[go: up one dir, main page]

WO2025160469A1 - Procédés de profilage de répertoires d'immunoglobulines - Google Patents

Procédés de profilage de répertoires d'immunoglobulines

Info

Publication number
WO2025160469A1
WO2025160469A1 PCT/US2025/013042 US2025013042W WO2025160469A1 WO 2025160469 A1 WO2025160469 A1 WO 2025160469A1 US 2025013042 W US2025013042 W US 2025013042W WO 2025160469 A1 WO2025160469 A1 WO 2025160469A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptides
amino acid
cdr
residues
immunoglobulin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/013042
Other languages
English (en)
Inventor
Jimmy GOLLIHAR
Andrew Horton
Jagannath SWAMINATHAN
Edward Marcotte
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Erisyon Inc
Houston Methodist Research Institute
University of Texas System
University of Texas at Austin
Original Assignee
Erisyon Inc
Houston Methodist Research Institute
University of Texas System
University of Texas at Austin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Erisyon Inc, Houston Methodist Research Institute, University of Texas System, University of Texas at Austin filed Critical Erisyon Inc
Publication of WO2025160469A1 publication Critical patent/WO2025160469A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • An organism s antibody reportoire, the collection of antibodies produced and circulating at a given time, can potentially provide a wealth of knowledge that may have diagnostic value and/or facilitate development of antibody-based therapeutics.
  • the present application addresses this need with method of profiling immunoglobulin repertoires which provide, in some embodiments, quantitative information which cannot be or is not obtained with the same level of confidence, from existing methods of profiling immunoglobulin repertoires.
  • the step of obtaining or having obtained comprises 1) digesting or having digested immunoglobulin molecules isolated from a biological sample from the subject; and 2) enriching or having enriched the digested immunoglobulin molecules for CDR-H3 peptides.
  • methods of profiling an immunoglobulin repertoire comprising the steps of: a) obtaining or having obtained a biological sample from the subject; b) isolating immunoglobulin molecules from the biological sample; c) digesting said isolated immunoglobulin molecules; d) enriching for CDR-H3 peptides; and e) fluoro sequencing said enriched CDR-H3 peptides, thereby obtaining information regarding the subject’s immunoglobulin repertoire.
  • the biological sample comprises blood or serum. In some embodiments, the biological sample comprises tumor-infiltrating B-cells. In some embodiments, the biological sample comprises a spleen, bone marrow, or saliva sample. In some embodiments, the biological sample comprises cerebrospinal fluid.
  • the immunoglobulin molecules are isolated from the biological sample by a method employing particles coated with immunoglobulin-binding polypeptides.
  • the immunoglobulin-binding polypeptides comprise protein A, protein G, or a combination thereof.
  • the particles comprise protein A/G beads.
  • said isolated immunoglobulin molecules are contacted with an agent that modifies one or more amino acid residues.
  • the agent modifies an amino acid residue to resemble another amino acid residue.
  • the agent modifies cysteine residues to resemble lysine residues, e.g., 2-bromoethylamine.
  • the step of digesting comprises digesting with an enzyme, e.g., an endoprotease.
  • the endoprotease is selected from the group consisting of trypsin and lysC.
  • the endoprotease cleaves C-terminal to a lysine, e.g., C-terminal to the amino acid sequence ASTK.
  • the step of enriching comprises using an isotype-selective binder, e.g., an IgG-selective binder.
  • the IgG-selective binder is capable of selectively binding the amino acid sequence ASTK.
  • the isotype- selective binder comprises an antibody or antigen-binding fragment thereof. In some embodiments, the isotype- selective binder is immobilized to a solid support. In some embodiments, the solid support comprises a material selected from the group consisting of sepharose resin and glass. In some embodiments, the solid support comprises beads. In some embodiments, the solid support comprises magnetic beads.
  • the solid support is attached to a identifier unique to the subject.
  • the identifier is unique to the biological sample from the subject.
  • the identifier comprises an oligonucleotide.
  • the identifier comprises a peptide.
  • the obtained information comprises quantitative information.
  • fluoro sequencing is conducted at single molecule resolution.
  • the obtained information comprises sequence information at the single molecule level.
  • step of fluorosequencing comprises labeling a subset of amino acid residues in said enriched CDR-H3 peptides with a fluorophore.
  • the subset of amino acid residues comprises lysine residues, cysteine residues, aspartic acid residues, glutamic acid residues, methionine residues, tyrosine residues, and any combination of the foregoing.
  • the subset of amino acid residues comprises tyrosine residues.
  • each type amino acid residue in the subset is labeled with a different fluorophore.
  • each fluorophore’ s emission spectrum is distinguishable from each other.
  • the step of fluorosequencing comprises counting fluorotypes.
  • IgG immunoglobulin G
  • methods of profiling an immunoglobulin G (IgG) repertoire specific to a subject comprising the steps of: a) digesting IgG molecules isolated from a biological sample from the subject with trypsin; b) enriching for CDR-H3 peptides using an ASTK-selective binder; c) fluorescently labeling at least tyrosine amino acid residues in said CDR-H3 peptides; and d) fluorosequencing said enriched CDR-H3 peptides by a method comprising counting fluorotypes at single molecule resolution, thereby obtaining quantitative information regarding the subject’s IgG repertoire.
  • provided methods further comprise a step of e) comparing fluorotypes to sequences in a reference database.
  • Figure 1 depicts the structure of an IgG antibody, including heavy chain complementarity determining regions (CDR-H1-3) and the heavy chain framework domains (FR-H-1 to 4).
  • Figure 2 depicts a plot from a motif analysis of IgG sequences, which shows a distinct lysine next to a conserved “ASTK” motif specific to the heavy chain FR4 domain on IgG molecules.
  • Figure 3 illustrates an embodiment of a fluoro sequencing method as described further herein.
  • the illustrated method employs chemical labeling of specific amino acid residues on peptides with fluorescent dyes.
  • Figure 4 depicts a schematic of a pipeline for methods disclosed herein for assessing a subject’s IgG repertoire.
