Classifications

• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B15/00—ICT specially adapted for analysing two-dimensional [2D] or three-dimensional [3D] molecular structures, e.g. structural or functional relations or structure alignment
  • G16B15/30—Drug targeting using structural data; Docking or binding prediction
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
  • G16B35/20—Screening of libraries

Definitions

• Mimetic molecules are polymers that interact with a binding partner in a way that recapitulates the network of interactions used by another ligand. By mimicking the structural and/or energetic interactions at a ligand/receptor interface, the functional properties of the ligand can be recapitulated.
• an antibody mimetic is a molecule that mimics the antigen binding activity of a specific antibody but is not identical (and in some embodiments, not related, e.g., exhibits no significant sequence similarity) in peptide sequence to that specific antibody or its paratope.
• mimetic proteins There are numerous applications of mimetic proteins ranging from utility as drug design and discovery tools to therapeutics and vaccines. To date, there has been no reliable and scalable method for identifying mimetic proteins.
• the present disclosure relates to the process of discovery of multiple classes of mimetic molecules that can be used as therapeutics, therapeutic precursors, and/or novel reagents/tool compounds, e.g., useful for screening interaction partners.
• the present disclosure provides methods of identifying a functional mimic molecule having a target functional profile that comprises one or more functions of a model by (a) providing one or more variant libraries of one or more functional mold molecules of the model, 1 DB1/ 146222728.1 Attorney Docket No.: MTPH-001PC/123828-02-5123 wherein the variant library comprises a plurality of variants of the one or more functional mold molecules, (b) providing one or more candidate functional mimic molecules, (c) evaluating the interaction of the plurality of variant functional mold molecules with (i) the model and (ii) the one or more candidate functional mimic molecules, and comparing the evaluations of i) and ii), and (d) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in c).
• the present disclosure provides methods of identifying a functional mimic molecule having a target functional profile that comprises one or more functions of a model by (a) providing one or more functional mold molecules of the model, (b) providing one or more variant libraries of one or more candidate functional mimic molecules, the variant libraries comprising a plurality of variants of the one or more candidate functional mimic molecules, (c) evaluating the interaction of the one or more functional mold molecules with: i) the model; and ii) the plurality of variants of the one or more candidate functional mimic molecules, and comparing the evaluations of i) and ii), and (d) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in c).
• the present disclosure provides methods of identifying a functional mimic molecule having a target functional profile that comprises one or more functions of a model by (a) providing one or more variant libraries of one or more functional mold molecules of the model, the variant libraries comprising a plurality of variants of the one or more functional mold molecules, (b) providing one or more variant libraries of one or more candidate functional mimic molecules, the variant libraries comprising a plurality of variants of the one or more candidate functional mimic molecules, (c) evaluating and comparing the interactions of the plurality of variant functional mold molecules with (i) the model and (ii) the plurality of variants of the one or more candidate functional mimic molecules, and (d) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in c).
• the present disclosure further provides, in aspects, methods of identifying functional mimic molecules having target functional profiles that comprise one or more functions of a model by (a) evaluating and comparing the interaction of a plurality of variant functional mold molecules with (i) the model and (ii) one or more candidate functional mimic molecules, or a plurality of variant candidate functional mimic molecules thereof, and (b) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in (a).
• DB1/ 146222728.1 2 Attorney Docket No.: MTPH-001PC/123828-02-5123
• the identified mimic molecule exhibits a non-zero similarity to the functional profile of the model.
• the candidate functional mimic molecule is an immunoglobulin antibody, an antibody-like molecule, or an antigen-binding fragment thereof.
• the antibody or antibody-like molecule is selected from a bi-specific antibody, a tri-specific antibody, and an antibody-drug conjugate.
• the antigen-binding fragment is selected from a single-domain antibody (sdAb), a variable domain of heavy-chain antibodies (VHH), a single- chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a Fv, a Fab, a Fab′, a F(ab′) 2 , and a microprotein (cysteine knot protein, knottin).
• the functional mimic molecule is a functional mimic polypeptide that is an anti-idiotypic antibody, or fragment thereof, that binds the antigen-binding domain of the functional mold molecule or polypeptide, optionally the paratope.
• the candidate functional mimic molecule is a small molecule (e.g., a molecular glue).
• the candidate functional mimic molecule is a small molecule (e.g., molecular glue) conjugated (e.g., covalently or non-covalently) to another domain (e.g., cereblon, E3-ligase, or immunomodulatory drugs).
• the molecular glue is conjugated to another domain via click chemistry (e.g., a reaction selected from one or more of conjugate addition, strained ring opening, acylation/sulfonylation, aldehyde capture by ⁇ - effect nucleophiles, cycloaddition, and nucleophilic addition to activated alkynes).
• the candidate functional mimic molecule is a polymer, optionally selected from a polypeptide and a nucleic acid.
• the polymer is a nucleic acid that comprises DNA, RNA, or a combination thereof.
• the nucleic acid is selected from a DNA aptamer, a DNA optimer, a RNA aptamer, and a RNA optimer, any of which optionally comprise one or more non-canonical and/or modified nucleotides, optionally wherein the aptamer and/or optimer comprises an altered backbone.
• the nucleic acid is a ribozyme and/or riboswitch.
• the polymer is a polypeptide
• the polypeptide optionally comprises one or more non-canonical amino acids, such as L-isomers of canonical or non-canonical amino acids (L-amino acids) and/or D-isomers of canonical or non-canonical amino acids (D-amino acids), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-canonical amino acids, such as about: 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% non-canonical amino acids.
• L-isomers of canonical or non-canonical amino acids L-amino acids
• D-amino acids D-isomers of canonical or non-canonical amino acids
• DB1/ 146222728.1 3 Attorney Docket No.: MTPH-001PC/123828-02-5123
• the polypeptide is a binding agent.
• the binding agent is an immunoglobulin antibody, an antibody-like molecule, or an antigen-binding fragment thereof.
• the antibody or antibody-like molecule is selected from a bi-specific antibody, a tri-specific antibody, and an antibody-drug conjugate.
• the antigen-binding fragment is selected from a single-domain antibody (sdAb), a variable domain of heavy-chain antibodies (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a Fv, a Fab, a Fab′, a F(ab′)2, and a microprotein (cysteine knot protein, knottin).
• the binding agent is a non-immunoglobulin-based binding domain, optionally selected from an affimer, an affilin, an affitin, an affibody, an alphabody, an anticalin, an avimer, a DARPin, a fynomer, a gastrobody, a Kunitz domain, a monobody, a nanoCLAMP, a repebody, a pronectin, a centyrin, and an obody.
• the non-immunoglobulin-based binding domain is an enzyme.
• the non-immunoglobulin-based binding domain is a signaling protein, optionally wherein the signaling protein is selected from a cytokine, growth factor, or hormone.
• the non-immunoglobulin-based binding domain is a structural protein such as a receptor.
• the non-immunoglobulin-based binding domain is a ligand.
• the non-immunoglobulin-based binding domain is an extracellular domain.
• the non-immunoglobulin-based binding domain is a bacterial nucleic acid binding protein, such as a bacteriophage MS2 protein or a bacteriophage lambda N protein.
• the binding agent is a bifunctional molecule, such as a fusion protein or non- fusion protein conjugate, optionally wherein the binding agent is conjugated to a carbohydrate (e.g., polysaccharide), a lipid, or a small molecule.
• the binding agent is glycosylated, lapidated, or otherwise conjugated.
• the target functional profile comprises one or more functions of the mimic molecule or complex, relative to the model, optionally selected from one or more of the affinity, avidity, activity, specificity, and/or selectivity for a receptor or ligand; the molecular structure or surface features; and biological activity.
• the model is a protein, a lipid, a carbohydrate, a nucleic acid, or an inorganic molecule, or a combination thereof.
• the model comprises an antigen, optionally selected from a viral protein, a bacterial protein, a protozoan protein, an DB1/ 146222728.1 4 Attorney Docket No.: MTPH-001PC/123828-02-5123 Archean protein, a fungal protein, a mammalian protein, or an inorganic protein, a small molecule, a carbohydrate, a nucleic acid, a lipid, or a plastic.
• the model is a therapeutic molecule, such as a therapeutic antibody or antigen-binding fragment thereof, optionally wherein the antigen-binding fragment of the antibody is the paratope of the antibody.
• the variant library comprises variants across the entire mold molecule or functional mimic molecule or a subset (e.g., a contiguous or a non-contiguous domain or set) of residue positions or moieties of the mold molecule or functional mimic molecule.
• variant libraries of the present disclosure are systematically engineered, optionally by an alanine scan, a shotgun scan, or binomial mutagenesis.
• variant libraries of the present disclosure are randomly engineered or semi- systematically engineered by random mutation or random mutation of specific set of residue positions.
• the variant library is selected from a fixed amino acid substitution library, a subset of possible substitutions library, or a full-substitution library.
• the full substitution library comprises deletions and insertions.
• the variant library is a deep mutational scan (DMS) library.
• providing of one or more variant libraries of one or more functional mold molecules or functional mimic molecules further comprises generation of the one or more functional mold molecules or functional mimic molecules by a method selected from interactions reported in the scientific literature, phage display, immunization (e.g., of a mammal, such as a mouse, rat, rabbit, non-human primate, or human) with the model (optionally by isolating model-binding B-cells and performing BCR sequencing), obtaining model-binding B cells from a subject previously exposed (or suspected of being exposed) to the model, competitive-binding assays, depletion studies, and structure-based modeling (including de novo in silico molecule design).
• immunization e.g., of a mammal, such as a mouse, rat, rabbit, non-human primate, or human
• immunization e.g., of a mammal, such as a mouse, rat, rabbit, non-human primate, or human
• immunization e.
• the present disclosure provides functional mold molecules that exhibit a non- zero functional complementarity to a model or functional mimic molecule.
• the functional mold molecule is a small molecule (e.g., a molecular glue).
