# US 12,215,164 B2 · Immunotoxins with albumin binding domain

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- Number: US 12,215,164 B2
- Document ID: US12215164B2
- Status: Granted
- Kind: B2
- Application number: 18/471,137
- Filed: 2023-09-20
- Published: 2024-04-25
- Granted: 2025-02-04
- Earliest claimed: 2017-09-18
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The United States of America,as represented by the Secretary,Department of Health and Human Services
- Inventors: Ira H. Pastan; Junxia Wei; Masanori Onda; Tapan Bera; Mitchell Ho
- Web page: https://patentgrep.com/patent/US12215164B2

## CPC

- C07K 16/30: from tumour cells
- A61K 47/6829: {Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A}
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61K 47/6889: {Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment}
- C07K 14/21: from Pseudomonadaceae (F)
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 2317/31: multispecific
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/624: Disulfide-stabilized antibody (dsFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C07K 2319/32: fusions with soluble part of a cell surface receptor, "decoy receptors"
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/50: containing protease site
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- C07K 2319/70: containing domain for protein-protein interaction
- 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)

## Abstract

Disclosed is a molecule comprising: (a) a first domain, which comprises a targeting moiety; (b) a second domain, which comprises an albumin binding domain (ABD), (c) a third domain, which comprises a furin cleavage sequence (“FCS”), which FCS is cleavable by furin; and (d) a fourth domain, which comprises an optionally substituted Domain III from Pseudomonas exotoxin A (“PE”). Related nucleic acids, recombinant expression vectors, host cells, populations of cells, pharmaceutical compositions, methods of producing the molecule, methods of treating or preventing cancer in a mammal, and methods of inhibiting the growth of a target cell are also disclosed.

## Claims

### Claim 1

A nucleic acid comprising a nucleotide sequence encoding a molecule, the molecule comprising:
(a) a first domain, which comprises a targeting moiety, wherein the targeting moiety is not an affibody;
(b) a second domain, which comprises an albumin binding domain (ABD) and comprises the amino acid sequence of SEQ ID NO: 2,
(c) a third domain, which comprises a furin cleavage sequence (FCS) which FCS is cleavable by furin; and
(d) a fourth domain, which comprises a Domain III from Pseudomonas exotoxin A (PE) or a Domain III from Pseudomonas exotoxin A (PE) comprising a substitution of one or more amino acid residues;
wherein the second domain is positioned between the first domain and the third domain;
wherein the third domain is positioned between the second domain and the fourth domain; and
wherein the molecule optionally has (i) a substitution of one or more amino acid residues within one or more T-cell epitopes, (ii) a substitution of one or more amino acid residues within one or more B cell epitopes, (iii) a deletion of one or more contiguous amino acid residues of residues 1-273 and 285-394 as defined by SEQ ID NO: 1; or (iv) a combination of any of (i)-(iii).

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein the molecule further comprises:
(i) a linker positioned between the first domain and the second domain;
(ii) a linker positioned between the second domain and the third domain;
(iii) a linker positioned between the third domain and the fourth domain;
(iv) a combination of any two of (i)-(iii); or
(v) a combination of all three of (i)-(iii).

### Claim 3 (depends on 2)

The nucleic acid of claim 2, wherein the linker of any one or more of (i)-(iii) consist(s) of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine, serine, lysine, and alanine.

### Claim 4 (depends on 2)

The nucleic acid of claim 2, wherein the linker of any one or more of (i)-(iii) consist(s) of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine.

### Claim 5 (depends on 1)

The nucleic acid of claim 1, wherein the fourth domain comprises a PE amino acid sequence, wherein the PE amino acid sequence has a substitution of one or more of amino acid residues R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552, as defined by reference to SEQ ID NO: 1.

### Claim 6 (depends on 1)

The nucleic acid of claim 1, wherein the fourth domain comprises a PE amino acid sequence, wherein the PE amino acid sequence has one or more of the following amino acid substitutions: R427A, F443A, R456A, D463A, R467A, L477H, R490A, R494A, R505A, R538A, and L552E, as defined by reference to SEQ ID NO: 1.

