# US 12,595,292 B2 · Interleukin-2 receptor (IL2R) and interleukin-2 (IL2) variants for specific activation of immune effector cells

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- Number: US 12,595,292 B2
- Document ID: US12595292B2
- Status: Granted
- Kind: B2
- Application number: 17/439,367
- Filed: 2020-03-16
- Published: 2022-06-09
- Granted: 2026-04-07
- Earliest claimed: 2019-03-18
- Assignees: BioNTech Cell & Gene Therapies GmbH
- Applicants: BioNTech Cell & Gene Therapies GmbH
- Inventors: Ugur Sahin; Sina Fellermeier-Kopf; Alexander Muik; Matthias Birtel
- Web page: https://patentgrep.com/patent/US12595292B2

## CPC

- C07K 14/7155: {for interleukins [IL]}
- A61K 38/2013: {IL-2}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4202: {Receptors, cell surface antigens or cell surface determinants}
- A61P 35/00: Antineoplastic agents
- C07K 14/55: IL-2
- C12N 5/0636: {T lymphocytes}
- A61K 2239/59: Reproductive system, e.g. uterus, ovaries, cervix or testes
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- 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)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to variants of the alpha subunit of interleukin-2 receptor (IL2R) and interleukin-2 (IL2). In one embodiment, the IL2 variants described herein have amino acid substitutions at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαβγ, reducing its ability to bind and activate the heterotrimeric receptor complex. Conversely, the corresponding IL2Rα variants described herein have amino acid substitutions compensating for such reduced ability of IL2 variants to bind to and activate IL2Rαβγ, preferably at amino acid residues contacted by IL2 amino acid residues that are substituted in the IL2 variants described herein.

## Claims

### Claim 1

A system comprising:
(i) a receptor polypeptide comprising a mutein of the alpha subunit of interleukin-2 receptor (IL2R), wherein the alpha subunit of IL2R has the amino acid sequence according to SEQ ID NO: 2 or 4, or of a functional variant of the alpha subunit of IL2R having at least 95% sequence identity to SEQ ID NO: 2 or 4 and having modifications only in positions which are not conserved between homologous IL2R amino acid sequences, wherein the alpha subunit of IL2R or the functional variant thereof is substituted at at least one position,
(ii) a ligand polypeptide comprising a mutein of IL2, wherein the IL2 has the amino acid sequence according to SEQ ID NO: 1, or of a functional variant of IL2 having at least 95% sequence identity to SEQ ID NO: 1 and having modifications only in positions which are not conserved between homologous IL2 amino acid sequences, wherein the IL2 or the functional variant thereof is substituted at at least one position,
wherein the substitutions are such that
(a) the mutein under (ii) binds to and activates IL2R comprising the mutein under (i) as alpha subunit,
(b) binding to and/or activation of IL2R comprising the mutein under (i) as alpha subunit by the mutein under (ii) exceeds binding to and/or activation of IL2R comprising the alpha subunit of IL2R or the functional variant thereof as alpha subunit by the mutein under (ii), and
(c) acidic amino acid residues are substituted by basic amino acid residues and basic amino acid residues are substituted by acidic amino acid residues,
wherein
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 1 (glutamic acid) relative to wild type human alpha subunit of IL2R according to SEQ ID NO: 2 and numbered in accordance with the wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 35 (lysine) relative to wild type human IL2 according to SEQ ID NO: 1 and numbered in accordance with the wild type human IL2;
and/or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 29 (glutamic acid) relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 43 (lysine) relative to wild type human IL2 and numbered in accordance with wild type human IL2;
and/or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 38 (lysine) relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 61 (glutamic acid) relative to wild type human IL2 and numbered in accordance with wild type human IL2.

### Claim 2 (depends on 1)

The system of claim 1, wherein
(a) binding to and/or activation of IL2R comprising the mutein under (i) as alpha subunit by the mutein under (ii) exceeds binding to and/or activation of IL2R comprising the mutein under (i) as alpha subunit by IL2 or the functional variant thereof, or
(b) binding to and/or activation of IL2R comprising the alpha subunit of IL2R or the functional variant thereof as alpha subunit by IL2 or the functional variant thereof exceeds binding to and/or activation of IL2R comprising the alpha subunit of IL2R or the functional variant thereof as alpha subunit by the mutein under (ii), or
(c) binding to and/or activation of IL2R comprising the alpha subunit of IL2R or the functional variant thereof as alpha subunit by IL2 or the functional variant thereof exceeds binding to and/or activation of IL2R comprising the mutein under (i) as alpha subunit by IL2 or the functional variant thereof.

