EP3762427A1

EP3762427A1 - Anti-pd-1 vaccine composition

Info

Publication number: EP3762427A1
Application number: EP19714322.5A
Authority: EP
Inventors: Lucille DESALLAIS; Jean-Pierre Salles; Jean-François ZAGURY
Original assignee: Peptinov Sas; CNAM Conservatoire National des Arts et Metiers
Current assignee: Peptinov Sas; CNAM Conservatoire National des Arts et Metiers
Priority date: 2018-03-05
Filing date: 2019-03-05
Publication date: 2021-01-13
Also published as: WO2019170684A1; JP2021516992A; US20210371496A1; FR3078536A1; CN112771078A

Abstract

The present invention relates to a polypeptide which comprises or consists of: - a first sequence consisting of at least 8 contiguous amino acid residues selected from within the sequence extending from amino acid residues 123 to 137 of the PD-1 protein, and at most 30 contiguous amino acid residues selected from within the complete sequence of the PD-1 protein; and/or - a second sequence consisting of at least 8 contiguous amino acid residues selected from within the sequence extending from amino acid residues 66 to 81 of the PD-1 protein, and at most 30 contiguous amino acid residues selected from within the complete sequence of the PD-1 protein; and/or - a third sequence consisting of at least 8 contiguous amino acid residues selected from within the sequence extending from amino acid residues 95 to 110 of the PD-1 protein, and at most 30 contiguous amino acid residues selected from within the complete sequence of the PD-1 protein; and/or - a fourth sequence consisting of at least 8 contiguous amino acid residues selected from within the sequence extending from amino acid residues 22 to 33 of the PD-1 protein, and at most 30 contiguous amino acid residues selected from within the complete sequence of the PD-1 protein.

Description

ANTI-PD-1 VACCINE COMPOSITION

Field of the invention

The present invention provides polypeptides useful for eliciting an immune response directed against PD-1 protein.

Technical background

It is now established that the intensity of the natural immune response against cancer antigens is correlated with better prognosis for patients for many types of neoplasia. Clinical observations, supported by extensive experimental evidence, have helped define the concept of cancer immunoassay, in which emerging tumors are usually eradicated by the immune system, except in circumstances where cancer cells have evolved. to escape immune detection.

Cancer Immunotherapy - a direct application of the concept of immuno-surveillance - which has experienced spectacular growth and success over the past decade, has revolutionized the clinical management of a wide range of malignancies previously associated with poor prognosis

At the forefront of the development of immunotherapy are monoclonal antibodies, blockers of immune-checkpoint blockers (ICBs), which are very successful in oncology because of their wide activity on many types tumors, the durability of their responses and their treatment capabilities in metastatic chemo-resistant tumors.

Among the control point blocking strategies, the two most important (in terms of clinical success to date) are its targeting by monoclonal antibodies specific for cytotoxic T-cell-associated protein 4 (CTLA-4) and interaction between the programmed cell death protein 1 (PD-1) and the ligand of this programmed cell death protein 1 (PD-L1). In particular, the inhibition of PD-L1 signaling has been proposed as a means of improving T cell immunity for the treatment of cancer (anfi-fumoral immunity) but also in the treatment of infections (acute persistent infection and chronic). To date, four CBIs targeting the PD-1 / PD-L1 axis have been approved by the US Federal Drug Administration (FDA) for review (see for example, see Abdin et al (2018) Cancers 10 32, ):

(1) pembrolizumab, an anti-PD-1 monoclonal antibody (mAb) approved for people with unresectable metastatic melanoma or advanced non-small-cell metastatic lung carcinoma (NSCLC) whose tumors express PD-1 L1;

(2) nivolumab, an anti-PD-1 monoclonal antibody (mAb) approved for unresectable or metastatic melanoma, advanced metastatic NSCLC progressing with or after platinum-based chemotherapy, and advanced renal cell carcinoma, including metastatic ;

(3) atezolizumab, a recently approved anti-PD-Ll monoclonal antibody (mAb) for the treatment of locally advanced or metastatic urothelial carcinoma that does not respond to platinum derivative chemotherapy; e †

(4) Avelumab, a recently approved anti-PD-Ll monoclonal antibody (mAb) for the treatment of metastatic Merkel cell carcinoma.

In addition, these already approved BCIs in a number of indications could also be recognized in the future as being useful in the treatment of other forms of cancer.

In addition, PD-L1 targeting ICBs have been shown to be highly effective in melanoma, NSCLC and renal cell carcinoma.

However, all the products marketed or developed as IBB targeting the PD-1 / PD-L1 axis are monoclonal antibodies e † are therefore affected by the same limitations as other monoclonal antibody treatments: a high cost The need for frequent re-administration is the development of an immune response against the administered monoclonal antibodies.

It is therefore an object of the present invention to overcome these disadvantages.

Summary of the invention

The present invention results from the unexpected finding by the inventors that polypeptides derived from sequences extending residues of amino acids 22 to 33, 66 to 81, 95 to 110, e 123 to 137 of the human PD-1 protein allowed the production of antibodies neutralizing the PD-1 protein by mice to which they were administered.

