# US 2020/0399338 A1 · IL-15 Conjugates and Uses Thereof

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- Number: US 2020/0399338 A1
- Document ID: US20200399338A1
- Status: Published application
- Kind: A1
- Application number: 17/001,965
- Filed: 2020-08-25
- Published: 2020-12-24
- Earliest claimed: 2018-02-26
- Assignees: Synthorx, Inc.
- Applicants: Synthorx, Inc.
- Inventors: Carolina E. CAFFARO; Jerod PTACIN; Marcos MILLA
- Web page: https://patentgrep.com/patent/US20200399338A1

## CPC

- A61K 38/2086: {IL-13 to IL-16}
- C07K 14/5443: {IL-15}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61P 35/00: Antineoplastic agents
- C07K 1/1075: {by covalent attachment of amino acids or peptide residues}
- C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids}
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C12N 2310/3513: Protein; Peptide
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are interleukin (IL) 15 conjugates and use in the treatment of one or more indications. Also described herein include pharmaceutical compositions and kits comprising one or more of IL-15 conjugates.

## Claims

### Claim 1

An isolated and modified interleukin 15 (IL-15) polypeptide comprising at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-15 polypeptide and interleukin 15 receptor α (IL-15Rα) but retains significant binding with interleukin 15 receptor βγ (IL-15Rβγ) signaling complex to form an IL-15/IL-15Rβγ complex, wherein the reduced binding to IL-15Rα is compared to binding between a wild-type IL-15 polypeptide and IL-15Rα.

### Claim 2

An isolated and modified interleukin 15 (IL-15) polypeptide comprising at least one unnatural amino acid at a position on the polypeptide that enhances half-life, wherein the enhanced half-life is compared to a half-life of a wild-type IL-15 protein.

### Claim 3

An isolated and modified interleukin 15 (IL-15) polypeptide comprising at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-15 polypeptide and interleukin 15 receptor α (IL-15Rα) but retains significant signaling potency with interleukin 15 receptor βγ (IL-15Rβγ) signaling complex, wherein the reduced binding to IL-15Rα is compared to binding between a wild-type IL-15 polypeptide and IL-15Rα.

### Claim 4 (depends on 3)

The isolated and modified IL-15 polypeptide of claim 3, wherein the signaling potency is compared to a signaling potency between a wild-type IL-15 polypeptide and IL-15Rβγ.

### Claim 5 (depends on 4)

The isolated and modified IL-15 polypeptide of claim 4, wherein a difference in receptor signaling potency between the modified IL-15/IL-15Rβγ complex and the wild-type IL-15/IL-15Rβγ complex is less than 1000-fold, less than 500-fold, less than 200-fold, less than 100-fold, less than 50-fold, less than 10-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.

### Claim 6 (depends on 4)

The isolated and modified IL-15 polypeptide of claim 4, wherein a difference in receptor signaling potency between the modified IL-15/IL-15Rβγ complex and the wild-type IL-15/IL-15Rβγ complex is greater than 10-fold, greater than 20-fold, greater than 30-fold, greater than 40-fold, greater than 50-fold, greater than 100-fold, greater than 200-fold, greater than 300-fold, greater than 400-fold, or greater than 500-fold.

### Claim 7 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the residue position of the at least one unnatural amino acid is selected from N1, W2, V3, N4, I6, S7, D8, K10, K11, E13, D14, L15, Q17, S18, M19, H20, I21, D22, A23, T24, L25, Y26, E28, S29, D30, V31, H32, P33, S34, C35, K36, V37, T38, K41, L44, E46, Q48, V49, S51, L52, E53, S54, G55, D56, A57, S58, H60, D61, T62, V63, E64, N65, 167, 168, L69, N71, N72, S73, L74, S75, S76, N77, G78, N79, V80, T81, E82, S83, G84, C85, K86, E87, C88, E89, E90, L91, E92, E93, K94, N95, I96, K97, E98, L100, Q101, S102, V104, H105, Q108, M109, F110, I111, N112, T113, and S114, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 8 (depends on 1, 3)

The isolated and modified IL-15 polypeptide of claim 1 or 3, the residue position of the at least one unnatural amino acid is selected from
D22, A23, T24, L25, Y26, L44, E46, Q48, V49, E53, E89, E90, and E93, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1;
Y26, E46, V49, E53, and L25, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1;
A23, T24, E89, and E93, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1;
D22, L44, Q48, and E90, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1;
L25, E53, N77, and S83, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1; or
L25 and E53, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 9 (depends on 2)

