c). In the case that the HLA-B antigen peptides as defined under item (e) are linked to each other via a linker, suitable linkers are known to the person skilled in the art from the prior art.

That one of the aforementioned amino acid sequences, in particular as defined in items (a) to … (e), may further comprise up to (but not more than) 1 to 30, more preferably 1 to 20, most preferably 1 to 15 amino acids in addition to the HLA-B antigen peptide, has the advantage that the stability of the HLA-B antigen peptides is increased thereby. In particular, this increases the stability after administration to a subject, as they may be undergoing partial degradation from the C- and/or N-terminus after application.

In a particular embodiment of the present invention, one of the aforementioned amino acid sequences, in particular as defined in items (a) to (c), comprises of preferably up to (but not more than) 3 HLA-B antigen peptides, more preferably up to two HLA-B peptides, and further comprise up to (but not more than 1 to 20, more preferably 1 to 15 most preferably 1 to 10 amino acids. This has the particular advantage that more than one viral epitope can be targeted with a single amino acid sequence in accordance with the present invention, thus increasing a potential B/T cell response. The use of compounds, constructs, proteins or polypeptides as defined under (e) according to the invention, which consist of at least two identical or different peptide sequences of HLA antigen peptides, has the further advantage

PMCR GmbH

PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung that the longer amino acid sequences of the compounds, constructs, proteins or polypeptides result in a longer retention time in the tissue of the subject after application, whereby the compounds, constructs, proteins or polypeptides after application to the subject can, e.g. by endogenous enzymes into smaller fragments (pharmaceutically active form, comprising at least 6 to 17 amino acids), which have a biologically desired function in the sense of the invention, by macrophages and dendritic cells. That is, the individual fragments of the oligopeptide exhibit activity as HLA-A and/or HLA-B antigen peptides and thus contribute to the activation of T cells.

In a particular embodiment, any HLA-B peptide sequence may be a humanized and/or sequence optimized sequence as further described herein.

In a particularly preferred embodiment, the HLA-A and/or HLA-B antigen peptides of the present invention are selected in such a way, that they match or correspond to coronavirus, preferably betacoronaviruses, more preferably sarbecovirus, most preferably SARS-CoV-2 epitope regions, preferably spike S1 domain, S2 domain, nucleocapsid protein, envelope protein and/or ORF1ab polyprotein, which are highly conserved across different virus variants, preferably in at least 2 different virus variants, more preferably in at least 4 different virus variants, most preferably in at least 6 different virus variants. This has the technical effect, that the combination preparation relating to the aforementioned antigen peptides is less susceptible against RNA mutations in new strains and variants of the SARS-CoV-2 epitope, such as the

Alpha (B.1.1.7), or Beta (B.1.351), or Gamma (P.1), or Delta (B.1.617.2) and/or Omicron variant (B.1.1.529) and thus allowing for a better (broadband) immunization. Correct sequence matching can be achieved by comparing the amino acid sequences of the different coronavirus variants and identifying the regions, preferably with respect to the spike S1 domain, S2 domain, nucleocapsid protein, envelope protein and/or ORF1ab polyprotein, that are (i) identical to each other or (ii) having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to each other or (iii) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to each other and screening these regions to identify appropriate

HLA-A and/or HLA-B antigen sequences. It has been found to be particularly advantageous to compare not only different variants of a subspecies of coronavirus among themselves (e.g., different mutants of SARS-CoV 2) but rather different variants within the family (coronavirus), at least within the genus (betacoronaviruses), but at least different mutants within a subgenus (sarbecovirus). This allows the HLA-A and/or HLA-B antigen peptides and/or antigen polypeptides according to the invention to be used not only for the treatment of a specific virus types (such as SARS-CoV 1, SARS-CoV 2, MERS-CoV), but moreover for the use in the

PMCR GmbH

PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung broad-spectrum immunization, preferably for use in the therapeutic or prophylactic treatment of a family-spanning, preferably at least subfamily-spanning, particularly preferably genus- spanning, especially subgenus-spanning coronavirus infectious disease.

In a preferred embodiment of the invention, the nucleocapsid protein, for example of the SARS-

CoV 2 was aligned against 5 different coronaviruses within the coronavirus family, preferably with a (multiple) sequence alignment tool, such as COBALT tool (Human coronavirus 229E,

UNIPROT reference: P15130; Human coronavirus OC43, UNIPROT reference: P33469,

Human coronavirus HKU1 (isolate N5), UNIPROT reference: Q0ZMES; Human coronavirus

HKU1 (isolate N1), UNIPROT reference: Q5MQC6; and severe acute respiratory syndrome coronavirus 2, UNIPROT reference: Q6S8E1) according to a conservation algorithm based on the relative entropy threshold of the residue. SARS-CoV-2 nucleocapsid protein positions 59- 84, 86-96, 99-123, 129-142, 155-170, 284-320, 321-336, 342-360 are highly conserved. These regions are highly preferable as target for HLA-A and/or HLA-B antigen peptide, preferably as described herein, corresponding to MHC class | complexes in accordance with the present invention.

In an alternative embodiment of the invention, the SARS-CoV-2 spike protein was aligned against 8 different coronaviruses within the coronavirus family (Human coronavirus 229E,

UNIPROT reference: P15423; Human coronavirus OC43, UNIPROT reference: P36334,

Human coronavirus HKU1 (isolate N5), UNIPROT reference: Q0ZME7; Human coronavirus

HKU1 (isolate N1), UNIPROT reference: Q5MQDO; Severe acute respiratory syndrome coronavirus, UNIPROT reference: P59594; Human Coronavirus NL63, UNIPROT reference:

Q6Q152; MERS-CoV, UNIPROT reference: K9N5Q8; and Human coronavirus HKU1 (isolate

N2), UNIPROT reference: Q14EB0). SARS-CoV-2 spike protein positions 152-211, 470-503, 534-570, 769-847, 939-1063 and 1176-1273 are highly conserved. These regions are highly preferable as target for HLA-A and/or HLA-B antigen peptide, preferably as described herein, corresponding to MHC class | complexes in accordance with the present invention.

Preferably, a monovalent amino acid sequence (or a polypeptide comprising only an amino acid sequence of the invention) used in the invention binds to a T cell receptor of endogenous

T cells with a 3-fold increased specific affinity compared to the corresponding wild-type HLA peptide sequence. It should be noted that "can specifically bind to" and "specifically binds to" are used synonymously herein and refer to the ability to bind specifically to the corresponding specified entity.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

It is possible to combine amino acid sequences belonging to different classes used in the invention into a single polypeptide of the invention. In particular, it has been demonstrated that the combination of HLA-A and/or HLA-B antigen peptides in a single polypeptide of the invention have unique binding properties (cf. Fig. 3).

By one skilled in the art, the specific activity (KD) of the polypeptides of the invention comprising more than one component of the amino acid sequence of the HLA-A and/or HLA-

B antigen peptides corresponding to the class | MHC complexes can be determined according to one of the detection methods described above/below, wherein the compounds, constructs, proteins or polypeptides of the invention preferably have a specific activity similar to the specific activity of each of their components; i.e., a specific activity similar to the specific activity of each of the (individual components of the) amino acid sequences of MHC class | contained in the compounds, constructs, or polypeptides of the invention. Some specific, but not limiting, examples of the above preferred compounds, constructs, or polypeptides are compounds, constructs, or polypeptides that either. i) a wild-type HLA antigen peptide corresponding to class | MHC complexes (HLA-A and/or HLA-B or HLA-C peptide, respectively); or ii) an antigen corresponding to class | MHC complexes (HLA-A and/or HLA-B antigen) and having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37; 59 —79 or ill) an antigen corresponding to class | MHC complexes (HLA-A and/or HLA-B antigen) and/or class || MHC complexes having an amino acid sequence having at least 80% sequence identity (as defined herein) with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-37, 59 — 79; or iv) an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence selected from the group of SEQ ID

NOs: 1-37, 59 — 79; or (v) compounds, constructs, proteins or polypeptides consisting of at least two identical or different HLA antigen peptide sequences in which the HLA peptides are optionally linked together by suitable linkers; or a suitable combination thereof.

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It should be noted that the last preceding paragraphs also generally apply to all amino acid sequences of the invention comprising one or more amino acid sequences for the HLA antigen peptides defined above.

Of course, all of the above HLA antigen peptides corresponding to Class | and Class II MHC complexes can be used and are effective for treating a coronavirus infectious disease as disclosed herein, particularly for treating SARS-CoV-2.

Some specific, but not limiting, examples of such compounds, constructs, proteins or polypeptides of the invention are set forth, for example, in Tables 1-4 or are apparent to those skilled in the art based on the present disclosure.

According to another preferred embodiment of the present invention, the HLA antigen peptide is present (in each case) as a single-membered amino acid sequence, i.e., not as an element of compounds, constructs, or polypeptides consisting of at least two identical or different amino acid sequences for HLA antigen peptides, in which the HLA antigen peptides are connected to each other by suitable linkers.

Itis also part of the present invention that an amino acid sequence of the invention can bind to two or more class | MHC complexes, epitopes, components, domains or subunits of a class

MHC complex. In such a case, the MHC complexes, epitopes, components, domains or subunits of an MHC complex to which the amino acid sequences and/or polypeptides of the invention bind may be substantially the same or different (and in the latter case, the amino acid sequences and polypeptides of the invention may bind to such different complexes, epitopes, components, domains or subunits of a class | MHC complex, including combinations thereof, with an affinity and/or specificity that may be the same or different).

It is also expected that the polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, components and fragments of a class | MHC complex. Also in such a case, the amino acid sequences and polypeptides of the invention may bind to such analogs, variants, mutants, components and fragments with an affinity and/or specificity equal to or different from the affinity and specificity with which the amino acid sequences of the invention bind to the wild-type of the class | MHC complex.

It is also within the scope of the present invention that the amino acid sequences and polypeptides of the invention bind to some analogs, variants, or mutants of a class | MHC complex, but not to other.