The present invention therefore relates to a polypeptide comprising or consisting of:

a first sequence consisting of at least 8 contiguous amino acid residues selected within the sequence extending from amino acid residues 123 to 137 of the PD-1 protein and of at most 30 acid residues contiguous amines selected from the complete sequence of the PD-1 protein, or a variant sequence having at least 75% identity with the first sequence; and or

a second sequence consisting of at least 8 contiguous amino acid residues chosen from the sequence extending from amino acid residues 66 to 81 of the PD-1 protein and of at most 30 acid residues contiguous amines selected from the complete sequence of the PD-1 protein, or a variant sequence having at least 75% identity with the second sequence; and or

a third sequence consisting of at least 8 contiguous amino acid residues selected within the sequence extending from amino acid residues 95 to 110 of the PD-1 protein and at most 30 residues of contiguous amino acids selected from the complete sequence of the PD-1 protein, or a variant sequence having at least 75% identity with the third sequence; and or

a fourth sequence consisting of at least 8 contiguous amino acid residues selected from the sequence extending from amino acid residues 22 to 33 of the PD-1 protein and of at most 30 acid residues contiguous amines selected from the complete sequence of the PD-1 protein, or a variant sequence having at least 75% identity with the fourth sequence;

provided that the polypeptide is different from the PD-1 protein e † that it does not consist of a portion of more than 30 contiguous amino acid residues of the PD-1 protein, e † provided that polypeptides consisting respectively of variant sequences of the first, second, third, and fourth sequences elicit an immune response directed against the PD-1 protein.

In a preferred embodiment, the invention relates more particularly to a polypeptide comprising or consisting of:

a first sequence consisting of SEQ ID NO: 1, 50, 51, 52, 53, or 54, or a variant sequence having at least 75% identity with the first sequence; and or

a second sequence consisting of SEQ ID NO: 2, 55, 56, or 57, or a variant sequence having at least 75% identity with the second sequence; and or

a third sequence consisting of SEQ ID NO: 3 or 58, or a variant sequence exhibiting at least 75% identity with the third sequence; and or

a fourth sequence consisting of SEQ ID NO: 4, or a variant sequence exhibiting at least 75% identity with the fourth sequence; provided that polypeptides consisting respectively of variant sequences of the first, the second, the third and the fourth sequence elicit an immune response directed against the PD-1 protein.

The present invention also relates to a nucleic acid encoding a polypeptide as defined above, or the complement thereof.

The present invention also relates to:

at least one polypeptide as defined above, or

at least one nucleic acid as defined above,

for use in fife medicine, especially vaccine. In a particular embodiment of the invention, the medicament, in particular the vaccine, as defined above also comprises at least one other compound intended for the prevention or treatment of a disease linked or due to the expression of PD-1 protein or PD-L1 protein, cancer or infectious disease.

The present invention also relates to a pharmaceutical composition, in particular a vaccine composition, comprising, with the filler of active substance:

at least one polypeptide as defined above, or

at least one nucleic acid as defined above, optionally in combination with at least one pharmaceutically acceptable vehicle. In a particular embodiment of the invention, the pharmaceutical composition, in particular vaccine, as defined above also comprises at least one other compound intended for the prevention or treatment of a disease linked or due to the expression of PD-1 protein or PD-L1 protein, cancer or infectious disease.

The present invention also relates to the use of a polypeptide as defined above, for the preparation of an antibody, an antibody fragment or an aptamer.

The present invention also relates to a method for preparing an antibody, an antibody fragment or an aptamer comprising a step of administering a polypeptide as defined above to an antibody producing organism or an affinity screening step of an antibody, an antibody fragment or an aptamer which binds to the polypeptide as defined above.

The present invention also relates to an antibody, an antibody fragment, or an anti-PD-1 aptamer specifically directed against the polypeptide as defined above, provided that the polypeptide does not comprise more than two amino acid residues. in addition to the first, second, third, or fourth sequences or their respective variant sequences.

The present invention also relates to an antibody, an antibody fragment, or an aptamer as defined above for use as a medicament. In a particular embodiment of the invention, the medicament as defined above also comprises at least one other compound intended for the prevention or treatment of a disease linked or due to the expression of the PD-1 protein. or PD-L1 protein, cancer or infectious disease.

The present invention also relates to a pharmaceutical composition comprising, as active ingredient, an antibody, an antibody fragment, or an aptamer as defined above, optionally in combination with a pharmaceutically acceptable vehicle. In a particular embodiment of the invention, the pharmaceutical composition as defined above also comprises at least one other compound intended for the prevention or treatment of a disease linked or due to the expression of the PD-protein. 1 or PD-L1 protein, cancer or infectious disease. The present invention also relates to a polypeptide as defined above, a nucleic acid as defined above, or a pharmaceutical composition as defined above, for use in a method for eliciting an immune response directed against the protein PD-1 in an individual. In a particular embodiment of the invention, the polypeptide, nucleic acid or pharmaceutical composition is used in combination with at least one other compound useful for eliciting an immune response directed against the PD-1 protein.

The present invention also relates to a method for eliciting an immune response directed against the PD-1 protein in an individual, comprising administering to the individual an effective amount of a polypeptide as defined above, a nucleic acid as defined above, or a pharmaceutical composition as defined above. In a particular embodiment of the invention, the polypeptide, nucleic acid or pharmaceutical composition is administered in combination with at least one other compound useful for eliciting an immune response directed against the PD-1 protein.

The present invention also relates to the use of a polypeptide as defined above or a nucleic acid as defined above for the preparation of a medicament for eliciting an immune response directed against the PD-1 protein in an individual. In a particular embodiment of the invention, the medicament also comprises at least one other compound useful for eliciting an immune response directed against the PD-1 protein.

The present invention also relates to a polypeptide as defined above, a nucleic acid as defined above, a pharmaceutical composition as defined above, or an antibody, an antibody fragment or an apfamer as defined herein. above, for use in a method of preventing or treating a PD-1 related or PD-related disease in an individual. In a particular embodiment of the invention, the polypeptide, the nucleic acid, the pharmaceutical composition, or the antibody, the antibody fragment or the apfamer is used in combination with at least one other therapy intended for the prevention or treatment of PD-1-related disease, cancer or infectious disease.

