WO2025160469A1 - Methods for profiling immunoglobulin repertoires - Google Patents

Abstract

Methods of profiling an immunoglobulin (e.g., IgG) repertoire specific to a subject, comprising fluorosequencing CDR-H3 peptides.

Description

METHODS FOR PROFILING IMMUNOGLOBULIN REPERTOIRES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of and priority to U.S. Provisional Patent Application No. US 63/625,266 filed January 25, 2024, the entire contents of which are hereby incorporated by reference for all purposes.

BACKGROUND

[0002] 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.

[0003] Despite strides in improved sample preparation and sensitivity, current methods to assess a given subject’s antibody repertoire are limited due to (i) high sequence diversity (~10⁸) and concentration range (~10'⁶M to 10'¹²M) of antibody species, (ii) difficulty in constructing an accurate protein reference database, and (iii) challenges in matching readouts (e.g., mass spectra) to unique peptides due to the presence of highly conserved sequences with intervening hypervariable sequences within immunoglobulin molecules. All these factors make it challenging to obtain well-quantified and deep identities of antibodies.

[0004] Thus, there remains a need for improved immunoglobulin (e.g., IgG) profiling methods.

SUMMARY

[0005] 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.

[0006] In one aspect, provided are method of profiling an immunoglobulin repertoire specific to a subject, the method comprising the steps of: a) obtaining or having obtained a composition enriched for CDR-H3 peptides; and b) fluoro sequencing said CDR-H3 peptides, thereby obtaining information regarding the subject’s immunoglobulin repertoire. [0007] In some embodiments, 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.

[0008] In one aspect, provided are methods of profiling an immunoglobulin repertoire, the method 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.

[0009] In some embodiments, 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.

[0010] In some embodiments, the immunoglobulin molecules are isolated from the biological sample by a method employing particles coated with immunoglobulin-binding polypeptides. In some embodiments, the immunoglobulin-binding polypeptides comprise protein A, protein G, or a combination thereof. In some embodiments, the particles comprise protein A/G beads.

[0011] In some embodiments, prior to the step of digesting, said isolated immunoglobulin molecules are contacted with an agent that modifies one or more amino acid residues. In some embodiments, the agent modifies an amino acid residue to resemble another amino acid residue. In some embodiments, the agent modifies cysteine residues to resemble lysine residues, e.g., 2-bromoethylamine.

[0012] In some embodiments, the step of digesting comprises digesting with an enzyme, e.g., an endoprotease. In some embodiments, the endoprotease is selected from the group consisting of trypsin and lysC. In some embodiments, the endoprotease cleaves C-terminal to a lysine, e.g., C-terminal to the amino acid sequence ASTK. In some embodiments, the step of enriching comprises using an isotype-selective binder, e.g., an IgG-selective binder. In some embodiments, the IgG-selective binder is capable of selectively binding the amino acid sequence ASTK. In some embodiments, 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.

[0013] In some embodiments, the solid support is attached to a identifier unique to the subject. In some embodiments, the identifier is unique to the biological sample from the subject. In some embodiments, the identifier comprises an oligonucleotide. In some embodiments, the identifier comprises a peptide.

[0014] In some embodiments, the obtained information comprises quantitative information. [0015] In some embodiments, fluoro sequencing is conducted at single molecule resolution. In some embodiments, the obtained information comprises sequence information at the single molecule level.

[0016] In some embodiments, wherein the step of fluorosequencing comprises labeling a subset of amino acid residues in said enriched CDR-H3 peptides with a fluorophore.

[0017] In some embodiments, 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. In some embodiments, the subset of amino acid residues comprises tyrosine residues. In some embodiments, each type amino acid residue in the subset is labeled with a different fluorophore. In some embodiments, each fluorophore’ s emission spectrum is distinguishable from each other. In some embodiments, the step of fluorosequencing comprises counting fluorotypes.

[0018] In one aspect, provided are methods of profiling an immunoglobulin G (IgG) repertoire specific to a subject, the method 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.

[0019] In some embodiments, provided methods, further comprise a step of e) comparing fluorotypes to sequences in a reference database.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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). [0021] 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.

[0022] 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.

[0023] Figure 4 depicts a schematic of a pipeline for methods disclosed herein for assessing a subject’s IgG repertoire.

[0024] Figure 5A depicts the enrichment ratios of precursor abundance for various peptides across the F(ab’) region, observed across three samples.

[0025] Figure 5B depict an ion intensity trace which showing increased counts for two rabbit clones (R36 and R33) as compared to an IgG control.

[0026] 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.

[0027] Figure 6B depicts a histogram of CDR-H3 length distributions. The median peptide length was 14 amino acid residues.

[0028] 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.)

[0029] Figure 7 depicts proposed reactions (Mannich-type reactions and diazonium reactions) that will be screened for tyrosine residue conjugation.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0030] 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). In many embodiments of the disclosed methods, 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.

[0031] The presently disclosed methods encompass the inventors’ recognition that 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. For example, IgG molecules include a characteristic ASTK amino acid sequence C-terminal to the CDR-H3. In some embodiments, 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.