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Formulation of the pharmaceutical combination

According to a particularly preferred embodiment of the present invention, the pharmaceutical composition consists exclusively of a carrier liquid (preferably water, a pharmaceutically acceptable saline solution and/or pharmaceutical DMSO. Such compositions of carrier fluids are known to those skilled in the art and are, for example, in the range of preferably 5-30%

DMSO and 70-95% water. Particularly preferably, the percentage of DMSO is 25% and that of water 75%. A pharmacologically effective amount of preferably at least one and particularly up to 12 peptide complexes is dissolved or suspended in the carrier liquid and mixed with an adjuvant selected from the following 5 adjuvants. An adjuvant preferably comprises CpG (oligonucleotide of cytidine and guanosine), c-di-AMP (bis-(3',5")-cyclic-adenosine), alum (usually a mixture of aluminum sulfate, potassium or sodium hydroxide, and phosphate),

Pam3Cys-GDPKHPKSF N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteinyl-

GDPKHPKSF), and poly-IC. Other suitable adjuvants and compositions of carrier fluids are known to the skilled person from the prior art.

The pharmaceutical composition and suitable dosage forms for application of the pharmaceutically active antigen polypeptides and/or HLA antigen peptides are prepared according to standard procedures known in the prior art and are readily applicable to any new or improved process for their preparation.

Another object of the present invention is the (medicament) formulation in their dosage form containing the combination of active ingredients according to the invention and optionally further active ingredients and/or excipients.

Preferred drug formulations are transdermal therapeutic systems, solutions, injections, emulsions, suspensions, easily reconstitutable dry preparations, powders, or sprays.

Particularly preferred drug formulations are injections or solutions.

Alternatively, the drug formulation is present in a suitable application device, preferably as a lyophilizate in a syringe, which allows in situ reconstitution with a pharmaceutically acceptable solution (e.g., saline).

In one embodiment, the antigen polypeptide corresponding to at least one HLA antigen peptide corresponding to MHC class | complexes or the amino acids is formulated or is to be formulated as aliquid, a solid, or a combination thereof.

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Preferably, the (medicament) pharmaceutical composition/formulation according to the invention is suitable for intravenous, intramuscular, subcutaneous, intrathecal, epidural, transdermal or nasal administration, with (medicament) formulations for subcutaneous or intravenous administration being particularly preferred. In a particularly preferred embodiment of the invention, the pharmaceutical composition is applicated subcutaneous. This has the technical advantage of reduced antigen peptide degradation and a depot forming effect and thus, the antigen polypeptide is slowly delivered to the subject..

In one embodiment, the HLA antigen peptide corresponding to MHC class | complexes and/or the antigen polypeptides are formulated or are to be formulated for injection.

In one embodiment, the HLA antigen peptide corresponding to MHC class | complexes and/or the antigen polypeptides are formulated or are to be formulated for intramuscular administration.

In one embodiment, the pharmaceutical composition/formulation is a medical composition. In one embodiment, the pharmaceutical composition/formulation is a vaccine.

In one embodiment, the HLA antigen peptide and/or the antigen polypeptide is formulated or is to be formulated as particles.

In one preferred embodiment, each single HLA antigen peptide described herein, preferably each antigen polypeptide, if it is part of the pharmaceutical composition, for the therapeutic or prophylactic treatment of a coronavirus infectious disease, especially for the prophylactic treatment, may be administered at an absolute concentration per dose of the pharmaceutical composition (i.e., administration dose) in a range from 10 to 1,000 pg, preferably from 100 to 700 pg, preferably from 300 to 600 pg, such as up to 150 ug, up to 200 pg, up to 250 ug, up to 350 ug, up to 400 pg, up to 450 ug, up to 500 pg or up to 550 ug may be administered. In one embodiment, the invention relates to a single dose administration. In one embodiment, the invention relates to the administration of a priming dose followed by one or more booster doses. The booster dose or the first booster dose may be administered 7 to 28 days or 14 to 24 days following administration of the priming dose.

In one preferred embodiment, each single HLA antigen peptide described herein, preferably each antigen peptide, if it is part of the pharmaceutical composition, for the therapeutic or prophylactic treatment of a coronavirus infectious disease, especially for the therapeutic treatment, may be administered at an absolute concentration per dose of the pharmaceutical

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung composition (i.e., administration dose) of at least 100 pg and particularly preferably at least 500 pg, alternatively preferably at an absolute concentration of at least 600 ug, i.e., containing at least in the range of 700 to 1,200 ug, preferably from 800 to 1,200 pg, is particularly preferred, as this greatly intensifies the therapeutic effect. This is particularly advantageous if the immune system of the subject to be treated is already weakened by other pre-disease, e.g., cardiovascular disease, diabetes, chronic cardiopulmonary disease, hypertension, chronic renal disease, chronic pulmonary disease, AIDS/HIV, any malignancy, lymphoma or metastatic solid tumor and possibly associated pretreatment thereof, or if the subject already suffers from a corona infection. Irrespective of the above, an increased absolute concentration per HLA antigen peptide is also preferred, as this substantially minimizes the influence of degradation of the HLA antigen peptides (e.g., by ligases) after their administration to the subject.

In one embodiment, administration of the pharmaceutical composition as described herein, more preferably administration of the pharmaceutical composition with an administration dose as described herein, most preferably after single dose administration, particularly after second dose administration, most particularly after third dose administration of the pharmaceutical composition with an administration dose as described herein results in an IFNy activity in the blood serum of the subject in a range of at least 50 to 500, more preferably a range of at least 100 to 400, most preferably a range of 100 to 250, determined via IFNy ELISpot analysis as described herein.

In one embodiment, administration of the pharmaceutical composition as described herein, more preferably administration of the pharmaceutical composition with an administration dose as described herein, most preferably after single dose administration, particularly after second dose administration, most particularly after third dose administration of the pharmaceutical composition with an administration dose as described herein results in an IFNy cell count per 100.000 cells in the blood serum of the subject in a range of at least 25 to 800 cells, more preferably a range of at least 50 to 600 cells, most preferably a range of 50 to 500 cells, determined via IFNy ELISpot analysis as described herein.

In one embodiment, the pharmaceutical composition/formulation is a kit. Thus, in one embodiment, the antigen peptide corresponding to MHC class | complexes or the antigen polypeptide and optionally the carrier liquid, a pharmaceutically acceptable adjuvant, carrier, diluent and/or excipient are in separate vials.

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Methods known in the prior art for the preparation of pharmaceutical compositions or dosage forms are found in, for example, "Remington's Pharmaceutical Sciences". Pharmaceutical compositions for parenteral administration may contain, for example, excipients, sterile water, or saline, polyalkylene glycols, such as polyethylene glycols, oils of plant, animal, or microbial origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems for therapeutic anti-prion compounds, for example, include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

In other embodiments, the present invention also relates to a pharmaceutical composition, a kit (or parts thereof), a method for determining/identifying a pharmacologically active HLA antigen peptide corresponding to class | MHC complexes and/or antigen peptide, a method for preparing a formulation according to the invention, and the use of a formulation according to the invention for the preparation of a pharmaceutical composition for the treatment and prevention of coronavirus, more preferred of SARS-CoV-2 infections.

Pharmaceutical compositions are prepared by dissolving or suspending the determined HLA antigen peptides and/or antigen polypeptides in a carrier fluid (i.e., a pharmacologically acceptable vehicle), optionally with the addition of other excipients such as wetting agents, dyes, permeation enhancers, resorption enhancers, preservatives, antioxidants, light stabilizers.

The carrier liquid is preferably selected from the group consisting of sodium chloride injection solution, Ringer's injection solution, isotonic dextrose, sterile water, dextrose solution, lactated

Ringer's injection solution, distilled water or mixtures thereof, for local injection.

It is particularly advantageous for good solubility of the determined HLA antigen peptides if their amino acid sequence has the lowest possible number of hydrophobic amino acids.

Preferably, dimethyl sulfoxide (DMSO), Diethylene glycol diethyl ether, ethanol, phosphatidylcholines, propylene glycol dipelargonates (DPPG), or glycolyzed ethoxylated glycerides are suitable permeation promoters.

According to a preferred embodiment of the present invention, the pharmaceutical composition comprises water, a pharmaceutically acceptable saline solution, and/or DMSO. For example,

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung pharmaceutical compositions comprising a mixture of, for example, 25% DMSO and 75% water are suitable for application.

Process for the Determination of pharmaceutically active peptides

Also encompassed by the present invention is a method for determining pharmaceutically active HLA antigen peptides and/or antigen polypeptides for use in the treatment or prophylaxis of SARS-CoV-2 infectious diseases or in a pharmaceutical composition according to the invention, said method comprising the following steps: (a) Bioinformatic expert system characterization and prediction of immunogenic, ADE-avoiding and B/T cell response balancing SARS-CoV-2 peptides. Bioinformatic analyses of mechanistic

ADE hypotheses are used to clarify open questions about molecular mechanisms of ADE.

Bioinformatic 3D modeling and binding prediction can test the effect of different antibodies on the conformational change of the SARS-CoV-2 spike protein and select peptides that are less likely to trigger ADE. Artificial intelligence is used for information processing, and the mathematical modeling and biotechnological advancements based on it are used for experimental analysis on the causality of a pharmaceutical composition of the vaccine. This allows existing knowledge, including that within the existing COVID-19 Disease Map

Collaboration, to be extracted from the extensive literature and tested for gaps through modeling: Knowledge gaps can be narrowed by simulation-based hypothesis testing to the point where experimental testing of the remaining variants becomes feasible.