The present invention also relates to a method of preventing or treating a disease related to or due to expression of the PD-1 protein or the PD-L1 protein in an individual, comprising administering to the individual an effective amount of a polypeptide as defined above, a nucleic acid as defined above, a pharmaceutical composition such as as defined above, or an antibody, an antibody fragment or an apfamer as defined above. In a particular embodiment of the invention, the method comprises at least one other therapy for the prevention or treatment of a disease related to the expression of the PD-1 protein or of the PD-L1 protein, d 'a cancer or an infectious disease.

The present invention also relates to the use of a polypeptide as defined above, a nucleic acid as defined above, or an antibody, an antibody fragment or an apfamer such as as defined above, for the preparation of a medicament for the prevention or treatment of a disease related or due to the expression of the PD-1 protein or the PD-L1 protein in an individual. In a particular embodiment of the invention, the medicament comprises at least one other compound intended for the prevention of a disease linked or due to the expression of the PD-1 protein or of the PD-L1 protein, of cancer or an infectious disease.

The present invention also relates to products containing:

a polypeptide or a nucleic acid as defined above, and

at least one other compound intended for the prevention or treatment of a disease linked or due to the expression of the PD-1 protein or the PD-L1 protein, of a cancer or of an infectious disease,

as a combination product for simultaneous, separate or spread use over time for the prevention or treatment of a disease related to or due to expression of PD-1 protein or PD-L1 protein, cancer or an infectious disease in an individual.

Description of the invention

As a preliminary, it will be recalled that the term "comprising" means "including", "containing" or "encompassing", that is, when an object "includes" one or more elements, other elements that those mentioned can also be included in the object. On the other hand, the expression "consisting of" means "consisting of", that is, when an object "consists of" In one or more elements, the object can not include other elements than those mentioned.

polypeptide

Definition of PD-J protein

PD-1 protein is the programmed cell death protein (PDM1), also called PDC1 or differentiation cluster 279 (cluster of differentiation 279, CD279), and is well-known to humans. job.

Preferred species and sequences of the PD-J protein

Preferably, the PD-1 protein according to the invention is selected from the group consisting of human PD-1 protein, mouse PD-1 protein, monkey PD-1 protein, PD-1 protein, 1, bovine PD-1 protein, pig PD-1 protein, sheep PD-1 protein, goat PD-1 protein, camel PD-1 protein, and the like. wild or domestic, camel PD-1 protein, dog PD-1 protein, e. of PD-1 cat protein. Particularly preferably, the PD-1 protein is the human PD-1 protein.

Preferably:

the human PD-1 protein (hPD-1) es † as described in the UniProt / Swisspro † database under the reference Q151 16 e † es consisting of SEQ ID NO: 5,

mouse PD-1 protein (mPD-1) as described in the UniProt / Swisspro † database under the reference Q02242 e † es † consisting of SEQ ID NO: 6,

the monkey PD-1 protein as described in the Genbank database as NP_001 107830 e † es † consisting of SEQ ID NO: 7, the horse PD-1 protein as described in the database. Genbank data under the reference XP_023498583.1 or XP_023498582.1 e † es † consisting of SEQ ID NO: 8 or 9,

the dog PD-1 is as described in the SwissProf database under the reference A0A024FCJ9 e † es consisting of SEQ ID NO: 10, the chat PD-1 as described in the Genbank database under the reference NP_001 138982.1 † † consisting of SEQ ID NO: 1 1.

bovine PD-1 protein as described in the Genbank database as BAX73992.1; DAA30855.1 or NP_001076975.1 and es † consisting of SEQ ID NO: 12 or 13 or 14,

the pig PD-1 es as described in the Genbank database under the reference NP_001 191308.1 or XP_020930289.1 and es † consisting of SEQ ID NO: 15 or 16,

sheep protein PD-1 as described in the Genbank database under the reference XP_012031617.1 and es † consisting of SEQ ID NO: 17.

goat PD-1 protein † as described in the Genbank database under the reference XP_013818552.2 or XP_01 7899964.1 and es † consisting of SEQ ID NO: 18 or 19.

the domestic camel PD-1 protein as described in the Genbank database under the reference XP_010946391 .1 or XP_010946392.1 and es † consisting of SEQ ID NO: 20 or 21.

the wild camel PD-1 protein as described in the Genbank database under the reference XP_014412330.1 or XP_014412331 .1 and es † consisting of SEQ ID NO: 22 or 23.

the camel PD-1 protein † as described in the Genbank database under the reference XP_010986792.1 or XP_010986793.1 and es † consisting of SEQ ID NO: 24 or 25.

Numbering of amino acid residues

As used herein, the amino acid residue numbering of the PD-1 protein begins on the first amino acid residue, typically a methionine (M), forming the N-terminus of complete PD-1. encoded by the open reading frame of the PD-1 gene, that is, including its signal peptide. In addition, the amino acid residue numbering of the PD-1 protein used herein is defined by reference to the human PD-1 protein. It is thus easy for the mistrusting man to determine the amino acid residue of a PD-1 protein corresponding to a position number to which it is referred to according to the invention: it is sufficient to align the sequence of the PD-1 protein for which the residue is to be determined amino acid corresponding to a position number with a sequence of the human PD-1 protein, in particular SEQ ID NO: 5, so as to optimize the percentage identity between the two aligned sequences, and then to identify the residue of the amino acid corresponding to the position number sought as being that which is aligned with the amino acid residue of the sequence of the human PD-1 protein which bears that position number.

