WO2022182662A1

WO2022182662A1 - Compositions and methods for mapping antigen-binding molecule affinity to antigen regions of interest

Info

Publication number: WO2022182662A1
Application number: PCT/US2022/017326
Authority: WO
Inventors: Wyatt James MCDONNELL; Bruce Alexander ADAMS; David Benjamin JAFFE; Michael John Terry STUBBINGTON
Original assignee: 10X Genomics, Inc.; 10X genomics Ltd
Priority date: 2021-02-23
Filing date: 2022-02-22
Publication date: 2022-09-01

Abstract

The present disclosure relates generally to compositions, methods, and systems for the characterization of antibodies produced by a population of cells, such as B cells, using single-cell immune profiling methodologies. This characterization permits the identification of the antibodies that bind to regions of interest, or the mapping of antibodies according to their binding to specific regions of interest, of an antigen. Understanding the binding characteristics of antibodies at a region of interest level facilitates the identification and production of immunotherapeutic molecules having desired properties.

Description

COMPOSITIONS AND METHODS FOR MAPPING ANTIGEN-BINDING MOLECULE AFFINITY TO ANTIGEN REGIONS OF INTEREST

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent application Serial Number 63/152,820 filed February 23, 2021, which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing file entitled 057862_572001WO_Sequence_Listing_ST25.txt, with a file size of about 1.73 MB (1,815,746 bytes) and created on February 22, 2022, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.

BACKGROUND

[0003] The identification of residues dictating interaction between an immune cell receptor and it cognate ligands is an important process in the discovery, characterization, and development of immunotherapeutic molecules. The design of effective immunotherapies is assisted by knowing which residues are required for an antigen to be bound and recognized by the immune system (epitope) and which residues in an immune cell receptor recognize the antigen (paratope). This information is also required as evidence of mechanism of action for regulatory approval of immunotherapeutic molecules and can be used to differentiate, or provide an underlying basis for an advantageous property over, other immunotherapeutic molecules in its class.

[0004] While the elucidation of the structural regions of both antibody and antigen have improved, epitope mapping remains a relatively low-throughput, laborious and difficult to parallelize process. The highest resolution approaches to epitope mapping, such as electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, deuterium exchange mass spectroscopy (HDX-MS) and X-ray crystallography are prohibitively expensive and dependent on availability of pure and stable crystal compounds or protein. Further, HDX-MS is remarkably susceptible to fluctuations in pH and temperature, and therefore requires a substantial number of experimental controls and replicates, and considerable skill, to interpret its exchange data. HDX-MS can also be difficult to interpret if the antigen or antibody of interest are highly glycated, which is a common feature of both antibodies and their targets.

[0005] Other approaches to epitope mapping, such as competition binning and competition enzyme-linked immunosorbent assay (ELISA), can provide readily interpretable, easily reproducible, and consistent measurement of degree to which sets of antibodies and antigens displace each other in a concentration dependent manner. However, these approaches are difficult to scale in throughput due to input requirements of highly purified antibody and antigen. Residue-level information can also be difficult to interpret in expressed proteins and peptides due to the potential for residue alterations to impact protein folding

[0006] There is a need for high-throughput methods and reagents that reliably and rapidly characterize and identify immunotherapeutic molecules, such as antibodies, according to their binding to an epitope or region of interest.

SUMMARY

[0007] Provided herein are, inter alia , methods and kits. The methods and kits are useful for: (i) identifying or characterizing an antibody, or antigen-binding fragment thereof; (ii) identifying an antibody, or antigen-binding fragment thereof, having binding affinity to a region of interest of a target antigen; and (ii) mapping binding affinity of an antibody, or antigen-binding fragment thereof, to a region of interest of a target antigen. The disclosure also provides for partitions and for isolated antibodies, or antigen-binding fragments thereof, that specifically bind to a spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0008] In one aspect, the description provide for a method. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, (ii) a target antigen and (iii) a fragment of the target antigen. The target antigen is coupled to a first reporter oligonucleotide and the fragment of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to: the target antigen, the fragment of the target antigen or both the target antigen and the fragment of the target antigen. The partitioning the reaction mixture provides a partition. The partition includes:

(i) a partitioned cell bound to the target antigen, the fragment of the target antigen, or both the target antigen and the fragment of the target antigen; and (ii) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first or second reporter oligonucleotide or a reverse complement thereof and the partition- specific barcode sequence or reverse complement thereof. The second barcoded nucleic acid molecule includes a nucleic acid encoding the antibody, or antigen-binding fragment thereof, expressed by the cell or the reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0009] In an aspect, the description provides for a method for identifying or characterizing an antibody, or antigen-binding fragment thereof. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, (ii) a target antigen and (iii) a fragment of the target antigen. The target antigen is coupled to a first reporter oligonucleotide and the fragment of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to: the target antigen, the fragment of the target antigen or both the target antigen and the fragment of the target antigen. The partitioning the reaction mixture provides a partition. The partition includes:

