# US 2022/0033795 A1 · Compositions and methods for treatment of pain

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- Number: US 2022/0033795 A1
- Document ID: US20220033795A1
- Status: Published application
- Kind: A1
- Application number: 17/402,323
- Filed: 2021-08-13
- Published: 2022-02-03
- Earliest claimed: 2015-08-27
- Assignees: President and Fellows of Harvard College; Massachusetts Institute of Technology
- Applicants: President and Fellows of Harvard College; Massachusetts Institute of Technology
- Inventors: R. John Collier; Isaac Chiu; Bradley L. Pentelute
- Web page: https://patentgrep.com/patent/US20220033795A1

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- A61K 39/07: Bacillus
- A61K 39/08: Clostridium, e.g. Clostridium tetani
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- C07K 14/32: from Bacillus (G)
- C07K 14/33: from Clostridium (G)
- C12N 9/50: Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)}
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Embodied herein are engineered fusion proteins that bind and target nociceptor neurons, compositions comprising these engineered fusion proteins, and methods for treatment of pain using these engineered fusion proteins or compositions containing the engineered fusion proteins. The engineered fusion proteins contain domains derived from protein toxins such as the anthrax toxin, clostridial botulinum family of toxins, disulphide-containing toxins, and AB component type toxins.

## Claims

### Claim 1

A fusion protein comprising:
a) a botulinum neurotoxin (BTx) or a tetanus neurotoxin (TTx), and
b) an anthrax toxin protective antigen (PA), or a C-terminal receptor-binding domain of PA,
wherein part (a) and (b) are linked or fused together.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the BTx or TTx comprises a BTx or TTx enzymatic moiety and the translocation signal.

### Claim 3 (depends on 2)

The fusion protein of claim 2, wherein the BTx enzymatic moiety or translocation signal is selected from the BTx light chain and heavy chain domains of any one of BTx/A, BTx/B, BTx/C, BTx/D, BTx/E, BTx/F, BTx/G, and a non-Clostridial botulinum-like toxin.

### Claim 4 (depends on 2)

The fusion protein of claim 2, wherein the BTx or TTx enzymatic moiety or translocation signal is selected from the respective LC enzymatic moiety or HN translocation peptide found in Table 1.

### Claim 5 (depends on 1)

The fusion protein of claim 1, wherein the part (a) and (b), or the enzymatic moiety and translocation peptide are linked by a linker peptide.

### Claim 6 (depends on 5)

The fusion protein of claim 5, wherein the linker peptide is 1-20 amino acids long.

### Claim 7 (depends on 6)

The fusion protein of claim 6, wherein the linker peptide is stable in human serum for at least 1 minute.

### Claim 8 (depends on 7)

The fusion protein of claim 7, wherein the linker peptide comprises at least one amino acid that is Gly or Ser.

### Claim 9 (depends on 6)

The fusion protein of claim 6, wherein the linker peptide does not comprise Lys and/or Arg.

### Claim 10 (depends on 1)

The fusion protein of claim 1, wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain, a PAd2 and a PAd4 domain, or PA63.

### Claim 11 (depends on 1)

The fusion protein of claim 1, wherein the PA or the C-terminal receptor-binding domain of PA is resistant to cleavage by a protease.

### Claim 12 (depends on 11)

The fusion protein of claim 11, wherein the the PA or the C-terminal receptor-binding domain of PA is resistant to furin cleavage, or Lys C cleavage or both.

### Claim 13

A fusion protein comprising:
a) a non-cytotoxic protease, which protease is capable of cleaving a SNARE protein in a nociceptor neuron; and
b) a protein capable of binding to an anthrax toxin protective antigen (PA) or a fragment thereof,
wherein the PA or fragment binds a receptor expressed on the nociceptor neuron.

### Claim 14 (depends on 13)

The fusion protein of claim 13, wherein the non-cytotoxic protease comprises a clostridial neurotoxin L-chain.

### Claim 15 (depends on 14)

The fusion protein of claim 14, wherein the clostridial neurotoxin is botulinum neurotoxin (BTx) or tetanus neurotoxin (TTx).