• the functional mold molecule is a small molecule conjugated to another domain (e.g., cereblon, E3-ligase, imide drugs (IMiDs), e.g., thalidomide, pomalidomide, or DB1/ 146222728.1 5 Attorney Docket No.: MTPH-001PC/123828-02-5123 lenalidomide).
• the molecular glue is conjugated to another domain via click chemistry (e.g., a reaction selected from one or more of conjugate addition, strained ring opening, acylation/sulfonylation, aldehyde capture by ⁇ -effect nucleophiles, cycloaddition, and nucleophilic addition to activated alkynes).
• the functional mold molecule is a molecular glue.
• a molecular glue is a type of small molecule stabilizer of protein-protein interactions that encourages two proteins to come together that normally wouldn’t interact by changing the surface of their target proteins.
• molecular glue-induced protein proximity can either be stabilized/activated or destabilized/inactivated, which is dependent on the property of the small molecules and the involved proteins.
• a molecular glue comprises a molecule selected from one or more of plant hormones, auxin, jasmonate, immunomodulatory imide drugs (IMiDs) (e.g., CC-122, CC-220, CC-885, thalidomide, pomalidomide, and lenalidomide).
• IiDs immunomodulatory imide drugs
• a molecular glue comprises cereblon and/or E3-ligase.
• the functional mold molecule is a Proteolysis Targeting Chimera (PROTAC), which is a heterobifunctional molecule that forms a ternary complex with the target protein and E3-ligase by making two distinct small molecule– protein interactions.
• the functional mold molecule is a polymer, such as a nucleic acid or a polypeptide, or a combination thereof.
• the functional mold molecule is a nucleic acid that is selected from a DNA aptamer, a DNA optimer, a RNA aptamer, or a RNA optimer.
• the functional mold molecule is a functional mold polypeptide comprising functional complementarity.
• the functional mold polypeptide binds the model.
• the present disclosure provides for evaluating and comparing interactions based on binding affinity.
• FIGURE 1 depicts candidate mimic binding to a commercial antibody Fab in a yeast surface display.
• the commercial antibody Fab was expressed on the surface of yeast and stained with a panel of anti-idiotypic antibodies discovered from a na ⁇ ve human scFv library.
• FIGURES 2A-D show commercial antibody WT and DMS library binding to the antigen.
• Figure 2A depicts the presence of human kappa light chain, confirming the surface expression of wildtype commercial antibody (solid) and commercial antibody libraries (dashed and dotted) on yeast.
• Figure 2B shows that after gating for Fab expression, the libraries (dashed and dotted lines) exhibited a greater phenotypic range of binding to antigen relative to WT (solid).
• Figures 2C-D show that both libraries with mutations of either heavy chain or mutations in the light chain exhibit antigen binding correlated with expression, except for those variants that appear to lose binding altogether.
• FIGURES 3A-B show the validation of the commercial antibody DMS assay.
• Figure 3A shows a scatterplot of variants from two replicate selections against antigen. Replicates are strongly correlated.
• Figure 3B shows the mapping of sensitive sites, as identified by DMS to the paratope of the commercial antibody for antigen. The paratope is strongly enriched for sensitive sites.
• FIGURES 4A-C show the result of scoring and clustering the anti-idiotypic antibodies for antigen mimicry.
• Figure 4A shows the validation of the anti-idiotypic antibody DMS assays through a cumulative histogram of inter-replicate correlations.
• Figure 4B shows the correlations of per variant binding scores from DMS between anti-idiotypic antibodies and antigen. The most highly correlated anti-idiotypic antibodies display similar correlations to a replicate antigen selection.
• Figure 4C shows the result of clustering anti-idiotypic antibodies by DMS binding profiles.
• FIGURES 5A-D are summaries showing the binding of individual DMS library variants to (A) antigen and (B-C) three anti-idiotypic antibodies.
• Figure 5B shows an anti-idiotypic antibody that binds using a different combination of mold CDRs to antigen.
• Figure 5C shows DB1/ 146222728.1 7 Attorney Docket No.: MTPH-001PC/123828-02-5123 an anti-idiotypic antibody that binds using a similar set of CDRs but different residues.
• FIG. 5D shows an anti-idiotypic antibody that mimics antigen, as evinced through a similar set of sensitive sites indicating binding through the same set of residues.
• the Y axis denotes particular substitutions, and the X axis denotes the position along the peptide.
• FIGURES 6A-C depict cryogenic electron microscopy structures of anti-idiotypic antibodies in complex with a commercial antibody.
• Figure 6A shows an anti-drug antibody.
• Figure 6B shows a discovered mimic anti-idiotypic antibody bound to a similar region.
• Overlapping structures, Figure 6C shows a highly similar epitope footprint on the commercial antibody despite minimal sequence similarity between the anti-idiotypic antibodies.
• FIGURE 7 shows the results of competitive inhibition ELISA measurements of the mold antibody in Example 2 in complex with a known mimic antibody. Increasing titrations of the model protein, unlabeled known mimic antibody and the discovered antibody show that all three are able to competitively displace the known mimic.
• FIGURES 8A-C are summaries showing the binding of individual DMS variants to (A) a model protein, (B) a known mimic antibody, and (C) a discovered mimic antibody.
• Figure 8B shows the known mimic antibody has similar sensitive sites to mutation as the model protein, with a notable exception of less sensitivity to mutations in the CDR1 region.
• Figure 8C shows the discovered antibody binds similar to the known mimic antibody, but displays mutational sensitivity to binding more similar to the model protein in the CDR1 region.
• FIGURES 9A-D depict cryogenic electron microscopy structures of the broadly neutralizing antibody in complex with (A) the peptide epitope of the model protein, (B) the known discovered mimic antibody, and (C) the known mimic antibody.
• Figure 9A shows the epitope forms a single loop structure that binds to a paratope made up of heavy chain CDR1, heavy chain CDR3, and light chain CDR3 of the broadly neutralizing antibody.
• Figure 9B shows the CDR3 of the discovered antibody forms a similar loop structure to the epitope and binds the same paratope of the broadly neutralizing antibody mold.
• Figure 9C shows the complex of the known mimic antibody bound to the broadly neutralizing antibody mold. While the paratope is similar, the epitope loop of known mimic adopts a dissimilar conformation.
• Figure 9D displays the overlayed structures.
• FIGURES 10A-D shows the competitive binding between the discovered antibodies in Example 3 and the model protein.
• Figure 10A shows a reduction in binding of the model DB1/ 146222728.1 8 Attorney Docket No.: MTPH-001PC/123828-02-5123 protein to an immobilized form of the extracellular domain of the receptor mold upon increasing concentrations of a discovered mimic antibody.
• Figure 10B shows similar results for a second discovered mimic antibody.
• FIG 10C shows the inhibition of mimic antibody in panel A binding to the receptor mold presented on HEK-293 cells by co-expression of a surface bound form of the model protein.
• Figure 10D shows similar cell-surface inhibition results for the mimic antibody characterized in Figure 10B.
• FIGURES 11A-D are summaries showing binding of individual DMS variants of the receptor mimic presented on yeast surface display to (A) a model protein, (B, C) two discovered mimic antibodies, and (D) a known non-mimic antibody.
• Figures 11B and 11C show the two discovered mimic antibodies bind to the mold with similar mutational effect patterns and similar site-specific effects as the model.
• Figure 11D shows the known non-mimic antibody binds with a dissimilar pattern in residues 4-30.
• FIGURES 12A-C are line plots showing differential position averaged DMS scores relative to the known non-mimic antibody.
• Figure 12A shows the results for the model protein.
• the model protein contact point residues for the receptor mold were calculated from a published crystal structure of the complex and are highlighted in the grey regions.
• the model protein DMS scores are increased in the known contact residues relative to the non-mimic antibody.
• Figure 12B displays the results for one discovered mimic antibody, showing a similar increase in scores in the known contact regions for the model protein.
• Figure 12C displays the results for a second discovered mimic antibody, which likewise showed increase DMS scores in the known contact regions of the model protein.
• FIGURE 13 depicts scatterplots that show correlations between individual DMS mutation scores between the model protein, the two discovered mimic antibodies and the known non- mimic antibody. Pearson’s correlation coefficients for the model protein to the two discovered mimic antibodies was 0.81 and 0.84, respectively. The two sequence-divergent mimic antibodies show a Pearson’s correlation coefficient of 0.88. The known non-mimic antibody had correlation coefficients between 0.67 – 0.68 with the model protein and discovered mimics, showing it has less similar site-specific mutational effects.
• the present disclosure is based, in part, on the surprising discovery of a process that facilitates the discovery and identification of multiple classes of mimetic molecules (e.g., mimetic DB1/ 146222728.1 9 Attorney Docket No.: MTPH-001PC/123828-02-5123 immunoglobulin molecules), which are useful as therapeutics, therapeutic precursors, and/or novel reagents/tool compounds.
• mimetic DB1/ 146222728.1 9 Attorney Docket No.: MTPH-001PC/123828-02-5123 immunoglobulin molecules
• the present disclosure provides, in part, a process for identifying mimetic molecules (“mimics”) that is analogous, without wishing to be bound by theory, to the manufacturing process of casting a replica.
• the present disclosure describes a method of using an anti-antibody as a mold.
• the process of the present disclosure provides distinct advantages in overcoming limitations of the original context for a binding domain by inserting a pharmacophore of an endogenous molecule onto a functional skeleton of a molecule, such as the antigen-binding portion of an immunoglobulin or immunoglobulin-type molecule, such as the paratope of another molecule to copy the endogenous molecule’s function with the advantages of the other molecule (e.g., for an antibody, long half-life and straightforward engineering and manufacturing).
• the present disclosure provides methods of identifying a functional mimic molecule having a target functional profile that comprises one or more functions of a model by (a) providing one or more variant libraries of one or more functional mold molecules of the model, wherein the variant library comprises a plurality of variants of the one or more functional mold molecules, (b) providing one or more candidate functional mimic molecules, (c) evaluating the interaction of the plurality of variant functional mold molecules with (i) the model and (ii) the one or more candidate functional mimic molecules, and comparing the evaluations of i) and ii), and (d) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in c).