(i) a partitioned cell bound to the target antigen, the fragment of the target antigen, or both the target antigen and the fragment of the target antigen; and (ii) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules may be generated in the partition. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first or second reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. The second barcoded nucleic acid molecule includes a nucleic acid encoding the antibody, or antigen-binding fragment thereof, expressed by the cell or the reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0010] In another aspect, the description provides for a method for identifying an antibody, or antigen binding fragment thereof, having binding affinity for a region of interest of a target antigen. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, (ii) a target antigen, and (iii) a fragment of the target antigen. The target antigen is coupled to a first reporter oligonucleotide and the fragment of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to: the target antigen, the fragment of the target antigen or both the target antigen and the fragment of the target antigen. The partitioning the reaction mixture provides a partition. The partition includes: (i) a partitioned cell bound to the target antigen, the fragment of the target antigen, or both the target antigen and the fragment of the target antigen; and (ii) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules may be generated in the partition. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first or second reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. The second barcoded nucleic acid molecule includes a nucleic acid encoding the antibody, or antigen-binding fragment thereof, expressed by the cell or the reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0011] In yet another aspect, the disclosure provides for a method for mapping binding affinity of an antibody, or an antigen-binding fragment thereof, to a region of interest of a target antigen. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, (ii) a target antigen, and (iii) a fragment of the target antigen. The target antigen is coupled to a first reporter oligonucleotide and the fragment of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to: the target antigen, the fragment of the target antigen or both the target antigen and the fragment of the target antigen. The partitioning the reaction mixture provides a partition.

The partition includes (i) a partitioned cell bound to the target antigen, the fragment of the target antigen, or both the target antigen and the fragment of the target antigen; and (ii) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules may be generated in the partition. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first or second reporter oligonucleotide or a reverse complement thereof and the partition- specific barcode sequence or reverse complement thereof. The second barcoded nucleic acid molecule includes a nucleic acid encoding the antibody, or antigen-binding fragment thereof, expressed by the cell or the reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0012] In yet another aspect, the disclosure provides a method. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes:

(i) a plurality of cells expressing antibodies or antigen fragments thereof, and (ii) a plurality of non-overlapping fragments of the target antigen. A first fragment of the non-overlapping fragments of the target antigen is coupled to a first reporter oligonucleotide and a second fragment of the non-overlapping fragments of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to the first fragment.

The partitioning provides a partition. The partition includes (i) a partitioned cell bound to the first fragment, and (ii) a plurality of nucleic acid barcode molecules having a partition- specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. The second barcoded nucleic molecule includes a sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0013] In a further aspect, the disclosure provides a method for identifying or characterizing an antibody, or antigen-binding fragment thereof. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes: (i) a plurality of cells expressing antibodies or antigen fragments thereof, and (ii) a plurality of non-overlapping fragments of the target antigen. A first fragment of the non-overlapping fragments of the target antigen is coupled to a first reporter oligonucleotide and a second fragment of the non-overlapping fragments of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to the first fragment.

The partitioning provides a partition. The partition includes (i) a partitioned cell bound to the first fragment, and (ii) a plurality of nucleic acid barcode molecules having a partition- specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules may be generated in the partition. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. The second barcoded nucleic molecule includes a sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0014] In an additional aspect, the disclosure provides a method for identifying an antibody, or antigen-binding fragment thereof, having binding affinity to a region of interest of a target antigen. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes: (i) a plurality of cells expressing antibodies or antigen fragments thereof, and (ii) a plurality of non-overlapping fragments of the target antigen. A first fragment of the non-overlapping fragments of the target antigen is coupled to a first reporter oligonucleotide and a second fragment of the non-overlapping fragments of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to the first fragment. The partitioning provides a partition. The partition includes (i) a partitioned cell bound to the first fragment, and (ii) a plurality of nucleic acid barcode molecules having a partition-specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules may be generated in the partition. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. The second barcoded nucleic molecule includes a sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0015] In another aspect, the disclosure provides a method for mapping binding affinity of an antibody, or an antigen-binding fragment thereof, to a region of interest of a target antigen. In the method, a reaction mixture is partitioned into a plurality of partitions. The reaction mixture includes: (i) a plurality of cells expressing antibodies or antigen fragments thereof, and (ii) a plurality of non-overlapping fragments of the target antigen. A first fragment of the non-overlapping fragments of the target antigen is coupled to a first reporter oligonucleotide and a second fragment of the non-overlapping fragments of the target antigen is coupled to a second reporter oligonucleotide. The reaction mixture includes a cell bound to the first fragment. The partitioning provides a partition. The partition includes (i) a partitioned cell bound to the first fragment, and (ii) a plurality of nucleic acid barcode molecules having a partition-specific barcode sequence. Barcoded nucleic acid molecules are generated. The barcoded nucleic acid molecules may be generated in the partition. The barcoded nucleic acid molecules include a first and a second barcoded nucleic acid molecule. The first barcoded nucleic molecule includes a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. The second barcoded nucleic molecule includes a sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

[0016] In a further aspect, the disclosure provides a partition. The partition includes a cell expressing an antigen-binding molecule. The antigen-binding molecule is bound to an antigen and a fragment of the antigen. The antigen is coupled to a first reporter oligonucleotide and the fragment of the antigen is coupled to a second reporter oligonucleotide.