  • Figure 5A depicts the enrichment ratios of precursor abundance for various peptides across the F(ab’) region, observed across three samples.
  • Figure 5B depict an ion intensity trace which showing increased counts for two rabbit clones (R36 and R33) as compared to an IgG control.
  • Figure 6A depicts frequencies of various amino acid residues in the CDR-H3 region based on an analysis of four data sets. Approximately 12% of the residues are tyrosine residues; the total frequency labelable amino acid residues was greater than 40%. In certain embodiments, the one or a combination of the following amino acid residues are labeled: aspartic acid, glutamic acid, asparagine, glutamine, arginine, serine, threonine, and tyrosine.
  • Figure 6B depicts a histogram of CDR-H3 length distributions. The median peptide length was 14 amino acid residues.
  • Figure 6C depicts the results of an analysis of the uniqueness of fluorotypes, showing that more than 85% of fluorotypes uniquely map to CDR-H3 peptides (as determined by the sequences of B cells.)
  • Figure 7 depicts proposed reactions (Mannich-type reactions and diazonium reactions) that will be screened for tyrosine residue conjugation.
  • the present disclosure overcomes analytical challenges with current techniques for profiling immunoglobulin repertoires by combining a peptide sequencing technique, e.g., a fluoro sequencing technique, with use of a reagent that allows for selective enrichment of peptides which comprise the heavy chain complementarity determining region 3 of immunoglobulin molecules (CDR-H3).
  • a peptide sequencing technique e.g., a fluoro sequencing technique
  • this selective enrichment enables the identification of CDR-H3 peptide sequences without having to identify sequences from other immunoglobulin regions, which may produce interfering peptides.
  • immunoglobulins have a characteristic sequence within or near the C-terminus of the CDR-H3 (e.g. within the heavy chain framework region 4 (FR4)). Such characteristics may vary depending in the immunoglobulin isotype.
  • IgG molecules include a characteristic ASTK amino acid sequence C-terminal to the CDR-H3.
  • cleavage with certain proteases such as lysC protease can be used to obtain a fragment which contains the CDR-H3 region. (See Figure 2).
  • a reagent which selectively binds to the characteristic sequence can then be used to enrich for CDR-H3 peptides.
  • Provided methods integrate steps of CDR-H3 peptide enrichment with a single molecule peptide sequencing technology.
  • Provided methods allow at least four significant benefits over existing solutions - (i) detection of peptides at single molecule sensitivity (4-6 orders of magnitude more sensitive), which enables identifying more antibody clonotypes and at lower concentrations; (ii) intrinsically quantitative measurements due to the counting of individual peptide molecules (iii) ability to read the positions of post-translational modified amino acids and (iv) discovering antibody sequences directly from human making them ideal therapeutic candidates as they are pre-selected for safety, solubility, high expression yields, and other developability criteria.
  • CDR-H3 peptide refers to a peptide which is 1) smaller than an immunoglobulin heavy chain variable region (which typically comprises heavy chain complementary determining regions 1-3 (CDR-H1, CDR-H2, and CDR-H3) and heavy chain framework regions FR1, FR2, FR3, and FR4) and 2) comprises the entirety of an immunoglobulin heavy chain CDR3 (CDR-H3).
  • CDR-H3 peptides may also comprise the FR4 (framework 4) region of an immunoglobulin heavy chain or a portion thereof.
  • the length of the CDR-H3 peptide is from 8 to 60 amino acid residues, e.g., from 10 to 55 amino acid residues, from 10 to 55 amino acid residues, from 10 to 55 amino acid residues, or from 10 to 55 amino acid residues.
  • the term “collective signal” refers to the combined signal that results from multiple labels (e.g., a first label and a second label) attached to an individual peptide molecule. In some embodiments, the “collective signal” refers to the combined signal from all of the labels attached to an individual peptide molecule.
  • Edman degradation generally refers to methods comprising chemical removal of amino acids from peptides or proteins.
  • Edman degradation denotes terminal (e.g., N- or C-terminal) amino acid removal.
  • Edman degradation refers to N-terminal amino acid removal through isothiocyanate (e.g., phenyl isothiocyanate) coupling and cyclization with the terminal amine group of an N- terminal residue, such that the N-terminal amino acid is removed from a peptide.
  • Edman degradation broadly encompasses N-terminal amino acid functionalizations leading to N-terminal amino acid removal.
  • Edman degradation encompasses C-terminal amino acid removal.
  • Edman degradation comprises terminal amino acid functionalization (e.g., N-terminal amino acid isothiocyanate functionalization) followed by enzymatic removal (e.g., by an ‘Edmanase’ with specificity for chemically derivatized N- terminal amino acids).
  • fluorescence refers to the emission of light of a particular wavelength by a substance that has absorbed light of a different wavelength.
  • fluorescence provides a non-destructive means of tracking and/or analyzing biological molecules based on the fluorescent emission at a specific wavelength.
  • Proteins including antibodies
  • peptides including nucleic acid, oligonucleotides (including single stranded and double stranded primers) may be “labeled” with a variety of extrinsic fluorescent molecules referred to as fluorophores.
  • fluorescein such as carboxyfluorescein
  • fluorophores such as proteins (such as antibodies for immunohistochemistry) or nucleic acids.
  • fluorescein may be conjugated to nucleoside triphosphates and incorporated into nucleic acid probes (such as “fluorescent-conjugated primers”) for in situ hybridization.
  • a molecule that is conjugated to carboxyfluorescein is referred to as “F AM-labeled.”
  • fluorescent signature refers to a fluorescent signature of a given peptide or polypeptide as obtained from a fluoro sequencing experiment.
  • the fluorescent signature refers to a pattern of fluorescence within a peptide or polypeptide being fluorosequenced, the pattern including (1) identities or probable identities of fluorescently labelled amino acid residues whose labels were detected during the fluorosequencing and (2) positional information regarding the aforementioned fluorescently amino acid residues.
  • fluorotype refers to a set of fluorosequences that match a given pattern. For example, multiple peptides or polypeptides may have the same fluorescent signature in a given fluorosequencing experiment; these peptides or polypeptides would be said to have the same fluoro type.
