# US 12,110,611 B2 · Conditionally active biological proteins

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- Number: US 12,110,611 B2
- Document ID: US12110611B2
- Status: Granted
- Kind: B2
- Application number: 17/573,389
- Filed: 2022-01-11
- Published: 2022-04-28
- Granted: 2024-10-08
- Earliest claimed: 2015-02-24
- Assignees: BioAtla, Inc.
- Applicants: Bioatla, Inc.
- Inventors: Jay M. Short; Hwai Wen Chang; Gerhard Frey
- Web page: https://patentgrep.com/patent/US12110611B2

## CPC

- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/005: {constructed by phage libraries}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/30: from tumour cells
- 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}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/31: multispecific
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/90: characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- 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)
- 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)

## Abstract

Methods of generating conditionally active biologic proteins, in particular therapeutic or diagnostic proteins, which are more active at an aberrant condition than at a normal physiological condition. The methods include discovery methods using libraries of proteins and assays employing physiological concentrations of components of bodily fluids. The conditionally active biologic proteins may be further evolved, conjugated to other molecules, masked, reduced in activity by attaching a cleavable moiety. Criteria for selecting starting proteins for the discovery methods, as well as formats of the proteins are also disclosed.

## Claims

### Claim 1

A method of preparing one or more conditionally active antibodies or antibody fragments, wherein the conditionally active antibody or antibody fragment exhibits a decrease in activity in an assay at a normal physiological pH compared to a same activity in an assay at an aberrant pH, the method comprising steps of:
i. selecting a template antibody or antibody fragment from a wild-type antibody or antibody fragment, a mutated antibody or antibody fragment, and a fragment of a wild-type antibody or antibody fragment with a desired property;
ii. evolving a DNA which encodes the template antibody or antibody fragment using one or more evolutionary techniques to create mutant DNAs;
iii. expressing the mutant DNAs to obtain mutant antibodies or antibody fragments at least one of which exhibits a decrease in activity in the assay at the normal physiological pH compared to the same activity in the assay at the aberrant pH; and
iv. screening each mutant antibody or antibody fragment obtained from step (iii) by conducting assays to determine the activity at the normal physiological pH and the activity at the aberrant pH and identifying the one or more conditionally active antibodies or antibody fragments that exhibits a decrease in activity in the assay at the normal physiological pH compared to the same activity in the assay under the aberrant pH,
wherein the assay under the normal physiological pH and the assay under the aberrant pH are performed in assay solutions containing at least one component at substantially the same concentration, said at least one component being selected from hydrogen sulfide at a concentration of 100 μM-100 mM and bicarbonate at a concentration of 25-100 mM.

### Claim 2 (depends on 1)

The method of claim 1, wherein the normal physiological pH is from 7.2 to 7.6 and the aberrant pH is from 6.2 to 6.8.

### Claim 3 (depends on 1)

The method of claim 1, wherein the activity is binding activity.

### Claim 4 (depends on 3)

The method of claim 3, wherein the ratio of the binding activity of the one or more conditionally active antibodies or antibody fragments identified in step (iv) at the aberrant pH compared to the binding activity at the normal physiological pH is at least 2:1.

### Claim 5 (depends on 1)

The method of claim 1, wherein said method further comprises a step of conjugating the one or more conditionally active antibodies or antibody fragments selected in step (iv) to an agent selected from cytokines, interleukins, enzymes, hormones, growth factors, cytotoxic agents, chemotherapy drugs, radioactive particles and diagnostic agents.

### Claim 6 (depends on 1)

The method of claim 1, wherein the assay solutions for the assay under the normal physiological pH and the assay under the aberrant pH contain no serum.

### Claim 7 (depends on 1)

The method of claim 1, wherein said at least one component is hydrogen sulfide at a concentration of 100 μM-100 mM.

### Claim 8 (depends on 1)

The method of claim 1, wherein said at least one component is bicarbonate at a concentration of 25-100 mM.