[0017] The disclosure, in yet another aspect, provides a partition in which the partition includes a cell expressing an antigen-binding molecule. The antigen-binding molecule is bound to a first and a second fragment of an antigen. The first and the second fragment of the antigen are coupled to a first and a second reporter oligonucleotide, respectively,

[0018] In another aspect, the disclosure provides a kit. The kit includes instructions for use. The kit also includes a target antigen and a fragment of the target antigen. The target antigen and the fragment of the target antigen are coupled, directly or indirectly, to reporter oligonucleotides. The kit is for: (i) identification of an antibody, or antigen-binding fragment thereof, that has binding affinity for a region of interest of the target antigen, or (ii) mapping binding affinity for at least one region of interest of the target antigen by the antibody, or antigen-binding fragment thereof, or (iii) characterizing the antibody, or antigen binding fragment thereof.

[0019] The disclosure, in another aspect, provides for a kit. In this aspect, the kit includes instructions for use and a plurality of fragments of a target antigen. The plurality of fragments are coupled, directly or indirectly to reporter oligonucleotides. The kit is for: (i) identification of an antibody, or antigen-binding fragment thereof, that has binding affinity for a region of interest of the target antigen, or (ii) mapping binding affinity for at least one region of interest of the target antigen by the antibody, or antigen-binding fragment thereof, or (iii) characterizing the antibody or antigen-binding fragment thereof.

[0020] Moreover, the disclosure provides for an isolated antibody, or antigen-binding fragment thereof, that specifically binds to a spike (S) protein of SARS-CoV-2. The antibody, or antigen-binding fragment thereof, includes a set of complementary-determining regions (CDR): a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3. The heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 include the amino acid sequences of any of SEQ ID NOs: 861-1075, SEQ ID NOs: 1076-1290, and SEQ ID NOs: 1291-1505, respectively.

[0021] The disclosure also provides for an isolated antibody, or antigen-binding fragment thereof, that specifically binds to a spike (S) protein of SARS-CoV-2. The antibody, or antigen-binding fragment thereof, includes a set of complementary-determining regions (CDR): a light chain CDR1, a light chain CDR2 and a light chain CDR3. The light chain CDR1, light chain CDR2 and light chain CDR3 include the amino acid sequences of any of SEQ ID NOs : 1506-1720, SEQ ID NOs: 1721-1935, and SEQ ID NOs: 1936-2150, respectively.

[0022] The foregoing is merely a summary and is illustrative only. It is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows an exemplary scheme for antigen-specific enrichment of B cells by using fluorescence-activated cell sorting (FACS) technique. In these experiments, cells were initially gated on being single, live (7AAD^negatlve) and PE-Cy7-CD19+ and then sorted on their PE and/or APC status directly into master mixed and water. In this figure, Y axis represents PE signal (some combination of trimerized S (SARS-2) WT+S1 NTD+RBD antigen+ and/or HSA+ control cells). X axis represents APC signal (trimerized S D614G (SARS-2), S2 ECD and/or HSA control antigen+ cells). Numbers adjacent to each gate name represent the fraction of events relative to the parent population (single, live, CD 19+ cells) for that gate.

[0024] FIG. 2 schematically summarizes the results of a representative analysis performed to illustrate clonotype enrichment based on affinity to receptor-binding domain (RBD) of a SARS-CoV spike (S) protein, but not full-length spike protein, using relative KD.

[0025] FIG. 3 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on affinity to both spike protein (wildtype and mutant) and the spike protein RBD, using relative KD.

[0026] FIG. 4 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on affinity for the N-terminal domain (NTD) of the spike protein SI subunit, but not spike protein RBD, using relative K_D.

[0027] FIG. 5 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on affinity for the spike protein extracellular domain (ECD) S2-ECD, using relative K_D.

[0028] FIG. 6 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on reactivity to domains of spike protein, using relative K_D.

[0029] FIG. 7 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on reactivity to antigen presenting cell (APC), using relative K_D.

[0030] FIG. 8 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on reactivity to human serum albumin (HSA), using relative K_D.

[0031] FIG. 9 schematically summarizes the results of representative analysis performed to illustrate clonotype enrichment based on reactivity to biotin, using relative K_D.

[0032] FIG. 10 is a dimensional reduction of the antigen count, indicative of binding affinity, data provided as geometric mean EIMIs, available for antibodies in the donor sample described in Example 1. Each point at a particular location in panels A-F represents an exact clonotype and is representative of data of one or more cells bearing that antibody. Coloring of the points in panels A-F indicates geometric mean antigen UMI counts for the antigen named in the panel, which is indicative of binding affinity of the antibody for the antigen. Panel A provides geometric mean UMIs for spike protein. Panel B provides geometric mean UMIs for the spike protein NTD fragment. Panel C provides geometric mean UMIs for the spike protein RBD fragment. Panel D provides geometric mean UMIs for the spike protein ECD fragment. Panel E provides geometric mean UMIs for control antigen HSA. Panel F shows geometric mean UMIs associated with antibody expression, z.e., antibody expression by a cell bearing the antibody.

[0033] FIG. 11 is a visualization display of mapped binding affinities of the spike protein antibodies from the patient sample described in Example 1, according to their affinity for one or more spike protein antigens or antigen fragments (one or more of full-length spike protein, spike protein RBD fragment, spike protein NTD fragment, spike protein ECD fragment). Each small circle, or dot, represents an antigen-specific B cell expressing a single antibody. Each small circle, or dot, is color coded according to Ig isotype or subisotype.