ADE and B/T cell response balancing SARS-CoV-2 peptides was made using a variety of in silico methods, 3D modeling, and binding prediction test the effect of different antibodies on the conformational change of the SARS-CoV-2 spike protein and select peptides that are less likely to trigger ADE. Artificial intelligence such as the neural network NNAlign used in the publicly available NetMHC-4.0 server is used for information processing, and the mathematical modeling and biotechnological advancements based on it are used for experimental analysis on the causality of a pharmaceutical composition of the vaccine. From the dataset of the

SARS-CoV-2 virus genome (especially Wuhan HU-1, Wuhan HU-1, nucleoprotein, UNIPROT

Reference ID sequence: PODTC9, and spike protein, UNIPROT Reference ID sequence:

PODTC2) nonameric variants involving amino acid exchange were determined with respect to 12 of the highest affinities (specific activity toward T cell receptor [nM]) based on the patient's alleles. Also, polymers of 17 amino acids (oligopeptides) were determined under the affinity criterion for this purpose. This selection criterion is used to algorithmically determine the probability of presenting the respective antigen peptide corresponding to the MHC complexes

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung in vivo on the corresponding MHC complexes (a 15 second requirement for a possible cellular immune response). b) Mathematical model for causality analysis of the relationship between vaccine assembly and ADE.

The likelihood of certain peptides being presented as epitopes on MHC-I and eliciting a T cell response can be predicted by various algorithms, each of which has low to moderate specificity. Similarly, sequence-based predictions of 3D protein structures with a few angstroms of resolution are possible if the respective sequence forms stable secondary structures and has at least sufficient homology to sequences with known structure.

Bioinformatic predictability combines the evaluation of individual algorithms with the optimization of the parameters of the algorithms as well as the combination of algorithms. In addition, artificial intelligence optimizes the identification of significant predictive information and association. (c) An Al and machine learning-based system for recognizing information links in scientific texts, integrating tables and figures during evaluation, and identifying relevant answers to specific questions.

Natural language processing (NLP) such as Natural Language Toolkit (NLTK) is used to extract further Information fo reinforce the predictions from Steps (a) and {b). The combination of artificial intelligence and machine learning methods, for example "Natural Language

Processing” (NLP), structure recognition, deep learning etc, is used to learn patterns that allow information linkage to be identified or that allow tables and figures to be included in the evaluation. Thus, relevant information on molecular and cellular interaction networks between coronaviruses and the host can be extracted from the total amount of scientific literature and related to ADE.

Mathematical modeling approaches are nowadays successfully used to study cellular processes such as signal transduction, gene regulation and metabolism. Information on ADE collected using artificial intelligence is translated into a mathematical model that maps the molecular mechanisms. (d) Biotechnology platforms for high-resolution, conformation-specific SARS-CoV-2 antibody characterization, cellular immune response characterization, and vaccine design optimization.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

In the development of peptide immunizations, both B-cell activating and different T-cell activating peptides can be heuristically designed. Subsequently, screening with high-resolution analysis of antibodies induced by the vaccines is required to determine both neutralization capacity and epitope specificity.

In an alternative embodiment of the present invention, the amino acid sequences of the HLA antigen peptides of the SARS-CoV-2 corresponding to the MHC complexes (nonamers) with the highest allelic affinities (specific activity towards T cell receptor [nM]) were first selected from the dataset of the SARS-CoV-2 virus proteins (nucleoprotein, UNIPROT Reference ID sequence: PODTC9, and spike protein, UNIPROT Reference ID sequence: PODTC2) based on the amino acid sequences of the respective proteins. The highest conservative regions in relation with other human coronaviruses and non-variant mutations were selected from proteins with a combination of different bioinformatic methods to further analyze. To this scope, the Nucleocapsid protein was aligned against 5 coronaviruses with COBALT tool (Human coronavirus 229E, UNIPROT reference: P15130; Human coronavirus OC43, UNIPROT reference: P33469, Human coronavirus HKU1 (isolate N5), UNIPROT reference: Q0ZMES3;

Human coronavirus HKU1 (isolate N1), UNIPROT reference: Q5MQC6; and severe acute respiratory syndrome coronavirus 2, UNIPROT reference: Q6S8E1) according to a conservation algorithm based on the relative entropy threshold of the residue. SARS-CoV-2 nucleocapsid protein positions 59-84, 86-96, 99-123, 129-142, 155-170, 284-320, 321-336, 342-360 are highly conserved. These regions are used for further binding affinity prediction.

SARS-CoV-2 spike protein was aligned against 8 coronaviruses (Human coronavirus 229E,

UNIPROT reference: P15423; Human coronavirus OC43, UNIPROT reference: P36334,

N2), UNIPROT reference: Q14EB0). SARS-CoV-2 spike protein positions 152-211, 470-503, 534-570, 769-847, 939-1063 and 1176-1273 are highly conserved. then, the selection criterion is used to algorithmically determine the probability with which the respective HLA antigen peptide is presented in vivo on the corresponding MHC complexes of the SARS-CoV-2 virus (a first prerequisite for a possible T cell immune response). This has the technical effect, that regions with the highest conservative character have a lesser probability of mutations across new coronavirus, such as SARS-CoV-2 virus, and thus allowing for an immunization against new variants.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

Process for the preparation of a pharmaceutical composition

The present invention further comprises a method for preparing a pharmaceutical composition or formulation according to the invention, said method comprising the following steps: (a) determining at least one HLA antigen peptides corresponding to MHC class complexes and/or at least one antigen polypeptide exposed on the cell surface of cells of the SARS-CoV-2 of the patient or group of patients to be treated having the same haplotype, using the determination method according to the invention as described above; (b) synthesizing the HLA antigen peptide(s) determined in step (a) according to the MHC class | complexes and/or at least one antigen polypeptide, wherein the definition of each HLA antigen peptide is the same as defined above; and (c) preparing the pharmaceutical composition according to the invention comprising at least the HLA antigen peptides synthesized in step (b) corresponding to the MHC class | complexes and/or at least one antigen polypeptide and an adjuvant as defined herein.

Also encompassed by the invention is a pharmaceutical composition comprising an antigen peptide as defined herein and a pharmaceutically acceptable excipient.

The invention also relates to an HLA antigen peptide and/or antigen polypeptide of the invention for use in the preparation of a formulation (such as, without limitation, a pharmaceutical formulation as further described herein) for the treatment of SARS-CoV-2 infectious disease, either in vitro (e.g. in an invitro or cellular detection method) or in vivo (such as in a unicellular or multicellular organism and more particularly in a mammal and more particularly in a human being, such as a human being at risk of contracting or suffering from

SARS-CoV-2 infectious disease of the inventions).

Detailed Description and Definitions

In the present description and claims, the following terms are defined as follows:

In the context of the present invention, the features of the invention defined by "comprising" are intended to include the more limited description of "consisting of" or "consisting essentially of" the same features of the present invention.

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

The term "and/or" is used to specifically disclose the two features or components together or separately. Therefore, the term "and/or" as used, for example, in the phrase "I and/or II" in the present disclosure includes "I and II", "I or II", "I" and "II".

For purposes of the present invention, an HLA antigen peptide is one if it is a SARS-CoV-2 exclusive HLA antigen peptide.

An immunogenic HLA antigen peptide is also referred to herein as an "epitope."

A pharmaceutical composition is herein to be understood as a so-called informatic that can be administered to a person or a group of persons having at least one identical HLA allele and that contains the combination of HLA antigen peptides according to the invention in the concentration disclosed herein, wherein an HLA antigen peptide represents an information carrier. This means that a sequence-specific activation of the immune system, in particular of

T cells (by HLA antigen peptides corresponding to MHC class | complexes) and/or B cells (by

HLA antigen peptides corresponding to MHC class Il complexes) is induced by the arrangement of amino acids in the amino acid sequence of the HLA antigen peptides ("code").

The primary objective of the present invention is to induce targeted activation and training of the immune system, in particular of T cells and B cells against SARS-CoV-2 viruses. For this reason, the pharmaceutical composition according to the invention is preferably applied intradermally. This has the particular advantage that the immune system or the T cells, which recognize the applied HLA antigen peptides, process this information applied in the form of antigen peptides and consequently specifically recognize and lyse body cells affected by viruses which present these HLA antigen peptides on their surface. At least one HLA antigen peptide within the scope of the present invention is particularly formulated for intradermal administration. The expression/term "composition" therefore refers to the provision of at least one HLA antigen peptide and an adjuvant in a pharmaceutical formulation that allows good applicability and includes solutions, in particular injection solutions, concentrates for the preparation of injection preparations, or powders for the preparation of injection preparations.

As used herein, the term "class | HLA antigen peptide (corresponding to MHC complexes)” means a peptide sequence that is bound to or immunogenic for the class | MHC complex (in humans, the HLA complex). The class | HLA protein complex is used for antigen presentation onthe cell surface and comprises a heavy chain with 3 domains (a1, a2, and a3) and the B2- microglobulin (2M).

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung

As such, the polypeptides and pharmaceutical compositions of the present invention (as defined herein) may be used for use in the prevention and treatment of SARS-CoV-2 (also referred to herein as "infectious disease"). In general, the "infectious diseases of the invention" may be defined as diseases and disorders that can be appropriately prevented and/or treated by appropriate administration of either an HLA antigen peptide or a pharmaceutical composition of the invention (and, more particularly, a pharmaceutically effective amount thereof) to a subject (i.e., a person having the disease or disorder, or at least one symptom thereof, and/or who is at risk of acquiring or developing such disease or disorder).

For purposes of the present invention, a "peptide sequence” (e.g., an HLA antigen peptide) having a "native sequence" comprises a peptide sequence having the same (i.e., unmodified) amino acid sequence as a naturally occurring peptide sequence in the subject. Such a peptide sequence with a "native sequence" can be isolated from nature or produced recombinantly or synthetically. In particular, the term peptide sequence having a "native sequence" includes naturally occurring truncated or secreted forms of the peptide sequence (e.g., an extracellular domain sequence), naturally occurring variants (e.g., alternatively spliced forms), and naturally occurring allelic variants of the peptide sequence.

In this context, the pharmaceutical composition is preferably applied subcutaneously ("under the skin"), or intradermally ("into the skin"), or intramuscularly ("into the muscle"). The application may be intramuscular as an injection into the thigh (vastus lateralis muscle), preferably into the upper arm (deltoid muscle).

As further described herein, the amino acid sequences used in the invention are single variable

HLA antigen peptide domains ("HLAs" or "HLA complex"). A single variable HLA antigen peptide domain is (as further defined herein) a region within the amino acid sequence of a protein that can be distinguished from its surrounding sequence based on defined characteristics.