First, second, third, and fourth sequences

Preferably, the first sequence, the second sequence, the third, and the fourth sound sequence consist of at least 8, 9, 10, 11, 12 contiguous amino acid residues respectively selected within the sequence extending amino acid residues 123 to 137, 66 to 81, and 95 to 110, of the PD-1 protein or its † consisting respectively of at least the sequence extending from amino acid residues 123 to 137, 66 to 81, 95 to 110, and 22 to 33 of the PD-1 protein.

Also preferably, the first sequence, the second sequence, the third and the fourth sequence according to the invention are respectively constituted by at most 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 residues. of contiguous amino acids selected from the complete sequence of the PD-1 protein, or its † consisting respectively of at most amino acid residues 123 to 137, 66 to 81, 95 to 110, and 22 to 33 of the PD-1 protein.

In a preferred manner:

the first block according to the invention consists of SEQ ID NO: 1, 50, 51, 52, 53 or 54,

the second sequence according to the invention consists of SEQ ID NO: 2, 55, 56 or 57,

the third sequence according to the invention consists of SEQ ID NO: 3 or 58, and

the fourth sequence consists of SEQ ID NO: 4.

The amino acid residues of the human PD-1 protein corresponding to these sequences are shown in the following table:

Variant sequences

A variant sequence according to the invention, which has at least 75% identity with one of the first, second, third, and fourth sequences above, preferably has at least 80%, 85%, 90%, 95% or 98% identity with one of the first, second, third, and fourth sequences above.

As used herein, the percentage identity between two peptide sequences can be determined by performing optimal alignment over the entire length of the sequences, by determining the number of aligned positions for which the amino acids are identical in each sequence and by dividing this number by the total number of amino acids in the longest of the two sequences. The optimal alignment is the one that gives the highest percentage of identity between the two sequences.

Also preferably, a variant sequence according to the invention has at least 75%, 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, 57 or 58.

The variant sequence according to the invention is such that a polypeptide consisting of the variant sequence must make it possible to elicit an immune response directed against the PD-1 protein; that is to say that the administration of such a peptide, optionally cyclized by formation of at least one inter-cysteine disulfide bridge, if necessary after adding one or two cysteines within the peptide, and / or at its N-terminal end and / or at its C-terminal end, the peptide optionally being linked to a carrier molecule, in particular a carrier protein, such as KLH (Keyhole Limpet Hemocyanin), to an animal, such as a mouse, a rat or a rabbit, causes the production of antibodies directed against a PD-1 protein, in particular a PD-1 protein of the same species. than the one to which the sequence with which the variant sequence belongs has the highest percentage of identity. Those skilled in the art are well aware of how to determine whether an antibody is directed against PD-1, in particular by implementing an ELISA test. Preferably, the antibodies elicited by administration of the peptide are blocking or neutralizing, that is to say they prevent the PD-1 protein from exercising all or part, especially at least 10%, 25%, 50 %, 75%, of its activity, for example measured in vitro. As used herein, the activity of PD-1 is preferably a binding to the PD-L1 protein, which can be measured as in Example 2 which follows.

Polypeptide length

The polypeptide according to the invention preferably comprises at most 200, 150, 100, 90, 80, 70, 60, 50, 40 or 30 amino acid residues. It is different from the PD-1 protein and does not consist of a portion of more than 30 contiguous amino acid residues of the PD-1 protein. As is well understood by those skilled in the art, this does not exclude that it may consist of two or more portions of the PD-1 protein of not more than 30 contiguous amino acid residues, since these portions are not arranged to reconstitute a portion of the PD-1 protein of more than 30 contiguous amino acids.

As will be clear to the skilled man, the polypeptide according to the invention can include several repetitions, for example 2, 3, 4, 5, 10 or 20 repetitions, respectively of the first, second, third, e † fourth sequences e † of the variant sequence according to the invention.

Sequences in addition to the sequences of the first, second, third, and fourth sequences and variant sequences

Moreover, the polypeptide according to the invention may also comprise additional sequences not originating from the PD-1 protein.

These additional sequences may in particular provide physicochemical characteristics allowing improved structural presentation or improved solubility of the polypeptide according to the invention relative to a similar polypeptide but which will not include these additional sequences. The additional sequences may also comprise one or more peptide linker sequences, that is to say peptide linker, which are useful for binding in particular to a carrier molecule. Such peptide linker sequences typically comprise from 1 to 10, especially from 4 to 6, amino acid residues.

Moreover, these sequences not originating from the PD-1 protein may also comprise epitopes belonging to other proteins, making it possible to elicit or generate an immune response directed against these other proteins.

In addition, the polypeptide according to the invention may comprise exogenous (s) T epitope sequences, preferably universal, which makes it possible to enhance the immunogenicity of the polypeptide according to the invention.

The polypeptide according to the invention may also comprise at least one sequence of a carrier protein, for example a virus-like particle (VLP), as is described in particular in the international application WO 05/117983 for TNF. .

Cyclization of the polypeptide

The polypeptide according to the invention may be linear subform or cyclized subform. Preferably, the polypeptide according to the invention is in cyclized form. This cyclization may be of any type known to those skilled in the art.

The choice of the cyclization strategy according to the invention can in particular take into account the best antigenic presentation of the epitopes contained in the polypeptide according to the invention, and relate only to a part of the polypeptide (cyclization within the sequence). . Thus, as used herein, when the polypeptide of the invention is in a cyclized form, only a portion of the polypeptide may be included in one cycle while the remainder of the polypeptide is in linear form.