The isolated and modified IL-15 polypeptide of claim 2, wherein the residue position of the at least one unnatural amino acid is selected from:
E13, D14, L15, Q17, S18, M19, H20, I21, S34, C35, K36, V37, T38, K41, L44, S51, L52, S54, G55, D56, A57, S58, H60, V63, I67, N71, S73, L74, S75, S76, N77, G78, N79, V80, T81, E82, S83, G84, C85, K86, E87, C88, L91, E92, K94, N95, I96, K97, E98, L100, Q101, and F110, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1;
D14, Q17, S18, K41, S51, L52, G55, D56, A57, S58, S75, S76, N77, N79, V80, T81, S83, G84, E92, K94, N95, K97, and E98, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1;
E13, L15, M19, H20, K36, V37, T38, S54, H60, 167, N71, G78, K86, E87, and Q101, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1; or
I21, S34, C35, L44, V63, S73, L74, E82, C85, C88, L91, 196, L100, and F110, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 10 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the at least one unnatural amino acid:
is a lysine analogue;
comprises an aromatic side chain;
comprises an azido group;
comprises an alkyne group; or
comprises an aldehyde or ketone group.

### Claim 11 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the at least one unnatural amino acid does not comprise an aromatic side chain.

### Claim 12 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine.

### Claim 13 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the at least one unnatural amino acid is incorporated into the modified IL-15 polypeptide by an orthogonal tRNA synthetase/tRNA pair.

### Claim 14 (depends on 13)

The isolated and modified IL-15 polypeptide of claim 13, wherein the orthogonal tRNA of the orthogonal synthetase/tRNA pair comprises at least one unnatural nucleobase.

### Claim 15 (depends on 13)

The isolated and modified IL-15 polypeptide of claim 13, wherein orthogonal tRNA of the orthogonal synthetase/tRNA pair comprise an unnatural base pair.

### Claim 16 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the modified IL-15 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid.

### Claim 17 (depends on 16)

The isolated and modified IL-15 polypeptide of claim 16, wherein the conjugating moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide.

### Claim 18 (depends on 17)

The isolated and modified IL-15 polypeptide of claim 17, wherein the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.

### Claim 19 (depends on 17)

The isolated and modified IL-15 polypeptide of claim 17, wherein the water-soluble polymer comprises a PEG molecule.

### Claim 20 (depends on 19)

The isolated and modified IL-15 polypeptide of claim 19, wherein the PEG molecule is a linear PEG.

### Claim 21 (depends on 19)

The isolated and modified IL-15 polypeptide of claim 19, wherein the PEG molecule is a branched PEG.

### Claim 22 (depends on 17)

The isolated and modified IL-15 polypeptide of claim 17, wherein the water-soluble polymer comprises a polysaccharide.

### Claim 23 (depends on 22)

The isolated and modified IL-15 polypeptide of claim 22, wherein the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).

### Claim 24 (depends on 17)

The isolated and modified IL-15 polypeptide of claim 17, wherein the lipid comprises a fatty acid.

### Claim 25 (depends on 24)

The isolated and modified IL-15 polypeptide of claim 24, wherein the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.

### Claim 26 (depends on 24)

The isolated and modified IL-15 polypeptide of claim 24, wherein the fatty acid is a saturated fatty acid.

### Claim 27 (depends on 17)

The isolated and modified IL-15 polypeptide of claim 17, wherein the protein comprises an albumin, a transferrin, or a transthyretin.

### Claim 28 (depends on 16)

The isolated and modified IL-15 polypeptide of claim 16, wherein the conjugating moiety comprises a TLR agonist.

### Claim 29 (depends on 17)

The isolated and modified IL-15 polypeptide of claim 17, wherein the protein comprises an antibody or its binding fragments thereof.

### Claim 30 (depends on 29)

The isolated and modified IL-15 polypeptide of claim 29, wherein the antibody or its binding fragments thereof comprises an Fc portion of an antibody.

### Claim 31 (depends on 17)

The isolated and modified IL-15 polypeptide of claim 17, wherein the peptide comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.

### Claim 32 (depends on 16)

The isolated and modified IL-15 polypeptide of claim 16, wherein the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the modified IL-15 through a linker.