### Claim 16 (depends on 14)

The fusion protein of claim 14, wherein the clostridial neurotoxin L-chain is selected from Table 1.

### Claim 17 (depends on 13)

The fusion protein of claim 13, wherein the PA-binding receptor expressed on the nociceptor neuron is ANTXR2 (CMG2).

### Claim 18 (depends on 13)

The fusion protein of claim 13, wherein the protein capable of binding to PA is:
i) an anthrax toxin lethal factor (LF); or
ii) an anthrax toxin edema factor (EF).

### Claim 19 (depends on 18)

The fusion protein of claim 18, wherein PA binding domain of LF is the N-terminal domain of LF, (abbreviated as LFPABD or LFn).

### Claim 20 (depends on 18)

The fusion protein of claim 18, wherein PA binding domain of EF is the N-terminal domain of EF, (abbreviated as EFPABD or EFn).

### Claim 21

A fusion protein comprising:
a) a disulfide-containing peptide toxin (these include the channel blocking toxins having a cysteine-knot motif), which is capable of blocking ion channels in a nociceptor neuron; and
b) a targeting moiety (TM) that is capable of binding to a binding site on the nociceptor neuron, wherein the nociceptor neuron expresses said ion channels therein (e.g., sodium or calcium or both sodium and calcium);

### Claim 22 (depends on 21)

The fusion protein of claim 21, wherein the disulfide-containing peptide toxin comprises a cysteine knot motif.

### Claim 23 (depends on 21)

The fusion protein of claim 21, wherein the disulfide-containing peptide toxin is a conotoxin, an agatoxin, a delta-palutoxin, a huwentotoxin or a ProTx II toxin.

### Claim 24 (depends on 21)

The fusion protein according to claim 21, wherein the TM is selected from the group consisting of:
i) an anthrax toxin protective antigen (PA);
ii) a C-terminal receptor-binding domain of PA;
iii) a nociceptor neuron-binding protein.

### Claim 25 (depends on 24)

The fusion protein according to claim 24, wherein the PA or C-terminal receptor-binding domain of PA interacts with and binds the ANTXR2 (CMG2) receptor expressed on the nociceptor neuron.

### Claim at position 26

26-29. (canceled)

### Claim 30 (depends on 24)

The fusion protein of claim 24, wherein the nociceptor neuron-binding protein is an antibody.

### Claim 31 (depends on 30)

The fusion protein of claim 30, wherein the antibody specifically binds to nerve growth factor receptor, the ANTXR2 receptor, or an ion-channel protein present on nociceptor neurons.

### Claim 32 (depends on 31)

The fusion protein of claim 31, wherein the ion-channel protein is selected from Nav1.7, Nav1.8 or Nav1.9.

### Claim 33

A fusion protein comprising:
a) a disulfide-containing peptide toxin (this are channel blocking toxin having a cysteine-knot motif), which is capable of blocking sodium or calcium or both sodium and calcium channels in a nociceptor neuron; and
b) a protein capable of binding to an anthrax toxin protective antigen (PA) or a fragment thereof,
wherein the fragment binds a receptor expressed on the nociceptor neuron.

### Claim at position 31

34-37. (canceled)

### Claim 38

A fusion protein comprising:
a) an AB toxin;
b) an anthrax toxin protective antigen (PA) or a fragment thereof, wherein the PA or fragment binds a receptor expressed on the nociceptor neuron; and
c) a Translocation domain (TL) that is capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptor neuron.

### Claim 39 (depends on 38)

The fusion protein of claim 38, wherein the AB toxin is selected from
a. Ricin toxin,
b. Cholera toxin A-part and B-part;
c. Pseudomonas aeruginosa Exotoxin A A-part and B-part;
d. Shiga toxin A-part and B-part; and
e. Diphtheria toxin A-part and B-part.

### Claim 40

(canceled)

### Claim 41 (depends on 38)

The fusion protein of claim 38, wherein the PA fragment is a C-terminal receptor-binding domain of PA.

### Claim 42 (depends on 38)

The fusion protein of claim 38, wherein the TL is
i) a clostridial neurotoxin translocation domain;
ii) a holotoxin; or
iii) a variant form of the holotoxin that has the toxin receptor-binding function of the AB toxin negated.