  • fluorotype represents one or more antibody clones with identical amino acid patterns in their CDR-H3 sequences.
  • polypeptide generally has its art-recognized meaning of a polymer of at least three amino acids, e.g., linked to each other by peptide bonds.
  • polypeptide is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides.
  • protein sequences generally tolerate some substitution without destroying activity.
  • Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • the term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acid residues, less than about 60 amino acids residues, less than about 55 amino acids residues, less than about 50 amino acids residues, less than about 45 amino acids residues, less than about 40 amino acids residues, less than about 35 amino acids residues, less than about 30 amino acids residues, less than about 25 amino acids residues, less than 20 amino acids residues, or less than 10 amino acids residues.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • “profiling an IgG repertoire” of a subject refers to cataloging the IgG sequences circulating in a subject.
  • profiling an immunoglobulin repertoire comprises cataloging CDR-H3 sequences from immunoglobulins circulating in a subject.
  • the term “single molecule sensitivity,” “at single molecule resolution,” “at the single molecule level,” and similar phrases generally refers to the acquisition of data (including, for example, amino acid sequence information) from individual peptide molecules in a mixture of diverse peptide molecules.
  • the mixture of diverse peptide molecules may be immobilized on a solid surface (including, for example, a glass slide, or a glass slide whose surface has been chemically modified). This may include the ability to simultaneously record the fluorescent intensity of multiple individual (e.g., single) peptide molecules distributed across the glass surface.
  • Optical devices are commercially available that may be applied in this manner.
  • a microscope equipped with total internal reflection illumination and an intensified charge-couple device (CCD) detector is available.
  • Imaging with a high sensitivity CCD camera allows the instrument to simultaneously record the fluorescent intensity of multiple individual (e.g., single) peptide molecules distributed across a surface.
  • Image collection may be performed using an image splitter that directs light through two band pass filters (one suitable for each fluorescent molecule) to be recorded as two side-by-side images on the CCD surface.
  • Using a motorized microscope stage with automated focus control to image multiple stage positions in the flow cell may allow millions of individual single peptides (or more) to be sequenced in one experiment.
  • the term “support” generally refers to an entity to which a substance (e.g., molecular construct) may be immobilized.
  • the solid may be a solid or semi-solid (e.g., gel) support.
  • a support may be a bead, a polymer matrix, an array, a microscopic slide, a glass surface, a plastic surface, a transparent surface, a metallic surface, a magnetic surface, a multi-well plate, a nanoparticle, a microparticle, or a functionalized surface.
  • the support may be planar.
  • the support may be non-planar, such as including one or more wells.
  • a bead may be, for example, a marble, a polymer bead (e.g., a polysaccharide bead, a cellulose bead, a synthetic polymer bead, a natural polymer bead), a silica bead, a functionalized bead, an activated bead, a barcoded bead, a labeled bead, a PCA bead, a magnetic bead, or a combination thereof.
  • a bead may be functionalized with a functional motif.
  • Suitable functional motifs include a capture reagent (e.g., pyridinecarboxyaldehyde (PCA)), a biotin, a streptavidin, a strep-tag II, a linker, or a functional group that may react with a molecule (e.g., an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, or an aldehyde dithiolane.
  • the functional group may couple specifically to an N-terminus or a C-terminus of a peptide.
  • the functional group may couple specifically to an amino acid side chain.
  • the functional group may couple to a side chain of an amino acid (e.g., the acid of a glutamate or aspartate, the thiol of a cysteine, the amine of a lysine, or the amide of a glutamine, or asparagine).
  • the functional group may couple specifically to a reactive group on a particular species, such as a label.
  • the functional motif may be reversibly coupled and cleaved.
  • a functional motif may also irreversibly couple to a molecule.
  • the biological sample will be a sample that is expected to contain, or suspected of containing, immunoglobulin molecules, such as IgG molecules.
  • immunoglobulin molecules such as IgG molecules.
  • biological samples include blood (e.g., peripheral blood), serum, spleen, bone marrow, cerebrospinal fluid, tumor biopsies, and combinations thereof.
  • the biological sample comprises tumor-infiltrating B-cells.
  • Isolation of isolated immunoglobulin molecules can be accomplished using any of a variety of means, including, for example, using particles e.g., beads) coated with immunoglobulin-binding polypeptides.
  • particles may be coated with polypeptides having affinity for immunoglobulin molecules or specific isotypes of immunoglobulin molecules, such as IgG.
  • protein A/G has a selected affinity for IgG.
  • the particles comprises protein A/G beads.
  • isolated immunoglobulin molecules are contacted with an agent that chemically modifies one or more amino acid residues.
  • the agent reduces and/or alkylates the amino acid residue.
  • the agent modifies an amino acid residue to resemble or become another amino acid residue.
  • 2-bromoethylamine can be used to reduce and alkylate cysteine residues such that the residues then resemble lysine residues.
  • Digestion of isolated immunoglobulin molecules can be accomplished using any of a variety of means, including, for example, use of endoproteases, including endoproteases with known recognition sites.
  • endoproteases with known recognition sites which include relatively rare amino acid residues within CDR-H3 sequences are used.
  • cysteine and lysine resides are present in abundances of less than 1% within CDR- H3 sequences.
  • the endoprotease cleaves C-terminal to a residue within a characteristic sequence that is adjacent to CDR-H3 regions. As described in Example 1, such characteristic sequences may vary depending on the immunoglobulin isotype.
  • the endoprotease cleaves C-terminal to a lysine residue, e.g., a lysine residue within the amino acid sequence ASTK.
  • the endoprotease comprises trypsin, lysC, or a combination thereof.
  • Enrichment for CDR-H3 peptides from digested can be accomplished using any of a variety of means, including, for example use of a binder selective for an immunoglobulin isotype, e.g., IgG.