[0032] 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.

Definitions

[0033] The terms “about” or “approximately,” when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0034] As used herein, the term “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. In some embodiments, 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.

[0035] As used herein, 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.

[0036] As used herein, the term “Edman degradation” generally refers to methods comprising chemical removal of amino acids from peptides or proteins. In some cases, Edman degradation denotes terminal (e.g., N- or C-terminal) amino acid removal. In some embodiments, 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. In some embodiments, Edman degradation broadly encompasses N-terminal amino acid functionalizations leading to N-terminal amino acid removal. In some cases, Edman degradation encompasses C-terminal amino acid removal. In some cases, 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).

[0037] As used herein, the term “fluorescence” refers to the emission of light of a particular wavelength by a substance that has absorbed light of a different wavelength. In some embodiments, 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, nucleic acid, oligonucleotides (including single stranded and double stranded primers) may be “labeled” with a variety of extrinsic fluorescent molecules referred to as fluorophores. Isothiocyanate derivatives of fluorescein, such as carboxyfluorescein, are a non-limiting example of fluorophores that may be conjugated to proteins (such as antibodies for immunohistochemistry) or nucleic acids. In some embodiments, fluorescein may be conjugated to nucleoside triphosphates and incorporated into nucleic acid probes (such as “fluorescent-conjugated primers”) for in situ hybridization. In some embodiments, a molecule that is conjugated to carboxyfluorescein is referred to as “F AM-labeled.”

[0038] As used herein, the term “fluorosequence” refer 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.

[0039] As used herein, the term “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. In some embodiments, such as some embodiments involving fluorosequencing of peptides (e.g., CDR-H3 peptides) obtained from immunoglobulin molecules (e.g., IgG molecules), each fluorotype represents one or more antibody clones with identical amino acid patterns in their CDR-H3 sequences.

[0040] The term “polypeptide,” as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids, e.g., linked to each other by peptide bonds. Those of ordinary skill in the art will appreciate that the term “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. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. 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. Useful modifications include, e.g., terminal acetylation, amidation, methylation, glycosylation etc. In some embodiments, 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. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0041] As used herein, the phrase “profiling an immunoglobulin repertoire” or “profiling an antibody repertoire” of a subject, and like terms, refer to cataloging the various immunoglobulin sequences circulating in a subject, e.g., as determined by assessing a biological sample from the subject. Thus, “profiling an IgG repertoire” of a subject refers to cataloging the IgG sequences circulating in a subject. As described herein, in many embodiments, profiling an immunoglobulin repertoire comprises cataloging CDR-H3 sequences from immunoglobulins circulating in a subject.

[0042] As used herein, 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. In some embodiments, 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. For example, 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.

[0043] As used herein, 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. As non-limiting examples, 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. As an alternative, 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. Some non-limiting examples of 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. In some examples of functionalized beads, the functional motif may be reversibly coupled and cleaved. A functional motif may also irreversibly couple to a molecule.

Methods

Samples

[0044] Any of a variety of biological samples may be used in accordance with methods disclosed herein. Generally, the biological sample will be a sample that is expected to contain, or suspected of containing, immunoglobulin molecules, such as IgG molecules. Non-limiting examples of such biological samples include blood (e.g., peripheral blood), serum, spleen, bone marrow, cerebrospinal fluid, tumor biopsies, and combinations thereof. In some embodiments, the biological sample comprises tumor-infiltrating B-cells.

Isolation

[0045] 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. For example, particles may be coated with polypeptides having affinity for immunoglobulin molecules or specific isotypes of immunoglobulin molecules, such as IgG. For example, protein A/G has a selected affinity for IgG. In some embodiments, the particles comprises protein A/G beads. Modification and/or Digestion

[0046] In some embodiments, prior to the step of digestion, isolated immunoglobulin molecules are contacted with an agent that chemically modifies one or more amino acid residues. For example, in some embodiments, the agent reduces and/or alkylates the amino acid residue. In some embodiments, the agent modifies an amino acid residue to resemble or become another amino acid residue. For example 2-bromoethylamine can be used to reduce and alkylate cysteine residues such that the residues then resemble lysine residues.

[0047] 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. In some embodiments, endoproteases with known recognition sites which include relatively rare amino acid residues within CDR-H3 sequences are used. For example, cysteine and lysine resides are present in abundances of less than 1% within CDR- H3 sequences.

[0048] In some embodiments, 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.

[0049] In some embodiments, such as in embodiments wherein the immunoglobulin molecules of interest are IgG molecules, the endoprotease cleaves C-terminal to a lysine residue, e.g., a lysine residue within the amino acid sequence ASTK. In some embodiments, the endoprotease comprises trypsin, lysC, or a combination thereof.

Enrichment

[0050] 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. In embodiments in which an isotype- selective binder is used, 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.

[0051] For example, in some embodiments, the isotype-selective binder is an IgG-selective binder which selectively binds the amino acid sequence ASTK.

