# US 9,872,904 B2 · Photoimmunoconjugates for use in photodynamic therapy

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- Number: US 9,872,904 B2
- Document ID: US9872904B2
- Status: Granted
- Kind: B2
- Application number: 14/381,386
- Filed: 2012-03-21
- Published: 2015-05-21
- Granted: 2018-01-23
- Earliest claimed: 2012-03-21
- Assignees: Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung e.V.; Rheinisch-Westfaelische Technische Hochschule Aachen
- Applicants: Stefan Barth; Mehmet K. Tur; Ahmad Hussain
- Inventors: Stefan Barth; Mehmet K. Tur; Ahmad Hussain
- Web page: https://patentgrep.com/patent/US9872904B2

## CPC

- A61K 41/0071: {PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61P 35/00: Antineoplastic agents
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/30: from tumour cells
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C07K 2317/622: Single chain antibody (scFv)
- C12Y 201/01063: Methylated-DNA-[protein]-cysteine S-methyltransferase (2.1.1.63), i.e. O6-methylguanine-DNA methyltransferase
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

A compound comprising a photosensitizer covalently coupled to a protein selected from the group consisting of antibodies or their derivatives or fragments thereof, synthetic peptides such as scFv, mimotopes which bind CD antigens, cytokine receptors, interleukin receptors, hormone receptors, growth factor receptors, more particularly tyrosine kinase growth factor receptor of the ErbB family, wherein the photosensitizer is coupled to the binding protein via O6-alkylguanine-DNA alkyltransferase (hAGTm), a modified human DNA repair protein.

## Claims

### Claim 1

A compound comprising:
a photosensitizer covalently coupled to
a binding protein selected from the group consisting of an antibody or its antigen-binding fragment thereof; and
wherein the binding protein binds a growth factor receptor,
the photosensitizer is coupled to the binding protein via O6-alkylguanine-DNA alkyltransferase (hAGTm), a modified human DNA repair protein, and
wherein the binding protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

### Claim 2 (depends on 1)

The compound of claim 1, wherein the photosensitizer is coupled at the active site of the O6-alkylguanine-DNA alkyltransferase.

### Claim 3 (depends on 1)

The compound of claim 1, wherein the photosensitizer is selected from the group consisting of porphyrins, chlorophylls and dyes.

### Claim 4 (depends on 1)

The compound of claim 1, specifically targeting an internalizing and disease-specific cell surface receptor.

### Claim 5 (depends on 1)

The compound of claim 1, wherein the binding protein is an scFv antibody.

### Claim 6

A compound comprising:
a binding protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
wherein the binding protein binds a growth factor receptor, and
wherein the binding protein is covalently coupled to a modified human DNA repair protein called O6-alkylguanine-DNA alkyltransferase (hAGTm).

### Claim 7 (depends on 6)

The compound of claim 6, wherein the binding protein is encoded by the polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

### Claim 8

A compound comprising:
a photosensitizer covalently coupled to
a binding protein comprising a protein encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 5;
wherein the binding protein binds a growth factor receptor, and
wherein the photosensitizer is coupled to the binding protein via O6-alkylguanine-DNA alkyltransferase (hAGTm), a modified human DNA repair protein.

### Claim 9 (depends on 1)

A method for manufacturing the compound of claim 1 comprising the step of fusing O6-alkylguanine-DNA alkyltransferase (hAGTm) with the binding protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 to form a fusion protein.

### Claim 10 (depends on 9)

The method of claim 9 wherein the binding protein encoded by the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 is inserted into a SifI and NotI-digested site of eukaryotic expression vector pMS-SNAP providing an N-terminal binding ligand and a C-terminal SNAP-tag sequence.

### Claim 11 (depends on 9)

The method of claim 9 wherein the fusion protein is expressed in human embryonic kidney cell line.

### Claim 12 (depends on 9)

The method of claim 9 wherein the fusion protein is purified from cell-free supernatant by an affinity chromatography.

### Claim 13 (depends on 1)

The compound of claim 1, wherein the photosensitizer is a porphyrin derivative of the formula

### Claim 14 (depends on 13)

A method of manufacturing the compound of claim 13, comprising reacting the carboxyl groups of the porphyrin derivative with an activated ester or a coupling agent, followed by reacting with O6-benzylguanine, O2-benzylcytosine or a coenzyme A (CoA).

### Claim 15 (depends on 14)

The method of claim 14, wherein O6-benzylguanine, O2-benzylcytosine or CoA is coupled to a linker molecule and/or the activated ester is formed by succinimides, or the coupling agent is a carbodiimide.

### Claim 16 (depends on 1)

A medicament comprising the compound of claim 1 and a pharmaceutically acceptable adjuvant for improving or rendering a pharmaceutical effect associated with photoimmunotherapy.

### Claim 17 (depends on 1)

A method for treating cancer in a subject by photoimmunotherapy, comprising administering to the subject the compound of claim 1.

### Claim 18 (depends on 1)

The compound of claim 1, wherein the binding protein is scFv.

### Claim 19 (depends on 1)

The compound of claim 1, wherein the growth factor receptor is a tyrosine kinase growth factor receptor of the ErbB family.

### Claim 20 (depends on 1)

The compound of claim 1, wherein the binding protein is encoded by the polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

### Claim 21 (depends on 6)

The compound of claim 6, wherein the binding protein is scFv.

### Claim 22 (depends on 6)

The compound of claim 6, wherein the growth factor receptor is a tyrosine kinase growth factor receptor of the ErbB family.