  • the isotype- selective binder may be selective for an amino acid sequence which is characteristic of CDR- H3 peptides digested from immunoglobulin molecules of that isotype (or a subsequence thereof). Such a characteristic sequence may vary depending on the isotype. See, e.g., Table 1 in Example 1, and may be found in a region immediately adjacent to the CDR-H3 region, such as the framework 4 (FR4) region.
  • the isotype-selective binder is an IgG-selective binder which selectively binds the amino acid sequence ASTK.
  • the isotype-selective binder comprises an antibody or an antigen-binding fragment thereof. Labeling
  • certain amino acid residues or sets of residues are labeled with distinct fluorophores. During fluorosequencing, these labels are detected and interpreted as representing a particular amino acid residue or subset of amino acid residues, and a granular peptide sequence, probable sequence, and/or partial peptide sequence is determined.
  • At least two, at least three, or at least four different fluorophores are used, each fluorophore’s emission spectrum being distinguishable from that of the others being used. In some embodiments, two, three, or four different fluorophores are used.
  • the amino acid residues which are labeled are selected from the group consisting of lysine residues, cysteine residues, aspartic acid residues, glutamic acid residues, methionine residues, tyrosine residues, and any combination of the foregoing. In some embodiments, at least the tyrosine residues are labeled. In some embodiments, tyrosine residues and at least one other type of amino acid residue is labeled, each with different fluorophores.
  • any of a variety of conjugation methods may be used.
  • the conjugation method chosen may depend on the amino acid residue or subset of amino acid residues which are desired to be labeled.
  • Szijj, Peter A., et al. (“Tyrosine bioconjugation-an emergent alternative.” Organic & Biomolecular Chemistry 18.44 (2020): 9018-9028.) describes a variety of methods to label tyrosine amino acid residues specifically.
  • Such tyrosine conjugation methods may comprise chemical reactions such Mannich type reaction, and/or reactions using diazonium, diazodicarboxyamides, transition metals, sulfurfluoride or triazole exchange, or chemical O- glycosylation of tyrosine.
  • Enzyme-based or ribozyme-catalyzed methods of labeling tyrosines may also be used.
  • serine and threonine residues can be selectively labeled using selective oxidation to generate aldehyde and ketones and/or nucleophilic displacement of the hydroxyl group on serine and threonine residue with thiocyanate.
  • each fluorophore used in the labeling scheme is solventstable.
  • the fluorophores that are used collectively span the visible spectra.
  • the fluorophore is an Alexa Fluor® dye, an Atto dye, a Janelia Fluor® dye, a carbopyronine derivative, a Rhodamine derivative, or any combination thereof.
  • the fluorophore is Alexa Fluor® 405, AlexaFluoi® 448, Alexa Fluor® 555, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Atto390, Atto425, Atto488, Atto495, Atto514, Atto532, Atto550, Atto643, Atto647N, Atto647, Atto655, Atto680, Atto700, AttoRho-12, (5)6-napthofluorescein, Oregon GreenTM 488, Oregon GreenTM 514, JFX554, 00488-NHS, 00488-Azide, 00488- Tetrazine, 00514-NHS,
  • the fluorophore is Texas Red, Janelia Fluor® 525, Janelia Fluor® 549, Janelia Fluor® 555, AlexaFluor® 448, Alexa Fluor® 555, Atto495, Atto643, Atto647N, Rhodamine, tetramethylrhodamine, or any combination thereof.
  • the fluorophore is Texas Red, Janelia Fluor® 549, Alexa Fluor® 555, Atto643, or any combination thereof.
  • solid supports and/or peptides are attached to identifiers (e.g., nucleic acid-based and/or peptide-based identifiers) unique to each subject and/or each biological sample.
  • identifiers e.g., nucleic acid-based and/or peptide-based identifiers
  • Such unique identifiers can be read by any of a variety of techniques, e.g., fluorescence in situ hybridization (FISH) or DNA sequencing. Use of such identifiers can ensure, for example, that each sample's unique identity remains associated with fluorosequences which are obtained.
  • Fluoro sequencing may include successively removing amino acid residues from a particular terminus (e.g., the N-terminus) of the each peptide (e.g., CDR-H3 peptide) through a technique such as chemical cleavage, Edman degradation, or other forms of enzymatic cleavage following or preceding subject peptide detection. Sequential removal of amino acid residues may generate sequence or position- specific information. For example, a reduction in fluorescence following an N- terminal amino acid residue removal step may indicate that an amino acid labeled with a particular fluorophore, and thus that a specific type of amino acid residue or subset of amino acid residues, was disposed at a peptide’s N- terminal at that round of removal.
  • a reduction in fluorescence following an N- terminal amino acid residue removal step may indicate that an amino acid labeled with a particular fluorophore, and thus that a specific type of amino acid residue or subset of amino acid residues, was disposed at a peptide
  • Removal of each amino acid residue may be carried out with a variety of different techniques including, for example, Edman degradation or proteolytic cleavage.
  • Edman degradation may be used remove the terminal amino acid residue.
  • an enzyme may be used remove the terminal amino acid residue.
  • These terminal amino acid residues may be removed from either the C-terminus or the N-terminus of the peptide chain. In embodiments where Edman degradation is used, the amino acid residue at the N-terminus of the peptide chain is removed.
  • a fluorophore of the present disclosure may be configured to withstand conditions for removing one or more of amino acid residues from a peptide.
  • potential fluorophores that may be used in the instant polymers and methods include, for example, those which emit a fluorescence signal in the red to infrared spectra such as an Alexa Fluor® dye, an Atto dye, Janelia Fluor® dye, a rhodamine dye, or other similar dyes.
  • a fluorophore may include a fluorescent peptide (e.g., green fluorescent protein or a variant thereof) or an optically detectable material, such as a carbon nanotube, a nanorod, or a quantum dot.
  • Peptide (e.g., CDR-H3) detection or imaging may include immobilizing peptides on a surface.
  • peptides are immobilized such that each individual peptide is spatially distinguishable from other peptides e.g., in discrete spots), such that fluorescence signals from one individual peptide are spatially distinguishable from fluorescence signals from other individual peptide.