• the present disclosure provides methods of identifying a functional mimic molecule having a target functional profile that comprises one or more functions of a model by (a) providing one or more functional mold molecules of the model, (b) providing one or more variant libraries of one or more candidate functional mimic molecules, the variant libraries comprising a plurality of variants of the one or more candidate functional mimic molecules, (c) DB1/ 146222728.1 10 Attorney Docket No.: MTPH-001PC/123828-02-5123 evaluating the interaction of the one or more functional mold molecules with: i) the model; and ii) the plurality of variants of the one or more candidate functional mimic molecules, and comparing the evaluations of i) and ii), and (d) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in c).
• the present disclosure provides methods of identifying a functional mimic molecule having a target functional profile that comprises one or more functions of a model by (a) providing one or more variant libraries of one or more functional mold molecules of the model, the variant libraries comprising a plurality of variants of the one or more functional mold molecules, (b) providing one or more variant libraries of one or more candidate functional mimic molecules, the variant libraries comprising a plurality of variants of the one or more candidate functional mimic molecules, (c) evaluating and comparing the interactions of the plurality of variant functional mold molecules with (i) the model and (ii) the plurality of variants of the one or more candidate functional mimic molecules, and (d) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in c).
• the present disclosure further provides, in aspects, methods of identifying functional mimic molecules having target functional profiles that comprise one or more functions of a model by (a) evaluating and comparing the interaction of a plurality of variant functional mold molecules with (i) the model and (ii) one or more candidate functional mimic molecules, or a plurality of variant candidate functional mimic molecules thereof, and (b) identifying one or more functional mimic molecules having the target functional profile on the basis of the evaluation in (a).
• Mimetic (Mimic) Molecules is or comprises an immunoglobulin antibody, an antibody-like molecule, an antibody format, or an antigen-binding fragment thereof.
• the antibody or antibody-like molecule is selected from a bi-specific antibody, a tri-specific antibody, and an antibody-drug conjugate.
• the immunoglobulin antibody or antibody-like molecule is monoclonal or polyclonal.
• the present disclosure provides mimetic (mimic) immunoglobulin molecules that interact with a binding partner in a way that recapitulates the network of interactions used by another ligand.
• the mimic immunoglobulin molecule can therefore recapitulate the DB1/ 146222728.1 11 Attorney Docket No.: MTPH-001PC/123828-02-5123 functional properties of the model ligand by mimicking the structural and/or energetic interactions at a ligand/receptor interface.
• the present disclosure provides functional mimic immunoglobulin molecules that exhibit a non-zero similarity to the functional profile of a model.
• the functional mimic immunoglobulin molecule exhibits a target functional profile having a non-zero similarity to the functional profile of a model, the functional profile comprising one or more of: a structure and a binding profile (e.g., activity and/or affinity, avidity, specificity, and/or selectivity).
• the functional mimic immunoglobulin molecule is or comprises an antibody, an antibody format, or an antigen-binding fragment.
• the antibody is selected from a bi-specific antibody, a tri-specific antibody, and an antibody-drug conjugate.
• the antibody is monoclonal or polyclonal.
• the present disclosure provides that the functional mimic immunoglobulin molecule is an anti-idiotypic antibody, or fragment thereof, that binds the antigen-binding domain of the functional mold immunoglobulin molecule.
• the anti-idiotypic antibody, or fragment thereof binds the paratope of the functional mold immunoglobulin molecule.
• an anti-idiotypic antibody binds to the idiotype of another antibody (e.g., an antibody drug).
• an idiotype is a specific combination of idiotopes present within an antibody’s complement determining regions (CDRs).
• a single idiotope is a specific region within an antibody’s Fv region which binds to the paratope (antigenic epitope binding site) of a different antibody.
• an idiotope is synonymous with an antigenic determinant of an antibody.
• the present disclosure provides that the functional mimic polypeptide is an anti-idiotypic antibody, or fragment thereof, that binds the antigen-binding domain of the functional mold polypeptide.
• the anti-idiotypic antibody, or fragment thereof binds the paratope of the functional mold polypeptide.
• an anti-idiotypic antibody binds to the idiotype of another antibody (e.g., an antibody drug).
• an idiotype is a specific combination of idiotopes present within an antibody’s complement determining regions (CDRs).
• CDRs complement determining regions
• a single idiotope is a specific region within an antibody’s Fv region which binds to the paratope (antigenic epitope binding site) of a different antibody.
• an idiotope is synonymous with an antigenic determinant of an antibody.
• DB1/ 146222728.1 12 Attorney Docket No.: MTPH-001PC/123828-02-5123
• an antibody refers to a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, and antibody fragments.
• antibodies are proteins or polypeptides that exhibit binding specificity to a specific antigen.
• Intact antibodies are heterotetrametric glycoproteins, composed of two identical light chains and two identical heavy chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
• VH variable domain
• Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain.
• Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains.
• Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
• an antibody fragment comprises an antigen binding or variable region of an intact antibody.
• the present disclosure contemplates antibody fragments selected from a single-domain antibody (sdAb), a variable domain of heavy-chain antibodies (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a Fv, a Fab, a Fab′, a F(ab′)2, and a microprotein (cysteine knot protein, knottin).
• an antigen comprises any molecule that has the ability to generate antibodies either directly or indirectly.
• a CDR is a complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs or CDR regions in the variable portion of an immunoglobulin. In embodiments, a CDR refers to all three heavy chain CDRs, or all three light chain CDRs or both all heavy and all light chain CDRs, if appropriate.
• Each variable region comprises three hypervariable regions also known as complementarity determining regions (CDRs) flanked by four relatively conserved framework regions (FRs).
• CDRs complementarity determining regions
• FRs relatively conserved framework regions
• the three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity, as the CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
• CDRs of interest can be derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived.
• the antibody is a chimeric antibody. In embodiments, the antibody is a humanized antibody.
• CDRs are based on sequence variability (Wu and Kabat, J. Exp. Med. 132:211-250, 1970). There are six CDRs--three in the variable heavy chain, or VH, and are typically designated H-CDR1, H-CDR2, and H-CDR3, and three CDRs in the variable light chain, or VL, and are typically designated L-CDR1, L-CDR2, and L-CDR3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
• a hypervariable region (“HVR” or “HV”) is a region of an antibody variable domain that is variable in structure as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-917, 1987). There are six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Chothia and Lesk refer to structurally conserved HVs as canonical structures.
• Another method of describing the regions that form the antigen-binding site has been proposed by Lefranc (Lefranc et al., Developmental & Comparative Immunology 27:55-77, 2003) based on the comparison of V domains from immunoglobulins and T-cell receptors (Lefranc et al., Developmental & Comparative Immunology 27:55-77, 2003).
• the antigen-binding site can also be delineated based on Specificity Determining Residue Usage (SDRU), according to Almagro (Almagro, Mol. Recognit.17:132-43, 2004), where SDRU refers to amino acid residues of an immunoglobulin that are directly involved in antigen contact.
• SDRU Specificity Determining Residue Usage
• the present disclosure contemplates that the functional mimic molecules can be any suitable class of molecule.
• the present disclosure provides mimetic (mimic) molecules that interact with a binding partner in a way that recapitulates the network of interactions used by another ligand.
• the mimic molecule can therefore recapitulate the functional properties of the model ligand by mimicking the structural and/or energetic interactions at a ligand/receptor interface.
• DB1/ 146222728.1 14 Attorney Docket No.: MTPH-001PC/123828-02-5123
• the present disclosure provides functional mimic molecules that exhibit a non-zero similarity to the functional profile of a model.
• the functional mimic molecule exhibits a target functional profile having a non-zero similarity to the functional profile of a model, the functional profile comprising one or more of: a structure, a binding profile (e.g., activity and/or affinity, specificity, and/or selectivity), a signaling profile (activity, specificity, and/or selectivity), an enzymatic profile (activity and/or processivity, specificity, and/or selectivity).
• the candidate functional mimic molecule is a small molecule (e.g., a molecular glue).
• the candidate functional mimic molecule is a small molecule conjugated to another domain (e.g., cereblon, E3-ligase, or immunomodulatory drugs).
• the molecular glue is conjugated to another domain via click chemistry (e.g., a reaction selected from one or more of conjugate addition, strained ring opening, acylation/sulfonylation, aldehyde capture by ⁇ -effect nucleophiles, cycloaddition, and nucleophilic addition to activated alkynes).
• the candidate functional mimic molecule is a molecular glue.
• a molecular glue is a type of small molecule stabilizer of protein-protein interactions that encourages two proteins to come together that normally wouldn’t interact by changing the surface of their target proteins.
• molecular glue-induced protein proximity can either be stabilized/activated or destabilized/inactivated, which is dependent on the property of the small molecules and the involved proteins.
• a molecular glue comprises a molecule selected from one or more of plant hormones, auxin, jasmonate, immunomodulatory imide drugs (IMiDs), e.g., CC-122, CC-220, CC-885, thalidomide, pomalidomide, and lenalidomide.
• IiDs immunomodulatory imide drugs
• a molecular glue comprises cereblon and/or E3-ligase.
• the functional mold molecule is a Proteolysis Targeting Chimera (PROTAC), which is a heterobifunctional molecule that forms a ternary complex with the target protein and E3-ligase by making two distinct small molecule–protein interactions.
• the mimic molecule is or comprises a polymer, such as a polypeptide or a nucleic acid, or a combination thereof.
• the mimic molecule is or comprises a nucleic acid that is a polynucleotide.
• the nucleic acid is or comprises DNA, RNA, or a combination thereof.
• the mimic molecule is a nucleic acid that is a DNA aptamer, a DNA optimer, a RNA aptamer, or a RNA optimer.
• DNA or RNA aptamers comprise short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and live cells. Aptamers can assume a variety of shapes due to their tendency to form helices and single-stranded loops and exhibit targeted binding with high selectivity and/or specificity.
• Aptamers recognize and bind targets as determined by the aptamer’s tertiary structure, wherein the aptamer recognizes and binds the target via three-dimensional, shape-dependent interactions, as well as hydrophobic interactions, base-stacking, and intercalation.