Depending on the functional groups present in the polypeptide, this cyclization can be carried out in several different ways, for example: from its C-terminus at its N-terminus, from its N-terminus to a side chain, a side chain at its C-terminal end or between two side chains. Among the various methods of cyclizing polypeptides, it is possible to mention lactamization, lactonization or the formation of a disulfide bridge. In particular, during the formation of an inter-cysteine disulfide bridge, that is to say between the radicals -SH of two cysteines, the cysteines may already be present in the variant sequence according to the invention or in the first, second, third and fourth sequences according to the invention, or else be added within these sequences, as well as at their N-terminus and / or or C-terminal.

Post-translational modifications, amino acid analogues

In addition, the polypeptide according to the invention may comprise post-translational modifications, such as glycosylations, methylations, acylations, in particular by fatty acids, or phosphorylations. In particular, the N-terminus of the polypeptide according to the invention may be acetylated and the C-terminus may be modified by amidation.

The polypeptide according to the invention may also comprise one or more analogs or derivatives of amino acids, don † non-natural or non-standard amino acids, in particular norleucine (Nie).

Carrier molecule

Also preferably, the polypeptide according to the invention is fixed or bound, in particular by covalent bonding, to a carrier molecule, in particular a carrier protein.

In particular, the carrier molecule may be Keyhole Limpet Hemocyanin (KLH) protein, hepatitis B surface antigen (HBsAg), bovine serum albumin (BSA), tetanus toxoid (TT) and toxoid of diphtheria (DT).

The toxoid of diphtheria (DT) according to the invention is preferably selected from the group consisting of CRM 197, CRM 176, CRM 228, CRM 45, CRM 9, CRM 102, CRM 103, and CRM 107.

Particularly preferably, the carrier molecule is CRM 197.

The binding of the polypeptide according to the invention to a carrier molecule, in particular a carrier protein, can be carried out using a heterobifunctional coupling agent, such as the Ny-maleimidobutyryloxysuccinimide ester (GMBS) and the derivative. sulfo-GMBS, m-maleimidobenzoyl-n-hydroxysuccinimide ester (MBS) and the sulfo-MBS derivative succinimidyl 4- (N-maleimidome † hyl) cyclohexane-1-carboxylate (SMCC), a carbodiimide, bisdiazonium-benzidine (BDB) or glutaraldehyde. When the GMBS, MBS or SMCC are used, they are preferably attached to a cysteine (C), which if not present in a first, a second, a third or a fourth sequence, or a sequence variant according to the invention may be added, in particular at its N-terminal or C-terminal end. Furthermore, when a cysteine is present in a first, a second, a third or a fourth sequence according to the invention at an undesired position, it is possible to implement, instead, a variant sequence in which cysteine is substituted with another amino acid, such as serine.

When BDB is used, it is preferably attached to a tyrosine (Y), which if not present in a first, a second, a third or a fourth sequence, or a variant sequence according to the invention, may be added, especially at its N-terminus or C-terminus. Moreover, when a tyrosine is present in a first, a second, a third or a fourth sequence according to the invention at an undesired position, it is possible to implement, instead, a variant sequence in which the tyrosine is substituted by another amino acid, such as phenylalanine (F).

Moreover, the binding of the polypeptide according to the invention to a carrier molecule, in particular a carrier protein, can also be carried out using a peptide linker or peptide linker, which binds to the polypeptide according to the invention. one side and the carrier molecule on the other side, optionally via a heterobifunctional coupling agent as defined above. Such peptide bonds typically comprise from 1 to 10, especially from 4 to 6, amino acid residues.

Most preferably, the polypeptide according to the invention is attached to the CRM 197 carrier protein according to a construction represented by a formula selected from the group consisting of the following formulas:

or :

CRM 197 denotes the carrier protein,

GMB denotes N-g-maleimidobutyryl,

- Acetyl + indicates that the N-terminus is acetylated,

- Amide + indicates that the C-terminus is modified by amidation,

cyclo () indicates a lacfame-type cyclization between the side chains of amino acid residues at C-terminal and N-terminus,

- cycloS-S () indicates a disulfide bridge cyclization between the sulfhydryl groups of the cysteines present in C-ferminal and in N-terminal,

the brackets ([X] n) indicate that one or more polypeptides are attached to the carrier protein, and

the underlined part represents the polypeptide according to the invention, of which SEQ ID NO is indicated in the right-hand column. Thus, very particularly preferably the polypeptide according to the invention consists of a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 26 to 49.

Preparation of the polypeptide

The polypeptide according to the invention may be prepared by any method known in the state of the art and in particular by chemical synthesis. It is also possible to prepare it by expression of the nucleic acid according to the invention in eukaryotic or prokaryotic cells.

Polypeptide activity

The polypeptide according to the invention, if necessary linked to a carrier molecule, is immunogenic, that is to say it can elicit or cause an immune reaction, in particular of humoral type, that is to say the production of antibodies by an individual, in particular a mammalian type, to which it is administered. In particular, the polypeptide according to the invention makes it possible to elicit an immune response directed against the PD-1 protein, in particular anti-PD-1 antibodies, preferably blocking or neutralizing anti-PD-1 antibodies, that is, that is to say that they prevent the PD-1 protein from exercising all or part, especially at least 10%, 25%, 50%, 75%, of its activity, for example measured in vitro. As used herein, the activity of PD-1 is preferably a binding to the PD-L1 protein, which can be measured as shown in Example 2 which follows.