[0052] In some embodiments, the isotype-selective binder comprises an antibody or an antigen-binding fragment thereof. Labeling

[0053] In certain embodiments, 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.

[0054] In some embodiments, 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.

[0055] In some embodiments, 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.

[0056] Non-limiting examples of labeling schemes are depicted in Figure 6C, for example,:

• Scheme 1: First fluorophore: aspartic acid and glutamic acid residues; second fluorophore: tyrosine residues

• Scheme 2: First fluorophore: serine and threonine residues; second fluorophore: aspartic acid and glutamic acid residues

• Scheme 3: Scheme 2, plus third fluorophore: arginine residues

• Scheme 4: Scheme 1, plus third fluorophore: arginine residues

• Scheme 5: Scheme 1, plus third fluorophore: serine and threonine residues

• Scheme 6: Scheme 5, plus fourth fluorophore

• Scheme 7: First fluorophore: serine and threonine residues; second fluorophore: tyrosine residues; third fluorophore: arginine residues; fourth fluorophore: asparagine and glutamine residues

[0057] To label a particular amino acid residue or subset of amino acid residues, 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. For example, 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. As another non-limiting example, 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.

[0058] In some embodiments, each fluorophore used in the labeling scheme is solventstable. In some embodiments, the fluorophores that are used collectively span the visible spectra.

[0059] In some embodiments, the fluorophore is an Alexa Fluor® dye, an Atto dye, a Janelia Fluor® dye, a carbopyronine derivative, a Rhodamine derivative, or any combination thereof. [0060] In some embodiments, 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, Janelia Fluor® 479, Janelia Fluor® 525, Janelia Fluor® 549, Janelia Fluor® 555, Janelia Fluor® 579, SF554, Texas Red, JFX 554, JFX 650, CF® 398, CF® 430, CF® 568, CF® 633, CF® 640R, CF® 680R, SarafluorTM 488B, SarafluorTM 650B, Rhodamine, Rhodamine 110 (5 -CR110), Rhodamine 6G, Rhodamine B, carboxyrhodamine B, tetramethylrhodamine (TMR), Rhodamine 101, Rhodamine Si, fluorescein, 5 -carb oxy fluorescein, napthofluorescein, 6-JOE-N3, 7- hydroxycoumarin-N3, Cy3 Cy5, Cy3B, Cy5B, Cy488, DylightTM405, DylightTM488, iFluor® 710, DY350XE, DY351XE, DY360XE, DY370XE, DY376XE, DY380XE, DY396XE, DY720, BodipyTM493, BodipyTMFE, BodipyTM650, PB430 (Phoxbri^Ait 430), Pyrene-N3, Eucifer Yellow, Nanohoop 6, Nanohoop 8, Etemeon 394, HilyteTM 405, HilyteTM 488, HilyteTM 647, or any combination thereof.

[0061] In some embodiments, 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.

[0062] In some embodiments, the fluorophore is Texas Red, Janelia Fluor® 549, Alexa Fluor® 555, Atto643, or any combination thereof.

[0063] In some embodiments, 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. 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.

Fluorosequencing

[0064] 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. 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. Alternatively or additionally, 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.

[0065] A fluorophore of the present disclosure may be configured to withstand conditions for removing one or more of amino acid residues from a peptide. Some non-limiting examples of 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. Examples of each of these dyes which are capable of withstanding the conditions of removing the amino acid residues include Texas Red, Alexa Fluor® 405, Rhodamine B, tetramethyl rhodamine, Janelia Fluor® 525, Janelia Fluor® 549, Janelia Fluor® 555, Alexa Fluor® 448, Alexa Fluor® 555, Atto495, Atto643, Atto647N, AttoRhol2, Rhodamine, tetramethylrhodamine, and (5)6- napthofluorescein. 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.

[0066] Peptide (e.g., CDR-H3) detection or imaging may include immobilizing peptides on a surface. In some embodiments, 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. In some embodiments, the surface is optically transparent across the visible spectrum and/or the infrared spectrum. In some embodiments, the surface possesses a low refractive index (e.g., a refractive index between 1 .3 and 1 .6). In some embodiments, 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. In some embodiments, the surface is chemically resistant to organic solvents. In some embodiments, 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. For example, a 20 nm thick, optically transparent fluoropolymer surface made of Cytop® may be used in the methods described herein. In some embodiment, surfaces used herein are further derivatized with a variety of fluoroalkanes that will sequester peptides for sequencing and modified targets for selection. Alternatively or additionally, an aminosilane modified surfaces may be used in the methods described herein.

[0067] In some embodiments, methods comprise immobilizing the peptides on the surface of beads, resins, gels, quartz particles, glass beads, or a combination thereof. In some nonlimiting examples, 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.

[0068] 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. In some embodiments, fluorosequencing comprises imaging at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, at least 10⁸ or more peptides or polypeptides (e.g., imaging a portion of a surface comprising at least 10³ to at least 10⁸ 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. For example, 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.