• Aptamers comprising about 15 to about 120 nucleotides can be selected in vitro from a randomized pool of oligonucleotides (about 10 14 - about 10 15 molecules).
• aptamers or aptamer sequences comprise a degenerate sequence, and can further comprise fixed sequences flanking the degenerate sequence.
• an aptamer further comprises both native and modified DNA and RNA bases, e.g.
• nucleic acids of the present disclosure comprise non-canonical and/or modified nucleotides.
• the nucleic acid comprises a chemical modification.
• the chemical modification is a nucleobase modification, a backbone modification, and/or a sugar modification.
• nucleobase modification replaces A, C, T, G, or U.
• the nucleobase modification is selected from pseudouridine, N1-methyl-pseudouridine, 5- methylcytidine (m5C), 2'-thiouridine (s2U), N6'-methyladenosine (m6A), and 5'-fluoro-2'- deoxyuridine.
• the backbone modification is selected from phosphorothioate, phosphorodithioate, methylphosphonate, and methoxypropylphosphonate.
• the sugar modification is selected from 2'-methoxy (2'-OMe), 2'-O- methoxyethyl (2'-O-MOE), 2'-fluoro (2'-F), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, constrained ethyl (cEt), peptide nucleic acid (PNA), glycol nucleic acid (GNA), unlocked nucleic acid (UNA)and locked nucleic acid (LNA).
• nucleic acid molecules of the present disclosure may include one or more modified nucleotides. Exemplary modified nucleotides are described in US Patent No. 8,278,036, which is hereby incorporated by reference in its entirety.
• the modified nucleotides may be selected from one or more of pseudouridine, N1- DB1/ 146222728.1 16 Attorney Docket No.: MTPH-001PC/123828-02-5123 methylpseudouridine, 5-methylcytidine, or N6-methyladenosine, 5-hydroxycytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-hydroxyuridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, pseudouridine, 2-thiouridine, 4- thiouridine, 5-azauridine, 5- aminouridine, 5-methyluridine, 2-thiopseudouridine, 4- thiopseudouridine, 5- hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine,
• nucleic acids are chemically modified.
• the chemical modification is selected from pseudouridine, N1-methyl-pseudouridine, 5-methylcytidine (m5C), 2'-thiouridine (s2U), N6'-methyladenosine (m6A), and 5'-fluoro-2'-deoxyuridine, phosphorothioate, phosphorodithioate, methylphosphonate, methoxypropylphosphonate, 2'- methoxy (2'-OMe), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'-F), 2'-arabino-fluoro (2'-Ara- F), 2'-O-benzyl, constrained ethyl (cEt), peptide nucleic acid (PNA), glycol nucleic acid (GNA), unlocked nucleic acid (UNA)and locked nucleic acid (LNA).
• pseudouridine N1-methyl-ps
• DNA or RNA aptamers or optimers of the present disclosure comprise non- canonical base pairing, which occurs when nucleobases hydrogen bond, or base pair, to one another in schemes other than the standard Watson-Crick base pairs (e.g., adenine (A)— thymine (T) in DNA; adenine (A)—uracil (U) in RNA; and guanine (G)—cytosine (C) in both DNA and RNA).
• non-canonical DNA structures can include, but are not limited to, A-DNA, B-DNA, Z-DNA, hairpin/cruciform, triplex, G-quadruplex, and/or i-motif.
• the contemplated mimic molecule is or comprises a peptide nucleic acid that includes an altered deoxyribose phosphate backbone.
• PNAs Peptide nucleic acids
• PNAs are synthetic mimics of DNA in which the deoxyribose phosphate backbone is replaced by repetitive units of N-(2-aminoethyl) glycine to which the purine and pyrimidine bases are attached via a methyl carbonyl linker.
• Peptide nucleic acids can hybridize with complementary DNAs or RNAs with remarkably high affinity and specificity, due to their uncharged and flexible polyamide backbone.
• Peptide nucleic acids hybridize to complementary DNA or RNA in a sequence-dependent manner, according to the Watson–Crick hydrogen bonding scheme.
• a peptide nucleic acid can bind in either parallel or antiparallel manner.
• Peptide nucleic acids are able to adopt both A-type and B-type structures when associating DB1/ 146222728.1 17 Attorney Docket No.: MTPH-001PC/123828-02-5123 with RNA and DNA, respectively, whereas PNA–PNA duplexes form an unusual helix conformation, called P-type and are characterized by a large pitch of 18 base pairs.
• Peptide nucleic acids, and their unique properties as DNA mimics can be used in drug design (e.g., gene therapy drug design), as well as in prognostics, diagnostics, and disease monitoring.
• the nucleic acid is or comprises a ribozyme and/or riboswitch.
• a riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule.
• the present disclosure contemplates that the mimic molecule is or comprises a polypeptide—that is, the mimic is a functional mimic polypeptide.
• the functional mimic polypeptide comprises one or more non-canonical or unnatural amino acids.
• a non-canonical or unnatural amino acid is a non-proteinogenic amino acid that is either found naturally in organisms or is synthetically made in a laboratory.
• a non-canonical or unnatural amino acid is an amino acid that is not located in the genetic code of naturally occurring organisms.
• a non-canonical or unnatural amino acid is selected from selenocysteine, pyrrolysine, N-formylmethionine ⁇ -alanine, GABA and ⁇ - Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t
• the functional mimic polypeptide comprises one or more L-amino acids (L- isomer of a canonical or non-canonical amino acid) and/or D-amino acids (D-isomer of a canonical or non-canonical amino acid).
• the functional mimic polypeptide comprises non-canonical or unnatural amino acid comprising a L-amino acid or a D-amino acid.
• the present disclosure contemplates a functional mimic polypeptide that is a binding agent.
• the binding agent is or comprises an immunoglobulin antibody, an antibody-like molecule, or an antigen-binding fragment thereof.
• the functional mimic polypeptide is a binding agent that comprises a non- immunoglobulin-based binding domain.
• the non-immunoglobulin-based DB1/ 146222728.1 18 Attorney Docket No.: MTPH-001PC/123828-02-5123 binding domain is selected from an affimer, an affilin, an affitin, an affibody, an alphabody, an anticalin, an avimer, a DARPin, a fynomer, a gastrobody, a Kunitz domain, a monobody, a nanoCLAMP, a repebody, a pronectin, a centyrin, and an obody.
• the functional mimic polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises an enzyme, or a fragment thereof. In embodiments, the functional mimic polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises a signaling protein, or a fragment thereof. In embodiments, the signaling protein is selected from a cytokine, growth factor, or hormone. In embodiments, the functional mimic polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises a structural protein such as a receptor, or a fragment thereof. In embodiments, the functional mimic polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises a ligand.
• the functional mimic polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises an extracellular domain. In embodiments, the functional mimic polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises an extracellular domain. In embodiments, the functional mimic polypeptide is or comprises a bacterial nucleic acid binding protein, such as a bacteriophage MS2 protein, which binds to a small RNA hairpin in its genomic RNA, or a bacteriophage lambda N protein, which binds to a boxB hairpin loop. In embodiments, the binding agent is bifunctional. For example, in embodiments, the binding agent comprises a fusion protein or a non-fusion protein conjugate.
• the binding agent is conjugated to a carbohydrate (e.g., polysaccharide), a lipid, or a small molecule.
• the binding agent is glycosylated, lapidated, or otherwise conjugated.
• the present disclosure provides methods for identifying one or more functional mimic molecules (e.g., functional mimic polypeptides).
• functional mimic molecules e.g., functional mimic polypeptides
• ADA anti-drug antibody
• phage display e.g., a mammal, such as a mouse, rat, rabbit, non- human primate (NHP), or human
• immunization e.g., of a mammal, such as a mouse, rat, rabbit, non- human primate (NHP), or human
• model-binding B cells from a DB1/ 146222728.1 19
• MTPH-001PC/123828-02-5123 subject previously exposed, or suspected of being exposed, to the model
• competitive binding assays depletion studies
• structure-based modeling e.g., de novo in silico molecule design
• immunization of a mammal with the model further comprises isolating model-binding B-cells and performing B cell receptor (BCR) sequencing.
• BCR sequencing employ high-throughput single-cell sequencing to obtain the VH and VL sequences for antibodies from individual human, rat, and mouse B cells.
• Model Molecule or Complex The present disclosure provides, in embodiments, for a model molecule or complex that comprises a surface to be mimicked.
• a model is a reference molecule or complex to be functionally mimicked by a functional mimic molecule.
• a model is non-limiting and can be any molecule or complex.
• a model may be a polypeptide or a non-polypeptide (e.g., a small molecule or nucleic acid).
• a model can be a protein, a peptide, a lipid, a carbohydrate, a nucleic acid, or an inorganic molecule.
• the model comprises an antigen, which can be selected from a viral protein, a bacterial protein, a protozoan protein, an Archean protein, a fungal protein, a mammalian protein, or an inorganic protein, a small molecule, a carbohydrate, a nucleic acid, a lipid, or a plastic.
• the model is a therapeutic molecule.
• the model can be a therapeutic antibody or antigen-binding fragment thereof.
• the antigen- binding fragment of the antibody is the paratope of the antibody.
• Mold Molecule The present disclosure contemplates mold molecules that bind to the surface to be mimicked (the “model”), and which are bound by candidate mimic molecules.
• a functional mold molecule is a molecule that exhibits some non-zero functional complementarity to a model and/or a functional mimic polypeptide.
• functional complementarity refers to binding.
• a functional mold polypeptide is or comprises an antibody that binds the model (e.g., antigen), and the functional mimic polypeptide is or comprises an anti-idiotype antibody DB1/ 146222728.1 20 Attorney Docket No.: MTPH-001PC/123828-02-5123 (or fragment thereof) that binds the antigen-binding domain of the functional mold polypeptide, specifically the paratope.
• the functional mold molecule is a functional mold polypeptide, such as an antibody, and the functional complementarity comprises binding to the model (e.g., an antigen).