Nucleic acid

The nucleic acid according to the invention is RNA or DNA, preferably DNA. It is preferred that the nucleic acid according to the invention is operably linked to a prokaryotic and / or eukaryotic promoter sequence, in particular that of a mammal or a virus. Moreover, the nucleic acid according to the invention can be included in a vector, such as a plasmid or a virus.

Antibodies, antibody fragments and aptamers

The antibodies, antibody fragments, and aptamers according to the invention are said to be specifically directed against a polypeptide as defined above when they show essentially no binding to another polypeptide, which does not does not include the polypeptide defined above, under conditions allowing the binding of antibodies, antibody fragment, and apfamers according to the invention to the polypeptides against which they are † specifically directed.

The antibody according to the invention may be polyclonal or monoclonal, preferably monoclonal. Moreover, as used herein, the "antibody fragments" comprise at least one antigen-binding part of the antibody don † they are derived †, and its † in particular of Fab, Fab 'type, F (ab ') 2, disulfide stabilized Fv (dsFv), dimerized (diabody) V region, trimerized, tetramerized or pentamerized, single chain Fv (scFv), complementarity determining region (CDR).

The antibodies can be of any species, in particular human, mouse, ra †, rabbit or camelide. Moreover, when they are not human, they can also be humanized, that is to say that the constant parts of these antibodies are replaced partially or entirely by corresponding human constant parts.

The antibodies according to the invention can be obtained by immunization of an animal using a polypeptide according to the invention according to techniques well known to those skilled in the art.

As used herein, aptamers are nucleic acids, particularly RNAs, capable of binding specifically to a molecular target, such as a protein. Aptamers can in particular be obtained by using the SELEX technique well known to those skilled in the art, from the polypeptides according to the invention.

Therapeutic use

diseases

Preferably, the disease linked or due to the expression of the PD-1 protein or of the PD-L1 protein according to the invention is a cancer or an infectious disease.

More preferably, the disease linked to or due to the PD-1 protein or the PD-L1 protein is selected from the group consisting of:

- unresectable metastatic melanoma, advanced non-small-cell metastatic lung carcinoma (NSCLC), advanced metastatic NSCLC progressing particularly with or after platinum-based chemotherapy, advanced renal cell carcinoma, including metastatic, and locally advanced or metastatic urothelial carcinoma especially not responding to chemotherapy with platinum derivatives, prostate cancer, breast cancer, colorectal cancer, or any other cancer where PD-1 / PD-L1 is involved in the suppression of an anti-tumor immune response,

- bacterial infections, such as pneumonia, meningitis, toxic shock syndrome, food poisoning, gastritis, ulcers, gonorrhea, boils, abscesses, impetigo, ear infections, tonsillitis, infections the urinary tract and genital infections broncho-pulmonary,

- viral infections, such as influenza, measles, hepatitis B, hepatitis C, infections with the human immunodeficiency virus (HIV), infections with herpes virus such as cytomegalovirus or Epsfein-Barr virus, herpes, e infections with human papillomavirus (HPV), e †

fungal infections, such as blastomycosis, coccidioiodomycosis, rhisfoplamosis, paracoccidioiodomycosis, candidiasis, cryptococcosis, aspergillosis, mucomycosis and pneumocystosis.

Individual

The person or individuals according to the invention are animals, preferably mammals or marsupials, more preferably humans, horses, cattle, pigs, sheep, goats, camels, camels, dogs or dogs. cats, most preferably humans. It will be preferred according to the invention that the polypeptide according to the invention is derived from a PD-1 protein belonging to the same species as the individual in which the polypeptide is to be used or administered.

Administration

Preferably, the polypeptide, the pharmaceutical composition, the drug or the product according to the invention is administered or in a form that can be administered orally, mucosally, in particular sublingually, parenterally, intraperitoneally, transcutaneously, intradermally, subcutaneously, intramuscularly. , intravenous or intra-arterial. doses

In the context of the invention, the polypeptide according to the invention may be administered in doses ranging for example from 1 ng to 1 g, preferably from 1 μg to 1 mg.

Pharmaceutically acceptable vehicle

As used herein, a "pharmaceutically acceptable carrier" includes all of the compounds, including excipients, that can be administered to an individual in conjunction with a pharmacological active ingredient.

adjuvant

Moreover, especially when it is used in a vaccine or prophylactic context, the polypeptide according to the invention may be combined or combined with an adjuvant, or the pharmaceutical composition, the drug or the product according to the invention may comprise an adjuvant. The adjuvant can be of any type suitable for increasing the immune response of an individual, animal or human, to the administration of a polypeptide. It can thus be complete or incomplete Freund's adjuvant, Monfanide ISA 51 VG, aluminum hydroxide or aluminum phosphate or calcium phosphate for example, the monfanide ISA 51 VG and the hydroxides of aluminum or aluminum phosphate being preferred. The adjuvant may be combined with the polypeptide according to the invention by carrying out a 1/1 mixture by volume of an adjuvant solution and a solution comprising the polypeptide.

Other therapy

As used herein, the term "other therapy" designates a pharmacological therapy with at least one other compound different from the polypeptide according to the invention or a non-pharmacological therapy such as, for example, radiotherapy, in particular anti-cancer therapy.

Other compound

The other compound that is useful for eliciting an immune response directed against the PD-1 protein according to the invention may in particular be a polypeptide different from that of the invention derived from the PD-1 protein or a polypeptide derived from PD-L1. Moreover, the other compound intended for the prevention or treatment of a disease linked to or due to the PD-1 protein or the PD-L1 protein, a cancer or an infectious disease can be a compound anticancer chemotherapy, an anti-cancer immunotherapy compound, for example a monoclonal antibody, an antibiotic, an antiviral agent, especially of the interferon type, or an antimycotic agent.