### Claim at position 37

43-49. (canceled)

### Claim 50 (depends on 1)

A method for treatment of pain, the method comprising administering to a subject in need thereof the fusion protein of claim 1.

### Claim 51

A method of treating pain comprising administering to a subject in need thereof, native mature anthrax toxin protective antigen (PA) and anthrax toxin edema factor (EF), anthrax toxin lethal factor (LF) or any combination thereof.

### Claim at position 40

52-69. (canceled)

### Claim 70

A fusion protein comprising:
a) a botulinum neurotoxin N-terminal enzymatic domain of a botulinum neurotoxin (BTx) moiety (L chain of BTx), and
b) a N-terminal domain (LFn) of anthrax toxin lethal factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA; or the N-terminal domain (EFn) of anthrax toxin edema factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PAthe enzymatic domain,
wherein part (a) is linked N-terminally or C-terminally or both N-terminally and C-terminally to part (b).

### Claim 71 (depends on 70)

The fusion protein of claim 70 further comprising an amino acid sequence defining a belt corresponding to the N-terminal part of the BTx HN domain which is located at C-terminal side of the BTx moiety, and the belt is linked to the L chain.

### Claim 72 (depends on 71)

The fusion protein of claim 71, wherein the residue corresponding to the L chain junction with the belt of the BTx HN domain has been cleaved.

### Claim 73 (depends on 71)

The fusion protein of claim 71, wherein the Cys residues in the BTx L chain and the belt corresponding to the N-terminal part of the BTx HN domain have been changed to Ala, Ser, or Thr.

### Claim 74 (depends on 70)

The fusion protein of claim 70, further comprising at least one D-amino acid at the N-terminus of the fusion protein.

### Claim 75 (depends on 70)

The fusion protein of claim 70, further comprising a linker peptide between the BTx L moiety and the LFn or EFn domain.

### Claim at position 47

76-79. (canceled)

### Claim 80 (depends on 70)

A composition comprising claim 70.

### Claim 81

(canceled)

### Claim 82 (depends on 80)

The composition of claim 80 and a native anthrax toxin protective antigen (PA) protein.

### Claim at position 51

83-85. (canceled)

### Claim 86

A fusion protein comprising:
a) anthrax toxin protective antigen (PA); an anthrax toxin protective antigen C-terminal receptor binding domain (PAd4); or a nociceptor neuron-binding protein linked to,
b) a disulfide-containing peptide toxin.

### Claim 87 (depends on 86)

The fusion protein of claim 86, wherein the disulfide-containing peptide toxin is an inhibitor cysteine knot toxin.

### Claim 88

(canceled)

### Claim 89 (depends on 86)

The fusion protein of claim 86, further comprising a linker peptide between the PA, PAd4 or nociceptor-binding protein and the inhibitor cysteine knot toxin.

### Claim at position 56

90-104. (canceled)

### Claim 105

A fusion protein comprising a disulfide-containing peptide toxin operably linked N-terminally or C-terminally or both N-terminally and C-terminally, or chemically crosslinked at one or more sites to the N-terminal domain (LFn) of anthrax toxin lethal factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA; or the N-terminal domain (EFn) of anthrax toxin edema factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA.

### Claim 106 (depends on 105)

The fusion protein of claim 105, further comprising a linker peptide between the LFn and the toxin or the EFn and the toxin.

### Claim at position 59

107-115. (canceled)

### Claim 116

A fusion protein comprising an AB toxin fused to a linker peptide operably linked to a C-terminal receptor-binding domain of anthrax toxin protective antigen (PAd4 domain), wherein the fusion protein further comprises a translocation domain, a holotoxin or a mutant form of the holotoxin that has the toxin receptor-binding function of the AB toxin negated.

### Claim at position 61

117-121. (canceled)

### Claim 122 (depends on 116)

The fusion protein of claim 116, wherein 1-60 consecutive amino acids from the N-terminal side adjacent to the native PAd4 domain are further incorporated between the AB toxin and the PAd4.