Each small circle, or dot, is grouped into a larger bounded polygon by color, indicating binding specificity and/or cross-reactivity for an antigen or set of antigens/antigen fragments. For example, the bounded polygon labelled “ECD” depicts antigen-specific B cells that specifically bind the ECD fragment. For other example, the bounded polygon labeled Spike- NTD depicts antigen-specific B cells that specifically bind to the full Spike antigen and to the NTD fragment. An inset showing a zoomed-in perspective of the Spike-NTD grouping shows that small circles, or dots representing cells belonging to a single clonotype are clustered together. Thus, the visualization display can depict the assignment of single cells or individual antibodies into clonotype groupings. Methods of producing such visual displays are described in U.S. Patent Application Ser. No. 17/182,147, filed on Feb. 22, 2021, which is hereby incorporated by reference in its entirety.

[0034] Fig. 12 shows an exemplary microfluidic channel structure for partitioning individual biological particles in accordance with some embodiments of the disclosure.

[0035] FIG. 13 shows an exemplary microfluidic channel structure for the controlled partitioning of beads into discrete droplets.

[0036] FIG. 14 shows an exemplary microfluidic channel structure for delivering barcode carrying beads to droplets.

[0037] FIG. 15 shows an exemplary barcode carrying bead.

[0038] FIG. 16 illustrates another example of a barcode carrying bead.

[0039] FIG. 17 schematically illustrates an example microwell array.

[0040] FIG. 18 schematically illustrates an example workflow for processing nucleic acid molecules.

[0041] FIG. 19 schematically illustrates examples of labelling agents.

[0042] FIG. 20 depicts an example of a barcode carrying bead.

[0043] FIGS. 21A, 21B and 21C schematically depict an example workflow for processing nucleic acid molecules.

[0044] FIG. 22 depicts a block diagram illustrating an example of a computing system, in accordance with some example embodiments.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0045] The present disclosure generally relates to, inter alia , compositions, methods, partitions, kits, and systems for characterizing and identifying antibodies or antigen-binding fragments of antibodies, e.g., as having binding affinity for a region of interest of a target antigen or for mapping their binding affinity to a region of interest of a target antigen. Furthermore, the present disclosure provides isolated antibodies, or antigen-binding fragments thereof, that specifically bind severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or a region thereof, as well as methods of making and using such antibodies and antigen-binding fragments.

[0046] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

[0047] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0048] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, T, & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. etal. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. etal. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY : Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. etal. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference

DEFINITIONS

[0049] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0050] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0051] As used herein, “isolated” antigen-binding molecules, e.g., antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors, are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or antigen binding fragments.

[0052] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals. The term “non-human animals” includes all vertebrates, e.g, mammals, e.g, rodents, e.g, mice, non-human primates, and other mammals, such as e.g, rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit; and non mammals, such as amphibians, reptiles, etc. A subject can be a healthy individual, an asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer or infection), an individual having a pre-disposition to a disease, an individual that is in need of therapy for a disease, or an individual who has recovered from a disease. In any event, the subject may have been exposed to an antigen characteristic of the disease, such as an antigen capable of producing an antibody immune response associated with the disease.

[0053] A “variant” of a polypeptide, such as an immunoglobulin chain (e.g, VH, VL, HC, or LC), any protein or peptide antigen, or a fragment of any protein antigen, refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g, 70, 72,

74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99

99.5, 99.9%) identical or similar to a referenced amino acid sequence that is set forth herein; when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.

[0054] The term “barcode” is used herein to refer to a label, or identifier, that conveys or is capable of conveying information ( e.g ., information about an analyte in a sample, a bead, and/or a nucleic acid barcode molecule). A barcode can be part of an analyte or nucleic acid barcode molecule, or independent of an analyte or nucleic acid barcode molecule. A barcode can be attached to an analyte or nucleic acid barcode molecule in a reversible or irreversible manner. A particular barcode can be unique relative to other barcodes. Barcodes can have a variety of different formats. For example, barcodes can include polynucleotide barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences. A barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for or facilitates identification and/or quantification of individual sequencing-reads. In some embodiments, a barcode can be configured for use as a fluorescent barcode. For example, in some embodiments, a barcode can be configured for hybridization to fluorescently labeled oligonucleotide probes. Barcodes can be configured to spatially resolve molecular components found in biological samples, for example, at single-cell resolution ( e.g ., a barcode can be or can include a “spatial barcode”). In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes). In some embodiments, the two or more sub barcodes are separated by one or more non-barcode sequences. In some embodiments, the two or more sub-barcodes are not separated by non-barcode sequences.

[0055] In some embodiments, a barcode can include one or more unique molecular identifiers (UMIs). Generally, a unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier for a particular analyte, or for a nucleic acid barcode molecule that binds a particular analyte (e.g, mRNA) via the capture sequence.

[0056] A UMI can include one or more specific polynucleotides sequences, one or more random nucleic acid and/or amino acid sequences, and/or one or more synthetic nucleic acid and/or amino acid sequences. In some embodiments, the UMI is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the UMI has less than 80% sequence identity (e.g, less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g, 80% or more) of the nucleic acid molecules in the biological sample. These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by 1 or more nucleotides.

