# US 2024/0189403 A1 · Antigen-Presenting Polypeptides with Chemical Conjugation Sites and Methods of Use Thereof

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- Number: US 2024/0189403 A1
- Document ID: US20240189403A1
- Status: Published application
- Kind: A1
- Application number: 18/287,729
- Filed: 2022-04-20
- Published: 2024-06-13
- Earliest claimed: 2021-04-21
- Assignees: Cue Biopharma, Inc.
- Applicants: Cue Biopharma, Inc.
- Inventors: Ronald D. SEIDEL, III; John F. ROSS; Chee Meng LOW
- Web page: https://patentgrep.com/patent/US20240189403A1

## CPC

- A61K 39/0008: {Antigens related to auto-immune diseases; Preparations to induce self-tolerance}
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- C07K 14/495: Transforming growth factor [TGF]
- C07K 14/55: IL-2
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- 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 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C07K 2318/10: Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
- C07K 2318/20: Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
- 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)

## Abstract

The present disclosure provides antigen presenting polypeptide comprising a TGF-β MOD that is reversibly masked and acts as a TGF-β receptor agonist. The antigen presenting polypeptides comprising one or more chemical conjugation sites for incorporation of, for example, epitope containing polypeptides. The present disclosure provides nucleic acids comprising nucleotide sequences encoding antigen-presenting polypeptides comprising one or more chemical conjugation sites, as well as cells genetically modified with the nucleic acids. The antigen-presenting poly peptides and their epitope conjugates are useful for modulating the activity of a T-cell, and accordingly, the present disclosure provides methods of modulating activity of a T-cell in vitro and in vivo as a method of treatment of diseases and disorders including autoimmune diseases, allergies, GVHD, HGVD, and metabolic disorder.

## Claims

### Claim 1

An unconjugated TMAPP comprising:
(i) a presenting sequence or a presenting complex,
(ii) optionally at least one scaffold polypeptide sequence, and
(iii) a TGF-β sequence, a masking sequence that binds to a TGF-β sequence reversibly masking it, or at least one masked TGF-β MOD; wherein
(a) each presenting sequence comprises MHC Class II α1, α2, β1, and β2 domain polypeptide sequences;
(b) each presenting complex comprises a presenting complex first sequence and a presenting complex second sequence, wherein
the presenting complex first sequence and presenting complex second sequence comprises at least one of the α1, α2, β1, and β2 polypeptide sequences, and
the presenting complex first sequence and presenting complex second sequence together comprise the MHC Class II α1, α2, β1, and β2 domain polypeptide sequences, and
(c) each masked TGF-β MOD comprises a masking sequence and TGF-β sequence;
wherein the unconjugated TMAPP optionally comprises one or more independently selected additional MODs (wt. and/or variant) or pairs of additional MODs (both wt, both variant, or one wt. and one variant) (e.g., located at the N-terminus of a presenting sequence, presenting complex first and/or second sequences);
wherein a the unconjugated TMAPP comprises a chemical conjugation site for conjugation of an epitope presenting molecule, and optionally comprises an additional chemical conjugation site for the conjugation of a payload; and
wherein the unconjugated TMAPP optionally comprise one or more linker sequences that are selected independently.

### Claim 2 (depends on 1)

The unconjugated TMAPP of claim 1, comprising from N-terminus to C-terminus:
(i) a presenting sequence or a presenting complex,
(ii) optionally at least one scaffold polypeptide sequence, and
(iii) a TGF-β sequence, a masking sequence that binds to a TGF-β sequence reversibly masking it, or at least one masked TGF-β MOD.

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

The unconjugated TMAPP of claim 1 or 2, comprising:
(A) a presenting sequence that comprises, ordered from N-terminus to C-terminus
(i) the β1, β2, α1, and α2 domain polypeptide sequences,
(ii) the β1, α1, α2, and β2 domain polypeptide sequences, or
(iii) α1, α2, β1, and β2 domain polypeptide sequences; or
(B) a presenting complex wherein
(i): the presenting complex first sequence comprises, ordered from N-terminus to C terminus, the β1 and β2 domain polypeptide sequences; and the presenting complex second sequence comprises the α1, and α2 domain polypeptide sequences,
(ii) the presenting complex first sequence comprises, ordered from N-terminus to C terminus, the α1, and α2 domain polypeptide sequences; and the presenting complex second sequence comprises the β1 and β2 domain polypeptide sequences,
(iii) the presenting complex first sequence comprises, ordered from N-terminus to C terminus, the 1 α1, and α2 domain polypeptide sequences; and the presenting complex second sequence comprises the β2 domain polypeptide sequence,
(iv) the presenting complex first sequence comprises the β2 domain polypeptide sequence; and the presenting complex second sequence comprises, ordered from N-terminus to C terminus, the β1, α1, and α2 domain polypeptide sequences, or
(v) the presenting sequence or a presenting complex comprising a disulfide bond formed between one of MHC α1 or α2 domain polypeptide sequence and one of the β1 or β2 domain polypeptide sequences.