  • Peptides may be immobilized to the surface by any of a variety of methods, such as, for example, coupling a cysteine residue in the peptide, the peptide N- terminus, or the peptide C-terminus with the surface or with a reagent coupled to the surface.
  • Peptides may be immobilized, for example, by reacting the cysteine residue with the surface or with a capture reagent coupled to the surface. Detecting the immobilized peptide may include capturing an image including the peptide. The image may include a spatial address specific to the peptide. A plurality of peptides may be detected in a single image, wherein one or more of the peptides may include a spatial address within the image.
  • the surface is optically transparent across the visible spectrum and/or the infrared spectrum.
  • the surface possesses a low refractive index (e.g., a refractive index between 1 .3 and 1 .6).
  • the surface is between 10 to 50 nm thick, between 20 and 80 nm thick, between 50 and 200 nm thick, between 100 and 500 nm thick, between 200 and 800 nm thick, between 500 nm and 1 pm thick, between 1 and 5 pm thick, between 2 and 10 pm thick, between 5 and 20 pm thick, between 20 and 50 pm thick, between 50 and 200 pm thick, between 200 and 500 pm thick, or greater than 500 pm in thickness.
  • the surface is chemically resistant to organic solvents.
  • the surface is chemically resistant to strong acids such as trifluoroacetic acid or sulfuric acid.
  • any of large range of substrates like fluoropolymers (Teflon -AF (Dupont), Cytop® (Asahi Glass, Japan)), aromatic polymers (polyxylenes (Parylene, Kisco, Calif.), polystyrene, polymethmethylacrytate) and metal surfaces (Gold coating)), coating schemes (spin-coating, dip-coating, electron beam deposition for metals, thermal vapor deposition and plasma enhanced chemical vapor deposition) and functionalization methodologies (polyallylamine grafting, use of ammonia gas in PECVD, doping of long chain end -functionalized fluoroalkanes etc.) may be used in the methods described herein as a useful surface.
  • substrates like fluoropolymers (Teflon -AF (Dupont), Cytop® (Asahi Glass, Japan)
  • aromatic polymers polyxylenes (Parylene, Kisco, Calif.), polystyrene, polymethmethyla
  • a 20 nm thick, optically transparent fluoropolymer surface made of Cytop® may be used in the methods described herein.
  • surfaces used herein are further derivatized with a variety of fluoroalkanes that will sequester peptides for sequencing and modified targets for selection.
  • an aminosilane modified surfaces may be used in the methods described herein.
  • methods comprise immobilizing the peptides on the surface of beads, resins, gels, quartz particles, glass beads, or a combination thereof.
  • the methods contemplate using peptides that have been immobilized on the surface of Tentagel® beads, Tentagel® resins, or other similar beads or resins.
  • Surfaces may be coated with a polymer, such as polyethylene glycol. Surfaces may be amine functionalized or thiol functionalized.
  • a sequencing technique described herein may involve imaging peptides (e.g., CDR- H3 peptides) or polypeptides to determine the presence of one or more fluorophores on the labeled peptide.
  • Fluorosequencing may include imaging a plurality of peptides or polypeptides to determine the presence of one or more fluorophores on individual peptides from among a plurality of peptides.
  • fluorosequencing comprises imaging at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 or more peptides or polypeptides (e.g., imaging a portion of a surface comprising at least 10 3 to at least 10 8 peptides or polypeptides). These images may be taken after each removal of an amino acid residue and thus may enable determination of the location of the specific amino acid residue (or subset of amino acid residues) in the peptide.
  • a C-terminal immobilized peptide may comprise a sequence (from N- terminal to C-terminal) of KDDYAGGGAAGKDA (SEQ ID NO: 10, wherein 'K' denotes lysine, 'D' denotes aspartate, 'Y' denotes tyrosine, 'A' denotes alanine, and 'G' denotes glycine), and may comprise fluorophores coupled to each lysine and tyrosine residue.
  • a first image comprising the C-terminal immobilized peptide may indicate the presence of two lysines and one tyrosine in the peptide.
  • the N-terminal amino acid may be removed (e.g., by Edman degradation), such that a second image comprising the C -terminal immobilized peptide may indicate the presence of one lysine and one tyrosine in the peptide.
  • This process may be repeated until a sequence of KXXYXXXXXXXKX is identified for the peptide, wherein 'X' indicates a non-lysine, non-tyrosine amino acid, 'K' indicates a lysine, and 'Y indicates a tyrosine.
  • Fluorosequencing may comprise identifying the position of a specific amino acid residue in a peptide sequence.
  • Locations of specific amino acid residues in the peptide sequence or these results may be used to determine the entire list of amino acid residues in a peptide sequence.
  • Disclosed methods may involve determining the location of one or more amino acid residues in the peptide sequence and comparing these locations to known peptide sequences, which may identify the entire list of amino acid residues in the peptide sequence. This identification may be possible even in embodiments in which only a partial sequence is obtained by fluorosequencing.
  • Imaging methods may involve any of a variety of different spectrophotometric and microscopy methods, such as fluorimetry, diffuse reflectance, interferometric scattering Raman, resonance enhanced Raman, infrared absorbance, visible light absorbance, ultraviolet absorbance, and fluorescence.
  • Disclosed methods may employ such fluorescent techniques, such as fluorescence polarization, Forster resonance energy transfer (FRET), or time-resolved fluorescence.
  • FRET Forster resonance energy transfer
  • a spectrophotometric or microscopy method may be used to determine the presence of one or more fluorophores coupled to a single peptide.
  • imaging methods may be used to determine the presence or absence of a fluorophore on a specific peptide sequence. After repeated cycles of removing an amino acid residue and imaging a subject peptide, the position of the labeled amino acid residue may be determined in the peptide.
  • fluorescence pattern known a ‘fluorosequence’ is generated for each peptide.
  • ‘Fluorotypes’ a set of fluorosequences that match a given pattern - can be counted and used as quantitative markers for a peptide sequence or set of related peptide sequences. Fluorotypes can be contrasted across samples, and/or compared with information from a range of reference databases, whether derived from RNA sequencing or MS-MS analysis. Furthermore, these fluorotypes can guide the synthesis of therapeutic antibodies, paving the way for personalized treatments.