[0057] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0058] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0059] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0060] It is understood that aspects and embodiments of the disclosure described herein include "comprising", "consisting", and "consisting essentially of aspects and embodiments. As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0061] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. METHODS OF THE DISCLOSURE

Methods for characterizing antigen-binding molecules

[0062] As described in more detail below, one aspect of the disclosure relates to methods. The methods include new approaches for the identification and characterization of antigen-binding molecules, e.g ., antibodies and antigen-binding fragments. The methods for the identification and characterization of the antigen-binding molecules may characterize the antigen-binding molecules as binding a region of interest of a target antigen, may identify the antigen-binding molecule as having binding affinity to a region of interest of a target antigen, or may map the anti gen -binding molecule’s binding affinity to a region of interest of a target antigen.

[0063] The antigen-binding molecule, characterized and/or identified in the methods may be an antibody or an antigen-binding fragment of an antibody. If the antigen-binding molecule is an antibody, the antibody may be an antibody having an Immunoglobulin (Ig)A (e.g, IgAl or IgA2), IgD, IgE, IgG (e.g, IgGl, IgG2, IgG3 and IgG4) or IgM constant region. If the antigen-binding molecule is a fragment of an antibody, the fragment of the antibody may be any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. An antigen-binding fragment of an antibody may be one of: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) sdAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g, an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FWR3-CDR3-FWR4 peptide. Further, an antigen-binding fragment of an antibody may be an engineered molecule, such as a domain-specific antibody, single domain antibody, chimeric antibody, CDR-grafted antibody, diabody, triabody, tetrabody, minibody, nanobody (e.g, monovalent nanobodies, bivalent nanobodies, etc.), a small modular immunopharmaceutical (SMTP), or a shark immunoglobulin new antigen receptor (IgNAR) variable domain.

[0064] The target antigen may be any antigen for which the characterization and/or identification of antigen-binding molecule such as an antibody, or antigen-binding fragment thereof capable of binding or as having an affinity thereto is desirable. The target antigen may be an antigen associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal or prion agent. If the target antigen is associated with an infectious agent that is a viral agent, the viral agent may be an influenza virus, a coronavirus, a retrovirus, a rhinovirus, or a sarcoma virus. The viral agent may be severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), a SARS-CoV-2, a Middle East respiratory syndrome coronavirus (MERS- CoV)), or human immunodeficiency virus (HIV), influenza, respiratory syncytial virus, or Ebola virus. If the target antigen is associated with an infectious agent that is a viral agent, the target antigen may be corona virus spike (S) protein, e.g., SARS-CoV-2 S protein, an influenza hemagglutinin protein, an HIV envelope protein or any other a viral glycoprotein. Further, the target antigen may be associated with a tumor or a cancer. If the target antigen is associated with tumors or cancers, it may be, for example, epidermal growth factor receptor (EGFR), CD38, platelet-derived growth factor receptor (PDGFR) alpha, insulin growth factor receptor (IGFR), CD20, CD 19, CD47, or human epidermal growth factor receptor 2 (HER2). In addition, the target antigen may be an immune checkpoint molecule that may or may not be associated with tumors or cancers (e.g., CD38, PD-1, CTLA-4, TIGIT, LAG-3, VISTA, TIM-3), or it may be a cytokine (e.g., soluble cytokine), a GPCR, a cell-based co-stimulatory molecule, a cell-based co-inhibitory molecule, an ion channel, glycan, glycan conjugate, or a growth factor. Further still, the target antigen may be associated with a degenerative condition or disease (e.g., an amyloid protein or a tau protein).

[0065] In some embodiments, the target antigen, for which the characterization and/or identification of an antigen-binding molecule such as an antibody, or antigen-binding fragment thereof, having affinity thereto may be desirable, may be a target antigen of a length of at least 20 amino acid residues, at least 40 amino acid residues, at least 60 amino acid residues, at least 80 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, at least 700 amino acids, at least 800 amino acid residues, at least 900 amino acid residues, at least 1000 amino acid residues, at least 1100 amino acid residues, at least 1200 amino acid residues, at least 1300 amino acid residues, up to 40 amino acid residues, up to 60 amino acid residues, up to 80 amino acid residues, up to 100 amino acid residues, up to 200 amino acid residues, up to 300 amino acid residues, up to 400 amino acid residues, up to 500 amino acid residues, up to 600 amino acid residues, up to 700 amino acids, up to 800 amino acid residues, up to 900 amino acid residues, up to 1000 amino acid residues, up to 1100 amino acid residues, up to 1200 amino acid residues, or up to 1300 amino acid residues. The target antigen may be an antigen that includes one domain, at least one domain, two domains, at least two domains, three domains, at least three domains, four domains, at least four domains, five domains, at least five domains, six domains, at least six domains, seven domains, at least seven domains, eight domains, at least eight domains, nine domains, at least nine domains, ten domains, at least ten domains, at least thirty domains, at least forty domains, at least fifty domains, at least sixty domains, at least seventy domains, at least eighty domains, at least ninety domains or at least one hundred domains. The target antigen may be an antigen that includes at most two hundred domains, at most 175 domains, at most 150 domains, at most 125 domains, at most 100 domains, at most 75 domains, at most 50 domains, at most 25 domains, at most 20 domains, at most 15 domains, at most 10 domains, or at most 5 domains.