### Claim 33 (depends on 32)

The isolated and modified IL-15 polypeptide of claim 32, wherein the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.

### Claim 34 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the modified IL-15 polypeptide further comprises a modification at residue position N71, N72, or N77, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 35 (depends on 34)

The isolated and modified IL-15 polypeptide of claim 34, wherein the modification improves a CMC condition and/or potency.

### Claim 36 (depends on 1, 3)

The isolated and modified IL-15 polypeptide of claim 1 or 3, wherein the modified IL-15 polypeptide further comprises a modification at residue E13, D14, L15, Q17, S18, M19, H20, I21, S34, C35, K36, V37, T38, K41, L44, S51, L52, S54, G55, D56, A57, S58, H60, V63, I67, N71, S73, L74, S75, S76, N77, G78, N79, V80, T81, E82, S83, G84, C85, K86, E87, C88, L91, E92, K94, N95, I96, K97, E98, L100, Q101, or F110, wherein the modification is to improve half-life extension.

### Claim 37 (depends on 2)

The isolated and modified IL-15 polypeptide of claim 2, wherein the modified IL-15 polypeptide further comprises a modification at:
residue D22, A23, T24, L25, Y26, L44, E46, Q48, V49, E53, E89, E90, or E93, wherein the modification impairs interaction with IL-15Rα;
residue N1, N4, S7, D8, K11, D61, T62, E64, N65, I68, L69, or N72, wherein the modification impairs interaction with IL-15Rβ;
residue V3, I6, K10, E28, S29, D30, V31, H32, P33, S102, V104, H105, Q108, M109, I111, N112, T113, or S114, wherein the modification impairs interaction with IL-15Rγ; or
a combination thereof.

### Claim 38 (depends on 36, 37)

The isolated and modified IL-15 polypeptide of claim 36 or 37, wherein the modification is to a natural amino acid or to an unnatural amino acid.

### Claim 39 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the decrease in binding affinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% decrease in binding affinity to IL-15Rα relative to a wild-type IL-15 polypeptide.

### Claim 40 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the decrease in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more to IL-15Rα relative to a wild-type IL-15 polypeptide.

### Claim 41 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the modified IL-15 polypeptide is:
a functionally active fragment of a full-length IL-15 polypeptide;
a recombinant IL-15 polypeptide; or
a recombinant human IL-15 polypeptide.

### Claim 42 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the modified IL-15 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.

### Claim 43 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the N-terminal deletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.

### Claim 44 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the C-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from the C-terminus, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.

### Claim 45 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the modified IL-15 polypeptide with the decrease in binding affinity to IL-15Rα is capable of expanding CD8+ effector naïve and memory cell, Natural Killer (NK) cell, Natural killer T (NKT) cell populations, or a combination thereof.

### Claim 46 (depends on 16)

The isolated and modified IL-15 polypeptide of claim 16, wherein the conjugating moiety or the unnatural amino acid impairs or blocks the binding of IL-15 with IL-15Rα.

### Claim 47 (depends on 16)

The isolated and modified IL-15 polypeptide of claim 16, wherein the conjugating moiety or the unnatural amino acid extends half-life but does not impairs or blocks binding of IL-15 with IL-15 receptors.

### Claim 48 (depends on 16)

The isolated and modified IL-15 polypeptide of claim 16, wherein the conjugating moiety or the unnatural amino acid increases binding of IL-15 to IL-15Rβ and/or IL-15Rγ.

### Claim 49 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the enhanced half-life of the IL-15 is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, or longer than the plasma half-life of the wild-type IL-15.

### Claim 50 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein the enhanced half-life of the IL-15 is about 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, or 7 days.

### Claim 51 (depends on 1, 2, 3)

The isolated and modified IL-15 polypeptide of any one of the claims 1-3, wherein activation of CD8+ effector naïve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population via the IL-15Rβγ complex by the modified IL-15 polypeptide retains significant potency of activation of said cell population relative to a wild-type IL-15 polypeptide.

### Claim 52 (depends on 51)

The isolated and modified IL-15 polypeptide of claim 51, wherein the receptor signaling potency of the modified IL-15 polypeptide to the IL-15Rβγ complex is higher than a receptor signaling potency of the wild-type IL-15 polypeptide to the IL-15Rβγ complex.