• the functional mimic polypeptide is an anti-idiotypic antibody, or fragment thereof, that binds the antigen-binding domain of the functional mold polypeptide, specifically the paratope.
• the functional mold molecule is a small molecule (e.g., a molecular glue).
• the functional mold molecule is a small molecule conjugated to another domain (e.g., cereblon, E3-ligase, or immunomodulatory drugs).
• the molecular glue is conjugated to another domain via click chemistry (e.g., a reaction selected from one or more of conjugate addition, strained ring opening, acylation/sulfonylation, aldehyde capture by ⁇ - effect nucleophiles, cycloaddition, and nucleophilic addition to activated alkynes).
• the functional mold molecule is a molecular glue.
• a molecular glue is a type of small molecule stabilizer of protein-protein interactions that encourages two proteins to come together that normally wouldn’t interact by changing the surface of their target proteins.
• molecular glue-induced protein proximity can either be stabilized/activated or destabilized/inactivated, which is dependent on the property of the small molecules and the involved proteins.
• a molecular glue comprises a molecule selected from one or more of plant hormones, auxin, jasmonate, immunomodulatory imide drugs (IMiDs), e.g., CC-122, CC-220, CC-885, thalidomide, pomalidomide, and lenalidomide.
• IiDs immunomodulatory imide drugs
• a molecular glue comprises cereblon and/or E3-ligase.
• the functional mold molecule is a Proteolysis Targeting Chimera (PROTAC), which is a heterobifunctional molecule that forms a ternary complex with the target protein and E3-ligase by making two distinct small molecule–protein interactions.
• a functional mold molecule is non-limiting and can be any class of molecule, such as a polymer.
• the polymer is a polypeptide, a nucleic acid, or a combination thereof.
• the functional mold molecule is or comprises a nucleic acid that is a DNA aptamer, a DNA optimer, a RNA aptamer, or a RNA optimer.
• DNA or RNA aptamers comprise short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small DB1/ 146222728.1 21 Attorney Docket No.: MTPH-001PC/123828-02-5123 molecules, toxins, and live cells. Aptamers can assume a variety of shapes due to their tendency to form helices and single-stranded loops and exhibit targeted binding with high selectivity and/or specificity.
• Aptamers recognize and bind targets as determined by the aptamer’s tertiary structure, wherein the aptamer recognizes and binds the target via three- dimensional, shape-dependent interactions, as well as hydrophobic interactions, base-stacking, and intercalation.
• Aptamers comprising 15 to 120 nucleotides can be selected in vitro from a randomized pool of oligonucleotides (10 14 -10 15 molecules).
• aptamers or aptamer sequences comprise a degenerate sequence, and can further comprise fixed sequences flanking the degenerate sequence.
• an aptamer further comprises both native and modified DNA and RNA bases, e.g.
• nucleic acids of the present disclosure comprise non-canonical and/or modified nucleotides.
• nucleic acid comprises a chemical modification.
• the chemical modification is a nucleobase modification, a backbone modification, and/or a sugar modification.
• nucleobase modification replaces A, C, T, G, or U.
• the nucleobase modification is selected from pseudouridine, N1-methyl-pseudouridine, 5- methylcytidine (m5C), 2'-thiouridine (s2U), N6'-methyladenosine (m6A), and 5'-fluoro-2'- deoxyuridine.
• the backbone modification is selected from phosphorothioate, phosphorodithioate, methylphosphonate, and methoxypropylphosphonate.
• the sugar modification is selected from 2'-methoxy (2'-OMe), 2'-O- methoxyethyl (2'-O-MOE), 2'-fluoro (2'-F), 2'-arabino-fluoro (2'-Ara-F), 2'-O-benzyl, constrained ethyl (cEt), peptide nucleic acid (PNA), glycol nucleic acid (GNA), unlocked nucleic acid (UNA)and locked nucleic acid (LNA).
• nucleic acid molecules of the present disclosure may include one or more modified nucleotides. Exemplary modified nucleotides are described in US Patent No. 8,278,036, which is hereby incorporated by reference in its entirety.
• the modified nucleotides may be selected from one or more of pseudouridine, N1- methylpseudouridine, 5-methylcytidine, or N6-methyladenosine, 5-hydroxycytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-hydroxyuridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, pseudouridine, 2-thiouridine, 4- DB1/ 146222728.1 22 Attorney Docket No.: MTPH-001PC/123828-02-5123 thiouridine, 5-azauridine, 5- aminouridine, 5-methyluridine, 2-thiopseudouridine, 4- thiopseudouridine, 5- hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine,
• nucleic acids are chemically modified.
• the chemical modification is selected from pseudouridine, N1-methyl-pseudouridine, 5-methylcytidine (m5C), 2'-thiouridine (s2U), N6'-methyladenosine (m6A), and 5'-fluoro-2'-deoxyuridine, phosphorothioate, phosphorodithioate, methylphosphonate, methoxypropylphosphonate, 2'- methoxy (2'-OMe), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'-F), 2'-arabino-fluoro (2'-Ara- F), 2'-O-benzyl, constrained ethyl (cEt), peptide nucleic acid (PNA), glycol nucleic acid (GNA), unlocked nucleic acid (UNA)and locked nucleic acid (LNA).
• pseudouridine N1-methyl-ps
• DNA or RNA aptamers or optimers can include non-canonical and/or modified nucleotides.
• DNA or RNA aptamers or optimers of the present disclosure comprise non-canonical base pairing, which occurs when nucleobases hydrogen bond, or base pair, to one another in schemes other than the standard Watson-Crick base pairs (e.g., adenine (A)—thymine (T) in DNA; adenine (A)—uracil (U) in RNA; and guanine (G)—cytosine (C) in both DNA and RNA).
• non-canonical DNA structures can include, but are not limited to, A-DNA, B-DNA, Z-DNA, hairpin/cruciform, triplex, G-quadruplex, and/or i-motif.
• the contemplated functional mold molecule is or comprises a peptide nucleic acid that includes an altered deoxyribose phosphate backbone.
• PNAs Peptide nucleic acids
• PNAs are synthetic mimics of DNA in which the deoxyribose phosphate backbone is replaced by repetitive units of N-(2-aminoethyl) glycine to which the purine and pyrimidine bases are attached via a methyl carbonyl linker.
• Peptide nucleic acids can hybridize with complementary DNAs or RNAs with remarkably high affinity and specificity, due to their uncharged and flexible polyamide backbone. Peptide nucleic acids hybridize to complementary DNA or RNA in a sequence-dependent manner, according to the Watson–Crick hydrogen bonding scheme. In contrast to DNA, a peptide nucleic acid can bind in either parallel or antiparallel manner. Peptide nucleic acids are able to adopt both A-type and B-type structures when associating with RNA and DNA, respectively, whereas PNA–PNA duplexes form an unusual helix conformation, called P-type and are characterized by a large pitch of 18 base pairs.
• Peptide DB1/ 146222728.1 23 Attorney Docket No.: MTPH-001PC/123828-02-5123 nucleic acids, and their unique properties as DNA mimics, can be used in drug design (e.g., gene therapy drug design), as well as in prognostics, diagnostics, and disease monitoring.
• the functional mold molecule is or comprises a polypeptide—that is, the mold molecule is a functional mold polypeptide.
• the functional mold polypeptide comprises one or more non-canonical or unnatural amino acids.
• a non-canonical or unnatural amino acid is a non- proteinogenic amino acid that is either found naturally in organisms or is synthetically made in a laboratory.
• a non-canonical or unnatural amino acid is an amino acid that is not located in the genetic code of naturally occurring organisms.
• a non-canonical or unnatural amino acid is selected from selenocysteine, pyrrolysine, N-formylmethionine ⁇ - alanine, GABA and ⁇ -Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid,
• the functional mold polypeptide comprises one or more L-amino acids (L- isomer of a canonical or non-canonical amino acid) and/or D-amino acids (D-isomer of a canonical or non-canonical amino acid).
• the functional mold polypeptide comprises non-canonical or unnatural amino acid comprising a L-amino acid or a D-amino acid.
• the present disclosure contemplates a functional mold polypeptide that is a binding agent.
• the binding agent comprises an antibody or antibody format, or a fragment (e.g., an antigen-binding fragment) thereof.
• the binding agent comprises a non-immunoglobulin-based binding domain.
• an antibody refers to a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, and antibody fragments.
• antibodies are proteins or polypeptides that exhibit binding specificity to a specific antigen.
• Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, DB1/ 146222728.1 24 Attorney Docket No.: MTPH-001PC/123828-02-5123 depending on the heavy chain constant domain amino acid sequence.
• an antibody fragment comprises an antigen binding or variable region of an intact antibody.
• the present disclosure contemplates antibody fragments selected from a single-domain antibody (sdAb), a variable domain of heavy-chain antibodies (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a Fv, a Fab, a Fab′, a F(ab′) 2 , and a microprotein (cysteine knot protein, knottin).
• an antigen comprises any molecule that has the ability to generate antibodies either directly or indirectly.
• the functional mold polypeptide is a binding agent that comprises a non- immunoglobulin-based binding domain.
• the non-immunoglobulin-based binding domain is selected from an affimer, an affilin, an affitin, an affibody, an alphabody, an anticalin, an avimer, a DARPin, a fynomer, a gastrobody, a Kunitz domain, a monobody, a nanoCLAMP, a repebody, a pronectin, a centyrin, and an obody.
• the functional mold polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises an enzyme, or a fragment thereof. In embodiments, the functional mold polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises a signaling protein, or a fragment thereof. In embodiments, the signaling protein is selected from a cytokine, growth factor, or hormone. In embodiments, the functional mold polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises a structural protein such as a receptor, or a fragment thereof. In embodiments, the functional mold polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises a ligand.
