# US 12,181,480 B2 · Polyspecificity reagents, methods for their preparation and use

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- Number: US 12,181,480 B2
- Document ID: US12181480B2
- Status: Granted
- Kind: B2
- Application number: 17/111,403
- Filed: 2020-12-03
- Published: 2021-05-27
- Granted: 2024-12-31
- Earliest claimed: 2013-04-29
- Assignees: Adimab, LLC
- Applicants: Adimab, LLC
- Inventors: Piotr Bobrowicz; Amber D. Hanna; Jerry M. Thomas
- Web page: https://patentgrep.com/patent/US12181480B2

## CPC

- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
- G01N 2500/10: involving cells
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The present invention relates, inter alia, to polyspecificity reagents, methods of making the same, and methods of using the same in, inter alia, the selection, screening, enrichment, and identification of non-polyspecific, and thus developable, polypeptides.

## Claims

### Claim 1

A method of generating an enriched plurality of developable polypeptides from a non-enriched plurality of polypeptides, the method comprising:
(a) providing the plurality of polypeptides;
(b) contacting the plurality of step (a) with a polyspecificity reagent, wherein the polyspecificity reagent comprises a composition of:
(i) a soluble cellular fraction; and,
(ii) a membrane cellular fraction;
thereby producing a reagent-plurality mixture;
(c) detecting from the reagent-plurality mixture of step (b) one or more polyspecific polypeptides from among the plurality of polypeptides that interacts with the polyspecificity reagent; and
(d) separating from the non-enriched plurality of polypeptides the one or more polyspecific polypeptides detected in step (c), thereby generating the enriched collection of developable polypeptides.

### Claim 2 (depends on 1)

The method of claim 1, the method further comprising contacting either:
the plurality of polypeptides;
the reagent-plurality mixture; or
both the plurality of polypeptides and the reagent-plurality mixture;
with at least one test binding partner.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polyspecificity reagent comprises at least one detergent.

### Claim 4 (depends on 1)

The method of claim 1, wherein the polyspecificity reagent comprises at least one detergent selected from the group consisting of beta-mercaptoethanol (B-ME), (Cyclohexyl-beta-D-maltoside (Cymal-6), Sodium Cholate, Polyoxyethylene(23) lauryl ether (Brij35), Decyl Maltose Neopentyl Glycol (DM-NG), n-Dodecyl β-D-maltoside (DDM), n-octyl-β-D-glucoside (OG), and 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).

### Claim 5 (depends on 1)

The method of claim 1, wherein the polypeptides comprise antibodies, human antibodies, humanized antibodies, full length IgGs, multispecific antibodies, bispecific antibodies, antibody fragments, or combinations thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the polyspecificity reagent is obtained from a collection of cells in culture.

### Claim 7 (depends on 1)

The method of claim 1, wherein the polyspecificity reagent is obtained from:
(a) a collection of mammalian cells in culture; or
(b) a collection of insect cells in culture.

### Claim 8 (depends on 5)

The method of claim 5, wherein the polypeptides comprise human antibodies.

### Claim 9 (depends on 5)

The method of claim 5, wherein the polypeptides comprise full-length IgGs.

### Claim 10 (depends on 6)

The method of claim 6, wherein preparation of the polyspecificity reagent comprises:
preparing the soluble cellular fraction;
preparing the membrane cellular fraction; and
combining the soluble cellular fraction and the membrane cellular fraction.

### Claim 11 (depends on 10)

The method of claim 10, wherein:
the soluble cellular fraction is a soluble cytoplasmic polyspecific reagent (SCP), wherein the SCP comprises supernatant collected after centrifugation of a homogenized mixture of the collection of cells in culture; and
the membrane cellular fraction is soluble membrane polyspecific reagent (SMP), wherein the SMP comprises an enriched membrane fraction collected and solubilized after centrifugation of the homogenized mixture of the collection of cells in culture.