[0066] In further embodiments, the target antigen may be a protein or peptide as expressed by a cell, e.g., full-length target antigen that may or may not include its leader sequence and may or may not have undergone a similar cell processing step.

[0067] The region of interest of the target antigen, e.g., for which an antibody or antigen-binding fragment thereof, may be characterized as having a binding affinity to or to which it may be mapped, may be of fewer amino acid residues in length than the full-length target antigen. The region of interest of the target antigen may include or may be an epitope of the target antigen, e.g, a linear or conformational or cryptic epitope. The region of interest of the target antigen may include or may be a domain of the target antigen. A domain of a target antigen may also be referred to as a unit or portion an antigen that is self-stabilizing and folds independently of the remainder of the antigen. Domains of antigens may be determined by Hydrophobicity/Kyte-Doolittle plots, which can identify extracellular vs. intracellular domains of proteins. Domains of antigens may also be determined using tools such as InterPro or PROSITE (https://www.ebi.ac.uk/interpro/) or protein BLAST, each of which is capable of identifying protein domains via sequence similarities shared by proteins having similar structures and/or functions. The region of interest of the target antigen may be a 10-200, 20-200, a 20-180, a 20-160, a 20-140, a 20-120, a 20-100, a 20-80, a 20-60, a 20- 40, a 40-200, a 40-180, a 40-160, a 40-140, a 40-120, a 40-100, a 40-80, a 40-60, 60-200, a 60-180, a 60-160, a 60-140, a 60-120, a 60-100, a 60-80, a 80-200, a 80-180, a 80-160, a 80- 140, a 80-120, a 80-100, a 100-200, a 150-100, or a 25-175 amino acid residue peptide region of the target antigen. The region of interest of the target antigen may be selected as it may be or may include one or more epitopes or domains of the target antigen that are involved in a signaling pathway, that interact with other proteins or peptides, or that result in or prevent a conformational change in the target antigen.

[0068] In the methods provided herein, a reaction mixture may be partitioned into a plurality of partitions. The partitioning of the reaction mixture may also be referred to as the compartmentalization or depositing of the reaction mixture into discrete compartments or partitions, where each partition maintains separation of its own contents from the contents of other partitions.

[0069] The reaction mixture, which may partitioned, may include: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, (ii) a target antigen, and (iii) a fragment of the target antigen. Alternatively, the reaction mixture, which may be partitioned, may include: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, and (ii) a plurality of non-overlapping fragments of a target antigen.

[0070] The plurality of cells expressing antibodies, or antigen-binding fragments thereof, in the reaction mixture may be a plurality of cells that includes cells of B cell lineage, e.g. memory B cells, which express an antibody as a cell surface receptor. In other examples, cells of the plurality of cells can be or include an engineered cell having been engineered to express antibodies or antigen-binding fragments thereof and that express the antibody or antigen-binding fragment thereof as a cell surface receptor. The plurality of cells expressing antibodies, or antigen-binding fragments thereof, may be obtained from a subject, e.g. , a mammal such as a human. If the plurality of cells is obtained from a subject, the plurality of cells may include cells obtained from a sample of the subject. The sample of the subject may be obtained by biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a plasma or serum sample.

[0071] If the plurality of cells in the reaction mixture is obtained from a sample of the subject, the sample may have been processed prior to its inclusion in the reaction mixture.

The processing of the sample may include steps such as filtration, selective precipitation, purification, centrifugation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort cells of different types, e.g. , to separate B cells from other cell types, including the separation of B cell subpopulations such as memory B cells. A separation process can be a positive selection process, a negative selection process (e.g, removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g, removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells). [0072] In an embodiment, the reaction mixture which may be partitioned may include: (i) a target antigen and a fragment of a target antigen, or (ii) a plurality of non-overlapping fragments of a target antigen, in addition to the plurality of cells. As discussed above, a target antigen may be any antigen of interest. It may be associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal or prion agent. The target antigen may be associated with a tumor or cancer. Further, the target antigen may be associated with an inflammatory or an autoimmune disease. Further still, the target antigen may be associated with a degenerative condition or disease. As used herein, an “antigen” is not limited to proteins, fats, and/or sugars that is foreign to the subject but may include self-antigens, e.g., amyloid or tau protein. Non-limiting examples of target antigens include, corona virus spike (S) protein, an influenza hemagglutinin protein, an HIV envelope protein, a viral glycoprotein, EGFR, CD38, PDGFR-alpha, IGFR, CD20, CD 19, CD47, HER2, CD38, PD-1, CTLA-4, TIGIT, LAG-3, VISTA, TIM-3 a cytokine, a GPCR, a cell-based co-stimulatory molecule, a cell-based co-inhibitory molecule, an ion channel, or a growth factor.

