# US 12,214,048 B2 · Combination of an immune checkpoint modulator and a complex comprising a cell penetrating peptide, a cargo and a TLR peptide agonist for use in medicine

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- Number: US 12,214,048 B2
- Document ID: US12214048B2
- Status: Granted
- Kind: B2
- Application number: 17/569,261
- Filed: 2022-01-05
- Published: 2022-04-21
- Granted: 2025-02-04
- Earliest claimed: 2016-03-16
- Assignees: Amal Therapeutics SA
- Applicants: Amal Therapeutics SA
- Inventors: Madiha Derouazi; Elodie Belnoue
- Web page: https://patentgrep.com/patent/US12214048B2

## CPC

- A61K 47/66: the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
- A61K 38/17: from animals; from humans {(enzyme inhibitors A61K38/005)}
- A61K 38/18: Growth factors; Growth regulators
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 39/0011: {Cancer antigens}
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61K 40/4214: {Receptors for cytokines}
- A61K 47/42: Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34)
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/642: {the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate}
- A61K 47/6425: {the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell}
- A61P 35/00: Antineoplastic agents
- C07K 2/00: Peptides of undefined number of amino acids; Derivatives thereof
- C07K 7/06: having 5 to 11 amino acids
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/475: Growth factors; Growth regulators
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 15/625: {containing a sequence coding for a signal sequence}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- A61K 2039/55516: {Proteins; Peptides}
- A61K 2039/55572: {Lipopolysaccharides; Lipid A; Monophosphoryl lipid A}
- C07K 2317/75: Agonist effect on antigen
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides a combination of an immune checkpoint modulator and a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist for use in medicine, in particular in the prevention and/or treatment of cancer. Moreover, the present invention also provides compositions, such as a pharmaceutical compositions and vaccines, which are useful in medicine, for example in the prevention and/or treatment of cancer.

## Claims

### Claim 1

A combination comprising:
(i) an immune checkpoint modulator, and
(ii) a complex comprising:
a) a cell penetrating peptide;
b) at least one antigen or antigenic epitope; and
c) at least one TLR peptide agonist, wherein the TLR peptide agonist is a TLR2 or TLR4 peptide agonist, and
wherein components a)-c) are covalently linked.

### Claim 2 (depends on 1)

The combination according to claim 1, wherein the complex is a recombinant polypeptide or a recombinant protein.

### Claim 3 (depends on 1)

The combination according to claim 1, wherein the cell penetrating peptide:
(i) has a length of 5 to 50 amino acids in total; and
(ii) has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO: 3, or a variant thereof wherein zero 1, 2, 3, 4, or 5 amino acids have been substituted, deleted, and/or added without abrogating said peptide's cell penetrating ability, or a variant thereof.

### Claim 4 (depends on 3)

The combination according to claim 3, wherein the cell penetrating peptide has an amino acid sequence comprising the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or a variant thereof sharing at least 90% sequence identity with at least one of SEQ ID NOs: 6, 7, 8, or 11.

### Claim 5 (depends on 4)

The combination according to claim 4, wherein the cell penetrating peptide has an amino acid sequence comprising the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18).

### Claim 6 (depends on 4)

The combination according to claim 4, wherein the cell penetrating peptide has an amino acid sequence consisting of the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18).

### Claim 7 (depends on 1)

The combination according to claim 1, wherein the at least one antigen or antigenic epitope comprises at least one pathogen epitope, at least one tumor epitope, or a combination thereof.

### Claim 8 (depends on 1)

The combination according to claim 1, wherein the complex comprises more than one antigen or antigenic epitope positioned consecutively in the complex.

### Claim 9 (depends on 1)

The combination according to claim 1, wherein the at least one TLR peptide agonist comprises the amino acid sequence according to SEQ ID NO: 15 or 47 or a variant thereof sharing at least 90% sequence identity with SEQ ID NO: 15 or 47 without abrogating said peptide's TLR agonist ability.

### Claim 10 (depends on 9)

The combination according to claim 9, wherein the at least one TLR peptide agonist comprises the amino acid sequence according to SEQ ID NO: 15 or 47.

### Claim 11 (depends on 1)

The combination according to claim 1, wherein the immune checkpoint modulator is an activator or an inhibitor of one or more immune checkpoint point molecule(s) selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, GITR, TNFR and FasR/DcR3; or an activator or an inhibitor of one or more ligands thereof.

### Claim 12 (depends on 11)

The combination according to claim 11, wherein the immune checkpoint modulator is an inhibitor of an inhibitory checkpoint molecule.

### Claim 13 (depends on 11)

The combination according to claim 11, wherein the immune checkpoint modulator is an activator of a stimulatory or costimulatory checkpoint molecule.

### Claim 14 (depends on 11)

The combination according to claim 11, wherein the immune checkpoint modulator is a modulator of CD40, CTLA-4, PD-L1, PD-L2, PD-1 and/or IDO.