### Claim 53 (depends on 51)

The isolated and modified IL-15 polypeptide of claim 51, wherein the receptor signaling potency of the modified IL-15 polypeptide the IL-15Rβγ complex is lower than a receptor signaling potency of the wild-type IL-15 polypeptide to the IL-15Rβγ complex.

### Claim 54

An isolated and modified interleukin 15 (IL-15) polypeptide comprising at least one unnatural amino acid, wherein the at least one unnatural amino acid is at a residue position that selectively decreases the binding affinity of the modified IL-15 polypeptide with interleukin 2/interleukin 15 receptor β (IL-2/IL-15Rβ), interleukin 15 receptor γ (IL-15Rγ), or a combination thereof, but does not affect the interaction with the interleukin 15 receptor α (IL-15R α).

### Claim 55 (depends on 54)

The isolated and modified IL-15 polypeptide of claim 54, wherein the modified IL-15 polypeptide has a decreased binding affinity to IL-15Rβ.

### Claim 56 (depends on 55)

The isolated and modified IL-15 polypeptide of claim 55, wherein the residue position of the at least one unnatural amino acid is selected from N1, N4, S7, D8, K11, D61, T62, E64, N65, I68, L69, and N72, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 57 (depends on 55)

The isolated and modified IL-15 polypeptide of claim 55, wherein the residue position of the at least one unnatural amino acid is selected from:
N4, S7, K11, and D61;
D8, E64, N65, I68, and N72; or
N1, T62, and L69.

### Claim 58 (depends on 54)

The isolated and modified IL-15 polypeptide of claim 54, wherein the modified IL-15 polypeptide has a decreased binding affinity to IL-15Rγ.

### Claim 59 (depends on 58)

The isolated and modified IL-15 polypeptide of claim 58, wherein the residue position of the at least one unnatural amino acid is selected from V3, I6, K10, E28, S29, D30, V31, H32, P33, S102, V104, H105, Q108, M109, I111, N112, T113, and S114, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 60 (depends on 58)

The isolated and modified IL-15 polypeptide of claim 58, wherein the residue position of the at least one unnatural amino acid is selected from:
V3, K10, S29, D30, H32, H105, Q108, M109, I111, N112, T113, and S114;
E28, P33, S102, and V104; or
I6 and V31.

### Claim 61 (depends on 54, 55, 56, 57, 58, 59, 60)

The isolated and modified IL-15 polypeptide of any one of the claims 54-60, wherein the modified IL-15 polypeptide further comprises a modification to improve half-life extension.

### Claim 62 (depends on 61)

The isolated and modified IL-15 polypeptide of claim 61, wherein the modification is at residue position E13, D14, L15, Q17, S18, M19, H20, I21, S34, C35, K36, V37, T38, K41, L44, S51, L52, S54, G55, D56, A57, S58, H60, V63, I67, N71, S73, L74, S75, S76, N77, G78, N79, V80, T81, E82, S83, G84, C85, K86, E87, C88, L91, E92, K94, N95, I96, K97, E98, L100, Q101, or F110, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 63 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62)

The isolated and modified IL-15 polypeptide of any one of the claims 54-62, wherein the modified IL-15 polypeptide further comprises a modification at residue position N71, N72, or N77, wherein the residue positions correspond to the positions as set forth in SEQ ID NO: 1.

### Claim 64 (depends on 61, 62, 63)

The isolated and modified IL-15 polypeptide of any one of the claims 61-63, wherein the modification is to an unnatural amino acid.

### Claim 65 (depends on 61, 62, 63)

The isolated and modified IL-15 polypeptide of any one of the claims 61-63, wherein the modification is to a natural amino acid.

### Claim 66 (depends on 63)

The isolated and modified IL-15 polypeptide of claim 63, wherein the modification improves a CMC condition and/or potency.

### Claim 67

An interleukin 15 (IL-15) conjugate comprising:
an isolated and purified IL-15 polypeptide; and
a conjugating moiety that binds to the isolated and purified IL-15 polypeptide at an amino acid position selected from N4, E46, D61, E64, N65, I68 and L69, wherein the numbering of the amino acid residues corresponds to SEQ ID NO: 1.

### Claim 68 (depends on 67)

The IL-15 conjugate of claim 67, wherein the amino acid residue is mutated to cysteine or lysine.

### Claim 69 (depends on 67)

The IL-15 conjugate of claim 67, wherein the amino acid residue selected from N4, E46, N65, and L69 is further mutated to an unnatural amino acid.