In addition, especially when it is used in a vaccine frame or included in a vaccine or a vaccine composition, the polypeptide according to the invention can be combined with other antigens intended to elicit an immune response. against a different target of the PD-1 protein, for example the PD-L1 protein. This type of combination is useful for the preparation of multivalent vaccines.

As used herein, the expression "in combination" or "combination product" means that the polypeptide as defined above and the other compound as defined above can be combined within one and the same pharmaceutical composition or the same drug, e † therefore be administered together, or be administered separately, that is to say, according to separate routes of administration and / or separate administration regimes, subject to when they are administered separately the periods of prophylactic or therapeutic activity of the polypeptide as defined above e † of the other compound as defined above overlap in whole or in part.

Thus, when the polypeptide and the other compound are administered separately, the polypeptide as defined above will preferably be administered within 24 hours, more preferably within 2 hours, and still more preferably within 1 hour. Depending on the administration of the other compound as defined above, its administration may be continued in the following days. Conversely, the other compound as defined above will preferably be administered within 24 hours, more preferably within 2 hours, even more preferably within 1 hour, following administration of the polypeptide as defined above. His administration will eventually be continued on the following days. In another preferred embodiment of the invention, when the polypeptide as defined above and the other compound as defined above are administered separately, they are administered essentially simultaneously.

The invention is further illustrated with the aid of Figures e † of the nonlimiting Examples which follow. Description of figures

Figure 1

FIG. 1 represents the relative amount of anfi-huPD-1 antibody present in the serum diluted to 1 / 500th of SWISS mice immunized with peptides PPV-10-01, PPV-10-02, PPV-10-03, PPV. -10-04 and PPV-10-05 measured by ELISA (ordinate axis, optical density (OD) at 450nm). The horizontal line at 0.2 OD unit represents the significance threshold.

Figure 2

FIG. 2 represents the percentage of neutralization of the PD1 / PD-L1 interaction obtained with purified rabbit antibodies (n = 4 / group) immunized with peptides PPV-10-01, PPV-10-02, PPV-10 -03, PPV-10-04 and PPV-10-05.

EXAMPLES

Example 1: Recognition of the whole human PD-1 protein fhPD-1) by sera of mice immunized with peptides derived from hPD-1.

Four peptides derived from the human PD-1 protein were produced by solid phase chemical synthesis and then cyclized by adding cysteines to the ends of the epitope of interest and forming a disulfide bridge. They were then coupled via a peptide linkage containing a C-ferminal cysteine to a carrier protein, CRM 197 (C-Reactive Material 197), using the GMBS coupling agent.

For each conjugate, SWISS mice (Janvier Labs, Genesf-Saint-Denis, France) free of specific pathogens were immunized subcutaneously with 100 μg peptide equivalent derived from human PD-1 (PPV-1). 10-01, PPV-10-02, PPV-10-03, PPV-10-04 or PPPV-10-05, see Table 1) emulsified with Montanide ISA 51 VG adjuvant (n = 8 per conjugate). The mice received four subcutaneous injections spaced 15 days apart (D0, D15, D30 and D45).

Table 1: Peptides derived from human PD-1 used for immunization

The amino acid residues are annotated from the sequence of the human PD-1 protein (Swissprot Q 151 16)

The relative amount of anti-PD-1 antibody was evaluated in mouse sera on D54 by ELISA (1: 500 dilution). It is observed that all the conjugates tested generate the production of antibodies recognizing the human PD-1 protein (FIG.

Example 2 Neutralization of the biological activity of human PD-1 with antibodies purified from the serum of rabbits immunized with the peptides derived from

PD-1 human

Neutralizing capacity of purified IgG from rabbit serum (n = 4 / group) respectively immunized with peptides PPV-10-01, PPV-10-02, PPV-10-03, PPV-10-04 e † PPV 10-05, was evaluated in a PD-1 / PD-L1 interaction neutralization cell (Promega, J 1250). This is based on the interaction between 2 cell lines:

a Jurkat effector cell line expressing the human PD-1 gene and the luciferase gene placed under the control of the NFAT-RE response element,

a CHO-K1 cell line expressing the human PD-L1 gene and a surface protein making it possible to activate the TCRs in an antigen-dependent manner.

When both lines are co-cultured, the interaction of PD-1 protein with PDL-1 protein inhibits TCR signaling and luciferase expression (no luminescence will be emitted). On the other hand, the addition of anti-PD-1 antibodies neutralizing the interaction of PD-1 with PD-L1 will raise the inhibitory signal, lead to the activation of the TCR and the emission of luminescence.

Description of the experiment carried out:

Plating (J l): Inoculation of a 96-well flat-bottomed plate treated for cell culture with CHO-K1 cells. Incubate the plate for 20h at 37 ° C.

Incubation of samples (J2): Distribution on the plate containing CHO-K1 cells, antibody samples to be tested as well as Jurkat effector cells. The plate is then incubated for 6 hours at 37 ° C.

Addition of Bio-GIo ™ Revelation Reagent to each well. Incubation for 30 minutes at room temperature. Measurement of luminescence using a luminometer. IgG from rabbits immunized with peptides PPV-10-01, PPV-10-02, PPV-10-03, PPV-10-04 and PPV-10-05 were found to neutralize the interaction of PD-1 protein. with the PD-L1 protein to varying degrees, from about 30% to about 60% (Figure 2).