Amino acid sequences, or regions within the amino acid sequence of a protein of the invention, that are HLAs are also referred to herein as "HLAs of the invention." Some preferred examples of single variable HLA antigen peptide domains suitable for use in the invention are apparent from the further description herein, and particularly include HLA-A and/or HLA-B antigen peptides corresponding to MHC class | complexes.

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The following terms are used to describe sequence relationships between two or more amino acid sequences or polypeptide sequences: "reference sequence," "amino acid exchange," "sequence identity," "percentage of sequence identity," and "substantial identity."

In the context of the present invention, the term “coronavirus” is used preferably to describe a orthocoronavirinae, more preferably to describe betacoronaviruses, particularly preferably to describe a sarbecovirus, most preferably SARS-CoV-2, especially a wild-type (WT) or mutant

SARS-CoV-2, such as the Alpha (B.1.1.7), or Beta (B.1.351), or Gamma (P. 1), or Delta (B.1.617.2) and/or Omicron variant (B.1.1.529).

In the context of the present invention, the term "amino acid exchange" refers to the exchange of one amino acid for another amino acid within the amino acid sequence of the HLA antigen peptide to be synthesized relative to the wild-type of that HLA antigen peptide (i.e., native HLA antigen peptide). "Sequence identity," "percentage of sequence identity," or identity or similarity with respect to said amino acid sequence is defined herein as the percentage of amino acid residues in the amino acid sequence of the polypeptide that is identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side chain characteristics, see below) to the amino acid sequence of the wild type.

According to a preferred embodiment of the present invention, the amino acid substitution for the HLA antigen peptide corresponding to the class | and/or class II MHC complexes comprises at least one substitution at any position within the amino acid sequence relative to the wild type of that HLA peptide.

For the purpose of comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated or determined by dividing the number of amino acids in the first amino acid sequence that are identical to amino acids at corresponding positions in the second amino acid sequence, by [the total number of amino acids in the first amino acid sequence] and multiplying by [100%], wherein each deletion, insertion, substitution or addition of an amino acid in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference to a single amino acid (position).

The term "disease" refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one’s perspective on life, and one’s personality.

In the present context, the term "treatment" or "treating" relates to the management and care of a subject or an individual for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

The term "therapeutic treatment” relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.

The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

The amino acids used herein are abbreviated according to the generally accepted 15 one- letter code of the IUPAC Nomenclature Commission. Where two amino acids are separated by a hyphen (/), this indicates that at a specific amino acid position in the amino acid sequence concerned, the wild-type amino acid (left side of the hyphen) has been replaced by another amino acid (right side of the hyphen). In the present invention, the amino acids (IUPAC one- letter code) S, T, C, N, Q, Y are considered hydrophilic acids; the amino acids (IUPAC one-

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PMC-0005-P-LU 17.08.2022 Luxemburgische Patentanmeldung letter code) A, F, G, |, L, M, P, V, W are considered hydrophobic acids; the amino acids (IUPAC one-letter code) D, E, are considered acidic; the amino acids (IUPAC one-letter code) R, K, H are considered basic acids.

The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.

The term "individual" (also referred to herein as "person" or "subject" or "patient") is used interchangeably with the term "subject" to mean any mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that is being treated for an abnormal physiological condition or has been diagnosed with a disease caused by SARS-CoV-2.

The terms "individual" and "subject" as used in the present invention include mammals, such as a rodent, a cloven-hoofed animal, an odd-toed ungulate, or a primate. In particularly preferred embodiments, the subject is a human. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In some embodiments, the term "subject" includes humans of age of at least 50, at least 55, at least 60, at least 65, at least 70, or older. In some embodiments, the term "subject" includes humans of age of at least 65, such as 65 to 80, 65 to 75, or 65 to 70.

For the purposes of the invention, the totality of all HLA antigen peptides presented on the cell surface via MHC molecules is referred to as the (HLA) ligandome. It is believed that more than 10° HLA molecules are expressed on the cell surface and the number of identical HLA proteins presented can vary from a few to up to 10,000 copies per cell. Consequently, approximately 10,000 different HLA proteins are presented on a cell in varying proportions.

The ligandome is influenced by various physiological, intrinsic as well as pathological factors such as cell type or tissue type, infection or transformation of the cell, or simply the current state of the cell, which depends on nutrient situation or external stress factors, resulting in changes in the HLA proteins presented.

At the beginning of the analysis of the HLA ligandome, for example, Edman degradation can be used to gain initial insights into the presented peptides. On the one hand, it is possible in this way to determine the general peptide motif of an allele via pool sequencing, and on the other hand, individual peptide sequences can already be determined via the analysis of individual reversed phase high performance liquid chromatography (RP-HPLC) fractions.

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Alternatively, or complementarily, the analysis of the ligandome can be performed by using modern mass spectrometers in proteomics, with which it is possible to unambiguously determine the sequences of many individual ligands. Two methods are used for the ionization of the peptides or proteins required for this purpose: Electrospray ionization (ESI) and matrix- assisted laser desorption/ionization (MALDI). In ESI, coupling with an RP-HPLC system is common. However, as mass spectrometers have become more sensitive, capillary electrophoresis (CE) has also been used as an analytical separation method.

In order to achieve a higher sample throughput and increased sensitivity in ligandome analysis, the so-called UHPLC systems (ultra-high performance liquid chromatography) can be used.

These HPLC systems use only 2.2 to 1.7 um diameter materials as packing material for separation columns, resulting in improved speed, efficiency, and chromatographic separation.

In ESI mass spectrometry, the direct coupling of HPLC and ESI interface allows on-line separation of the sample, which, in combination with an autosampler, enables a fully automated measurement procedure. Because of the continuous solvent flow from the HPLC, samples can be measured in a relatively short time. ESI mass spectrometry uses a wide range of instruments for analysis such as quadrupole time-of-flight mass spectrometers, linear quadrupole ion traps, triple quadrupoles, or ion trap-orbitrap hybrid systems. This advantageously allows the identification of hundreds of HLA peptides in one measurement.

IFNy ELISpot analysis (for protocol see A. Lalvani, BR. Brookes, S. Hambleton, W. J. Britton,

A. VS. Hill, A. J. McMichael, J Exp Med 1887; 186 (6): 659-865) was performed ex vivo (without further in vitro culturing for expansion) using PBMCs depleted of CD4+ and enriched for CD8+ T cells (CD8+ effectors), or depleted of CD8+ and enriched for CD4+ T cells (CD4+ effectors). Tests were performed in duplicate and with a positive and negative control,

Multiscreen filter plates (Merck Millipore) pre-coated with IFNy-specific antibodies (ELISpotPro kit, Mabtech). 267.000 Cells were used per plate. Plates were scanned using an AID Classic

Robot ELISPOT Reader and analyzed by AID ELISPOT 7.0 software (AID Autoimmun

Diagnostika). Antigen Peptides (except negative and positive control group) were done in duplicates, spot counts were taken as mean values of each duplicate.

To provide antigen peptides, in principle, synthetic or isolated HLA antigen peptides derived from the cumulative ranking can be used for the preparation of the (medicament) formulations of the present invention and for the preparation of the pharmaceutical composition (as so- called informaticum).

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The term "active ingredient enhancer/adjuvant” refers to an excipient that triggers and/or enhances the effect of the HLA peptides in the first place. In principle, all commonly used adjuvants known to the skilled person are suitable for the preparation of a formulation according to the invention. Advantages and Description of the Process

However, it is convenient to use synthetic HLA peptides. Processes for the synthetic preparation of peptides are known to those skilled in the art. Examples of such production methods are the Merrifield solid-phase peptide synthesis, the Bailey peptide synthesis, and the N-carboxylic anhydride method.

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Brief description of the drawings

Herein shows:

Fig. 1: Schematic overview of the immunization scheme for CoV-WT-2

Four groups of wild-type mice, one reference group (no vac.), two groups (2,3) injected with a pharmaceutical composition in accordance with the present invention (SEQ IDs SEQ ID NOs: 38-49) and one group injected with an mRNA vaccine.

Fig. 2 Comparison of Group 1-4 INFy CD8+ ICS

Intracellular cytokine staining (ICS) with ELISpot analysis, CD8 T-cell responses, activity

IFNy+ CD8+. *- compared to no vac. reference group. Horizontal lines indicate median values.

Fig. 3 Comparison of Group 1-4 INFy CD8+ IL-2+ ICS

IFNy+ TNFa+ IL-2+ CD8+. (total) *- compared to no vac. reference group. Horizontal lines indicate median values.

Fig. 4 Comparison of Group 1-4 INFy CD4+ ICS

IFNy+ CD4+ *- compared to no vac. reference group. Horizontal lines indicate median values.

Fig. 5 Single peptide-specific CD8 T-cell ICS (Pep X)

Pep X, Intracellular cytokine staining (ICS), Single peptide-specific CD8 T-cell responses corresponding to peptides from SEQ ID NOs: 38-49, OVA: unrelated peptide (*- compared to no vac. reference group)

Fig. 6 Single peptide-specific CD8 T-cell ICS (Pep C)

Pep C, Intracellular cytokine staining (ICS), Single peptide-specific CD8 T-cell responses corresponding to peptides from SEQ ID NOs: 38-49, OVA: unrelated peptide (*- compared to no vac. reference group)

Fig. 7: Schematic overview of the immunization scheme for CoV-WT-2

Three groups of wild-type mice, one reference group (no vac.), one group injected with a pharmaceutical composition (SEQ ID NOs: 43, 44, 46, 47, 50-58) in accordance with the present invention and one group injected with an mRNA vaccine.

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Fig. 8 Single peptide-specific CD4 T-cell ICS

Intracellular cytokine staining (ICS), Single peptide-specific CD4 T-cell responses corresponding to peptides from SEQ ID NOs: 38-49, OVA: unrelated peptide (*- compared to no vac. reference group)

Fig. 9 Single peptide-specific CD8 T-cell ICS

Intracellular cytokine staining (ICS), Single peptide-specific CD4 T-cell responses corresponding to peptides from SEQ ID NOs: 43, 44, 46, 47, 50-58, OVA: unrelated peptide (*- compared to no vac. reference group)

Fig. 10 Single peptide-specific CD8 T-cell Human EliSpot

Intracellular cytokine staining (ICS), cells/100000 and INFy activation, single peptide-specific

CD8 T-cell responses corresponding to peptides from SEQ ID NOs: 43, 44, 46, 47, 50-58, neg = negative control.

