Conditionally active polypeptides and methods of generating them
Inventors
Short, Jay M. • Chang, Hwai Wen • Frey, Gerhard
Assignees
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Publication Number
US-11773509-B2
Publication Date
2023-10-03
Expiration Date
Abstract
A method of preparing a conditionally active polypeptide from a parent polypeptide, comprising steps of evolving a DNA encoding the parent polypeptide by increasing a net charge of the parent polypeptide using one or more techniques selected from increasing a total number of codons of charged amino acid residues in the DNA and decreasing a total number of codons of uncharged amino acid residues in the DNA to create mutant DNAs; expressing the mutant DNAs to obtain mutant polypeptides; and selecting the conditionally active polypeptide from the mutant polypeptides which exhibits a decrease in activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition. The conditionally active polypeptide, pharmaceutical compositions containing same, nanoparticle and drug conjugates thereof and uses thereof are also provided.
Core Innovation
The invention provides conditionally active antibodies or antibody fragments generated by evolving a parent antibody, single chain antibody, or antibody fragment. A DNA encoding the parent is evolved to increase the number of acidic amino acid residues, producing mutant DNAs that are expressed to obtain mutant antibodies or antibody fragments.
Conditionally active antibodies or antibody fragments are selected so that they are reversibly inactivated in a first assay at a first pH of 7.4. The selected variants exhibit a decrease in activity in the first assay at pH 7.4 compared to a second assay at a second pH of 6.0, including pH-dependent activity behavior under normal physiological conditions versus aberrant conditions.
The description further states assay factors that influence conditionality, including the presence of small, ionizable species in assay solutions and constraints on physicochemical properties such as pKa and molecular weight. Additional embodiments extend the conditional activity concept to antibody formats and engineered therapeutic contexts, including conditionally active antibody domains used in pharmaceutical compositions and engineered formats such as CAR-T, alongside discussion of masking conditional activity.
Claims Coverage
The document contains one independent claim directed to preparing a conditionally active antibody or antibody fragment, with dependent claims refining the mutation strategy and the assay media used for the pH-dependent selection. The independent claim is anchored by a reversible inactivation and activity decrease relationship at two specified pH values.
Evolving DNA to increase acidic residues
Evolving a DNA encoding a parent antibody, single chain antibody, or antibody fragment to increase the number of acidic amino acid residues, to create mutant DNAs.
Expressing mutant DNAs to obtain mutant antibodies
Expressing the mutant DNAs to obtain mutant antibodies or antibody fragments.
Selecting reversibly inactivated, pH-decreased activity variants
Selecting the conditionally active antibody or antibody fragment from the mutant antibodies or antibody fragments, where the conditionally active antibody or antibody fragment is reversibly inactivated in a first assay at a first pH of 7.4 and exhibits a decrease in activity in the first assay at the first pH of 7.4 compared to the same activity in a second assay at a second pH of 6.0.
Assay-media physicochemical constraints
Carrying out the first and second assays in assay media containing at least one species with molecular weight less than 900 a.m.u. and a pKa within 3 units of a first pH of 7.4.
Target acidic-codon introduction to complementarity determining regions
Introducing one or more codons encoding acidic amino acid residues into a DNA region that encodes complementarity determining regions of a parent antibody, single-chain antibody, or antibody fragment.
Select conditionally active antibodies based on affinity, expression level, humanization
Selecting a conditionally active antibody or antibody fragment based on a property chosen from affinity, expression level, humanization, or combinations thereof.
Use acidic amino acid residues of Asp, Glu, or combinations
Using acidic amino acid residues that are Aspartic acid, Glutamic acid, or a combination of both.
The claim coverage centers on evolving antibody-encoding DNA to increase acidic amino acid residues, expressing mutant DNAs to generate mutant antibodies or fragments, and selecting variants that are reversibly inactivated and show reduced activity at pH 7.4 relative to pH 6.0, with dependent refinements concerning assay media, acidic codon targeting, residue type, and additional selection properties such as affinity, expression level, and humanization.
Stated Advantages
Provides conditionally active antibodies or antibody fragments that are reversibly inactivated in a first assay at pH 7.4 while exhibiting decreased activity at pH 7.4 compared to pH 6.0.
Improves mutant selection based on paired screening under normal physiological conditions versus aberrant conditions.
Enables screening hit rate and selectivity using assay media having small-ion and buffering species with pKa values near the aberrant pH.
Documented Applications
Producing pharmaceutical compositions and using conditionally active antibodies or antibody fragments in treating cancers and related diseases, including solid, CNS, and hematologic cancers.
Pharmaceutical compositions including conditionally active antibody or antibody fragment formats.
Nanoparticles and drug conjugates using conditionally active binding domains.
Engineered therapeutic formats including chimeric antigen receptor (CAR) and CAR-T using conditionally active binding domains.
Therapeutic targeting contexts and disease targeting contexts described as solid tumors, synovial fluid, brain diseases/disorders, and tumor microenvironment.
Oncolytic virus context described as including a diverse therapeutic context.
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