[0069] 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. A spectrophotometric or microscopy method may be used to determine the presence of one or more fluorophores coupled to a single peptide. Such 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.

Analysis and application of sequencing data

[0070] As peptides are sequenced, a 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.

[0071] In some embodiments, fluorotypes are counted, thereby allowing quantitative information regarding different CDR-H3 peptide species, which may therefore indicate antibody titers for multiple antibodies.

[0072] In some embodiments, 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. For example, 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

[0073] Embodiment 1. A method for profiling patient-specific IgG repertoire, the method comprising:

(a) collecting a sample comprising immunoglobulins;

(b) isolating said immunoglobulins;

(c) digesting said immunoglobulins to generate CDR3-FR4 heavy chain peptides;

(e) enriching said CDR3-FR4 heavy chain peptides;

(f) labeling said peptides; and

(g) determining the sequence of said peptides.

[0074] Embodiment 2. The method of embodiment 1, wherein sequence of said peptides is determined using fluorosequencing.

[0075] Embodiment 3. The method of embodiment 1, wherein the sample is peripheral blood.

[0076] 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.

[0077] Embodiment 5. The method of embodiment 1, wherein the immunoglobulins are IgG molecules.

[0078] Embodiment 6. The method of embodiment 1, wherein the immunoglobulins are isolated using protein A/G beads.

[0079] 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.

[0080] Embodiment 8. The method of embodiment 7, wherein the digestion is enzymatic using an enzyme selected from the group comprising of trypsin and lysC.

[0081] Embodiment 9. The method of embodiment 1, wherein the recognition site for digestion is lysine (K) in a ASTK sequence.

[0082] Embodiment 10. The method of embodiment 1, wherein the enrichment of CDR3- FR4 heavy chain peptides involves using a selective binder.

[0083] Embodiment 11. The method of embodiment 10, wherein the selective binder is a ASTK binder.

[0084] Embodiment 12. The method of embodiment 11, wherein said ASTK binder binds to ASTK sequence with varying affinity constants.

[0085] Embodiment 13. The method of embodiment 11, wherein said ASTK binder is immobilized to a solid support. [0086] Embodiment 14. The method of embodiment 13, wherein said solid support is selected from a group comprising sepharose resin, glass, beads, surfaces, magnetic beads.

[0087] Embodiment 15. The method of embodiment 12, wherein the ASTK binder is covalently bound to the solid support.

[0088] Embodiment 16. The method of embodiment 12, wherein the solid support comprises a barcode for sample multiplexing.

[0089] Embodiment 17. The method of embodiment 16, wherein said barcode is a DNA sequence.

[0090] Embodiment 18. The method of embodiment 16, wherein said barcode is a peptide chain.

[0091] Embodiment 19. The method of embodiment 16, wherein said barcode distinguishes individual samples.

[0092] Embodiment 20. The method of embodiment 19, wherein each barcode is associated with an individual patient's sample.

[0093] Embodiment 21. The method of embodiment 19, wherein multiple individual samples are pooled and associated with a single barcode.

[0094] Embodiment 22. The method of embodiment 19, wherein the barcode distinguishes longitudinal samples from the same individual.

[0095] Embodiment 23. The method of embodiment 1, wherein the labeling involves using labels on amino acids.

[0096] 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.

[0097] Embodiment 25. The method of embodiment 23, wherein the label is a fluorophore. [0098] Embodiment 26. The method of embodiment 25, wherein different amino acids are labeled with different fluorophores.

[0099] Embodiment 27. The method of embodiment 25, wherein fluorophores can span multiple channels.

[0100] Embodiment 28. The method of embodiment 1, wherein sample barcodes are read out through different assays on a flow cell.

[0101] Embodiment 29. The method of embodiment 28, wherein said assays are FISH (Fluorescent In-situ hybridization) with different DNA sequence fragments.

[0102] Embodiment 30. The method of embodiment 29, wherein each DNA sequence fragment contains a distinct fluorophore. [0103] Embodiment 31. The method of embodiment 2, further comprising determining fluorotypes.

[0104] Embodiment 32. The method of embodiment 31, wherein said fluorotypes are associated with counts.

[0105] Embodiment 33. The method of embodiment 31, wherein said fluorotypes are represented as relative percentages.

[0106] Embodiment 34. The method of embodiment 31, wherein fluorotypes are matched to a reference database.

[0107] Embodiment 35. The method of embodiment 34, wherein the reference database is obtained from RNA sequencing of a B-cell receptor repertoire.

[0108] Embodiment 36. The method of embodiment 35, wherein said RNA sequencing is individualized to the patient sample.

[0109] Embodiment 37. The method of embodiment 35, wherein said RNA sequencing is derived from different B-cell populations including PBMCs and memory cells.

[0110] Embodiment 38. The method of embodiment 34, wherein the reference database is obtained from MS-MS analysis.

[0111] Embodiment 39. The method of embodiment 31, further comprising synthesizing therapeutic antibodies based on the determined fluorotypes.