### Claim 7 (depends on 1)

The nucleic acid of claim 1, wherein the targeting moiety is a monoclonal antibody or an antigen binding portion thereof.

### Claim 8 (depends on 1)

The nucleic acid of claim 1, wherein the targeting moiety specifically binds to a cell surface marker selected from CD19, CD21, CD22, CD25, CD30, CD33, CD79b, BCMA, glypican 2 (GPC2), glypican 3 (GPC3), transferrin receptor, EGF receptor (EGFR), mutated EGFR, mesothelin, cadherin, and Lewis Y.

### Claim 9 (depends on 1)

The nucleic acid of claim 1, wherein the targeting moiety is an antibody selected from B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, MORAb-009, YP218, YP7, HN3, LH1, LH4, LH7, BM24, BM306, antigen binding portions of the foregoing, and an antigen binding portion of HA22.

### Claim 10 (depends on 1)

The nucleic acid of claim 1, wherein the fourth domain contains only Domain III.

### Claim 11 (depends on 1)

The nucleic acid of claim 1, wherein the molecule comprises PE-LR.

### Claim 12 (depends on 1)

The nucleic acid of claim 1, wherein the fourth domain is PE24.

### Claim 13

A nucleic acid comprising a nucleotide sequence encoding a molecule, the molecule comprising a sequence of Formula (I):
TM-R1k-ABD-R2m-FCS-R3p-R4q-PE functional domain III (Formula I)
wherein:
TM is a targeting moiety;
R1 is 1 to 20 amino acid residues;
k, m, p, and q are, independently, 0 or 1;
ABD is an albumin binding domain and comprises the amino acid sequence of SEQ ID NO: 2;
R2 is 1 to 20 amino acid residues;
FCS is a furin cleavage sequence of amino acid residues, which sequence is cleavable by furin and has an amino end and a carboxyl end;
R3 is 1 to 20 amino acid residues;
R4 is 1 or more contiguous residues of residues 365-394 of SEQ ID NO: 1; and
PE functional domain III is residues 395-613 of Pseudomonas exotoxin A (PE) SEQ ID NO: 1,
wherein the molecule has a deletion of amino acid residues 253-273 and 285-364 as defined by SEQ ID NO: 1; and
wherein the molecule optionally has (i) a substitution of one or more amino acid residues within one or more T-cell epitopes, (ii) a substitution of one or more amino acid residues within one or more B cell epitopes, (iii) a deletion of one or more contiguous amino acid residues of residues 1-252 as defined by SEQ ID NO:1; or (iv) a combination of any of (i)-(iii).

### Claim 14 (depends on 13)

The nucleic acid of claim 13, wherein the molecule has a substitution of one or more of amino acid residues R427, F443, R456, D463, R467, L477, R490, R494, R505, R538, and L552, as defined by reference to SEQ ID NO: 1, in PE functional domain III.

### Claim 15 (depends on 13)

The nucleic acid of claim 13, wherein the molecule has one or more of the following amino acid substitutions: R427A, F443A, R456A, D463A, R467A, L477H, R490A, R494A, R505A, R538A, and L552E, as defined by reference to SEQ ID NO: 1, in PE functional domain III.

### Claim 16 (depends on 13)

The nucleic acid of claim 13, wherein one or more of R′, R 2, and R 3 consist(s) of 1 to 20 amino acid residues selected, independently, from the group consisting of lysine, alanine, glycine, and serine.

### Claim 17 (depends on 13)

The nucleic acid of claim 13, wherein one or more of R′, R 2, and R 3 consist(s) of 1 to 20 amino acid residues selected, independently, from the group consisting of glycine and serine.

### Claim 18 (depends on 1)

A recombinant expression vector comprising the nucleic acid of claim 1.

### Claim 19 (depends on 13)

A recombinant expression vector comprising the nucleic acid of claim 13.

### Claim 20 (depends on 18)

A host cell comprising the recombinant expression vector of claim 18.

### Claim 21 (depends on 19)

A host cell comprising the recombinant expression vector of claim 19.

### Claim 22 (depends on 20)

A population of cells comprising at least one host cell of claim 20.

### Claim 23 (depends on 21)

A population of cells comprising at least one host cell of claim 21.