[0073] In the embodiment in which the reaction mixture includes, in addition to the plurality of cells: (i) a target antigen and a fragment of a target antigen, the target antigen may be a full-length version of the target antigen as expressed by a cell, e.g., full-length target antigen that may or may not include its leader sequence and may or may not have undergone a similar cell processing step. The fragment of the target antigen included in the reaction mixture, i.e., with the plurality of cells and the target antigen, may be identical in sequence to the target antigen but shorter in amino acid length. For example, the fragment of the target antigen may have an amino acid length that is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% that of the target antigen. As another example, the fragment of the target antigen may have an amino acid sequence length that is 75% or below, 70% or below, 65% or below, 60% or below, 55% or below, 50% or below, 45% or below, 40% or below, 35% or below, 30% or below, 25% or below, 20% or below, 15% or below, 10% or below, or 5% or below that of the target antigen. The fragment of the target antigen may be 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, 20-60, 20-40, 15-20, 40-200, 40-180, 40-160, 40-140, 40-120, 40-100, 40-80, 40-60, 60-200, 60-180, 60-160, 60-140, 60-120, 60-100, 60-80, 80-200, 80-180, 80-160, 80- 140, 80-120, 80-100, 100-200, 150-100, 25-175, 25-150, 25-125, 25-100, or 25-75 amino acid residues in length, so long as it is shorter in length than the full-length version of the target antigen. [0074] It will also be understood that a fragment of a target antigen, while of a shorter amino acid residue length than the target antigen, may also have one or more amino acid substitutions in its sequence relative to the target antigen. For example, a fragment of a length of greater than 200 amino acid residues may have one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 30, or 40 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. A fragment of a length of greater than 200 amino acids may have 1-40, 1-30, 1-20, 1-15, 1-10, or 1-5 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. A fragment of a length of greater than 200 amino acid residues may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding amino acid sequence of the target antigen. In another example, a fragment of a length of 100 to 200 amino acid residues may have one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, or 30 amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. A fragment of a length of 100 to 200 amino acids may have 1-30, 1-20, 1-15, 1-10, or 1-5 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. A fragment of a length of 100 to 200 amino acid residues may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet another example, a fragment of a length of less than 100 amino acid residues may have one, two, three, four, five, six, seven, eight, nine or 10 amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. A fragment of a length of less than 100 amino acids may have 1-10, 1-5, 1-4, or 1-3 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. A fragment of a length of less than 100 amino acid residues may have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet another example, a fragment of a length of less than 40 amino acid residues may have one or two amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. A fragment of a length of less than 40 amino acids may have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet a further example, a fragment of a length of less than 20 amino acid residues may have one amino acid residue substitution relative to its corresponding amino acid sequence of the target antigen. [0075] The fragment of the target antigen may include or may be an epitope of the target antigen known to be of importance. The fragment of the target antigen may include or may be a domain of the target antigen known to be of importance. An epitope or domain of importance of the target antigen may be an epitope or domain of the target antigen that mediates a process, e.g., affects a signaling pathway directly or by costimulation, is critical to host-pathogen interaction, or affects a conformational change. In some embodiments, the fragment is not complexed with a MHC molecule.

[0076] In the embodiment in which the reaction mixture includes, in addition to the plurality of cells: a plurality of non-overlapping fragments of the target antigen, then the plurality of non-overlapping fragments of the target antigen may include a first and a second non-overlapping fragment of the target antigen.

[0077] In some embodiments, the first and second fragments are completely non overlapping, e.g., for a full-length target antigen having a set of numbered amino acid residues, the first fragment encompasses a first subset of the numbered residues and the second fragment encompasses a second subset of the numbered residues that has no overlap with the first subset. In such embodiments, the first fragment encompasses a first subset of the numbered residues from the full length target antigen that does not include any members of the second subset of the numbered residues encompassed by the second fragment. In other words, in such embodiments there is no intersection of the first subset and the second subset of numbered amino acid residues. By way of example only, the completely non-overlapping fragments may be from different domains or regions or portions of the target antigen.

[0078] In some embodiments, a reaction mixture of the disclosure may comprise plurality of non-overlapping fragments that tile across the entirety or a portion of the full- length antigen. In some embodiments, there are as many completely non-overlapping fragments as required to tile across the entirety of the full length antigen. In such embodiments, the minimum length of a non-overlapping fragment of the target antigen is 20 amino acids.

[0079] In other embodiments, the first and second fragments are partially non overlapping, e.g., for a full-length target antigen having a set of numbered amino acid residues, the first fragment encompasses a first subset of the numbered residues and the second fragment encompasses a second subset of the numbered residues that partially overlaps with the first subset. In such embodiments, the first and second subsets each comprise a common subset of the numbered residues that is the intersection of the first and second subsets and distinct subsets of the numbered residues, e.g., the first subset further encompasses numbered residues from the full length target antigen that are not included in the second subset. In other words, there is an intersection of the first subset and second subset of numbered amino acid residues. By way of example only, the partially non-overlapping fragments of the target antigen may include consecutive amino acid residues that are identical, e.g., at their N- or C-terminus, and consecutive amino acid residue that are completely distinct, i.e., are non-overlapping to an extent. For example, for first and second partially non-overlapping fragments may each be 100 amino acid residues in length, of which the 20 C-terminal amino acid residues of the first fragment and the 20 N-terminal amino acid residues of the second fragment are identical, while the 80 N-terminal amino acid residues of the first and the 80 C-terminal amino acid residues of the second fragment are distinct.

[0080] It will be understood that non-overlapping fragments of the target antigen need not be of the same or of similar amino acid residue length. It will also be understood that the non-overlapping fragments, if non-overlapping to an extent, may include amino acid sequences of the same and/or different epitopes of the target antigen, as well as include amino acid sequence of the same and/or different domains of the target antigen.