Fig. 11 Single peptide-specific CD8 T-cell Human EliSpot

Intracellular cytokine staining (ICS), Total INFy Activity, single peptide-specific CD8 T-cell responses corresponding to peptides from SEQ ID NOs: 43, 44, 46, 47, 50-58, neg = negative control.

Fig. 12: INFy CD8+ ELiSpot responses of human PBMCs

ELISpot well plates corresponding to Fig 10/11 results of peptide activation with peptides from

SEQ ID NOs: 43, 44, 46, 47, 50-58, neg = negative control, pos = positive control. .

Examples

With reference to the following figures and embodiments, the present invention will be explained in more detail without limiting the invention thereto.

The following tables list HLA antigen peptides, all of which have been tested and are immunogenic. SEQ ID NOs: 38-58 list some preferred, but not limiting, examples of amino acid sequences of antigen peptides of the invention for the use as vaccination against SARS-CoV- 2, each of which is another embodiment of the present invention.

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Tables

Table 1, SARS-CoV-2 Spike Protein

HLA Isotype Subtype HLA Peptide | Reaction | Target Protein

Specific Value

Antigene-

HLA-B Antigen peptide (SEQ ID NO.:8) HLA-B2705/ | YRLFRKSNL | 66,99/ Chain A, SARS-CoV-2 spike glycoprotein (441-466)

TEE Je | fy [TEE

HLA-B Antigen peptide (SEQ ID NO.:10) HLA-B3901/ | YQPYRVVVL | 79,44/ Chain A, SARS-CoV-2 spike glycoprotein (497-521)

TERE Jee | ag [TE

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Table 2, SARS-CoV-2 Nucleocapside Protein

HLA Isotype Subtype HLA Peptide | Reaction Target Protein

Specific Value

Antigene-

HLA-B Antigen peptide (SEQ ID NO.:13) HLA-B0702/ | FPRGQGVPI | 3,82/ 1037,41 | Nucleocapsid phosphoprotein [Bat coronavirus

EEE sn | a

HLA-B Antigen peptide (SEQ ID NO.:14) HLA-B0702/ | KPRQKRTAT | 4,42/ 364,72 | Chain A, SARS-CoV-2 nucleocapsid protein (7-

TEE aa

HLA-B Antigen peptide (SEQ ID NO.:15) HLA-B5801/ | KAYNVTQAF | 12,51/40,35 | Chain A, SARS-CoV-2 nucleocapsid protein (7-

Hh ll

HLA-A Antigen peptide (SEQ ID NO.:16) HLA-A0201 | GMSRIGMEV | 50,61 nucleocapsid protein N [SARS coronavirus mmm EE mee TT

HLA-B Antigen peptide (SEQ ID NO.:17) HLA-B5801 | MEVTPSGTW | 1256,27 nucleocapsid protein N [SARS coronavirus mmm EE N

HLA-A Antigen peptide (SEQ ID NO.:18) HLA-A0301 | ASAFFGMSR | 292,41 nucleocapsid protein N [SARS coronavirus ll ha

HLA-A Antigen peptide (SEQ ID NO.:19) HLA-A2402/ | FAPSASAFF | 422,31/ nucleocapsid protein N [SARS coronavirus

EE [tes | Jaa Je TE

HLA-B Antigen peptide (SEQ ID NO.:20) HLA-B1501 | AQFAPSASA | 62,45 nucleocapsid protein N [SARS coronavirus mem me Ee

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Table 3, SARS-CoV-2 Envelope Protein

HLA Isotype Subtype HLA Peptide | Reaction Target Protein

Specific Value

Antigene-

Variant

HLA-A Antigen peptide (SEQ ID NO.:21) HLA-A0101 | VSLVKPSFY | 1007,63 envelope protein [SARS-CoV-2] (49-75)

HLA-A Antigen peptide (SEQ ID NO.:22) | HLA-A0201 | SLVKPSFYV envelope protein [SARS-CoV-2] (49-75)

HLA-A Antigen peptide (SEQ ID NO.:23) HLA-A0301 | SFYVYSRVK | 646,15 envelope protein [SARS-CoV-2] (49-75)

HLA-B Antigen peptide (SEQ ID NO.:24) HLA-B1501 | LVKPSFYVY | 14,11 envelope protein [SARS-CoV-2] (49-75)

HLA-B Antigen peptide (SEQ ID NO.:25) HLA-B5801 | NSSRVPDLL | 311,64 envelope protein [SARS-CoV-2] (49-75)

HLA-B Antigen peptide (SEQ ID NO.:26) HLA-B0801 | YVYSRVKNL | 250,69 envelope protein [SARS-CoV-2] (49-75)

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Table 4, SARS-CoV-2 Surface Protein

HLA Isotype Subtype HLA Peptide | Reaction Target Protein

Specific Value

Antigene-

Variant

HLA-A Antigen peptide (SEQ ID NO.:27) HLA-A0201 VLNDILSRL | 33,57 Chain A, Surface Glycoprotein [SARS-CoV-2] (972-988)

HLA-B Antigen peptide (SEQ ID NO.:28) HLA-B5801 ISSVLNDIL 518,51 Chain A, Surface Glycoprotein [SARS-CoV-2] (972-988)

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Table 5, SARS-CoV-2 ORF1ab Protein

HLA Isotype Subtype HLA Peptide | Reaction Target Protein

Specific Value

Antigene-

HLA-A Antigen peptide (SEQ ID NO.:31) | HLA-A2402/ | SFSASTSAF | 695,27/ ? ORF1ab polyprotein (SARS-CoV-2] (474-488)

EE a

HLA-B Antigen peptide (SEQ ID NO.:37) | HLA-B0702/ | HLRIAGHHL | 647,29/494,02 | ORF1ab polyprotein (SARS-CoV-2]

TEE Jae [TEETER

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Table 6, SARS-CoV-2 specific HLA antigen peptides

HLA antigen peptide Identification NO. | Target Protein Matching Epitope ere | amen

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Table 7, SARS-CoV-2 Polypeptide Combination medication

No. | Amino Acid Complex Identification Pep No.

CE ee PT 4 FPRGQGVPI SEQ-ID NO.: 41 | Pep-4

CT ee 6 [KFOPTNGVGYOPYAWWLSFELLHAP | SEQTDNO. #0 [Peps [3 | VSLUPSFYWSRVININGSRVDLLY | SEGIONO. 45 [Pep jp [WTGECVIGOSKWD [SEGTONOLA6 [Peps

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Example 1: Antigen response in Mice first trial 4 Groups of wild-type mice (C57BL/6) consisting of 5 mice (group 2-4) and 3 mice for the negative control (group 1) were administered different vaccine candidates (as following) for evaluation.

The Groups were administered the following compositions: a) Group 1 (no vac.): no vaccine b) Group 2 (Pep X): peptide mix (60 ug per single peptide, 12 peptides selected from SEQ

ID NOs: 38-49) with Pam3Cys (10 ug/dose) adjuvant c) Group 3 (Pep C): peptide mix (60 ug per single peptide, 12 peptides selected from SEQ

ID NOs: 38-49) with c-di-AMP (10 pg/dose) adjuvant d) Group 4 (mRNA): Comirnaty mRNA vaccine (5 ug /dose)

The antigen peptide formulations in accordance with the present invention (Groups 2 and 3) were administered subcutaneous (SC) three times in two-week intervals (see Fig. 1), the mRNA vaccine was administered intramuscular (IM) two times in a four-week interval (Group 4). After five weeks the immune response was determined, T cell CD4 and CD8 activity was examined by cytokines secreted in response to stimulation with the peptide antigens were determined by intracellular cytokine staining (ICS) with Intracellular cytokine staining with IFNy,

TNFa, and IL-2. Specific antibodies.

The immunization with a combination preparation in accordance with the present invention, particularly with Pep X and Pep C induced strong and multifunctional Spool2 and peptide mix specific CD8 T cell responses in comparison with the no vac. group 1 and even a greater response than the Comirnaty mRNA vaccine (Fig. 2 and Fig. 3). Immunization with Bis-(3’,5’)- cyclic-adenosine monophosphate (c-di-AMP) adjuvant (Pep C) induced a stronger CD8 T cell response than Pep X.

The immunization with a combination preparation in accordance with the present invention, particularly with Pep X and Pep C (Groups 2,3) also induced a stronger CD4 T cell response than the mRNA vaccination (Group 4) and the control group (Fig. 4). The CD8 T cell responses induced by the combination preparation, in particular comprising antigen peptide from SEQ ID

NOs.: 38-49 were mostly spike protein S2 domain specific responses (Fig. 5,6). In the Pep X composition, the peptide combination of Pep 6+7+9+10 (SEQ ID NO.: 43, 44, 46, 47) induced a moderate CD8 T cell reaction (Fig. 5), while Pep 6+7+9+10 (SEQ ID NO: 43, 44, 46, 47) in

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Pep- 5+11 (SEQ ID NO.: 42, 48) showed some CD8+ activity (Fig. 6).

Example 2: Antigen response in Mice second trial 3 Groups of wild-type mice (C57BL/6) consisting of 5 mice (group 2,3) and 3 mice for the negative control (group 1) were administered different vaccine candidates (as following) for evaluation.

The Groups were administered the following compositions: a) Group 1 (no vac.): no vaccine b) Group 2 (Pep2 C): peptide mix (60 pg per single peptide, 12 peptides selected from

SEQ ID NOs: 43, 44, 46, 47, 50-58) with c-di-AMP (10 ug/dose) adjuvant c) Group 3 (mRNA): Comirnaty mRNA vaccine (5 ug /dose)

The antigen peptide formulations in accordance with the present invention were administered subcutaneous (SC) three times in two-week intervals (see Fig. 7), the mRNA vaccine was administered intramuscular (IM) two times in a four-week interval (Group 3). After five weeks the immune response was determined, T cell CD8 activity was examined by cytokines secreted in response to stimulation with the peptide antigens were determined by intracellular cytokine staining (ICS) with Intracellular cytokine staining with IFNy and TNFy. Specific antibodies.