[0112] Embodiment 40. The method of embodiment 39, wherein amino acids not labeled are introduced during antibody synthesis based on underlying amino acid frequency.

[0113] Embodiment 41. The method of embodiment 39, wherein amino acids not labeled are introduced randomly during antibody synthesis.

[0114] Embodiment 42. The method of embodiment 39, wherein amino acids not labeled are introduced based on B-cell sequences during antibody synthesis.

[0115] Embodiment 43. The method of embodiment 39, wherein amino acids not labeled are introduced based on MS-MS analysis during antibody synthesis.

[0116] Embodiment 44. The method of embodiment 39, wherein amino acids not labeled are introduced based on data from a database during antibody synthesis.

[0117] Embodiment 45. The method of embodiment 44, wherein said database is Opig.

[0118] 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.

[0119] Embodiment 47. The ASTK binder of embodiment 46, wherein said peptides are generated after trypsin or lysC digestion. [0120] Embodiment 48. The ASTK binder of embodiment 46, wherein said peptides represent the terminal amino acid sequence of the CDR3-FR4 fragment.

[0121] 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.

[0122] Embodiment 50. The ASTK binder of embodiment 46, generated through a process of directed evolution.

[0123] 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.

[0124] Embodiment 52. A method for identifying patient-specific CDR3-FR4 Heavy chain peptides, the method comprising:

(a) obtaining a sample from a patient;

(b) processing the sample to yield immunoglobulin-derived peptides;

(c) selectively binding said peptides using a specific binder; and

(d) profiling the bound peptides through a sequencing technique.

[0125] Embodiment 53. A method for profiling peptides from a biological sample, comprising:

(a) obtaining said sample;

(b) processing the sample to yield a peptide mixture;

(c) selectively enriching specific peptides from the mixture using a binder; and

(d) sequencing the enriched peptides.

[0126] Embodiment 54. A method for characterizing immunoglobulin-derived peptides, comprising:

(a) treating a sample to release peptides;

(b) selectively binding said peptides with a custom binder; and

(c) analyzing the bound peptides.

[0127] Embodiment 55. A method for analyzing patient-derived peptides, comprising:

(a) processing a sample to yield peptides;

(b) utilizing a binder for specific peptide enrichment; and

(c) obtaining peptide sequences.

[0128] Embodiment 56. A method for profiling patient-specific IgG repertoire, the method comprising the steps of:

(a) obtaining a biological sample, wherein the sample is selected from the group consisting of peripheral blood, spleen, bone marrow, saliva, and infiltrating tumor B-cells; (b) isolating IgG molecules from said sample using Protein A/G beads;

(c) enzymatically digesting the isolated IgG molecules to target a sequence adjacent to CDR3, wherein the enzyme is selected from the group consisting of trypsin and lysC, and wherein the targeted sequence comprises the peptide sequence “ASTK”;

(d) selectively enriching CDR3-FR4 heavy chain peptides from the digested IgG molecules using an ASTK binder that binds to the “ASTK” sequence; and

(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.

[0129] Embodiment 57. A method for generating a patient-specific IgG peptide profile, comprising the steps of:

(a) collecting a biological sample wherein infiltrating tumor B-cells are included;

(b) employing Protein A/G beads to isolate IgG molecules from the sample;

(c) utilizing enzymes, including trypsin or lysC, to cleave the IgG molecules to reveal a terminal lysine residue C-terminal to the CDR-H3 - FR4 region;

(d) employing an ASTK binder, capable of binding to an “ASTK” sequence with varied affinity constants, to selectively enrich the CDR3-FR4 peptides; and

(e) implementing a fluorosequencing method wherein the enriched peptides are labeled using fluorophores corresponding to amino acids such as lysine, cysteine, aspartic and glutamic acid, methionine, and tyrosine, and wherein the resultant sequences are distinguished and categorized as fluorotypes.

[0130] Embodiment 58. A method for isolating and analyzing CDR3-FR4 heavy chain peptides, the method comprising:

(a) obtaining a sample selected from the group consisting of peripheral blood, bone marrow, and saliva;

(b) extracting IgG molecules using Protein A/G beads or other isolation methods compatible with different Ig classes;

(c) enzymatically digesting the extracted IgG molecules wherein trypsin targets the specific “ASTK” sequence;

(d) employing a custom ASTK binder, covalently bound to a solid support selected from the group consisting of sepharose resin, glass, beads, surfaces, and magnetic beads, to specifically bind and enrich CDR3-FR4 heavy chain peptides; and

(e) decoding the peptide sequences through a fluorosequencing procedure, wherein amino acids within the peptides are labeled with distinct fluorophores and the sequences are represented as fluorotypes, which are then matched with a reference database obtained from RNA sequencing of a B-cell receptor repertoire or MS-MS analysis.