  • fluorotypes are counted, thereby allowing quantitative information regarding different CDR-H3 peptide species, which may therefore indicate antibody titers for multiple antibodies.
  • identification of the peptide or protein is based on only partial amino acid sequence information, such as in embodiments wherein only a subset of amino acid residues (not all of the types of amino acid residues) are labeled before the fluoro sequencing.
  • Partial amino acid sequence information including for example, a pattern of a specific amino acid residue (e.g., lysine) within individual peptide molecules, may be sufficient to uniquely identify an individual peptide molecule.
  • a pattern of amino acids such as X-X-X- Tyr-X-X-X-X-Tyr-X-Tyr, which indicates the distribution of tyrosine molecules within an individual peptide molecule, may be searched against a known proteome of a given organism to identify the individual peptide molecule. Sequencing of peptides at single molecule resolution is not limited to identifying the pattern of tyrosine residues in an individual peptide molecule; sequence information for any amino acid residue (including multiple amino acid residues) may be used to identify individual peptide molecules in a mixture of diverse peptide molecules. Numbered embodiments
  • Embodiment 1 A method for profiling patient-specific IgG repertoire, the method comprising:
  • Embodiment 2 The method of embodiment 1, wherein sequence of said peptides is determined using fluorosequencing.
  • Embodiment 3 The method of embodiment 1, wherein the sample is peripheral blood.
  • Embodiment 4 The method of embodiment 1, wherein the sample is selected from the group comprising of: spleen, bone marrow, saliva, and infiltrating tumor B-cells.
  • Embodiment 5 The method of embodiment 1, wherein the immunoglobulins are IgG molecules.
  • Embodiment 6 The method of embodiment 1, wherein the immunoglobulins are isolated using protein A/G beads.
  • Embodiment 7 The method of embodiment 1, wherein the digestion of the immunoglobulins cleaves a terminal lysine residue C-terminal [from] the CDR-H3 - FR4 region.
  • Embodiment 8 The method of embodiment 7, wherein the digestion is enzymatic using an enzyme selected from the group comprising of trypsin and lysC.
  • Embodiment 9 The method of embodiment 1, wherein the recognition site for digestion is lysine (K) in a ASTK sequence.
  • Embodiment 10 The method of embodiment 1, wherein the enrichment of CDR3- FR4 heavy chain peptides involves using a selective binder.
  • Embodiment 11 The method of embodiment 10, wherein the selective binder is a ASTK binder.
  • Embodiment 12 The method of embodiment 11, wherein said ASTK binder binds to ASTK sequence with varying affinity constants.
  • Embodiment 13 The method of embodiment 11, wherein said ASTK binder is immobilized to a solid support.
  • Embodiment 14 The method of embodiment 13, wherein said solid support is selected from a group comprising sepharose resin, glass, beads, surfaces, magnetic beads.
  • Embodiment 15 The method of embodiment 12, wherein the ASTK binder is covalently bound to the solid support.
  • Embodiment 16 The method of embodiment 12, wherein the solid support comprises a barcode for sample multiplexing.
  • Embodiment 17 The method of embodiment 16, wherein said barcode is a DNA sequence.
  • Embodiment 18 The method of embodiment 16, wherein said barcode is a peptide chain.
  • Embodiment 19 The method of embodiment 16, wherein said barcode distinguishes individual samples.
  • Embodiment 20 The method of embodiment 19, wherein each barcode is associated with an individual patient's sample.
  • Embodiment 21 The method of embodiment 19, wherein multiple individual samples are pooled and associated with a single barcode.
  • Embodiment 22 The method of embodiment 19, wherein the barcode distinguishes longitudinal samples from the same individual.
  • Embodiment 23 The method of embodiment 1, wherein the labeling involves using labels on amino acids.
  • Embodiment 24 The method of embodiment 23, wherein said amino acids are selected from the group comprising of: lysine, cysteine, aspartic acid, glutamic acid, methionine, and tyrosine.
  • Embodiment 25 The method of embodiment 23, wherein the label is a fluorophore.
  • Embodiment 26 The method of embodiment 25, wherein different amino acids are labeled with different fluorophores.
  • Embodiment 27 The method of embodiment 25, wherein fluorophores can span multiple channels.
  • Embodiment 28 The method of embodiment 1, wherein sample barcodes are read out through different assays on a flow cell.
  • Embodiment 29 The method of embodiment 28, wherein said assays are FISH (Fluorescent In-situ hybridization) with different DNA sequence fragments.
  • FISH Fluorescent In-situ hybridization
  • Embodiment 30 The method of embodiment 29, wherein each DNA sequence fragment contains a distinct fluorophore.
  • Embodiment 31 The method of embodiment 2, further comprising determining fluorotypes.
  • Embodiment 32 The method of embodiment 31, wherein said fluorotypes are associated with counts.
  • Embodiment 33 The method of embodiment 31, wherein said fluorotypes are represented as relative percentages.
  • Embodiment 34 The method of embodiment 31, wherein fluorotypes are matched to a reference database.
  • Embodiment 35 The method of embodiment 34, wherein the reference database is obtained from RNA sequencing of a B-cell receptor repertoire.
  • Embodiment 36 The method of embodiment 35, wherein said RNA sequencing is individualized to the patient sample.
  • Embodiment 37 The method of embodiment 35, wherein said RNA sequencing is derived from different B-cell populations including PBMCs and memory cells.
  • Embodiment 38 The method of embodiment 34, wherein the reference database is obtained from MS-MS analysis.
  • Embodiment 39 The method of embodiment 31, further comprising synthesizing therapeutic antibodies based on the determined fluorotypes.
  • Embodiment 40 The method of embodiment 39, wherein amino acids not labeled are introduced during antibody synthesis based on underlying amino acid frequency.
  • Embodiment 41 The method of embodiment 39, wherein amino acids not labeled are introduced randomly during antibody synthesis.
  • Embodiment 42 The method of embodiment 39, wherein amino acids not labeled are introduced based on B-cell sequences during antibody synthesis.
  • Embodiment 43 The method of embodiment 39, wherein amino acids not labeled are introduced based on MS-MS analysis during antibody synthesis.
  • Embodiment 44 The method of embodiment 39, wherein amino acids not labeled are introduced based on data from a database during antibody synthesis.
  • Embodiment 45 The method of embodiment 44, wherein said database is Opig.