The CD4 T cell responses induced by the combination preparation, in particular comprising antigen peptide from SEQ ID NOs.: 43, 44, 46, 47, 50-58 were mostly spike protein S1 and S2 domain specific responses. The different peptides of the pharmaceutical composition show different CD4 and CD8 activation selectivity. While the peptides Pep 13+14 (SEQ ID NOs.: 50- 51) and Pep 16+21 (SEQ ID NOs.: 50-51) caused a moderate CD4 T cell reaction. (Fig. 8), the peptides Pep 6 (SEQ ID NO.: 43) and Pep 7 (SEQ ID NO.: 44) caused a CD8 T cell reaction (Fig. 9). The CD8 T cell responses induced by the combination preparation, in particular comprising antigen peptide from SEQ ID NOs: 43, 44, 46, 47, 50-58 were mostly a spike protein S2 domain specific response.

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Example 3: IFNy Activity of antigen peptides in human blood serum.

IFNy ELISpot analysis was performed ex vivo (without further in vitro culturing for expansion) using PBMCs from a healthy adult human. The cells of the healthy donor were pre-stimulated with the pool of Covid peptides. Then the cells were stimulated in the ELISpot with the individual peptides, all peptide wells were prepared as duplicates, except for blind test and positive control which were performed as single wells. 267,000 cells per well were used.

The positive counts per 100,000 cells and activation values times 10 for the antigen peptides as analyzed with ELISpot (Fig. 11) are noted below.

Peptide [SEQIDNO. | Cells/100,000 cells | Activation10____ neg }- | 258 860 (Pep17 ~~ |SEQIDNO.54 | 2491] 996]

Peptides 13, 14 and 17 showed especially high counts per 100,000 cells, while the IFNy activity was the highest for the antigen peptides Pep-6, Pep-7, Pep-21 and Pep-14 in descending order (Fig. 10), corresponding to the SEQ ID NOs.: 43, 44, 58, 51. The activation value is a measure of the cytokine activity and was calculated according to the following formula: yp, Semen The results are shown in Fig. 11, the effectiveness of immunization is (according to formula) the product of number of activated cells and activation formed, thus the most effective antigen peptides are (in descending order) Pep-14, Pep-17, Pep-13, Pep-6,

Pep-21, and Pep-7, corresponding to the SEQ ID NOs.: 51, 54, 50, 43, 58, 44. All of these peptides have an activation over 75, which is the threshold (3 times negative control) for a positive immunization effect.

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Example 4: Vaccination of a group of healthy Patients with risk of contracting a coronavirus

All subjects (hereinafter also referred to as patients) are adults, preferably human adults. The vaccine consisting of the pharmaceutical composition according to the invention is administered as described, preferably intramuscularly. The pharmaceutical composition comprises a pharmacologically effective amount (600 pg per peptide per vial) of the 12 antigen peptides (SEQ ID NO: 38-58) and the adjuvant c-di-AMP (bis-(3',5")-cyclic-adenosine dissolved or suspended in water:DMSO 75%:25% (vol%:vol%). The administration is repeated two times in two-week intervals (Fig. 1), for a total of three administrations in four weeks. This ensures an optimal CD4 and CD8 T cell response.

Example 5: Bioinformatic determination of pharmaceutically active HLA Antigen

Peptides and/or Antigen Polypeptides

Step (a): Bioinformatics expert system: In order to bioinformatically determining the amino acid sequences of the HLA antigen peptides of the SARS-CoV-2 corresponding to the MHC complexes (nonamers) with the highest allelic affinities (specific activity towards T cell receptor [nM]) were first selected from the dataset of the SARS-CoV-2 virus proteins (nucleoprotein,

UNIPROT Reference ID sequence: PODTC9, and spike protein, UNIPROT Reference ID sequence: PODTC2) based on the amino acid sequences of the respective proteins. The highest conservative regions in relation with other human coronaviruses and non-variant mutations were selected from proteins with a combination of different bioinformatic methods to further analyze. To this scope, the Nucleocapsid protein was aligned against 5 coronaviruses with COBALT tool (Human coronavirus 229E, UNIPROT reference: P15130;

Human coronavirus OC43, UNIPROT reference: P33469, Human coronavirus HKU1 (isolate

N5), UNIPROT reference: Q0ZMES3; Human coronavirus HKU1 (isolate N1), UNIPROT reference: Q5MQC6; and severe acute respiratory syndrome coronavirus 2, UNIPROT reference: Q6S8E1) according to a conservation algorithm based on the relative entropy threshold of the residue. SARS-CoV-2 nucleocapsid protein positions 59-84, 86-96, 99-123, 129-142, 155-170, 284-320, 321-336, 342-360 are highly conserved. These regions are used for further binding affinity prediction. SARS-CoV-2 spike protein was aligned against 8 coronaviruses (Human coronavirus 229E, UNIPROT reference: P15423; Human coronavirus

OC43, UNIPROT reference: P36334, Human coronavirus HKU1 (isolate N5), UNIPROT reference: Q0ZME7; Human coronavirus HKU1 (isolate N1), UNIPROT reference: Q5MQDO;

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Severe acute respiratory syndrome coronavirus, UNIPROT reference: P59594; Human

Coronavirus NL63, UNIPROT reference: Q6Q1S2; MERS-CoV, UNIPROT reference:

K9N5Q8; and Human coronavirus HKU1 (isolate N2), UNIPROT reference: Q14EB0). SARS-

CoV-2 spike protein positions 152-211, 470-503, 534-570, 769-847, 939-1063 and 1176-1273 are highly conserved. then, the selection criterion is used to algorithmically determine the probability with which the respective HLA antigen peptide is presented in vivo on the corresponding MHC complexes of the SARS-CoV-2 virus (a first prerequisite for a possible T cell immune response). ADE and B/T cell response balancing SARS-CoV-2 peptides was made using a variety of in silico methods, 3D modeling, and binding prediction test the effect of different antibodies on the conformational change of the SARS-CoV-2 spike protein and select peptides that are less likely to trigger ADE. Artificial intelligence such as the neural network NNAlign used in the publicly available IEDB server is used for information processing, and the mathematical modeling and biotechnological advancements based on it are used for experimental analysis on the causality of a pharmaceutical composition of the vaccine. From the dataset of the SARS-CoV-2 virus proteins (nucleoprotein, UNIPROT Reference ID sequence: PODTC9, and spike protein, UNIPROT Reference ID sequence: PODTC2) nonameric variants involving amino acid exchange were determined with respect to 12 of the highest affinities (specific activity toward T cell receptor [nM]) based on the patient's alleles.

Also, polymers of 17 amino acids (oligopeptides) were determined under the affinity criterion for this purpose. This selection criterion is used to algorithmically determine the probability of presenting the respective antigen peptide corresponding to the MHC complexes in vivo on the corresponding MHC complexes (a 15 second requirement for a possible cellular immune response).

Step (b): Bioinformatics expert system: Based on the new data sets identified in step (a) with the addition of the HLA-restricted ligands (the data set of the ligandome), a selection of HLA antigen peptides corresponding to the MHC class | complexes and corresponding to the MHC class Il complexes was made, which were the most promising epitope candidates for eliciting a cellular immune response, both individually and, most importantly, 12 in combination. This allows existing knowledge about ADE, including that within the existing COVID-19 Disease

Map Collaboration, to be extracted from the extensive literature about FIPV2,3, MERS4,5, and

SARS and tested for gaps through modeling: Knowledge gaps can be narrowed by simulation- based hypothesis testing to the point where experimental testing of the remaining variants becomes feasible.

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Step (c): Al and machine learning: Natural language processing (NLP) such as Natural

Language Toolkit (NLTK) is used lo extract further information to reinforce the predictions from steps (a) and (b), by learning patterns that allow information linkage to be identified or that allow tables and figures to be included in the evaluation. To further improve on the mathematical model, relevant information on molecular and cellular interaction networks between coronaviruses and the host can be extracted from the total amount of scientific literature and related to ADE. Biological processes like signal transduction, gene regulation and metabolism are accounted and integrated into the ADE prevention by artificial intelligence by translation into a mathematical model that maps the molecular mechanisms.

Step (d): Biotechnology platforms: The effectiveness of the peptide immunizations is tested with high throughput biotechnological methods such as rapid peptide synthesis (Merrifield solid-phase peptide synthesis, Bailey peptide synthesis, N-carboxylic anhydride method) and rapid immunogenicity assays (Western Blot, Electrochemiluminescent immunoassay (ECL),

ELISA techniques, intracellular cytokine staining (ICS), ELISPOT, AFM, MS, see Fig. 2-6).

These techniques ensure that conformation-specific SARS-CoV-2 antibody and a balanced

B/T-cell reaction as predicted with in silico methods (a)-(c) are archived by the pharmaceutical composition comprising of at least one HLA antigen peptide in accordance with the present invention is found.

