# US 10,501,510 B2 · Muteins of human tear lipocalin capable of binding lymphocyte-activation gene 3 (LAG-3) and methods of use thereof

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- Number: US 10,501,510 B2
- Document ID: US10501510B2
- Status: Granted
- Kind: B2
- Application number: 15/744,531
- Filed: 2016-07-15
- Published: 2019-01-31
- Granted: 2019-12-10
- Earliest claimed: 2015-07-15
- Assignees: Pieris Pharmaceuticals GmbH
- Applicants: Pieris Pharmaceuticals GmbH
- Inventors: Christine Rothe; Andrea Allersdorfer; Alexander Wiedenmann; Rachida Siham Bel Aiba; Shane Olwill; Timo Eichner
- Web page: https://patentgrep.com/patent/US10501510B2

## CPC

- C07K 14/47: from mammals
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 37/04: Immunostimulants
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- G01N 33/6893: {related to diseases not provided for elsewhere}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- G01N 2333/70503: Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N 2333/70539: MHC-molecules, e.g. HLA-molecules
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)

## Abstract

The present disclosure provides human tear lipocalin muteins that specifically bind to LAG-3, which can be used in pharmaceutical applications, for example, as anti-cancer agents and/or immune modulators for the treatment or prevention of human diseases such as cancer, infectious diseases, and autoimmune diseases. The present disclosure further shows the human lipocalin muteins can inhibit the binding of LAG-3 to MHC class II on cells overexpressing MHC class II. The present disclosure also concerns methods of making LAG-3 binding lipocalin muteins described herein as well as compositions comprising such lipocalin muteins. The present disclosure further relates to nucleic acid molecules encoding such lipocalin muteins and to methods for generation of such lipocalin muteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of these lipocalin muteins as well as compositions comprising one or more of such lipocalin muteins.

## Claims

### Claim 1

A mutein of human tear lipocalin that is capable of binding lymphocyte-activation gene 3 (LAG-3),
wherein the amino acid sequence of the mutein comprises at least 14 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1): Ser 14→Pro; Asp 25→Ser; Arg 26→Ser, Phe, Gly, Ala, Asp or Glu; Glu 27→Asp, Val or Thr; Phe 28→Cys or Asp; Pro 29→Phe, Leu or Trp; Glu 30→Trp, Asn or Tyr; Met 31→Ile, Val, Asp, Leu or Tyr; Asn 32→Asp, Glu, Tyr, Trp, Val, Thr or Met Leu 33→Asp, Glu or Pro; Glu 34→Val, Trp or His; Val 36→Ala; Asn 48→Asp; Lys 52→Glu, Ser, Arg or Asn; Val 53→Ala; Met 55→Ala or Val; Leu 56→Asp, Gln or Asn; Ile 57→Leu; Ser 58→Phe, Trp or Asp; Arg 60→Phe or Glu; Cys 61→Pro, Leu or Trp; Ala 66→Asn; Ala 79→Glu; Val 85→Ala; Ala 86→Asp; Cys 101→Ser or Phe; Glu 104→Tyr; Leu 105→Cys or Gly; His 106→Ala, Glu, Thr, Tyr, Gln or Val; Lys 108→Tyr, Phe, Thr or Trp; Val 110→Gly or Ala; Arg 111→Pro; Gly 112→Met or Thr; Lys 114→Trp or Ala; Lys 121→Thr; Ser 140→Gly; and Cys 153→Ser, and
wherein the mutein has at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-22.

### Claim 2 (depends on 1)

The mutein of claim 1, wherein the mutein is capable of binding LAG-3 with an affinity measured by Kd of about 500 nM or lower.

### Claim 3 (depends on 1)

The mutein of claim 1, wherein the mutein is capable of binding LAG-3 with an affinity measured by Kd of about 160 nM or lower.

### Claim 4 (depends on 1)

The mutein of claim 1, wherein the mutein is capable of binding LAG-3 with an affinity measured by Kd of about 30 nM or lower.