• the functional mold polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises an extracellular domain. In embodiments, the functional mold polypeptide comprises a non-immunoglobulin-based binding domain that is or comprises an extracellular domain. In embodiments, the functional mold polypeptide is or comprises a bacterial nucleic acid binding protein, such as a bacteriophage MS2 protein, which binds to a small RNA hairpin in its genomic RNA, or a bacteriophage lambda N protein, which binds to a boxB hairpin loop. DB1/ 146222728.1 25 Attorney Docket No.: MTPH-001PC/123828-02-5123 In embodiments, the binding agent is bifunctional.
• the binding agent comprises a fusion protein or a non-fusion protein conjugate.
• the binding agent is conjugated to a carbohydrate (e.g., polysaccharide), a lipid, or a small molecule.
• the binding agent is glycosylated, lapidated, or otherwise conjugated.
• the present disclosure provides methods for identifying one or more functional mold molecules (e.g., functional mold polypeptides).
• functional mold molecules e.g., functional mold polypeptides
• phage display e.g., immunization (e.g., of a mammal, such as a mouse, rat, rabbit, non-human primate (NHP), or human) with the model; obtaining model- binding B cells from a subject previously exposed, or suspected of being exposed, to the model; competitive binding assays; depletion studies; and/or structure-based modeling (e.g., de novo in silico molecule design).
• immunization e.g., of a mammal, such as a mouse, rat, rabbit, non-human primate (NHP), or human
• model- binding B cells from a subject previously exposed, or suspected of being exposed, to the model
• competitive binding assays e.g., de novo in silico molecule design
• structure-based modeling e.g., de novo in silico molecule design
• immunization of a mammal with the model further comprises isolating model-binding B-cells and performing B cell receptor (BCR) sequencing.
• BCR sequencing employ high-throughput single-cell sequencing to obtain the VH and VL sequences for antibodies from individual human, rat, and mouse B cells.
• the functional mold molecule is a functional mold polypeptide comprising functional complementarity.
• the functional mold polypeptide binds the model or the candidate mimic molecule.
• the functional mold molecule’s binding to the model or the candidate mimic molecule is measured via binding affinity interaction assays.
• the binding affinity interactions are measured by one or more of a chemical assay, an optical assay, and a radioactive assay.
• the chemical assay is selected from one or more of gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunoblotting (e.g., a western blot).
• the optical assay is selected from one or more of a fluorescence intensity assay, a fluorescent anisotropy assay, and a fluorescence resonance energy transfer (FRET) assay.
• the radioactive binding assay is a G protein-coupled receptor (GPCR)-radioligand binding assay.
• variant libraries of molecules are generated and/or provided.
• variant libraries of mimic DB1/ 146222728.1 26 Attorney Docket No.: MTPH-001PC/123828-02-5123 molecules; mold molecules; and/or both mimic molecules and mold molecules are generated and/or provided.
• a variant library is or comprises a collection of variants of some reference molecules, such as polypeptides, across the entire molecule (e.g., polypeptide), or in embodiments, a subset (e.g., a contiguous domain or a non-contiguous set) of positions (e.g., residues) of the molecule.
• the variant library comprises a collection of variants that is systematically engineered.
• the systematic protein engineering comprises a method selected from combinatorial alanine-scanning strategy, binomial mutagenesis, and shotgun scanning.
• the systematic protein engineering comprises site-directed mutagenesis.
• An alanine-scanning strategy is a form of site-directed mutagenesis that allows for systematic mapping of functional binding epitopes. Because substitution with alanine removes all side chain atoms past the ⁇ -carbon, the effects of individual alanine mutations can be used to infer the roles of individual side chains. Alanine-scanning mutagenesis provides a detailed map of a protein-binding interface. Binomial mutagenesis is a combinatorial mutagenesis technique that allows the effects of a given mutation to be studied in the context of many other mutations, thereby allowing for an analysis of additivity and potential residue-residue interactions.
• the randomization results in a set of mutant proteins with a binomial distribution of substitutions.
• the mutants are then classified into active and inactive classes, and subsets of both classes are then sequenced.
• the importance of the wild-type residue at a given position can be inferred from the frequency with which the mutation to alanine occurs in the set of active mutants. In principle, if the change is completely neutral, alanine and wild type should each occur in roughly half of the active mutants. A deleterious mutation will be recovered less frequently and vice versa.
• the frequency with which mutations occur at two positions simultaneously can also be compared with the frequency expected from the product of the single-position frequencies.
• Shotgun scanning is a general combinatorial method for rapidly mapping functional epitopes of proteins by combining the concepts of alanine-scanning mutagenesis and binomial mutagenesis with phage display technology. Specifically, shotgun scanning analyzes many side chains simultaneously and circumvents the need for protein purification and biophysical DB1/ 146222728.1 27 Attorney Docket No.: MTPH-001PC/123828-02-5123 analysis by instead deriving the energetic contributions of individual side chains from statistical analysis of DNA sequences.
• the variant library comprises a collection of variants that is random or semi- systematically engineered.
• the method employs fully random mutation and/or random mutation focused on a specific set of positions (e.g., residues) of the molecule.
• a variant is a molecule having one or more of: a substitution, an insertion, or a deletion at one or more positions, relative to a reference molecule, such as a polymer (e.g., a polypeptide).
• variants are those that have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues.
• a conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
• the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
• Conservative substitutions may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
• the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
• conservative substitutions are exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above.
• the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
• glycine and proline may be substituted for one another based on their ability to disrupt ⁇ -helices.
• non-conservative substitutions are exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
• the variant library comprises a collection of variant polypeptide molecules, and the variant library is one or more of: an alanine (or other fixed amino acid substitution) library, a subset of possible substitutions library, or a full-substitution (including deletions and insertions) library.
• DB1/ 146222728.1 28 Attorney Docket No.: MTPH-001PC/123828-02-5123
• the variant library comprises variation across the entirety of a reference molecule (e.g., a polypeptide).
• the variant library comprises variation across a subset of residues of a reference molecule (e.g., a polypeptide).
• the variant library covers at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more of the contiguous or non-contiguous average length of a functional molecule (e.g., a functional mold molecule or functional mimic molecule), wherein the average length of a functional molecule is at least about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150 monomers (e.g., amino acids or nucleotides), and/or comprises at least about 100, about 200, about 400, about 500, about
• the variant library relative to a reference polymer (e.g., polypeptide), comprises at least about 80%, about 85%, about 90%, about 95%, or about 100% single position substitutions to a specific residue (e.g., alanine), at least about 80%, about 85%, about 90%, about 95%, or about 100% single position substitutions to any residue (e.g., any non-identity residue of the 20 canonical amino acids, including deletions or insertions of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more residues), or combinations thereof, e.g., n% of all double substitutions, m% of all triple substitutions, or approximately following a negative binomial distribution with shape parameters approximately described by ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 1 ⁇ 0.4 ⁇ ⁇ _ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ / ⁇ .
• the variant library comprises one or more immunoglobulin variable domains (variable heavy, variable light, or variable heavy and variable light), or fragments thereof (e.g., hCDR1, hCDR2, hCDR3, including combinations thereof, including 1, 2, or all 3, e.g., hCDRs1-3; or lCDR1, lCDR2, lCDR3, including combinations thereof, including 1, 2, or all 3, e.g., lCDRs1-3).
• the fragment comprises 3 or 6 CDRs.
• the variant library comprises all possible single mutations according to any design and a distribution of about 2, about 3, about 4, or more multiple mutants.
• the variant library is a deep mutational scan (DMS) library.
• DMS deep mutational scan
• Deep mutational scanning is a method for multiplex measurement of functional consequences of protein variants. Specifically, DMS assays can investigate protein-ligand binding affinity by measuring the functional consequences of hundreds of thousands of variants of a protein simultaneously.
• a library of protein variants is first introduced into a model system. Model systems that have been used in deep mutational scanning include phage, bacteria, yeast, and cultured mammalian cells. A selection is applied for protein function or another molecular property of interest, altering the frequency of each variant according to its functional capacity.
• Selections can be growth-based or implement physical separation of variants into bins, as in phage display or flow sorting of cells.
• the frequency of each variant in each time point or bin is determined by using deep sequencing to count the number of times each variant appears.
• the variable region is either directly sequenced using a single-end or paired-end strategy, or a short barcode that uniquely identifies each variant in the population is sequenced instead. Barcoding enables accurate assessment of variable regions longer than a single sequencing read. Analysis of the change in each variant’s frequency throughout the selection yields a score that estimates the variant’s effect.
• DMS experiments one begins with a library of variants of a specific protein.
• Proteins that have high levels of a particular activity of interest are then enriched via one or more rounds of selection.
• the set of enriched sequences is then compared to the initial library, and protein sequences (or mutations within these sequences) are scored according to how much this enrichment procedure increases their prevalence.
• the present disclosure provides a variant library of functional mold molecules (e.g., polypeptides) and/or functional mimic molecules (e.g., candidate functional mimic polypeptides).
• providing one or more variant libraries of one or more functional mold molecules or functional mimic molecules further comprises generation of the one or more functional mold molecules or functional mimic molecules by a method selected from interactions reported in the scientific literature, phage display, ribosome display, mammalian cell display, immunization (e.g., of a mammal, such as a mouse, rat, rabbit, non-human primate, or human) with the model (optionally by isolating model-binding B-cells and performing BCR sequencing), obtaining model-binding B cells from a subject previously exposed (or suspected of being exposed) to the model, competitive-binding assays, depletion studies, and structure- based modeling (including de novo in silico molecule design).
• a method selected from interactions reported in the scientific literature phage display, ribosome display, mammalian cell display, immunization (e.g., of a mammal, such as a mouse, rat, rabbit, non-human primate, or human) with the model (
• the variant library is provided as a collection of one or more soluble proteins and/or cell surface displayed constructs and/or phage display.
• the soluble proteins of the variant library are full Ig, or fragments thereof.
• the cell surface displayed constructs comprises a yeast cell, a mammalian cell, an immortalized cell, or an insect cell.
• the yeast cell is selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Kluyveromyces lactis.
• the mammalian cell is a human cell.
• the mammalian cell is selected from a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, a human embryonic kidney (HEK293T) cell, a Vero cell, or a Spodoptera frugiperda 9 (Sf9) cell.