  • Embodiment 46 An ASTK binder for use in the method of embodiment 1, wherein said binder binds to peptide fragments comprising a terminal ASTK sequence.
  • Embodiment 47 The ASTK binder of embodiment 46, wherein said peptides are generated after trypsin or lysC digestion.
  • Embodiment 48 The ASTK binder of embodiment 46, wherein said peptides represent the terminal amino acid sequence of the CDR3-FR4 fragment.
  • Embodiment 49 The ASTK binder of embodiment 46, wherein said binder binds with high affinity and is selective for the ASTK sequence over other sequences.
  • Embodiment 50 The ASTK binder of embodiment 46, generated through a process of directed evolution.
  • Embodiment 51 A solid support for use in the method of embodiment 1, wherein said support has immobilized thereon an ASTK binder and optionally comprises a barcode for sample multiplexing.
  • Embodiment 52 A method for identifying patient-specific CDR3-FR4 Heavy chain peptides, the method comprising:
  • Embodiment 53 A method for profiling peptides from a biological sample, comprising:
  • Embodiment 54 A method for characterizing immunoglobulin-derived peptides, comprising:
  • Embodiment 55 A method for analyzing patient-derived peptides, comprising:
  • Embodiment 56 A method for profiling patient-specific IgG repertoire, the method comprising the steps of:
  • fluorotypes (e) analyzing the enriched peptides through fluorosequencing, wherein the peptides are labeled with specific fluorophores binding to designated amino acids, and wherein the sequences derived from fluorosequencing are termed fluorotypes.
  • Embodiment 57 A method for generating a patient-specific IgG peptide profile, comprising the steps of:
  • Embodiment 58 A method for isolating and analyzing CDR3-FR4 heavy chain peptides, the method comprising:
  • Embodiment 59 A method for profiling the IgG repertoire and generating therapeutic antibodies, the method consisting of:
  • Embodiment 60 A method for deciphering patient-specific IgG peptide sequences, comprising:
  • GTLVTVSSASTK SEQ ID NO: 1
  • FR4 fourth heavy chain framework region
  • N-termini of the CDR-H3 region in FR3 was identified to contain a conserved motif terminating with “..YCA” (see Figure 2).
  • EXAMPLE 2 Enrichment of ASTK peptides using ASTK-binding rabbit polyclonal antibodies
  • Monoclonal antibodies can also be generated against ASTK-containing peptides and validated, for example, as described below.
  • TBS PBS buffer with 0.1% Triton-x detergent
  • 50 mM glycine buffer pH 2.5
  • Peptides observed in the released solution will be identified and quantified for each condition by LC- MS/MS.
  • conditions providing enrichment of at least 10:1 of “ASTK” terminated peptides will be used. This optimized protocol will be used with the peptide library to evaluate the stability and reproducibility of the antibody-conjugated beads.
  • Conjugated beads will be used to validate the workflow for enriching CDR-H3 peptides in IgG antibodies from standard serum samples. Briefly, IgG molecules will be isolated from serum (Sigma, Cat #NIST® SRM® 909c) using a protein A bead kit (Thermo Fiser, catalog #44667), IgG concentration will be measured sing IgG EEISA assay (Life Technologies, Cat # 991,000), IgG molecules will be digested wit lysC (Promega, catalog # VAI 170), and CDR-H3 peptides will be enriched with ASTK- selective antibody beads.
  • IgG molecules will be isolated from serum (Sigma, Cat #NIST® SRM® 909c) using a protein A bead kit (Thermo Fiser, catalog #44667), IgG concentration will be measured sing IgG EEISA assay (Life Technologies, Cat # 991,000), IgG molecules will be digested wit lysC (Promega, catalog
  • Amounts of input IgG protein will be varied, and beads lacking ASTK-selective antibodies will be used as a negative control.
  • the workflow for enriching CDR-H3 peptides will be validated by comparing the LC-MS/MS results of the peptide species released from the two bead sets.
  • Preparing peptide samples for fluorosequencing by labeling side chains of acidic and tyrosine residues with distinct fluorophores A sample preparation workflow will be performed to (a) modify CDR-H3 peptides C-termini with an alkyne moiety and (b) label the side chains of acidic (aspartate/glutamate) and tyrosine residues with fluorophores AttoRho-12 and Atto643. (See Example 3 regarding labeling of tyrosine residues.) Additionally, (a) biological replicates on the input serum sample and (b) reversed dye replicates (where the dyes labeling the respective amino acid residues are swapped) will be performed.
  • the present Example describes an example workflow for profiling immunoglobulin repertoires. (See Figure 4.)
  • Serum collection can be used for pipeline development. Serum can also be obtained from human subjects, e.g., for further development and/or in practicing methods of the present disclosure.
  • the present Example describes an example workflow for profiling immunoglobulin repertoires. (See Figure 4.)
  • Serum collection can be used for pipeline development. Serum can also be obtained from human subjects, e.g., for further development and/or in practicing methods of the present disclosure.
  • IgG molecules are extracted from serum using a protein A bead-based isolation method (Thermo Fisher, catalog # 44667).
  • Cysteine capping and protease digestion Cysteine residues are reduced and alkylated with 2-bromoethylamine such that cysteine residues resemble lysine residues.
  • Extracted IgG molecules are digested using lysC enzyme (Promega, Cat # VAI 170) to generate fragments terminating in lysine residues.
  • CDR-H3 peptides are enriched using ASTK- selective binders (e.g., binders developed as described in Examples 2 or 3).
  • fluorescently labeled peptide samples are prepared by conjugating the side chains of specific amino acids (e.g., one or a combination of the following: tyrosine, aspartic/glutamic acid, methionine, tryptophan, or arginine) with unique fluorophores. Fluorosequencing is then performed at the single molecule level to acquire approximately 100 million individual peptide reads.
  • specific amino acids e.g., one or a combination of the following: tyrosine, aspartic/glutamic acid, methionine, tryptophan, or arginine
  • fluorotypes Data analysis to determine the fluorotypes.
  • a list of fluorescent patterns (fluorosequences) obtained from the fluorosequencing is into a table detailing observed fluorosequences and their counts. These fluorosequences, representing one or more antibody clones with identical amino acid patterns in their CDR-H3 sequences, are defined as fluorotypes. Fluorotypes can be matched to a reference database if accurate B-cell receptor (BCR) sequences are available or used to track CDR-H3 sequence variations in longitudinal studies and comparison with antigen-enriched samples.
  • BCR B-cell receptor