### Claim 5 (depends on 1)

The mutein of claim 1, wherein the mutein is capable of binding LAG-3 with an affinity measured by EC50 of about 109 nM or lower.

### Claim 6 (depends on 1)

The mutein of claim 1, wherein the mutein is cross-reactive with both human LAG-3 and cynomolgus LAG-3.

### Claim 7 (depends on 1)

The mutein of claim 1, wherein the mutein is capable of binding cynomolgus LAG-3 with an affinity measured by Kd of about 160 nM or lower.

### Claim 8 (depends on 1)

The mutein of claim 1, wherein the mutein is capable of interfering or competing with the binding of LAG-3 to major histocompatibility complex (MHC) class II.

### Claim 9 (depends on 1)

The mutein of claim 1, wherein the amino acid sequence of the mutein comprises one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1):
(a)
Arg 26 → Ser; Glu 27 → Asp; Phe 28 → Cys;
Pro 29 → Phe; Glu 30 → Trp; Met 31 → Ile;
Asn 32 → Glu; Leu 33 → Glu; Glu 34 → Trp;
Leu 56 → Asp; Ser 58 → Phe; Arg 60 → Phe;
Cys 61 → Trp; Cys 101 → Ser; Leu 105 → Cys;
His 106 → Ala; Lys 108 → Phe; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser;
(b)
Ser 14 → Pro; Asp 25 → Ser; Arg 26 → Gly;
Phe 28 → Asp; Asn 32 → Thr; Lys 52 → Asn;
Met 55 → Ala; Ser 58 → Asp; Ala 66 → Asn;
Ala 79 → Glu; Ala 86 → Asp; Cys 101 → Phe;
Leu 105 → Gly; Lys 108 → Thr; Val 110 → Ala;
Gly 112 → Thr; Lys 114 → Ala;
and
Lys 121 → Thr;
(c)
Arg 26 → Phe; Glu 27 → Val; Phe 28 → Cys;
Pro 29 → Leu; Glu 30 → Tyr; Met 31 → Asp;
Asn 32 → Val; Leu 33 → Pro; Leu 56 → Gln;
Ser 58 → Trp; Arg 60 → Glu; Cys 61 → Leu;
Cys 101 → Ser; Glu 104 → Tyr; Leu 105 → Cys;
His 106 → Val; Lys 108 → Tyr; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser;
(d)
Arg 26 → Glu; Glu 27 → Thr; Phe 28 → Cys;
Pro 29 → Trp; Glu 30 → Trp; Met 31 → Tyr;
Asn 32 → Val; Leu 33 → Asp; Glu 34 → His;
Leu 56 → Asn; Ile 57 → Leu; Ser 58 → Trp;
Arg 60 → Phe; Cys 61 → Trp; Cys 101 → Ser;
Leu 105 → Cys; His 106 → Gln; Lys 108 → Trp;
Arg 111 → Pro; Lys 114 → Trp;
and
Cys 153 → Ser;
(e)
Arg 26 → Ser; Glu 27 → Asp; Phe 28 → Cys;
Pro 29 → Phe; Glu 30 → Trp; Met 31 → Ile;
Asn 32 → Asp; Leu 33 → Asp; Glu 34 → Val;
Leu 56 → Asp; Ser 58 → Phe; Arg 60 → Phe;
Cys 61 → Trp; Cys 101 → Ser; Leu 105 → Cys;
His 106 → Ala; Lys 108 → Tyr; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser;
(f)
Arg 26 → Ser; Glu 27 → Asp; Phe 28 → Cys;
Pro 29 → Phe; Glu 30 → Trp; Met 31 → Ile;
Asn 32 → Asp; Leu 33 → Glu; Glu 34 → Val;
Leu 56 → Asp; Ser 58 → Phe; Arg 60 → Phe;
Cys 61 → Trp; Cys 101 → Ser; Leu 105 → Cys;
His 106 → Ala; Lys 108 → Tyr; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser;
(g)
Arg 26 → Ser; Glu 27 → Asp; Phe 28 → Cys;
Pro 29 → Phe; Glu 30 → Trp; Met 31 → Ile;
Asn 32 → Glu; Leu 33 → Glu; Glu 34 → Trp;
Val 36 → Ala; Asn 48 → Asp; Leu 56 → Asp;
Ser 58 → Phe; Arg 60 → Phe; Cys 61 → Trp;