### Claim 4

The unconjugated TMAPP of any preceding claim, wherein the presenting sequence or the presenting complex comprises:
an α1 and α2 domain polypeptide sequences each having at least 95% or 100% sequence identity to an α1 or α2 domain of a HLA DR alpha (DRA), DM alpha (DMA), DO alpha (DOA), DP alpha 1 (DPA1), DQ alpha 1 (DQA1), or DQ alpha 2 (DQA2) polypeptide sequence provided in any of FIG. 4, 9, 11, 13, 15, or 16; and
a β1 and β2 domain polypeptide sequences each having at least 95% or 100% sequence identity to β1 or β2 domain of a HLA DR beta 1 (DRB1), DR beta 3 (DRB3), DR beta 4 (DRB4), DR beta 5 (DRB5), DM beta (DMB), DO beta (DOB), DP beta 1 (DPB1), DQ beta 1 (DQB1), or DQ beta 2 (DQB2) polypeptide sequences provided in any of FIG. 5, 6, 7, 8, 10, 12, 14, 17 or 18.

### Claim 5

The unconjugated TMAPP of any preceding claim, wherein at least one presenting sequence or presenting complex comprises:
a α1 and α2 domain polypeptide sequences each having at least 95% or 100% sequence identity to an α1 or α2 domain of a HLA DR alpha (DRA) polypeptide sequence provided in FIG. 4; and
a β1 and β2 domain polypeptide sequences each having at least 95% or 100% sequence identity to a 1 or β2 domain of a HLA DR beta 1 (DRB1), DR beta 3 (DRB3), DR beta 4 (DRB4), or DR beta 5 (DRB5) β1 polypeptide sequences provided in any one of FIG. 5, 6, 7, or 8.

### Claim 6

The unconjugated TMAPP of any preceding claim, wherein the presenting sequence or a presenting complex comprises a disulfide bond formed between one of MHC α1 or α2 domain polypeptide sequence and one of the β1 or β2 domain polypeptide sequences.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The unconjugated TMAPP of any of claims 1 to 6, wherein the unconjugated TMAPP comprises a scaffold polypeptide sequence a scaffold sequence that is a non-interspecific sequence or interspecific sequence.

### Claim 8 (depends on 7)

The unconjugated TMAPP of claim 7, wherein the interspecific and non-interspecific sequence are selected from the group consisting of: immunoglobulin heavy chain constant regions (Ig Fc e.g., Ig CH2-CH3); collectin polypeptides, coiled-coil domains, leucine-zipper domains; Fos polypeptides; Jun polypeptides; Ig CH1; Ig CL κ; Ig CLλ; knob-in-hole without disulfide (“KiH”); knob-in hole with a stabilizing disulfide bond (“KiHs-s”); HA-TF; ZW-1; 7.8.60; DD-KK; EW-RVT; EW-RVTs-s; and A107 sequences.

### Claim 9 (depends on 7, 8)

The unconjugated TMAPP of any of claims 7 to 8 complexed to form a duplex or higher order unconjugated TMAPP comprising at least a first unconjugated TMAPP and a second unconjugated TMAPP:
(i) the first unconjugated TMAPP comprises a first scaffold polypeptide sequence; and
(ii) the second unconjugated TMAPP comprises a second scaffold polypeptide sequence;
wherein the first unconjugated TMAPP and the second unconjugated TMAPP are associated by binding interactions between the first scaffold polypeptide sequence and second scaffold polypeptide sequence, and wherein the interactions optionally including one or more interchain covalent bonds; and
wherein the duplex or higher order unconjugated TMAPP comprises at least one masked TGF-β MOD wherein the masking sequence and the TGF-β sequence are present in cis or in trans.

### Claim 10 (depends on 9)

The unconjugated TMAPP or unconjugated duplex TMAPP of claim 9, wherein the first scaffold polypeptide sequence and the second scaffold polypeptide sequence are interspecific sequences.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The unconjugated TMAPP or unconjugated duplex TMAPP of any of claims 1 to 10, comprising a masked TGF-β MOD in trans, wherein the masking sequence is located at the N-terminus of the presenting complex first sequence or the presenting complex second sequence, and the TGF-β sequence is located at the other of the presenting complex first sequence or the presenting complex second sequence.

### Claim 12 (depends on 10)

The unconjugated TMAPP or unconjugated duplex TMAPP of claim 10, comprising a masked TGF-β MOD in trans, wherein the masking sequence is located at the C-terminus of the first scaffold polypeptide sequence, and the TGF-β sequence is located at the C-terminus of the second scaffold polypeptide sequence.

### Claim 13

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, comprising a masked TGF-β MOD in cis.