[0131] Embodiment 59. A method for profiling the IgG repertoire and generating therapeutic antibodies, the method consisting of:

(a) drawing a biological sample, specifically peripheral blood or infiltrating tumor B-cells;

(b) isolating IgG molecules from the sample through Protein A/G beads;

(c) applying enzymes such as trypsin to digest the IgG molecules to reveal the “ASTK” sequence adjacent to CDR3;

(d) utilizing an ASTK binder, immobilized to a solid support which can support a barcode for sample multiplexing, to selectively bind and enrich the CDR3-FR4 heavy chain peptides;

(e) performing fluorosequencing, wherein the peptides are tagged with specific fluorophores, and the derived sequences are termed fluorotypes; and

(f) synthesizing therapeutic antibodies based on identified fluorotypes wherein unlabeled amino acids are introduced during synthesis using reference databases such as Opig.

[0132] Embodiment 60. A method for deciphering patient-specific IgG peptide sequences, comprising:

(a) procuring a biological sample, including compartments like spleen or saliva;

(b) isolating IgG molecules or other Ig class molecules from the sample;

(c) enzymatically processing the IgG molecules to highlight a terminal lysine residue adjacent to the CDR3 region;

(d) applying a custom ASTK binder, which is capable of binding to an “ASTK” sequence, and is immobilized to a solid support that may contain a barcode for sample distinction, to enrich the desired CDR3-FR4 heavy chain peptides; and

(e) executing a comprehensive fluorosequencing method wherein the enriched peptides undergo labeling with specific fluorophores, and the resultant fluorescence patterns, termed fluorosequences, are processed and interpreted as quantifiable fluorotypes. EXAMPLES

EXAMPLE 1: Analysis of CDR-H3-adjacent sequences

[0133] A distinct highly conserved sequence (GTLVTVSSASTK” (SEQ ID NO: 1)) was identified in the fourth heavy chain framework region (FR4) of IgG molecules. Additionally, the N-termini of the CDR-H3 region in FR3 was identified to contain a conserved motif terminating with “..YCA” (see Figure 2).

[0134] To extract peptides containing CDR-H3, it is proposed to (1) convert cysteine residues to lysine residues by treatment with 2-bromoethylamine, and (2) digest peptides with lysC protease, thereby cleaving just N-terminal to the CDR-H3 region.

[0135] Sequences in FR4, which is adjacent to the CDR-H3 region, were further analyzed across different isotype. Table 1 lists N-terminal motif sequences in heavy chain FR4 for each immunoglobulin isotype. Based on this analysis, an agent which binds to the “ASTK” motif with high specificity would enable selective enrichment of human-derived IgG isotypes. Additional isotype-specific affinity binders could be developed to catalog the diversity of their respective CDR-H3 peptides using information in Table 1.

Table 1: Heavy chain FR4 N-terminal motif sequences by immunoglobulin isotype

EXAMPLE 2: Enrichment of ASTK peptides using ASTK-binding rabbit polyclonal antibodies

[0136] To generate polyclonal antibodies that bind ASTK, rabbits were inoculated with antigenic peptide CVTVSSASTK (SEQ ID NO: 9). High antibody titers (1:512,000) were confirmed using an enzyme-liked immunosorbent (ELISA) assay. Purified custom antibody containing a highly ionizable CDR-H3 peptide fragment was alkylated and trypsin digested. Peptide fragments were incubated with three polyclonal antibodies (IgG control negative control, and two rabbit clones (R33 and R36)) cross-linked to agarose beads (AminoLink Plus Resin). Eluted peptides were quantified using tandem mass spectrometry. For peptides constituting the FR2 to FR4 region of the antibody and peptides observed across all the 3 samples, an approximately 5- to 8- fold increase was observed in the precursor abundance for the “ASTK” containing peptide fragment when compared to the other peptide fragments, whereas the IgG control did not exhibit this level of enrichment (Figures 5A and 5B).

EXAMPLE 3: Development and validation of ASTK-binding monoclonal antibodies

[0137] Monoclonal antibodies can also be generated against ASTK-containing peptides and validated, for example, as described below.

[0138] Optimize enrichment conditions of peptide library by monoclonal antibodies. Monoclonal antibodies will be covalently immobilized to magnetic beads (Dynabeads M270 epoxy, Thermo cat #1431 ID); conditions for optimal enrichment will be tested with the previously described peptide library. A fixed amount (10 pg) of peptides and varying (a) bead concentrations (5 pL. 10 pL, and 50 pL of slurry in 100 pL Phosphate Buffer Saline (PBS)) and (b) time/temperature at 30 min at room temperature (RT), four hours at RT, 16 hours at RT and overnight at 4 °C will be tested. Beads will be washed extensively with TBS (PBS buffer with 0.1% Triton-x detergent) followed by a 50 mM glycine buffer (pH 2.5) release. Peptides observed in the released solution will be identified and quantified for each condition by LC- MS/MS. In further experiments, 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.

[0139] Validate enrichment of CDR-H 3 peptides from serum using monoclonal antibodies. 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. 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.

EXAMPLE 4: Analysis of CDR-H3 sequences

[0140] In a typical fluoro sequencing method, peptides are sequenced through their first twenty amino acids.