Val 85 → Ala; Cys 101 → Ser; Leu 105 → Cys;
His 106 → Ala; Lys 108 → Phe; Arg 111 → Pro;
Lys 114 → Trp; Ser 140 → Gly;
and
Cys 153 → Ser;
(h)
Arg 26 → Ser; Glu 27 → Asp; Phe 28 → Cys;
Pro 29 → Phe; Glu 30 → Trp; Met 31 → Ile;
Asn 32 → Asp; Leu 33 → Glu; Glu 34 → Val;
Leu 56 → Asp; Ser 58 → Phe; Arg 60 → Phe;
Cys 61 → Trp; Cys 101 → Ser; Leu 105 → Cys;
His 106 → Glu; Lys 108 → Phe; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser;
(i)
Arg 26 → Ser; Glu 27 → Asp; Phe 28 → Cys;
Pro 29 → Phe; Glu 30 → Trp; Met 31 → Ile;
Asn 32 → Glu; Leu 33 → Glu; Glu 34 → Trp;
Val 36 → Ala; Lys 52 → Glu; Val 53 → Ala;
Leu 56 → Asp; Ser 58 → Phe; Arg 60 → Phe;
Cys 61 → Trp; Cys 101 → Ser; Leu 105 → Cys;
His 106 → Ala; Lys 108 → Phe; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser;
(j)
Arg 26 → Ser; Glu 27 → Asp; Phe 28 → Cys;
Pro 29 → Phe; Glu 30 → Trp; Met 31 → Val;
Asn 32 → Asp; Leu 33 → Glu; Glu 34 → Val;
Leu 56 → Asp; Ser 58 → Phe; Arg 60 → Phe;
Cys 61 → Trp; Cys 101 → Ser; Leu 105 → Cys;
His 106 → Ala; Lys 108 → Phe; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser;
(k)
Ser 14 → Pro; Asp 25 → Ser; Arg 26 → Gly;
Phe 28 → Asp; Met 31 → Leu; Asn 32 → Trp;
Lys 52 → Ser; Met 55 → Ala; Ser 58 → Asp;
Ala 66 → Asn; Ala 79 → Glu; Ala 86 → Asp;
Cys 101 → Phe; Leu 105 → Gly; His 106 → Tyr;
Lys 108 → Thr; Val 110 → Gly; Gly 112 → Met;
Lys 114 → Ala;
and
Lys 121 → Thr;
(l)
Ser 14 → Pro; Asp 25 → Ser; Arg 26 → Ala;
Phe 28 → Asp; Met 31 → Leu; Asn 32 → Val;
Lys 52 → Ser; Met 55 → Ala; Ser 58 → Asp;
Ala 66 → Asn; Ala 79 → Glu; Ala 86 → Asp;
Cys 101 → Phe; Leu 105 → Gly; Lys 108 → Thr;
Val 110 → Ala; Gly 112 → Thr; Lys 114 → Ala;
and
Lys 121 → Thr;
(m)
Ser 14 → Pro; Asp 25 → Ser; Arg 26 → Asp;
Phe 28 → Asp; Asn 32 → Thr; Lys 52 → Ser;
Met 55 → Ala; Ser 58 → Asp; Ala 66 → Asn;
Ala 79 → Glu; Ala 86 → Asp; Cys 101 → Phe;
Leu 105 → Gly; His 106 → Gln; Lys 108 → Thr;
Val 110 → Gly; Gly 112 → Met; Lys 114 → Ala;
and
Lys 121 → Thr;
(n)
Ser 14 → Pro; Asp 25 → Ser; Arg 26 → Glu;
Phe 28 → Asp; Asn 32 → Thr; Lys 52 → Ser;
Met 55 → Ala; Ser 58 → Asp; Ala 66 → Asn;
Ala 79 → Glu; Ala 86 → Asp; Cys 101 → Phe;
Leu 105 → Gly; Lys 108 → Thr; Val 110 → Gly;
Gly 112 → Met; Lys 114 → Ala;
and
Lys 121 → Thr;
(o)
Ser 14 → Pro; Asp 25 → Ser; Arg 26 → Gly;
Phe 28 → Asp; Asn 32 → Met; Lys 52 → Arg;
Met 55 → Val; Ser 58 → Asp; Ala 66 → Asn;
Ala 79 → Glu; Ala 86 → Asp; Cys 101 → Phe;
Leu 105 → Gly; His 106 → Gln; Lys 108 → Thr;
Val 110 → Gly; Gly 112 → Met; Lys 114 → Ala;
and
Lys 121 → Thr;
or
(p)
Arg 26 → Phe; Glu 27 → Val; Phe 28 → Cys;
Pro 29 → Leu; Glu 30 → Asn; Met 31 → Asp;
Asn 32 → Tyr; Leu 33 → Pro; Leu 56 → Gln;
Ser 58 → Trp; Arg 60 → Glu; Cys 61 → Pro;
Cys 101 → Ser; Glu 104 → Tyr; Leu 105 → Cys;
His 106 → Thr; Lys 108 → Tyr; Arg 111 → Pro;
Lys 114 → Trp;
and
Cys 153 → Ser.