### Claim 3 (depends on 1)

The system of claim 1, wherein
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 1 (glutamic acid) by lysine, position 29 (glutamic acid) by lysine and position 38 (lysine) by glutamic acid relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 35 (lysine) by glutamic acid, position 43 (lysine) by glutamic acid and position 61 (glutamic acid) by lysine relative to wild type human IL2 and numbered in accordance with wild type human IL2;
or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 29 (glutamic acid) by lysine and position 38 (lysine) by glutamic acid relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 43 (lysine) by glutamic acid and position 61 (glutamic acid) by lysine relative to wild type human IL2 and numbered in accordance with wild type human IL2;
or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 38 (lysine) by glutamic acid relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 61 (glutamic acid) by lysine relative to wild type human IL2 and numbered in accordance with wild type human IL2;
or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 29 (glutamic acid) by lysine relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 43 (lysine) by glutamic acid relative to wild type human IL2 and numbered in accordance with wild type human IL2;
or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 29 (glutamic acid) by lysine and position 38 (lysine) by glutamic acid relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 35 (lysine) by glutamic acid, position 43 (lysine) by glutamic acid and position 61 (glutamic acid) by lysine relative to wild type human IL2 and numbered in accordance with wild type human IL2;
or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 29 (glutamic acid) by lysine relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 43 (lysine) by glutamic acid and position 61 (glutamic acid) by lysine relative to wild type human IL2 and numbered in accordance with wild type human IL2.

### Claim 4 (depends on 1)

The system of claim 1, wherein the substitution in IL2 or the functional variant thereof reduces the affinity for IL2R comprising the wild type alpha subunit of IL2R as alpha subunit (IL2Rαβγ), and/or
wherein the substitution in IL2 or the functional variant thereof reduces the affinity for IL2R comprising the wild type alpha subunit of IL2R as alpha subunit (IL2Rαβγ) to a greater extent than for the βγ IL2 receptor complex (IL2Rβγ).

### Claim 5 (depends on 1)

The system of claim 1, wherein the mutein under (ii) has a decreased ability to stimulate regulatory T cells compared to wild type IL2, and/or wherein the mutein under (ii) further comprises one or more amino acid substitutions which enhance the affinity for IL2Rβγ.

### Claim 6 (depends on 1)

The system of claim 1, wherein the mutein under (ii) further comprises one or more amino acid substitutions which enhance the affinity for IL2Rβγ.

### Claim 7 (depends on 6)

The system of claim 6, wherein the one or more amino acid substitutions which enhance the affinity for IL2Rβγ comprise the following set of substitutions: 80F, 81D, 85V, 86V, 92F.

### Claim 8 (depends on 1)

The system of claim 1, wherein the ligand polypeptide is an extended pharmacokinetic (PK) polypeptide.

### Claim 9 (depends on 1)

The system of claim 1, wherein the modifications in the functional variant of the alpha subunit of IL2R and in the functional variant of IL2 are conservative amino acid substitutions.

### Claim 10 (depends on 1)

The system of claim 1, wherein the functional variant of the alpha subunit of IL2R and in the functional variant of IL2 each have 5 or fewer substitutions.

### Claim 11 (depends on 9)

The system of claim 9, wherein the functional variant of the alpha subunit of IL2R and in the functional variant of IL2 each have 5 or fewer substitutions.