[0141] These methods typically require sufficient labelable amino acids to produce distinct patterns. Furthermore, the accuracy of peptide matching depends on the database, which is challenging for immune-repertoire analysis, as antibodies mainly originate from long-lived plasma cells in the bone marrow, rather than B-cells that are circulating at the time of sampling. [0142] This Example tests whether the limitations of fluoro sequencing could affect the success of the proposed profiling methods disclosed herein.

[0143] Publicly deposited B-cell sequences in the Observed Antibody space (OAS) databases from multiple studies were used for simulations. The median length of CDR-H3 peptides was found to be 14 amino acids (Figure 6A) and has a biased frequency distribution of amino acids (Figure 6B). Simulating different potential combinations of labeled amino acids showed that more than 85% of peptides would be uniquely identifiable with an appropriate choice of labels (see schemes VI and VII in Figure 6C). These simulations indicate that fluorotypes and their associated counts would provide for a rich and new type of data.

EXAMPLE 5: Development of a tyrosine-specific bioconjugation method for fluorosequencing

[0144] As CDR-H3 regions contain approximately 12% tyrosine residues (Figure 6B), it is proposed that labeling tyrosine residues with fluorophores to map their positions would produce a rich diversity of fluorotypes. Out of nine different bioconjugation strategies to label tyrosine residues, two strategies - Mannich type reaction and diazonium - are proposed for evaluation due to their mild reaction conditions (pH 7.5; 1-2 h) and previous evidence of success with conjugating fluorophores. (See Figure 7.)

[0145] Selecting between Mannich-type and diazonium bioconjugation strategies for improved efficiency. To screen the two methods, two tyrosine-containing peptides with different hydrophobicity and which do not contain serine, threonine, lysine, or cysteine residues will be used. Yield (amount of tyrosine modified/total input peptide) and conversion efficiency (% of modified peptides) of the reactions between the peptides and conjugating reagent (R- azide; synthesized by X-chem) will be measured by LC-MS. For each of the methods, various conditions and reagents will be tested: (i) concentrations of the conjugating reagent (1, 2, 5, and 10 equivalents), (ii) different solvents (as they may impact resin swelling and reagent solubility), and (iii) buffer percentages (10, 25 and 50%). Conditions that maximize the efficiency of tyrosine coupling will be used in subsequent steps.

[0146] Screen conditions for improved selectivity of the reaction. Using different combinations of tyrosine-containing peptides with 1, 2, or 5 serine, threonine, and lysine residues, conditions will be optimized for tyrosine specificity (% of tyrosine label/all labeled residues). Minimum reagent amounts (between 1, 2, 5, and 10 equivalents) will be first established, then time (1, 2, and 6 h) and temperature (4 °C, RT, and 37 °C) conditions will be varied.

[0147] Label tyrosine residue with fluorophore and perform fluorosequencing. Under optimal conditions, tyrosine residues will be labeled with Atto643 fluorophore, and results will be compared with results with an identical control peptide, in which lysine residue were labeled. Achieving a >95% correlation will validate a tyrosine conjugation method for use in accordance with disclosed methods of profiling immunoglobulin repertoires. EXAMPLE 6: Fluorosequencing of CDR-H3 peptides from IgG molecules

[0148] Given the sequence diversity of CDR-H3 species and their wide concentration range, attention is give to maximizing individual peptide identification. Fluorescent patterns for up to -100 million reads per lane can be obtained with flow cells, thus providing adequate depth for identifying and quantifying the antibody-derived peptides. CDR-H3 peptides, isolated from standard serum and analyzed with LC-MS/MS (as described in the foregoing Examples), will be used for the fluoro sequencing study.

[0149] 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.

[0150] Fluorosequencing. Samples will be loaded into flow cells and fluorosequencing will be performed. Three technical replicates and three biological replicates will be performed on each sample.

[0151] Analysis of fluorosequencing results. Estimated errors for the following will be modeled: Edman inefficiency, missed labeling of tyrosine and acidic residues, missed cleavage rates of lysC enzymes, photobleaching/dye destruction rates. The resulting fluorosequences will be deconvolved to individual fluorotypes and their counts. Pearson correlation of the fluorotype counts across technical and biological replicates will demonstrate the reproducibility of the technology.

[0152] Comparison of fluorosequencing results with MS-MS observations. An MS- MS-derived list of enriched CDR-H3 peptides will be obtained and binned into classes with distinct tyrosine and acidic residue patterns, collating their peptide- spectrum match (PSM) counts. Correlation and concordant analysis (testing for the presence or absence of the different classes) will be performed, and the Kappa-Cohen correlation between fluorosequencing and MS-MS results will be calculated. EXAMPLE 8: Example workflow for profiling immunoglobulin repertoires

[0153] The present Example describes an example workflow for profiling immunoglobulin repertoires. (See Figure 4.)

[0154] Serum collection. Commercially available human serum (e.g., Sigma, catalog # H4522) 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.

EXAMPLE 8: Example workflow for profiling immunoglobulin repertoires

[0155] The present Example describes an example workflow for profiling immunoglobulin repertoires. (See Figure 4.)