### Claim 14

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, wherein the TGF-β sequence is:
(i) a TGF-β1 polypeptide optionally comprising a substitution of C77;
(ii) a TGF-β2 polypeptide optionally comprising a substitution of C77; or
(iii) is a TGF-β3 polypeptide optionally comprising a substitution of C77.

### Claim 15

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, wherein the TGF-β sequence is a TGF-β3 polypeptide optionally comprising a substitution of C77.

### Claim 16

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, wherein at least one masking sequence is a TGF-β receptor (TOR) polypeptide, anti-TGF-β antibody or antibody-related polypeptide/aa sequence.

### Claim 17 (depends on 16)

The unconjugated TMAPP or unconjugated duplex TMAPP of claim 16, wherein the TBR polypeptide comprises a TβRI, TβRII, or TβRIII aa sequence.

### Claim 18 (depends on 17)

The unconjugated TMAPP or unconjugated duplex TMAPP of claim 17, wherein the TBR polypeptide comprises:
(i) a TβRII isoform A ectodomain aa sequence;
(ii) a TβRII isoform B ectodomain aa sequence;
(iii) a TβRII isoform B Δ14 (14 aa N-terminal deletion) ectodomain aa sequence; or
(iv) a TβRII isoform B Δ25 (25 aa N-terminal deletion) ectodomain aa sequence.

### Claim 19

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, wherein at least one chemical conjugation site or additional chemical conjugation site is selected from the group consisting of:
a) an amino acid chemical conjugation site;
b) non-natural amino acids and/or selenocysteines;
c) a peptide sequence that acts as an enzyme modification sequence (e.g., sulfatase, transglutaminase or sortase sites);
d) carbohydrate or oligosaccharide; and
e) IgG nucleotide binding sites.

### Claim 20 (depends on 19)

The unconjugated TMAPP or unconjugated duplex TMAPP of claim 19, wherein the amino acid chemical conjugation site is a cysteine.

### Claim 21

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, wherein the additional MOD (wt. or variant) or the additional pair of MODs (wt. or variant) are selected independently from the group consisting of IL-2, PD-L1, 4-1BBL polypeptide sequences and variants of any thereof. For example, the unconjugated TMAPP or unconjugated duplex TMAPP may comprise at least one IL-2 MOD (wt. or variant) and/or at least one PD-L1 (wt. or variant) polypeptide sequence(s).

### Claim 22

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, wherein the additional MOD (wt. or variant) or the additional pair of MODs (wt. or variant) comprise at least one IL-2 MOD (wt. or variant) polypeptide sequence, or at least one pair of IL-2 MOD (wt. or variant) polypeptide sequences in tandem.

### Claim 23

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, wherein the additional MOD (wt. or variant) or the additional pair of MODs (wt. or variant) comprise at least one PD-L1 MOD (wt. or variant) polypeptide sequence.

### Claim 24

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim wherein the additional MOD (wt. or variant) or the additional pair of MODs (wt. or variant) comprise at least one 4-1BBL MOD (wt. or variant) polypeptide sequence.

### Claim 25

The unconjugated TMAPP or unconjugated duplex TMAPP of any preceding claim, comprising an epitope covalently bound directly, or indirectly through a linker, to the chemical conjugation site to form a TMAPP-epitope conjugate or duplex TMAPP-epitope conjugate.

### Claim 26 (depends on 25)

The TMAPP-epitope conjugate or duplex TMAPP-epitope conjugate of claim 25, wherein the peptide epitope is from about 8 aa to about 20 aa.

### Claim 27 (depends on 25, 26)

The TMAPP-epitope conjugate or duplex TMAPP-epitope conjugate of any of claim 25 or 26, wherein the epitope is an epitope of an epitope of an autoantigen, an epitope of a grafted tissue, or epitope of an allergen.

### Claim 28 (depends on 25, 26, 27)

A pharmaceutical composition comprising one or more TMAPP-epitope conjugates, or duplex TMAPP-epitope conjugates of any one of claims 25 to 27.

### Claim 29 (depends on 25, 26, 27, 28)

A method of treatment or prophylaxis of a patient or subject having a disease or condition comprising administering to a patient/subject an effective amount of one or more TMAPP-epitope conjugates, or duplex TMAPP-epitope conjugates of any of claims 25-27, or a pharmaceutical composition of claim 28.

### Claim 30 (depends on 29)

The method of claim 29, wherein the disease or condition is an autoimmune disease, an allergy, GVHD, HGVD, or a metabolic disorder.

### Claim 31 (depends on 29)

The method of claim 29, wherein the disease is and autoimmune disease selected from the group consisting of: Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune-associated infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, Crohn's disease, Goodpasture's syndrome, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), Grave's disease, Hashimoto's thyroiditis, autoimmune gastritis, inflammatory bowel diseases, irritable bowel disease or syndrome, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus (e.g., pemphigus vulgaris), pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus (SLE), vasculitis, and vitiligo.