### Claim 12

A system comprising:
(i) a receptor polypeptide comprising a mutein of the alpha subunit of interleukin-2 receptor (IL2R), wherein the alpha subunit of IL2R has the amino acid sequence according to SEQ ID NO: 2 or 4, or of a functional variant of the alpha subunit of IL2R having at least 95% sequence identity to SEQ ID NO: 2 or 4 and having only modifications which are conservative substitutions, wherein the alpha subunit of IL2R or the functional variant thereof is substituted at at least one position,
(ii) a ligand polypeptide comprising a mutein of IL2, wherein the IL2 has the amino acid sequence according to SEQ ID NO: 1, or of a functional variant of IL2 having at least 95% sequence identity to SEQ ID NO: 1 and having only modifications which are conservative substitutions, wherein the IL2 or the functional variant thereof is substituted at at least one position,
wherein the substitutions are such that
(a) the mutein under (ii) binds to and activates IL2R comprising the mutein under (i) as alpha subunit,
(b) binding to and/or activation of IL2R comprising the mutein under (i) as alpha subunit by the mutein under (ii) exceeds binding to and/or activation of IL2R comprising the alpha subunit of IL2R or the functional variant thereof as alpha subunit by the mutein under (ii), and
(c) acidic amino acid residues are substituted by basic amino acid residues and basic amino acid residues are substituted by acidic amino acid residues,
wherein
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 1 (glutamic acid) relative to wild type human alpha subunit of IL2R according to SEQ ID NO: 2 and numbered in accordance with the wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 35 (lysine) relative to wild type human IL2 according to SEQ ID NO: 1 and numbered in accordance with the wild type human IL2;
and/or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 29 (glutamic acid) relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 43 (lysine) relative to wild type human IL2 and numbered in accordance with wild type human IL2;
and/or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 38 (lysine) relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 61 (glutamic acid) relative to wild type human IL2 and numbered in accordance with wild type human IL2.

### Claim 13 (depends on 12)

The system of claim 12, wherein the functional variant of the alpha subunit of IL2R and in the functional variant of IL2 each have 5 or fewer substitutions.

### Claim 14

A system comprising:
(i) a receptor polypeptide comprising a mutein of the alpha subunit of interleukin-2 receptor (IL2R), wherein the alpha subunit of IL2R has the amino acid sequence according to SEQ ID NO: 2 or 4, or of a functional variant of the alpha subunit of IL2R having 5 or fewer substitutions to SEQ ID NO: 2 or 4, wherein the alpha subunit of IL2R or the functional variant thereof is substituted at at least one position,
(ii) a ligand polypeptide comprising a mutein of IL2, wherein the IL2 has the amino acid sequence according to SEQ ID NO: 1, or of a functional variant of IL2 having 5 or fewer substitutions, wherein the IL2 or the functional variant thereof is substituted at at least one position,
wherein the substitutions are such that
(a) the mutein under (ii) binds to and activates IL2R comprising the mutein under (i) as alpha subunit,
(b) binding to and/or activation of IL2R comprising the mutein under (i) as alpha subunit by the mutein under (ii) exceeds binding to and/or activation of IL2R comprising the alpha subunit of IL2R or the functional variant thereof as alpha subunit by the mutein under (ii), and
(c) acidic amino acid residues are substituted by basic amino acid residues and basic amino acid residues are substituted by acidic amino acid residues,
wherein
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 1 (glutamic acid) relative to wild type human alpha subunit of IL2R according to SEQ ID NO: 2 and numbered in accordance with the wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 35 (lysine) relative to wild type human IL2 according to SEQ ID NO: 1 and numbered in accordance with the wild type human IL2;
and/or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 29 (glutamic acid) relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 43 (lysine) relative to wild type human IL2 and numbered in accordance with wild type human IL2;
and/or
(i) the alpha subunit of IL2R is the human alpha subunit of IL2R and the alpha subunit of IL2R or the functional variant thereof is substituted at at least position 38 (lysine) relative to wild type human alpha subunit of IL2R and numbered in accordance with wild type human alpha subunit of IL2R, and
(ii) the IL2 is human IL2 and the IL2 or the functional variant thereof is substituted at at least position 61 (glutamic acid) relative to wild type human IL2 and numbered in accordance with wild type human IL2.

### Claim 15 (depends on 14)

The system of claim 14, wherein the functional variant of the alpha subunit of IL2R and in the functional variant of IL2 each have 4 or fewer substitutions.

### Claim 16 (depends on 14)

The system of claim 14, wherein the functional variant of the alpha subunit of IL2R and in the functional variant of IL2 each have 3 or fewer substitutions.