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Sequence Listing Information: LU102995

DTD Version: V1 _3

File Name: PMC-0005-P-LU.xml

Software Name: WIPO Sequence

Software Version: 2.1.2

Production Date: 2022-08-17

General Information:

Current application / Applicant file reference: PMC-0005-P-LU

Applicant name: PMCR GmbH

Applicant name / Language: en

Invention title: Immunization against coronavirus ( en )

Sequence Total Quantity: 79

Sequences:

Sequence Number (ID): 1

Length: 9

Molecule Type: AA

Features Location/Qualifiers: - source, 1..9 > mol type, protein > organism, severe acute respiratory syndrome coronavirus 2

Residues:

LLFNKVTLA 9

Sequence Number (ID): 2

Length: 9

Molecule Type: AA

Residues:

FIKQYGDCL 9

Sequence Number (ID): 3

Length: 9

Molecule Type: AA

Residues:

TLADAGFIK 9

Sequence Number (ID): 4

Length: 9

Molecule Type: AA

Residues: LU102995

KCYGVSPTK 9

Sequence Number (ID): 5

Length: 9

Molecule Type: AA

Residues:

CYGVSPTKL 9

Sequence Number (ID): 6

Length: 9

Molecule Type: AA

Residues:

RLFRKSNLK 9

Sequence Number (ID): 7

Length: 9

Molecule Type: AA

Residues:

NYNYLYRLF 9

Sequence Number (ID): 8

Length: 9

Molecule Type: AA

Residues:

YRLFRKSNL 9

Sequence Number (ID): 9

Length: 9

Molecule Type: AA

Residues: LU102995

PYRVVVLSF 9

Sequence Number (ID): 10

Length: 9

Molecule Type: AA

Residues:

YQPYRVVVL 9

Sequence Number (ID): 11

Length: 9

Molecule Type: AA

Residues:

RVVVLSFEL 9

Sequence Number (ID): 12

Length: 9

Molecule Type: AA

Residues:

YRFNGIGVT 9

Sequence Number (ID): 13

Length: 9

Molecule Type: AA

Residues:

FPRGQGVPI 9

Sequence Number (ID): 14

Length: 9

Molecule Type: AA

Residues: LU102995

KPRQKRTAT 9

Sequence Number (ID): 15

Length: 9

Molecule Type: AA

Residues:

KAYNVTQAF 9

Sequence Number (ID): 16

Length: 9

Molecule Type: AA

Residues:

GMSRIGMEV 9

Sequence Number (ID): 17

Length: 9

Molecule Type: AA

Residues:

MEVTPSGTW 9

Sequence Number (ID): 18

Length: 9

Molecule Type: AA

Residues:

ASAFFGMSR 9

Sequence Number (ID): 19

Length: 9

Molecule Type: AA

Residues: LU102995

FAPSASAFF 9

Sequence Number (ID): 20

Length: 9

Molecule Type: AA

Residues:

AQFAPSASA 9

Sequence Number (ID): 21

Length: 9

Molecule Type: AA

Residues:

VSLVKPSFY 9

Sequence Number (ID): 22

Length: 9

Molecule Type: AA

Residues:

SLVKPSFYV 9

Sequence Number (ID): 23

Length: 9

Molecule Type: AA

Residues:

SFYVYSRVK 9

Sequence Number (ID): 24

Length: 9

Molecule Type: AA

Residues: LU102995

LVKPSFYVY 9

Sequence Number (ID): 25

Length: 9

Molecule Type: AA

Residues:

NSSRVPDLL 9

Sequence Number (ID): 26

Length: 9

Molecule Type: AA

Residues:

YVYSRVKNL 9

Sequence Number (ID): 27

Length: 9

Molecule Type: AA

Residues:

VLNDILSRL 9

Sequence Number (ID): 28

Length: 9

Molecule Type: AA

Residues:

ISSVLNDIL 9

Sequence Number (ID): 29

Length: 9

Molecule Type: AA

Residues: LU102995

FSASTSAFV 9

Sequence Number (ID): 30

Length: 9

Molecule Type: AA

Residues:

ILASFSAST 9

Sequence Number (ID): 31

Length: 9

Molecule Type: AA

Residues:

SFSASTSAF 9

Sequence Number (ID): 32

Length: 9

Molecule Type: AA

Residues:

AARVVRSIF 9

Sequence Number (ID): 33

Length: 9

Molecule Type: AA

Residues:

SLPFGWLIV 9

Sequence Number (ID): 34

Length: 9

Molecule Type: AA

Residues: LU102995

IQASLPFGW 9

Sequence Number (ID): 35

Length: 9

Molecule Type: AA

Residues:

ASLPFGWLI 9

Sequence Number (ID): 36

Length: 9

Molecule Type: AA

Residues:

RIAGHHLGR 9

Sequence Number (ID): 37

Length: 9

Molecule Type: AA

Residues:

HLRIAGHHL 9

Sequence Number (ID): 38

Length: 26

Molecule Type: AA

Features Location/Qualifiers: - source, 1..26 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

LLFNKVTLAD AGFIKQYGDC LGDIAA 26

Sequence Number (ID): 39

Length: 19

Molecule Type: AA

Features Location/Qualifiers: - source, 1..19

> mol type, protein LU102995 > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

KCYGVSPTKL NDLCFTNVY 19

Sequence Number (ID): 40

Length: 26

Molecule Type: AA

Residues:

LDSKVGGNYN YLYRLFRKSN LKPFER 26

Sequence Number (ID): 41

Length: 9

Molecule Type: AA

Features Location/Qualifiers: - source, 1..9 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

FPRGQGVPI 9

Sequence Number (ID): 42

Length: 30

Molecule Type: AA

Features Location/Qualifiers: - source, 1..30 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

SAAEASKKPR QKRTATKAYN VTQAFGRRPE 30

Sequence Number (ID): 43

Length: 26

Molecule Type: AA

Features Location/Qualifiers: - source, 1..26 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide LU102995 > organism, synthetic construct

Residues:

KFQPTNGVGY QPYRVVVLSF ELLHAP 26

Sequence Number (ID): 44

Length: 17

Molecule Type: AA

Features Location/Qualifiers: - source, 1..17 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

MAYRFNGIGV TQNVLYE 17

Sequence Number (ID): 45

Length: 27

Molecule Type: AA

Features Location/Qualifiers: - source, 1..27 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

VSLVKPSFYV YSRVKNLNSS RVPDLLV 27

Sequence Number (ID): 46

Length: 17

Molecule Type: AA

Residues:

AATKMSECVL GQSKRVD 17

Sequence Number (ID): 47

Length: 17

Molecule Type: AA

Residues: LU102995

AISSVLNDIL SRLDKVE 17

Sequence Number (ID): 48

Length: 17

Molecule Type: AA

Residues:

FFGMSRIGME VTPSGTW 17

Sequence Number (ID): 49

Length: 15

Molecule Type: AA

Features Location/Qualifiers: - source, 1..15 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

AFASEAARVV RSIFS 15

Sequence Number (ID): 50

Length: 17

Molecule Type: AA

Residues:

TFEYVSQPFL MDLEGKQ 17

Sequence Number (ID): 51

Length: 14

Molecule Type: AA

Features Location/Qualifiers: - source, 1..14 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

GDCLGDIAAR DLIC 14

Sequence Number (ID): 52

Length: 21

Molecule Type: AA

Features Location/Qualifiers: - source, 1..21 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

LGKYEQYIKW PWYIWLGFIA G 21

Sequence Number (ID): 53

Length: 20

Molecule Type: AA

Features Location/Qualifiers: - source, 1..20 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

EVAKNLNESL IDLQELGKYE 20

Sequence Number (ID): 54

Length: 16

Molecule Type: AA

Features Location/Qualifiers: - source, 1..16 > mol type, protein > note, synthetic peptide used as antigen comprising at least one HLA antigen peptide > organism, synthetic construct

Residues:

DGYFKIYSKH TPINLV 16

Sequence Number (ID): 55

Length: 20

Molecule Type: AA

Residues:

GKEDLKFPRG QGVPINTNSS 20

Sequence Number (ID): 56

Molecule Type: AA

Residues:

RQGTDYKHWP QIAQF 15

Sequence Number (ID): 57

Length: 15

Molecule Type: AA

Residues:

KDLSPRWYFY YLGTG 15

Sequence Number (ID): 58

Length: 17

Molecule Type: AA

Residues:

TRAAEIRASA NLAATKM 17

Sequence Number (ID): 59

Length: 9

Molecule Type: AA

Residues:

AETRASANL 9

Sequence Number (ID): 68

Length: 9

Molecule Type: AA

Features Location/Qualifiers: - source, 1..9

> mol type, protein LU102995 > organism, severe acute respiratory syndrome coronavirus 2

Residues:

ASANLAATK 9

Sequence Number (ID): 61

Length: 9

Molecule Type: AA

Residues:

DCLGDIAAR 9

Sequence Number (ID): 62

Length: 9

Molecule Type: AA

Residues:

AYRFNGIGV 9

Sequence Number (ID): 63

Length: 9

Molecule Type: AA

Residues:

DGYFKIYSK 9

Sequence Number (ID): 64

Length: 9

Molecule Type: AA

Residues:

DILSRLDKV 9

Sequence Number (ID): 65

Length: 9

Molecule Type: AA

Features Location/Qualifiers: - source, 1..9

Residues:

DLSPRWYFY 9

Sequence Number (ID): 66

Length: 9

Molecule Type: AA

Residues:

EYVSQPFLM 9

Sequence Number (ID): 67

Length: 9

Molecule Type: AA

Residues:

FPRGQGVPI 9

Sequence Number (ID): 68

Length: 9

Molecule Type: AA

Residues:

FQPTNGVGY 9

Sequence Number (ID): 69

Length: 9

Molecule Type: AA

Residues:

GYFKIYSKH 9

Sequence Number (ID): 70

Length: 9

Molecule Type: AA

Features Location/Qualifiers: - source, 1..9

Residues:

GYOPYRVVV 9

Sequence Number (ID): 71

Length: 9

Molecule Type: AA

Residues:

IGVTQNVLY 9

Sequence Number (ID): 72

Length: 9

Molecule Type: AA

Residues:

KDLSPRWYF 9

Sequence Number (ID): 73

Length: 9

Molecule Type: AA

Residues:

KHWPQIAQF 9

Sequence Number (ID): 74

Length: 9

Molecule Type: AA

Residues:

KIYSKHTPI 9

Sequence Number (ID): 75

Length: 9

Molecule Type: AA

Features Location/Qualifiers: - source, 1..9

Residues:

LSPRWYFYY 9

Sequence Number (ID): 76

Length: 9

Molecule Type: AA

Residues:

NGVGYQPYR 9

Sequence Number (ID): 77

Length: 9

Molecule Type: AA

Residues:

NLNESLIDL 9

Sequence Number (ID): 78

Length: 9

Molecule Type: AA

Residues:

PYRVVVLSF 9

Sequence Number (ID): 79

Length: 9

Molecule Type: AA

Residues:

QYIKWPWYT 9

END

Claims

1. A pharmaceutical composition for use as a medicament, comprising a pharmacologically effective amount consisting of at least 2 antigen polypeptide sequence comprising at least one HLA-A and/or HLA-B antigen peptide, wherein the HLA antigen peptide corresponds to at least one amino acid sequence in the coronavirus, such as spike S1 domain, S2 domain, nucleocapsid protein, envelope protein and/or ORF1ab polyprotein, wherein the antigen polypeptide sequence comprises the following scaffold amino acid sequence: (a) a long peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which comprises one HLA-A antigen peptide or HLA-B antigen peptide as defined herein, wherein the amino acid sequence comprises, in addition to the HLA-A antigen peptide or HLA-B antigen peptide, up to 1 to 30 amino acids; and/or (b) a similarity peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a) or (b), wherein the HLA-A antigen peptide or HLA-B antigen peptide as defined herein having less than 100% sequence identity or similarity to the native HLA antigen peptide, such as having at least 85%, more preferably at least 90%, sequence identity; and/or (c) a substitution peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a) or (b), wherein the HLA-A antigen peptide or HLA-B antigen peptide as defined herein having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence of the native HLA antigen peptide; and/or (d) a tandem peptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of at least 2, identical or different HLA-A and/or HLA-B antigen peptide sequences, both as defined in items (a) to (c); and/or (e) an overlapping tandem peptide, i.e. a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of PMCR GmbH PMC-0005-P-LU 14.08.2023 Luxemburgische Patentanmeldung at least 2 identical or different HLA-A and/or HLA-B antigen peptide sequences, both as defined in items (a) to (c), in which the at least two HLA antigen peptides overlap in their amino acid sequence, characterized in that the HLA-A and/or HLA-B antigen peptide is an amino acid sequence selected from the group consisting of SEQ ID NO: 2 — 37, 59 — 79, and/or the antigen polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NOs 39 — 58.

2. The pharmaceutical composition according to claim 1, wherein the HLA-A and/or HLA-B antigen peptide corresponding to MHC class | complexes is selected to match a sequence of the viral epitope of the SARS-CoV-2 virus, such as the SARS-CoV-2 wild type Hu-1 and particularly mutants, such as the Alpha (B.1.1.7), or Beta (B.1.351), or Gamma (P. 1), or Delta (B.1.617.2) and/or Omicron variant (B.1.1.529).

3. The pharmaceutical composition according to claim 1 or 2 for use in the therapeutic or prophylactic treatment of a coronavirus infectious disease.

4. The pharmaceutical composition according to any claims 1 to 3, for use in the treatment in a subject or group of subjects suffering from or at risk of suffering from a coronavirus infectious disease.

5. The pharmaceutical composition of any one of the claims 1 to 4, further comprising at least one amino acid sequence comprising or consisting of an HLA-A or HLA-B antigen peptide, wherein the HLA peptide corresponds to at least one amino acid sequence in the coronavirus, such as spike S1 and S2 domain, nucleocapsid protein, envelope protein and/or ORF1ab polyprotein.

6. The pharmaceutical composition of any one of the claims 1 to 5, wherein the pharmaceutical composition comprises at least two antigen polypeptides, wherein the HLA-A and/or HLA-B antigen peptides of the at least two antigen polypeptides correspond PMCR GmbH PMC-0005-P-LU 14.08.2023 Luxemburgische Patentanmeldung to a combination of different parts of the coronavirus epitope selected from the spike S1 domain, S2 domain, nucleocapsid protein, envelope protein and/or ORF1ab polyprotein.

7. The pharmaceutical composition of any one of the claims 1 to 6, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier liquid and a pharmaceutically acceptable adjuvant, carrier, diluent and/or excipient.

8. The pharmaceutical composition according to any one of the preceding claims for the use set forth in claim 1, wherein the pharmaceutical composition is applied subcutaneously, intramuscularly or intradermally.

9. The pharmaceutical composition according to claim 8, wherein said subject is a human.

10. The pharmaceutical composition according to claims 1 to 9 for the use set forth in claim 1, wherein the HLA antigen peptides and/or antigen polypeptides corresponding to MHC class | complexes are immunogenic antigen peptides, determined by means of an immunogenicity assay, in particular (but not limited to) by means of Western blot, Electrochemiluminescent immunoassay (ECL), ELISA techniques, ELISPOT, ICS, AFM, MS or immunodetection with microscopic analysis.

11. The pharmaceutical composition according to any one of claims 1 to 10 for the use set forth in claim 1, further comprising at least one HLA-A antigen peptide and/or HLA-B antigen peptides corresponding to MHC class | complexes.

12. The pharmaceutical composition according to any one of claims 1 to 11 for the use as set forth in claim 1, wherein the antigen polypeptide corresponding to the respective subtype of the MHC class | complexes are selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs. 2-37, 39-58 or have at least one amino acid substitution with respect to these amino acid sequences. PMCR GmbH PMC-0005-P-LU 14.08.2023 Luxemburgische Patentanmeldung

13. The pharmaceutical composition according to any one of the preceding claims for use as recited in claim 1, wherein the pharmacologically effective amount of each individual antigen polypeptide in the composition is in an absolute concentration (i.e., administration dose) ranging from at least 10 to 1,000 ug.

14. The pharmaceutical composition according to any one of the preceding claims for the use set forth in the claim, wherein the pharmaceutical composition is administered as prophylaxis or a first-line therapy to the subjects or group of subjects having at least one identical HLA allele.

15. An antigen polypeptide for use as a medicament,comprising at least one HLA-A and/or HLA-B antigen peptide, wherein the HLA antigen peptide corresponds, preferably is identical to at least one amino acid sequence in the coronavirus, such as spike S1 and S2 domain, nucleocapsid protein, envelope protein and/or ORF 1ab polyprotein, wherein the antigen polypeptide sequence comprises the following scaffold amino acid sequence: (a) a long peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which comprises one HLA-A antigen peptide or HLA-B antigen peptide as defined herein; the amino acid sequence further comprises up to 1 to 30 amino acids; and/or (b) a similarity peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a) or (b), wherein the HLA-A antigen peptide or HLA-B antigen peptide as defined herein having less than 100% sequence identity or similarity to the native HLA antigen peptide, such as having at least 85%, more preferably at least 90%, sequence identity; and/or (c) a substitution peptide, i.e., a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a) or (b), wherein the HLA-A antigen peptide or HLA-B antigen peptide as defined herein having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence of the native HLA antigen peptide; and/or PMCR GmbH PMC-0005-P-LU 14.08.2023 Luxemburgische Patentanmeldung

(d) a tandem peptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of at least 2 identical or different HLA-A and/or HLA-B antigen peptide sequences, both as defined in items (a) to (c); and/or (e) an overlapping tandem peptide, i.e. a compound, construct, or polypeptide, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting of at least 2 identical or different HLA-A and/or HLA-B antigen peptide sequences, both as defined in items (a) to (c), in which the at least two HLA antigen peptides overlap in their amino acid sequence, wherein the antigen polypeptide of claim 15, wherein the antigen polypeptide is selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs. 39 — 58.

16. The antigen polypeptide of claim 15, wherein the antigen polypeptide sequence comprises the following scaffold amino acid sequence: (a) an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 — 58; and/or (b) an amino acid sequence having less than 100% sequence identity or similarity to the amino acid sequence according to item (a), such as at least 85%, more preferably at least 90%, sequence identity (as defined herein) to an amino acid sequence of SEQ ID NOs: 38 — 58; and/or (c) an amino acid sequence, wherein the amino acid sequence of which is as defined in item (a), comprising or consisting essentially of only one amino acid substitution relative to the amino acid sequence(s) selected from the group consisting of SEQ ID NOs: 38 — 58; and/or (d) a tandem peptide, i.e., a compound, construct, or polypeptide comprising or consisting of at least two identical or different peptide sequences of at least one sequence as defined in items (a) to (c) above, in which the amino acid sequences are optionally linked to each other by suitable linkers (so-called oligopeptides); and/or (e) an overlapping tandem peptide comprising or consisting of at least 2, more preferably 3, such as at least 4 identical or different HLA-A and/or HLA-B antigen peptide sequences (as defined herein), encoding for at least two, different HLA-A and/or HLA- PMCR GmbH PMC-0005-P-LU 14.08.2023 Luxemburgische Patentanmeldung

B antigen peptides (as defined herein), wherein the at least two HLA antigen peptides overlap in their amino acid sequence

17. The antigen polypeptide of claim 15, in particular for use in the therapeutic or prophylactic treatment of a coronavirus infectious diseases or for a pharmaceutical composition according to any one of claims 1 to 14.

18. The antigen polypeptide of any one of claims 15 to 17 for use as a medicament, in particular for use in the therapeutic or prophylactic treatment of a coronavirus infectious diseases, wherein the method comprising administering to the subject a treatment regimen including a pharmacologically effective amount of the antigen polypeptide.

19. The antigen polypeptide any one of claims 15 to 18 that are immunogenic in the patient or group of patients, determined by an immunogenicity assay, in particular by Western blot, Electrochemiluminescent immunoassay (ECL), ELISA techniques, ICS, ELISPOT, AFM, MS or immunodetection with microscopic analysis.

20. A method for preparing a pharmaceutical composition according to any one of claims 1 to 14, comprising the following steps: (a) determining at least one HLA antigen peptide corresponding to MHC class | complexes and/or at least one antigen polypeptide exposed on the cell surface of cells of the patient or group of patients to be treated with at least one identical HLA allele; (b) synthesizing the HLA antigen peptide corresponding to MHC class | complexes and/or the antigen polypeptide determined in step (a); (c) preparing the pharmaceutical composition according to the invention comprising at least one HLA antigen peptide corresponding to MHC class | complexes and/or at least one antigen polypeptide synthesized in step (b). PMCR GmbH PMC-0005-P-LU 14.08.2023 Luxemburgische Patentanmeldung