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des procédés de profilage d'un répertoire d'immunoglobuline (par exemple, IgG) spécifique à un sujet, comprenant le fluoroséquençage de peptides CDR-H3.
PCT/US2025/013042 2024-01-25 2025-01-24 Procédés de profilage de répertoires d'immunoglobulines Pending WO2025160469A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463625266P 2024-01-25 2024-01-25
US63/625,266 2024-01-25

Publications (1)

Publication Number Publication Date
WO2025160469A1 true WO2025160469A1 (fr) 2025-07-31

Family

ID=96545890

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/013042 Pending WO2025160469A1 (fr) 2024-01-25 2025-01-24 Procédés de profilage de répertoires d'immunoglobulines

Country Status (1)

Country Link
WO (1) WO2025160469A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130178370A1 (en) * 2011-11-23 2013-07-11 The Board Of Regents Of The University Of Texas System Proteomic identification of antibodies
US9090674B2 (en) * 2010-05-17 2015-07-28 The Board Of Regents Of The University Of Texas System Rapid isolation of monoclonal antibodies from animals
WO2022192661A1 (fr) * 2021-03-12 2022-09-15 Board Of Regents, The University Of Texas System Anticorps neutralisant le sras-cov-2 et leurs utilisations
WO2022251457A2 (fr) * 2021-05-26 2022-12-01 Board Of Regents, The University Of Texas System Compositions, procédés et utilité de codes-barres biomoléculaires conjugués
WO2023056414A1 (fr) * 2021-10-01 2023-04-06 Board Of Regents, The University Of Texas System Profilage structural de protéines natives à l'aide de fluoroséquençage, une technologie de séquençage de protéine à une seule molécule
WO2023091961A2 (fr) * 2021-11-17 2023-05-25 Erisyon Inc. Méthodes et systèmes de traitement d'échantillons automatisés

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9090674B2 (en) * 2010-05-17 2015-07-28 The Board Of Regents Of The University Of Texas System Rapid isolation of monoclonal antibodies from animals
US20130178370A1 (en) * 2011-11-23 2013-07-11 The Board Of Regents Of The University Of Texas System Proteomic identification of antibodies
WO2022192661A1 (fr) * 2021-03-12 2022-09-15 Board Of Regents, The University Of Texas System Anticorps neutralisant le sras-cov-2 et leurs utilisations
WO2022251457A2 (fr) * 2021-05-26 2022-12-01 Board Of Regents, The University Of Texas System Compositions, procédés et utilité de codes-barres biomoléculaires conjugués
WO2023056414A1 (fr) * 2021-10-01 2023-04-06 Board Of Regents, The University Of Texas System Profilage structural de protéines natives à l'aide de fluoroséquençage, une technologie de séquençage de protéine à une seule molécule
WO2023091961A2 (fr) * 2021-11-17 2023-05-25 Erisyon Inc. Méthodes et systèmes de traitement d'échantillons automatisés

Similar Documents

Publication Publication Date Title
US20220163536A1 (en) Identifying peptides at the single molecule level
US12379380B2 (en) Single-molecule protein and peptide sequencing
US20240192221A1 (en) Protein sequencing method and reagents
US20150087526A1 (en) Peptide identification and sequencing by single-molecule detection of peptides undergoing degradation
US10571473B2 (en) Methods of polypeptide identification, and compositions therefor
JP5350215B2 (ja) 複合タンパク質混合物中の被分析タンパク質および／または被分析ペプチドを検出および／または濃縮するための方法
JP2023527149A (ja) ポリペプチドの処理および分析のための方法、システムおよびキット
US20230104998A1 (en) Single-molecule protein and peptide sequencing
WO2025160469A1 (fr) Procédés de profilage de répertoires d'immunoglobulines
WO2024076928A1 (fr) Conjugués fluorophore-polymère et leurs utilisations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25745827

Country of ref document: EP

Kind code of ref document: A1