[0156] Serum collection. Commercially available human serum (e.g., Sigma, catalog # H4522) 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.

[0157] Immunoglobulin G protein isolation. IgG molecules are extracted from serum using a protein A bead-based isolation method (Thermo Fisher, catalog # 44667).

[0158] 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.

[0159] Enrich for CDR-H3 peptides. CDR-H3 peptides are enriched using ASTK- selective binders (e.g., binders developed as described in Examples 2 or 3).

[0160] Labeling of select amino acids with fluorophores and fluorosequencing. For fluorosequencing, 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.

[0161] 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.

OTHER EMBODIMENTS

[0162] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein.

Claims

WHAT IS CLAIMED IS:

1. A method of profiling an immunoglobulin repertoire specific to a subject, the method comprising the steps of: a) obtaining or having obtained a composition enriched for CDR-H3 peptides; and b) fluoro sequencing said CDR-H3 peptides, thereby obtaining information regarding the subject’s immunoglobulin repertoire.

2. The method of claim 1, wherein 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.

3. A method of profiling an immunoglobulin repertoire, the method 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.

4. The method of any one of claims 2-3, wherein the biological sample comprises blood or serum.

5. The method of any one of claims 2-4, wherein the biological sample comprises tumorinfiltrating B -cells.

6. The method of any one of claims 2-5, wherein the biological sample comprises a spleen, bone marrow, or saliva sample.

7. The method of any one of claims 2-6, wherein the biological sample comprises cerebrospinal fluid.

8. The method of any one of claims 2-7, wherein the immunoglobulin molecules are isolated from the biological sample by a method employing particles coated with immunoglobulin-binding polypeptides .

9. The method of claim 8, wherein the immunoglobulin-binding polypeptides comprise protein A, protein G, or a combination thereof.

10. The method of claim 9, wherein the particles comprise protein A/G beads.

11. The method of any one of claims 2-10, wherein, prior to the step of digesting, said isolated immunoglobulin molecules are contacted with an agent that modifies one or more amino acid residues.

12. The method of claim 11, wherein the agent modifies an amino acid residue to resemble another amino acid residue.

13. The method of claim 12, wherein the agent modifies cysteine residues to resemble lysine residues.

14. The method of claim 13, wherein the agent is 2-bromoethylamine.

15. The method of any one of claims 1-14, wherein the step of digesting comprises digesting with an enzyme.

16. The method of claim 15, wherein the enzyme is an endoprotease.

17. The method of claim 16, wherein the endoprotease is selected from the group consisting of trypsin and lysC.

18. The method of claim 16, wherein the endoprotease cleaves C-terminal to a lysine.

19. The method of claim 18, wherein the endoprotease cleaves C-terminal to the amino acid sequence ASTK.

20. The method of any one of claims 2-18, wherein the step of enriching comprises using an isotype-selective binder.

21. The method of claim 20, wherein the isotype- selective binder is an IgG-selective binder.

22. The method of claim 21, wherein the IgG-selective binder is capable of selectively binding the amino acid sequence ASTK.

23. The method of any one of claims 20-22, wherein the isotype-selective binder comprises an antibody or antigen-binding fragment thereof.

24. The method of claim 22 or 23, wherein the isotype- selective binder is immobilized to a solid support.

25. The method of claim 24, wherein the solid support comprises a material selected from the group consisting of sepharose resin and glass.

26. The method of claim 24 or 25, wherein the solid support comprises beads.

27. The method of claim 26, wherein the solid support comprises magnetic beads.

28. The method of any one of claims 24-27, wherein the solid support is attached to a identifier unique to the subject.

29. The method of claim 28, wherein the identifier is unique to the biological sample from the subject.

30. The method of claim 28 or 29, wherein the identifier comprises an oligonucleotide.

31. The method of any one of claims 28-30, wherein the identifier comprises a peptide.

32. The method of any one of claims 1-31, wherein the obtained information comprises quantitative information.

33. The method of any one of claims 1-32, wherein the fluorosequencing is conducted at single molecule resolution.

34. The method of any one of claim 1-33, wherein the obtained information comprises sequence information at the single molecule level.

35. The method of any one of claims 1-34, wherein the step of fluorosequencing comprises labeling a subset of amino acid residues in said enriched CDR-H3 peptides with a fluorophore.

36. The method of claim 35, wherein 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.

37. The method of claim 36, wherein the subset of amino acid residues comprises tyrosine residues.

38. The method of any one of claims 35 to 37, wherein each type amino acid residue in the subset is labeled with a different fluorophore.

39. The method of claim 38, wherein each fluorophore’s emission spectrum is distinguishable from each other.

40. The method of any one of claims 1-39, wherein the step of fluorosequencing comprises counting fluorotypes.

41. A method of profiling an immunoglobulin G (IgG) repertoire specific to a subject, the method 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.

42. The method of any one of claims 1-41, further comprising a step of e) comparing fluorotypes to sequences in a reference database.