### Claim 15 (depends on 11)

The combination according to claim 11 comprising more than one immune checkpoint modulator.

### Claim 16 (depends on 11)

The combination according to claim 11, wherein the complex is a recombinant polypeptide or a recombinant protein wherein:
a) the cell penetrating peptide has an amino acid sequence comprising the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or a variant thereof sharing at least 90% sequence identity with at least one of SEQ ID NOs: 6, 7, 8, or 11 without abrogating said peptide's cell penetrating ability; and
b) the at least one antigen or antigenic epitope is a peptide, polypeptide or protein.

### Claim 17 (depends on 11)

The combination according to claim 11, wherein the complex is a recombinant polypeptide or a recombinant protein wherein:
a) the cell penetrating peptide has an amino acid sequence consisting of the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or a variant thereof sharing at least 90% sequence identity with at least one of SEQ ID NOs: 6, 7, 8, or 11 without abrogating said peptide's cell penetrating ability; and
b) the at least one antigen or antigenic epitope is a peptide, polypeptide or protein.

### Claim 18 (depends on 1)

The combination according to claim 1, wherein the at least one TLR peptide agonist consists of the amino acid sequence according to SEQ ID NO: 15 or 47 or a variant thereof sharing at least 90% sequence identity with SEQ ID NO:15 or 47 without abrogating said peptide's TLR agonist ability.

### Claim 19 (depends on 18)

The combination according to claim 18, wherein the at least one TLR peptide agonist consists of the amino acid sequence according to SEQ ID NO: 15 or 47.

### Claim 20

A kit comprising:
(i) an immune checkpoint modulator; and
(ii) a complex comprising:
a) a cell penetrating peptide;
b) at least one antigen or antigenic epitope; and
c) at least one TLR peptide agonist, wherein the TLR peptide agonist is a TLR2 or TLR4 peptide agonist; and
wherein components a)-c) are covalently linked; and
(iii) a package insert or label having directions to treat cancer.

### Claim 21

A pharmaceutical composition comprising:
(i) an immune checkpoint modulator; and
(ii) a complex comprising:
a) a cell penetrating peptide;
b) at least one antigen or antigenic epitope; and
c) at least one TLR peptide agonist, wherein the TLR peptide agonist is a TLR2 or TLR4 peptide agonist; and
wherein components a)-c) are covalently linked.

### Claim 22

A combination comprising:
(i) an immune checkpoint modulator; and
(ii) a complex comprising:
a) a cell penetrating peptide;
b) at least one antigen or antigenic epitope; and
c) at least one TLR peptide agonist, wherein the TLR peptide agonist is a TLR2 or TLR4 peptide agonist;
wherein components a)-c) are covalently linked, and
wherein the complex is a recombinant polypeptide or a recombinant protein.

### Claim 23 (depends on 22)

The combination according to claim 22, wherein the cell penetrating peptide has an amino acid sequence comprising the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or a variant thereof sharing at least 90% sequence identity with at least one of SEQ ID NOs: 6, 7, 8, or 11.

### Claim 24 (depends on 23)

The combination according to claim 23, wherein the cell penetrating peptide has an amino acid sequence consisting of the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18).

### Claim 25 (depends on 22)

The combination according to claim 22, wherein the at least one antigen or antigenic epitope comprises at least one pathogen epitope, at least one tumor epitope, or a combination thereof.

### Claim 26 (depends on 22)

The combination according to claim 22, wherein the complex comprises more than one antigen or antigenic epitope positioned consecutively in the complex.

### Claim 27 (depends on 22)

The combination according to claim 22, wherein the at least one TLR peptide agonist comprises the amino acid sequence according to SEQ ID NO: 15 or 47 or a variant thereof sharing at least 90% sequence identity with SEQ ID NO:15 or 47 without abrogating said peptide's TLR agonist ability.

### Claim 28 (depends on 27)

The combination according to claim 27, wherein the at least one TLR peptide agonist consists of the amino acid sequence according to SEQ ID NO: 15 or 47.

### Claim 29 (depends on 22)

The combination according to claim 22, wherein the immune checkpoint modulator is an activator or an inhibitor of one or more immune checkpoint point molecule(s) selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, GITR, TNFR and FasR/DcR3; or an activator or an inhibitor of one or more ligands thereof.

### Claim 30 (depends on 29)

The combination according to claim 29, wherein the immune checkpoint modulator is a modulator of CD40, CTLA-4, PD-L1, PD-L2, PD-1 and/or IDO.

### Claim 31 (depends on 29)

The combination according to claim 29 comprising more than one immune checkpoint modulator.

### Claim 32 (depends on 30)

The combination according to claim 30, wherein:
a) the cell penetrating peptide has an amino acid sequence consisting of the amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18);
b) the at least one TLR peptide agonist consists of the amino acid sequence according to SEQ ID NO: 15 or 47; and
c) the at least one antigen or antigenic epitope is a peptide, polypeptide or protein.