# US 11,897,952 B2 · Mechanically interlocking complexes

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- Number: US 11,897,952 B2
- Document ID: US11897952B2
- Status: Granted
- Kind: B2
- Application number: 15/563,093
- Filed: 2016-03-30
- Published: 2018-03-29
- Granted: 2024-02-13
- Earliest claimed: 2015-03-30
- Assignees: City of Hope
- Applicants: City of Hope
- Inventors: John Williams; Krzysztof P. Bzymek; David Horne; Jun Xie
- Web page: https://patentgrep.com/patent/US11897952B2

## CPC

- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 7/06: having 5 to 11 amino acids
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/02: from eggs
- C07K 16/2869: {against hormone receptors (for antibodies against neuromediator receptors C07K16/286)}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/58: {raising an immune response against a target which is not the antigen used for immunisation}
- A61K 2039/64: {characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units}
- C07K 2317/55: Fab or Fab'
- C07K 2317/60: characterized by non-natural combinations of immunoglobulin fragments
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/00: Fusion polypeptide
- C07K 2319/01: containing a localisation/targetting motif
- 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

Provided herein are functionalized monoclonal antibodies (mAbs) including antibody fragments, including those where a Fab-binding molecule (Fab binding moiety) linked to a steric hindering molecule (steric hindering chemical moiety) is mechanically interlocked (e.g., through noncovalent conjugation) with the antibody or antibody fragment. Also provided are compositions that form highly stable and versatile drug delivery and diagnostic compositions.

## Claims

### Claim 1

A mechanically interlocked complex comprising a compound mechanically interlocked with a fragment antigen-binding (Fab) domain,
said Fab domain comprising a hole within a central cavity lined by amino acid residues of the VH, VL, CH1, and CL regions of said Fab domain, wherein said central cavity comprises a non-CDR binding site;
wherein said amino acid residues of the VL region are a threonine at position 40, an asparagine at position 41, and an aspartate at position 85, according to Kabat numbering, and
wherein said amino acid residues of the VH region are a seine or proline at position 40 and an isoleucine, tyrosine, methionine, phenylalanine, or a tryptophan at position 89, according to Kabat numbering;
said compound comprising a Fab binding moiety attached to a steric hindering chemical moiety through a chemical linker,
wherein said Fab binding moiety is bound to said non-CDR binding site, said chemical linker passes through said hole, and steric hindrance occurs between said steric hindering chemical moiety and amino acids lining said hole thereby mechanically interlocking said compound and said Fab;
wherein said compound has the formula:
R1-L1-R2
wherein R1 is
R3—X0-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-R4
wherein:
X0 is Ser or null;
X1 is Cys, Ser, Gly, β-alanine, diaminopropionic acid, β-azidoalanine, or null;
X2 is Gln or null;
X3 is Phe, Tyr, β,β′-diphenyl-Ala, His, Asp, 2-bromo-L-phenylalanine, 3-bromo-L-phenylalanine, 4-bromo-L-phenylalanine, Asn, Gln, a modified Phe, or a boronic acid-containing residue;
X4 is Asp or Asn;
X5 is Leu, β,β′-diphenyl-Ala, Phe, Trp, Tyr, tryptophan, or tyrosine, or a boronic acid-containing residue;
X6 is Ser or Cys;
X7 is Thr, Ser or Cys;
X8 is an amino acid comprising a side chain of the formula -L1A-L1-R2,
wherein L1A is substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene;
wherein L1 is said chemical linker of about 5 Å to about 15 Å in length; and
R2 is said steric hindering chemical moiety wherein the longest bond length distance is at least 10 Å;
X9 is Arg or Ala;
X10 is Leu, Gln, Glu, β,β′-diphenyl-Ala, Phe, Trp, Tyr, tryptophan, or tyrosine, or a boronic acid-containing residue;
X11 is Lys or Arg;
X12 is Cys, Gly, 7-aminoheptanoic acid, β-alanine, diaminopropionic acid, propargylglycine, isoaspartic acid, or null,
R3 and R4 are independently null, -L2-R5 or an amino acid peptide sequence optionally substituted with -L2-R5, wherein L2 is a covalent or non-covalent linker and R5 is a therapeutic agent, a diagnostic agent, or a detectable agent; and
wherein X1 and X12 are optionally joined together to form a cyclic peptidyl moiety.

### Claim 2 (depends on 1)

The mechanically interlocked complex of claim 1, wherein said non-CDR binding site is a peptide binding site comprising framework region amino acid residues and said Fab binding moiety is a peptidyl moiety.

### Claim 3 (depends on 2)

The mechanically interlocked complex of claim 2, wherein said peptidyl moiety binds to said peptide binding site with a KD of less than 10 nM.

### Claim 4 (depends on 2)

The mechanically interlocked complex of claim 2, wherein said peptidyl moiety binds to said peptide binding site with a KD of less than 1 nM.

### Claim 5 (depends on 2)

The mechanically interlocked complex of claim 2, wherein said peptidyl moiety binds to said peptide binding site with a τ1/2 of more than 2000 seconds.

### Claim 6 (depends on 1)

The mechanically interlocked complex of claim 1, wherein said compound and said Fab are bound together with a τ1/2 of more than 4500 seconds.

### Claim 7 (depends on 1)

The mechanically interlocked complex of claim 1, wherein said chemical linker is a substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene.

### Claim 8 (depends on 1)

The mechanically interlocked complex of claim 1, wherein L1A is —(CH2)3—NH(N)—NH—.

### Claim 9 (depends on 1)

The mechanically interlocked complex of claim 1, wherein R3 and R4 are independently null, -L2-R5 or a 1 to 100 amino acid peptide sequence optionally substituted with -L2-R5, wherein L2 is a covalent or non-covalent linker and R5 is a therapeutic agent, a diagnostic agent, or a detectable agent.

### Claim 10 (depends on 1)

The mechanically interlocked complex of claim 1, wherein R3 is a three amino acid peptide sequence optionally substituted with -L2-R5.

### Claim 11 (depends on 1)

The mechanically interlocked complex of claim 1, wherein the non-CDR binding site is formed by amino acids at positions 8, 9, 10, 38, 39, 40, 41 42, 43, 44, 45, 82, 83, 84, 85, 86, 87, 99, 100, 101, 102, 103, 104, 105, 142, 162, 163, 164, 165, 166, 167, 168, and 173 of the light chain and 6, 9, 38, 39, 40, 41, 42, 43, 44, 45, 84, 86, 87, 88, 89, 90, 91, 103, 104, 105, 106, 107, 108, 111, 110, 147, 150, 151, 152, 173, 174, 175, 176, 177, 185, 186, and 187 of the heavy chain of the Fab, according to Kabat numbering.

### Claim 12 (depends on 1)

A method of binding an antigen, said method comprising contacting an antigen with said mechanically interlocked complex of claim 1 and allowing said Fab to bind said antigen.