• the CHO cell is a CHO-K1, a CHO-DHB11, a CHO-DXB1, a CHO-S, or a CHO-DG44 cell.
• the CHO cell comprises or is selected from CHO-K1 (ATCC CCL-61) cells, SURE CHO-M cells (derivative of CHO-K1), or baby hamster kidney cells (BHK, ATCC CCL-10).
• the Vero cell comprises or is selected from a Vero cell, a Vero 76 cell, and a Vero E6 cell.
• the cell is or comprises a human cervical carcinoma cell (HELA, ATCC CCL-2), 293 (ATCC CRL-1573), 3T3 (ATCC CCL- 163), or a monkey kidney CV1 line (ATCC CCL-70), which can be transformed with SV40 (COS-7, ATCC CRL-1587).
• a target functional profile expressed by a molecule or complex of the disclosure.
• a target functional profile is or comprises a description of the functions of a molecule or complex (e.g., a functional mimic molecule), relative to a model molecule or complex.
• illustrative functions include, but are not limited to, affinity for a receptor or ligand; molecular structure or surface features, such as arrangements of charge, polarity, or hydrophobicity; and biological activity, such as modulation of cellular signaling, communication, or pathogen interactions.
• the target functional profile comprises functions selected from a binding profile (e.g., activity and/or affinity, specificity, and/or selectivity); a signaling profile (activity, specificity, and/or selectivity); and an enzymatic profile (activity and/or processivity, specificity, and/or selectivity).
• a binding profile e.g., activity and/or affinity, specificity, and/or selectivity
• a signaling profile e.g., activity, specificity, and/or selectivity
• an enzymatic profile activity and/or processivity, specificity, and/or selectivity
• the function is selected from one or more of the affinity, avidity, activity, specificity, and/or selectivity for a receptor or ligand; agonism, antagonism, and/or inhibition; the molecular structure or surface features; and biological activity.
• the molecular structure or surface features comprises arrangements of charge, polarity, and/or hydrophobicity.
• the biological activity comprises modulation of cellular signaling, communication, and/or pathogen interactions.
• the biological activity comprises transcription, translation, and/or post-translational modification.
• the functional mimic molecule exhibits a target functional profile having a non-zero similarity to the functional profile of a model, the functional profile comprising one or more of: a structure, a binding profile (e.g., activity and/or affinity, avidity, specificity, and/or selectivity), a signaling profile (activity, specificity, and/or selectivity), an enzymatic profile (activity and/or processivity, specificity, and/or selectivity).
• a binding profile e.g., activity and/or affinity, avidity, specificity, and/or selectivity
• a signaling profile activity, specificity, and/or selectivity
• an enzymatic profile activity and/or processivity, specificity, and/or selectivity
• binding affinity interactions of one or more mold-candidate mimic interactions or mold-model interactions can be measured by chemical assays, optical assays, radioactive assays.
• chemical binding assays can measure interactions between two molecules (e.g., protein binding another protein, a small molecule, or a nucleic acid).
• a chemical assay is selected from one or more of gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunoblotting.
• the immunoblot is a western blot.
• optical binding assays can measure interactions between two molecules (e.g., protein binding another protein, a small molecule, or a nucleic acid).
• the optical assay is selected from one or more of a fluorescence intensity assay, a fluorescent anisotropy assay, and a fluorescence resonance energy transfer (FRET) assay.
• the radioactive binding assay is a radioimmunoassay, such as a radioallergosorbent test.
• a radioimmunoassay is a heterogeneous assay that employs a radiolabeled drug, such as isotopes of iodine (e.g., 125 I, 131 I, or tritium ( 3 H)) as labels.
• the binding affinity interactions are measured individually by flow cytometry, biolayer interferometry, surface plasmon resonance, or enzyme-linked immunosorbent assay.
• the method of the present disclosure further comprises an interaction evaluation that provides a first vector comprising a model fingerprint and a second vector comprising a functional mimic molecule fingerprint, wherein the evaluation constitutes calculating a metric of similarity between the vectors.
• a fingerprint is a set of calculated characteristics for measuring an interaction between vectors.
• a fingerprint is a set of calculated characteristics for measuring an interaction between vectors using one or more of the methods described herein.
• the interaction evaluation is of binding affinity of the variant functional mold molecules and/or of the variant functional mimic molecules to i) the target and ii) the one or more functional mimic molecules and/or functional mold molecules, respectively.
• the interaction evaluation comprises evaluating binding affinity interactions, wherein the binding affinity interactions are measured individually.
• the individual measurement comprises one or more of flow cytometry, biolayer interferometry, surface plasmon resonance, and/or enzyme-linked immunosorbent assay.
• the interaction evaluation comprises evaluating binding affinity interactions, wherein the binding affinity interactions are measured as a pool, where candidate functional mimic molecules are conjugated to a fluorophore or magnetic bead and used to stain cells expressing variants of functional mold molecules as surface display constructs.
• bound and unbound populations of functional mimic molecules are separated by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting.
• FACS fluorescence-activated cell sorting
• antibody DNA sequences are amplified from the unsorted, unsorted, and/or bound cell populations by polymerase chain reaction, and the antibody variants comprising each population are determined by next-generation sequencing.
• the relative binding affinity of each functional mold molecule variant for a functional mimic molecule is determined by computing an enrichment score (as by the ordinary least squares regression). See, e.g., Rubin et al., “A statistical framework for analyzing deep mutational scanning data,” Genome Biology volume 18, Article number: 150 (2017).
• each functional mold molecule variant for a functional mimic molecule is determined by computing mean bin methods. See, e.g., Starr et al., “Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding,” Cell, Volume 182, Issue 5 (2020). For example, computing mean bin of a variant phenotype can reveal its binding affinity.
• the interaction evaluation comprises evaluating binding affinity interactions, wherein the binding affinity interactions are measured by cells expressing functional mold molecules surface display constructs stained with multiple concentrations of functional mimic molecules prior to sorting. Apparent dissociation constants can then be determined by fitting, for each variant, a Hill function to the number of observations, by sequencing, per concentration. See, e.g., Adams et al., “Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves,” eLIFE, 5:e23156 (2016).
• the interaction evaluation comprises evaluating binding affinity interactions, wherein the binding affinity interactions are measured as a pool wherein functional mold molecule variants are conjugated to unique DNA barcodes and used to stain cells expressing functional mimic molecules.
• relative affinities are determined by barcode enrichments after single cell sequencing. See, e.g., Stoeckius et al., “Simultaneous epitope and transcriptome measurement in single cells,” Nature Methods, Volume 14, pp.865–868 (2017).
• the disclosure provides for comparing the interaction of the plurality of variant functional mold molecules with i) the model and ii) the one or more candidate functional mimic molecules, in order to identify the functional mimic molecule having the target profile.
• the comparison comprises the correlation (e.g., Pearson’s rho, Spearman’s rho, or Kendall’s tau) between vectorized fingerprints.
• the comparison comprises a measure of distance (e.g., Euclidean distance or Manhattan distance) between vectorized fingerprints.
• the comparison comprises a collection of vectorized functional mimic molecule fingerprints that are clustered (e.g., as by k means clustering or affinity propagation), optionally wherein functional mimic molecules co-clustering with the model are considered similar.
• DB1/ 146222728.1 34 Attorney Docket No.: MTPH-001PC/123828-02-5123
• the comparison comprises clustering that is performed on a low dimensional embedding (e.g., principal components, independent components, t-distributed stochastic neighbor embedding, or uniform manifold approximation) of functional mimic molecule fingerprints.
• the comparison comprises fingerprints comprised of variants that include multiple mutations collapsed to single-mutation relative affinities through a global epistasis model. See, e.g., Otwinowski et al., “Inferring the shape of global epistasis,” PNAS, 115 (32) E7550-E7558.
• single mutant relative affinities can be compared using any of the approaches selected from the correlation between vectorized fingerprints; a measure of distance between vectorized fingerprints; a collection of vectorized functional mimic molecule fingerprints that are clustered; clustering that is performed on a low dimensional embedding of functional mimic molecule fingerprints; and fingerprints comprised of variants that include multiple mutations collapsed to single-mutation relative affinities through a global epistasis model.
• single mutant relative affinities can be converted into probability distributions such that each possible combination of amino acid sequence position and mutation is assigned a value representative of the probability of sampling a mutation at that position after sorting a population in which every position-mutation combination was equally represented prior to sorting.
• Probability distribution representations of fingerprints can be compared using measures of divergence such as earth mover’s distance or Kullback-Leibler divergence.
• similarity metrics are calculated in discrete or overlapping contiguous or dis- contiguous regions of the functional mold polypeptide or candidate functional mimic polypeptide amino acid sequence or structure and experimentally determined or predicted in silico, such that a functional mimic polypeptide is considered a partial mimic if it bears high similarity in a subset of regions.
• compositions and Formulations pertains to pharmaceutical compositions comprising the compositions, e.g. mimic molecules (functional mimic polymer), and a pharmaceutically acceptable carrier or excipient.
• a pharmaceutically acceptable carrier or excipient e.g. a pharmaceutically acceptable carrier or excipient.
• Any pharmaceutical compositions described herein can be administered to a patient as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle.
• Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
• pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• the pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.
• auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
• the pharmaceutically acceptable excipients are sterile when administered to a patient. Water is a useful excipient when any agent described herein is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions.
• suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents. Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R.
• compositions and/or additional therapeutic agents
• Any pharmaceutical composition (and/or additional therapeutic agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powder, frozen suspension, desiccated powder, or any other form suitable for use.
• the composition is in the form of a capsule.
• the composition is in the form of a tablet.
• the pharmaceutical composition is formulated in the form of a soft-gel capsule. In embodiments, the pharmaceutical composition is formulated in the form of a gelatin capsule. In embodiments, the pharmaceutical composition is formulated as a liquid.
• the present pharmaceutical compositions can also include a solubilizing agent.