### Claim 10 (depends on 1)

The mutein of claim 1, wherein the mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-22 or a variant thereof.

### Claim 11 (depends on 1)

The mutein according of claim 1, wherein the mutein has at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-22.

### Claim 12 (depends on 1)

The mutein of claim 1, wherein the mutein is conjugated to a compound selected from the group consisting of an organic molecule, an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a cytostatic agent, a toxin, a metal complex, a metal, and colloidal gold.

### Claim 13 (depends on 1)

The mutein of claim 1, wherein the mutein is fused at its N-terminus and/or its C-terminus, with or without an intervening linker, to a fusion partner selected from the group consisting of an antibody, an antibody fragment, a protein, a protein domain, a peptide, or a lipocalin mutein.

### Claim 14 (depends on 1)

The mutein of claim 1, wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein.

### Claim 15 (depends on 1)

The mutein of claim 1, wherein the mutein is capable of binding LAG-3 with an affinity measured by Kd of about 1 nM or lower.

### Claim 16 (depends on 1)

A diagnostic or analytical kit comprising a mutein of claim 1.

### Claim 17 (depends on 1)

An immunoconjugate or fusion protein comprising the mutein of claim 1 linked to a therapeutic agent.

### Claim 18 (depends on 1)

A pharmaceutical composition comprising a mutein of claim 1 and a pharmaceutically acceptable excipient.

### Claim 19 (depends on 1)

A method of binding LAG-3 comprising applying one or more muteins of claim 1 or one or more compositions comprising such muteins.

### Claim 20 (depends on 1)

A method of stimulating immune response in a subject, comprising administering to a subject one or more muteins of claim 1 or one or more compositions comprising such muteins.

### Claim 21 (depends on 1)

A method of inducing T lymphocyte proliferation in a subject, comprising administering to a subject one or more muteins of claim 1 or one or more compositions comprising such muteins.

### Claim 22 (depends on 1)

A method of interfering or competing with the binding of LAG-3 to WIC class II in a subject, comprising administering to a subject one or more lipocalin muteins of claim 1 or one or more compositions comprising such muteins.

### Claim 23 (depends on 1)

A method of detecting the presence of LAG-3, comprising the steps of
(a) contacting a sample suspected of containing LAG-3 with a mutein of claim 1 under conditions that allow the formation of a complex between the mutein and LAG-3, and
(b) detecting the complex of the mutein and LAG-3 for the presence of LAG-3.