### Claim 70 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)

A pharmaceutical composition comprising:
a modified IL-15 polypeptide as in any of claims 1-66 or an IL-15 conjugate as in any of claims 67-69; and
a pharmaceutically acceptable excipient.

### Claim 71 (depends on 70)

The pharmaceutical composition of claim 70, wherein the pharmaceutical composition is formulated for parenteral administration.

### Claim 72 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)

A method of treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a modified IL-15 polypeptide as in any of claims 1-66 or an IL-15 conjugate as in any of claims 67-69.

### Claim 73 (depends on 72)

The method of claim 72, wherein the proliferative disease or condition is a cancer.

### Claim 74 (depends on 73)

The method of claim 73, wherein the cancer is a solid tumor cancer.

### Claim 75 (depends on 74)

The method of claim 74, wherein the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.

### Claim 76 (depends on 73)

The method of claim 73, wherein the cancer is a hematologic malignancy.

### Claim 77 (depends on 76)

The method of claim 76, wherein the hematologic malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

### Claim 78 (depends on 72, 73, 74, 75, 76, 77)

The method of any one of the claims 72-77, further comprising administering an additional therapeutic agent.

### Claim 79 (depends on 78)

The method of claim 78, wherein the modified IL-15 polypeptide or the IL-15 conjugate and the additional therapeutic agent are administered simultaneously.

### Claim 80 (depends on 78)

The method of claim 78, wherein the modified IL-15 polypeptide or the IL-15 conjugate and the additional therapeutic agent are administered sequentially.

### Claim 81 (depends on 80)

The method of claim 80, wherein the modified IL-15 polypeptide or the IL-15 conjugate is administered prior to the additional therapeutic agent.

### Claim 82 (depends on 80)

The method of claim 80, wherein the modified IL-15 polypeptide or the IL-15 conjugate is administered after the administration of the additional therapeutic agent.

### Claim 83 (depends on 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The method as in any of claims 72-82, wherein the subject is a human.

### Claim 84 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)

A method of expanding effector T (Teff) cell, memory T (Tmem) cell, and Natural Killer (NK) cell populations, comprising:
a) contacting a cell with a modified IL-15 polypeptide as in any of claims 1-66 or an IL-15 conjugate of claims 67-69; and
b) interacting the IL-15 with IL-15Rβ and IL-15Rγ subunits to form an IL-15/IL-15Rβγ complex;
wherein the IL-15 conjugate has a decreased affinity to IL-15Rα subunit, and wherein the IL-15/IL-15Rβγ complex stimulates the expansion of Teff, Tmem, and NK cells.

### Claim 85 (depends on 84)

The method of claim 84, wherein the cell is a eukaryotic cell.

### Claim 86 (depends on 84, 85)

The method as in any of claims 84-85, wherein the cell is a mammalian cell.

### Claim 87 (depends on 84, 85, 86)

The method as in any of claims 84-86, wherein the cell is a human cell.

### Claim 88 (depends on 84, 85, 86, 87)

The method as in any of claims 84-87, wherein the IL-15 conjugate comprises an isolated and purified IL-15 polypeptide and a conjugating moiety that binds to the isolated and purified IL-15 polypeptide at an amino acid residue selected from N4, E46, N65, and L69, wherein the numbering of the amino acid residues corresponds to SEQ ID NO: 1.

### Claim 89 (depends on 84, 85, 86, 87, 88)

The method as in any of claims 84-88, wherein the decreased affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% decrease in binding affinity to IL-15Rα relative to a wild-type IL-15 polypeptide.

### Claim 90 (depends on 84, 85, 86, 87, 88)

The method as in any of claims 84-88, wherein the decreased affinity to IL-15Rα is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more relative to a wild-type IL-15 polypeptide.

### Claim 91 (depends on 84, 85, 86, 87, 88, 89, 90)

The method as in any of claims 84-90, wherein the conjugating moiety impairs or blocks the binding of IL-15 with IL-15Rα.

### Claim 92 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)

A kit comprising a modified IL-15 polypeptide as in any of claims 1-66, an IL-15 conjugate as in any of claims 67-69, or a pharmaceutical composition as in any one of claims 70-71.

### Claim 93 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66)

A kit comprising a polynucleic acid molecule encoding a modified IL-15 polypeptide as in any of claims 1-66.