### Claim at position 63

123-131. (canceled)

### Claim 132

A fusion protein comprising an N-terminal enzymatic domain (Chain A) together with the translocation/pore-forming domain from a tetanus neurotoxin (TTx) operably linked to a C-terminal receptor-binding domain of anthrax toxin protective antigen (PAd4 domain).

### Claim 133 (depends on 132)

The fusion protein of claim 132 further comprising a linker peptide between the TTx moiety and the PAd4 domain.

### Claim at position 66

134-138. (canceled)

### Claim 139 (depends on 132)

The fusion protein of claim 132, wherein 1-60 consecutive amino acids from the N-terminal side adjacent to the native PAd4 domain are further incorporated between the BTx moiety and the PAd4.

### Claim at position 68

140-148. (canceled)

### Claim 149 (depends on 132)

A nucleic acid encoding the fusion protein of claim 132.

### Claim 150 (depends on 149)

A vector comprising the nucleic acid of claim 149.

### Claim 151 (depends on 150)

The vector of claim 150, wherein the vector is a plasmid, a bacteriophage, a phagmid, a cosmid, a viral particle, or viral vector.

### Claim 152 (depends on 149)

A cell comprising the nucleic acid of claim 149.

### Claim 153 (depends on 152)

A method of producing fusion protein comprising:
a) culturing the cell of claim 152 in conditions such that the fusion protein is expressed; and
b) recovering the fusion protein.

### Claim 154 (depends on 153)

The fusion protein produced by the method of claim 153.

### Claim 155 (depends on 1)

The fusion protein of claim 1, which is glycosylated.

### Claim at position 76

156-164. (canceled)

### Claim 165 (depends on 132)

A use of fusion protein of claim 132 for the treatment of pain.

### Claim 166 (depends on 132)

A use of fusion protein of claim 132 for the manufacture of a medicament for the treatment of pain.

### Claim 167 (depends on 80)

A method for treatment of pain, the method comprising administering to a subject in need thereof the composition from claim 80.

### Claim 168 (depends on 167)

The method of claim 167, wherein the administering is performed by intrathecal infusion or intra-cerebroventricular infusion or by an epidural injection into the central nervous system, or by peripheral administration using intradermal injection, subcutaneous injection, intramuscular injection, intraneural injection, or intra-articular injection.

### Claim 169 (depends on 168)

The method of claim 168, wherein the pain is selected from diabetic neuropathic pain, cancer pain, fibromyalgia and other systemic pain disorders.

### Claim 170 (depends on 80)

A method for treatment of nerve, joint, skin, visceral, bladder, or muscle pain comprising administering peripherally by intradermal injection, subcutaneous injection, intramuscular injection, intraneural injection, or intra-articular injection to a subject in need thereof the composition from claim 80.

### Claim 171 (depends on 80)

A method for treatment of diabetic neuropathic pain, cancer pain, fibromyalgia or other systemic pain disorders comprising administering by epidural injection, intrathecal infusion or intra-cerebroventricular infusion into the central nervous system of a subject in need thereof the composition from claim 80.

### Claim 172 (depends on 167)

The method of claim 167, wherein the composition is administered separately before, simultaneously or after administering a composition comprising an anthrax protective antigen (PA) in a pharmaceutically acceptable carrier, excipient or diluent.

### Claim 173

(canceled)

### Claim 174 (depends on 80)

A method of manufacture of a pharmaceutical composition of claim 80 for the treatment of pain, wherein the pharmaceutical composition comprising fusion protein and a pharmaceutically acceptable carrier or excipient.

### Claim 175

A fusion protein comprising:
a. a botulinum neurotoxin (BTx) moiety, wherein the BTx moiety comprises an enzymatic moiety and a translocation domain and
b. a targeting moiety comprising a C-terminal receptor-binding domain of an anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain, wherein part (a) and (b) are linked or fused together.

### Claim 176

A nucleic acid encoding a fusion protein comprising:
a. a botulinum neurotoxin (BTx) moiety that comprises an enzymatic moiety and a translocation domain; and
b. a targeting moiety comprising a C-terminal receptor-binding domain of anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain,
wherein part (a) and (b) are linked or fused together.