• the agents can be delivered with a suitable DB1/ 146222728.1 36 Attorney Docket No.: MTPH-001PC/123828-02-5123 vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.
• the formulations comprising the present pharmaceutical compositions (and/or additional therapeutic agents) of the present disclosure may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).
• a carrier which constitutes one or more accessory ingredients.
• the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by
• any pharmaceutical compositions (and/or additional therapeutic agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
• Routes of administration include, for example: topical, oral, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, intravenous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, rectal, or by inhalation.
• Administration can be local or systemic.
• the administering is by an intravenous route.
• the mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition.
• compositions (and/or additional therapeutic agents) described herein are formulated in accordance with routine procedures as a composition adapted for administration.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
• the carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
• Dosage forms suitable for parenteral administration include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured DB1/ 146222728.1 37 Attorney Docket No.: MTPH-001PC/123828-02-5123 in the form of sterile solid compositions (e.g.
• lyophilized composition which can be dissolved or suspended in sterile injectable medium immediately before use.
• sterile injectable medium may contain, for example, suspending or dispersing agents known in the art.
• Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
• antibacterial agents such as benzyl alcohol or methyl para
• compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
• Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
• Compositions for topical delivery can be in the form of a cream, gel, ointment, lotion, spray, aqueous or oily suspensions, powders, or emulsions, for example.
• Increased skin permeability and penetration may be achieved by non-invasive methods, for example, with the use of any nanocarriers combined with any pharmaceutical composition (and/or additional therapeutic agents) described herein.
• the skin can act as a reservoir and can be used to deliver the compositions (and/or additional therapeutic agents) described herein for more extended periods in a sustained manner.
• Any pharmaceutical compositions (and/or additional therapeutic agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.
• Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropyl cellulose, hydropropylmethyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.
• Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for DB1/ 146222728.1 38 Attorney Docket No.: MTPH-001PC/123828-02-5123 use with the active ingredients of the agents described herein.
• the disclosure thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.
• Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
• a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
• compositions preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution. Definitions The following definitions are used in connection with the invention disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs. As used herein, “a,” “an,” or “the” can mean one or more than one.
• the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication.
• the language “about 50” covers the range of 45 to 55.
• the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.” This disclosure is further illustrated by the following non-limiting examples. EXAMPLES Without wishing to be bound by theory, the process for identifying mimics is analogous to the manufacturing process of casting a replica.
• Example 1 Identification and characterization of antibodies that mimic the epitope of a commercial antibody on an antigen The experiments of this example demonstrate the process of identifying mimics and discovering and characterizing antibodies that mimic the epitope of a commercial antibody on an antigen.
• the antigen’s commercial antibody epitope was used as the model and the commercial antibody was used as the mold.
• Biopanning and anti-drug antibody (ADA) repertoire mining were performed to identify a collection of anti-idiotypic antibodies that bind to the commercial antibody.
• Deep mutational scanning of the commercial antibody amino acid sequence was then used to characterize the effects of nearly all possible heavy chain mutations on binding to both the antigen and the anti- idiotypic antibodies.
• comparing the profiles of mutational effects between the antigen and anti-idiotypic antibodies allowed for identification of antibodies that bind to the commercial antibody in a manner that finely mimics the antigen.
• Figures 2A-D show commercial antibody WT and DMS library binding to the antigen.
• Figure 2A depicts the presence of human kappa light chain confirms the surface expression of wildtype commercial antibody (solid) and commercial antibody libraries (dashed and dotted) on yeast.
• Figure 2B shows that after gating for Fab expression, the libraries (dashed and dotted lines) exhibited a greater phenotypic range of binding to antigen relative to WT (solid).
• Figures 2C-D show that both libraries with mutations of either heavy chain or mutations in the light chain exhibit antigen binding correlated with expression, except for those variants that appear to lose binding altogether. Selections were performed in duplicate and an inter-replicate replicate Pearson’s correlation of 0.804 ( Figure 3A) was observed.
• each antibody was compared to the antigen by calculating a mimicry score as the Pearson’s correlation between binding score profiles DB1/ 146222728.1 41 Attorney Docket No.: MTPH-001PC/123828-02-5123 across variants (Figure 4B). Notably, the most positively correlated antibody displayed a mimicry score comparable to a replicate antigen selection.
• clustering was performed to group the anti-idiotypic antibodies by their binding profiles across commercial antibody mutants. Clustering identified groups of binding profiles that mimic the antigen’s, as well as more distant clusters ( Figure 4C). Further, exploration of representative binding profiles distinguished antibodies which bind the commercial antibody through distinct modes of interaction.
• Example 2 Identification and characterization of antibodies that mimic the epitope of a known broadly neutralizing antibody
• the experiments of this example demonstrate the process of identifying mimics and discovering and characterizing antibodies that mimic the epitope of a known broadly neutralizing antibody against an infectious-species antigen.
• the epitope of infectious species antigen was used as the model and the known broadly neutralizing antibody was used as the mold. Biopanning against the broadly neutralizing antibody was used to identify an anti-idiotypic antibody.
• Deep mutational scanning of the broadly neutralizing antibody was used to characterize the effects on binding the discovered antibody, the model, and a known anti- idiotypic mimic antibody for nearly all possible heavy and light chain mutations. Characterization of the mutational profiles confirmed that the identified antibody mimics the antigen model. Mimicry was further validated by the experimentally determined structures of the complexes. DB1/ 146222728.1 42 Attorney Docket No.: MTPH-001PC/123828-02-5123 Identification of candidate mimics Biopanning against the broadly neutralizing antibody was performed using a naive human scFv phage display library.
• Each antibody was bound to the library-expressing yeast population, and bound yeast were captured by magnetic bead.
• the bound and unbound populations were isolated, and the relative abundances of each variant in each population was measured by next generation sequencing.
• a binding score was calculated for each variant based on the relative enrichments in the bound and unbound populations.
• the library was also characterized for binding to the model antigen protein. Due to the lower affinity of broadly neutralizing antibody for the antigen protein (see Figure 7, grey diamonds), FACS sorting was performed, as described in Example 1.
• the binding profiles of the antibodies and antigen are depicted in Figures 8A-C. Comparison of the antigen protein ( Figure 8A) to the known mimic antibody profile ( Figure 8B) showed that key sensitive sites were conserved, with one notably different sensitive site in the middle of the CDR1 region.
• the discovered antibody (Figure 8C), showed highly similar sensitive sites to the known mimic, but displayed a similar pattern to the model in the CDR1 region. Despite having a highly divergent sequence, the discovered antibody had not only similar sensitive sites, but also similar effects for individual mutations, indicating antigen mimicry. Cryogenic electron microscopy of the two mimic antibodies in complex with the broadly neutralizing antibody was performed, and derived structures were compared to the published structure of the broadly neutralizing antibody in complex with the model epitope peptide.
• the epitope forms a single loop structure that interacts with the heavy chain CDR1, heavy chain DB1/ 146222728.1 43 Attorney Docket No.: MTPH-001PC/123828-02-5123 CDR3, and light chain CDR3 of the broadly neutralizing antibody (Figure 9A).
• the heavy chain CDR3 of the discovered mimic formed a highly similar loop which binds in the same location ( Figure 9B).
• the discovered mimic antibody and known mimic antibody were observed to bind to the same region of the broadly neutralizing antibody, although with different docking angles ( Figures 9C and 9D). Additionally, the structure of the loop mimicking the native epitope was different.
• Example 3 Identification and characterization of antibodies that mimic a native protein’s binding interaction with a cell surface receptor
• the experiments in this example demonstrate the process of identifying and characterizing mimics of a native protein (model) using its natural protein receptor as a mold. Biopanning against a soluble form of the receptor (mold) was performed to identify a set of antibodies. In vitro characterization of candidate mimics was performed to confirm binding to the soluble mold and to confirm competitive binding with the model in both soluble and cell- surface presented forms of the mold. Deep mutational scanning of the sequence of the soluble extracellular domain of the mold was used to characterize the binding of candidate mimic antibodies, as well as the model protein and a known non-mimic antibody.
• Comparison of mutational effect profiles enabled identification of antibodies that compete and finely mimic the native protein. Identification of mimics Multiple rounds of biopanning were performed against a soluble form of the extracellular domain of the mold using a naive human scFv phage display library. After three rounds of selection, 192 individual colonies were isolated, sequenced by Sanger sequencing and characterized by phage ELISA for binding to the mold protein and polyclonal IgG. 36 unique scFv sequences were selected and converted to IgG for production.
• Candidate mimics were characterized for competitive binding with the model to the mold by ELISA. The extracellular domain of the mold receptor as a human Fc fusion was immobilized on the surface and bound to the model protein.
• Antibodies showed strong binding in the absence of co-expressing model protein, but binding was fully inhibited when the model was present. Characterization of mimicry A yeast surface display deep mutational scanning variant DNA library of the extracellular domain of the mold receptor was constructed and validated, as described in Example 1. The deep mutational scanning library was used to characterize the effects of mold mutations on binding to the model protein, a known non-mimic antibody and discovered candidate mimic antibodies. The yeast library was bound to protein and sorted into bins gated on binding by FACS, as described in Example 1. Two discovered antibodies appeared to be mimics: the binding profiles (Figures 11B and 11C) were similar to that of the model, as determined by the conservation of sensitive sites (Figure 11A).
• the known non-mimic showed different sensitivities, particularly between residues 4 and 30 (Figure 11D).
• a differential comparison of the mean position scores with the known non-mimic antibody showed an increase in binding signal for the model protein in the receptor mold residues observed to interact with model protein in a published crystal structure of the complex, highlighted in grey ( Figure 12A).
• Discovered mimic antibodies showed increases in the same sites and a nearly identical overall pattern ( Figures 12B and 12C).
• Individual mutation effect scores were highly correlated between the model protein and the discovered mimic antibodies with Pearson’s correlation coefficients of 0.81 and 0.84 respectively ( Figure 13). Although divergent in sequence, the profiles of the two discovered antibody mimics were highly correlated, with a Pearson’s correlation coefficient equal to 0.88.

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