Claims

A polypeptide comprising or consisting of:

a fourth sequence consisting of at least 8 contiguous amino acid residues selected from the sequence extending from amino acid residues 22 to 33 of the PD-1 protein and of at most 30 acid residues contiguous amines selected from the complete sequence of the PD-1 protein, or a variant sequence having at least 75% identity with the fourth sequence; provided that the polypeptide is different from the PD-1 protein e † that it does not consist of a portion of more than 30 contiguous amino acid residues of the PD-1 protein, and

provided that polypeptides consisting respectively of variant sequences of the first, second, third and fourth sequences elicit an immune response directed against the PD-1 protein.

The polypeptide according to claim 1, wherein the first sequence, the second sequence, the third, and the fourth sequence consist of at least 12 contiguous amino acid residues respectively selected within the

RECTIFIED SHEET (RULE 91) ISA / EP sequence extending from amino acid residues 66 to 81, 95 to 110, 123 to 137, and 22 to 33 of the PD-1 protein.

The polypeptide according to claim 1 or 2, wherein the first sequence, the second sequence, the third and the fourth sequence are respectively at most amino acid residues 66 to 81, 95 to 110, 123 to 137, and 22 to 33 of the PD-1 protein.

The polypeptide according to one of claims 1 to 3, wherein the PD-1 protein is selected from the group consisting of human PD-1 protein, mouse PD-1 protein, PD-1 protein monkey, horse PD-1 protein, bovine PD-1 protein, pig PD-1 protein, sheep PD-1 protein, goat PD-1 protein, protein PD-1 camel and camel PD-1 protein, dog PD-1 protein, and cat PD-1 protein.

The polypeptide according to one of claims 1 to 4, wherein the PD-1 protein is human PD-1 protein.

Polypeptide according to one of Claims 1 to 5, in which:

the first sequence consists of SEQ ID NO: 1, 50, 51, 52 or 54,

the second sequence consists of SEQ ID NO: 2, 55, 56 or 57, and

the third sequence consists of SEQ ID NO: 3 or 58,

the fourth sequence consists of SEQ ID NO: 4.

The polypeptide according to one of claims 1 to 6, wherein the polypeptide is in cyclized form.

8. The polypeptide according to one of claims 1 to 7, wherein the polypeptide is linked to a carrier molecule.

9. Nucleic acid encoding a polypeptide as defined in one of claims 1 to 8, or the complement thereof.

RECTIFIED SHEET (RULE 91) ISA / EP

10. Pharmaceutical composition comprising, as active substance:

at least one polypeptide as defined in one of claims 1 to 8, or

at least one nucleic acid as defined in claim 9,

optionally in combination with at least one pharmaceutically acceptable carrier.

11. Use of a polypeptide as defined in one of claims 1 to 8, for the preparation of an antibody, an antibody fragment or an aptamer.

An antibody, antibody fragment, or anti-PD-1 aptamer specifically directed against the polypeptide as defined in one of claims 1 to 7, provided that the polypeptide does not comprise more than two amino acid residues. in addition to the first, second, third or fourth sequences or their respective variant sequences.

An antibody, antibody fragment, or aptamer according to claim 12 for use with a fife of medicine.

A polypeptide as defined in one of claims 1 to 8, a nucleic acid as defined in claim 9, or a pharmaceutical composition as defined in claim 10, for use in a method for eliciting a directed immune response against the PD-1 protein in an individual.

Polypeptide as defined in one of claims 1 to 8, nucleic acid as defined in claim 9, pharmaceutical composition as defined in claim 10, or antibody, antibody fragment or aptamer as defined in the present invention. claim 1 3 for use in a method of preventing or treating a disease related to or due to expression of the PD-1 protein or PD-L1 protein in an individual.

Polypeptide, nucleic acid, pharmaceutical composition, or antibody, antibody fragment or aptamer for use according to claim 15, wherein the disease related to or due to the expression of the PD-1 protein or the PD-1 protein. L1 is a cancer or an infectious disease.

RECTIFIED SHEET (RULE 91) ISA / EP

17. A polypeptide, nucleic acid, pharmaceutical composition, or antibody, antibody fragment or apfamer for use according to claim 15 or 16, wherein the disease linked or due to the expression of the PD-1 protein or the protein PD-L1, is selected from the group consisting of:

- unresectable metastatic melanoma, advanced non-small-cell metastatic lung carcinoma (NSCLC), advanced metastatic NSCLC progressing particularly with or after platinum-based chemotherapy, advanced renal cell carcinoma, including metastatic carcinoma, and urothelial carcinoma locally advanced or metastatic in particular not responding to chemotherapy based on platinum drifts, prostate cancer, breast cancer, colorectal cancer, or any other cancer where the PD-1-PD-L1 axis is involved in suppressing an anti-tumor immune response

- bacterial infections, such as pneumonia, meningitis, toxic shock syndrome, food poisoning, gastritis, ulcers, gonorrhea, boils, abscesses, impetigo, ear infections, tonsillitis, infections urinary and genital tract and bronchopulmonary infections,

- viral infections, such as influenza, measles, hepatitis B, hepatitis C, infections with the human immunodeficiency virus (HIV), infections with herpes virus such as cytomegalovirus or the Epsfein-Barr virus, herpes, and human papillomavirus (HPV) infections, and

fungal infections, such as blastomycosis, coccidioiodomycosis, histoplamosis, paracoccidioiodomycosis, candidiasis, cryptococcosis, aspergillosis, mucomycosis and pneumocystosis.

RECTIFIED SHEET (RULE 91) ISA / EP