[0081] Any given fragment of the non-overlapping fragments of the target antigen may be 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% that of the target antigen. As another example, the fragment of the target antigen may have an amino acid sequence length that is 75% or below, 70% or below, 65% or below, 60% or below, 55% or below, 50% or below, 45% or below, 40% or below, 35% or below, 30% or below, 25% or below, 20% or below, 15% or below, 10% or below, or 5% or below that of the target antigen. Any given fragment of the non-overlapping fragments of the target antigen may be 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, 20-60, 20-40, 15-20, 40-200, 40-180, 40-160, 40-140, 40-120, 40-100, 40-80, 40-60, 60-200, 60- 180, 60-160, 60-140, 60-120, 60-100, 60-80, 80-200, 80-180, 80-160, 80-140, 80-120, 80- 100, 100-200, 150-100, 25-175, 25-150, 25-125, 25-100, or 25-75 amino acid residues in length, so long as it is shorter in length than the full-length version of the target antigen.

[0082] Fragments of the non-overlapping fragments of the target antigen, may also have one or more substitutions in its amino acid sequence relative to the target antigen. In an example, any fragment of a length of greater than 200 amino acid residues may have one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 30, or 40 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any fragment of a length greater than 200 amino acids may have 1-40, 1-30, 1-20, 1-15, 1-10, or 1-5 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any fragment of a length of greater than 200 amino acid residues may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding amino acid sequence of the target antigen. In another example, any fragment of a length of 100 to 200 amino acid residues may have one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, or 30 amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. Any fragment of a length of 100 to 200 amino acids may have 1-30, 1-20, 1-15, 1-10, or 1-5 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any fragment of a length of 100 to 200 amino acid residues may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet another example, any fragment of a length of less than 100 amino acid residues may have one, two, three, four, five, six, seven, eight, nine or 10 amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. Any fragment of a length of less than 100 amino acids may have 1-10, 1-5, 1-4, or 1-3 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any fragment of a length of less than 100 amino acid residues may have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet another example, any fragment of a length of less than 40 amino acid residues may have one or two amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. Any fragment of a length of less than 40 amino acids may have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet a further example, any fragment of a length of less than 20 amino acid residues may have one amino acid residue substitution relative to its corresponding amino acid sequence of the target antigen.

[0083] Also contemplated herein are embodiments wherein the reaction mixture comprises a plurality of antigen fragments, wherein a first fragment of a target antigen and second fragment of the target antigen comprise the same subset of numbered amino acid residues of the target antigen having a set of numbered residues, and the first fragment and second fragment may comprise different amino acid substitutions. Such embodiments may be useful for positional mutagenesis within a particular region of interest of the target antigen. For example, in some cases, a reaction mixture may include the first and the second fragment of the target antigen, and the first and second fragment of the target antigen may both have an amino acid substitution at the same corresponding position of the target antigen amino acid sequence, but the substitution in the first and the second fragment at that position may be to a first and a second amino acid residue. Alternatively, the first and the second fragment of the target antigen may include first and second amino acid residue substitutions at first and second corresponding positions of the target antigen amino acid sequence.

[0084] If the reaction mixture, which may be partitioned, includes: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, (ii) a target antigen, and (iii) a fragment of the target antigen, then the target antigen and the fragment of the target antigen may be coupled to first and second reporter oligonucleotides, respectively. In such a reaction mixture, a cell of the plurality of cells may be bound to the target antigen coupled to the reporter oligonucleotide, or it may be bound to the fragment of the target antigen coupled to the second reporter oligonucleotide, or it may be bound to both the target antigen coupled to the first reporter oligonucleotide and the fragment of the target antigen coupled to the second reporter oligonucleotide. Further, the partitioning of such a reaction mixture, may provide a partition which includes (a) a cell bound to one of the target antigen, the fragment of the target antigen, or both the target antigen and the fragment of the target antigen, and (b) a plurality of nucleic acid barcode molecules having a partition-specific barcode sequence.

[0085] If the reaction mixture, which may be partitioned, includes: (i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, and (ii) a plurality of non overlapping fragments of a target antigen, then a first reporter oligonucleotide may be coupled to a first fragment of the non-overlapping fragments and a second reporter oligonucleotide may be coupled to a second fragment of the non-overlapping fragments. In such a reaction mixture, a cell of the plurality of cells may be bound to the first fragment coupled to the first reporter oligonucleotide, or it may be bound to the second fragment coupled to the second reporter oligonucleotide, or it may be bound to both the first fragment coupled to the first reporter oligonucleotide and the second fragment coupled to the second reporter oligonucleotide. Further, the partitioning of such a reaction mixture may provide a partition which includes (a) a cell bound to one of the first fragment of the target antigen, the second fragment of the target antigen, or both the first and the second fragment of the target antigen, and (b) a plurality of nucleic acid barcode molecules having a partition-specific barcode sequence.

[0086] A reporter oligonucleotide, bound to any of a target antigen, or any fragment of the target antigen, may be or include a nucleotide sequence that is specific for the target antigen to which it is coupled or the fragment of the target antigen to which it is coupled.

The reporter oligonucleotide may include nucleotide sequences including (a) a reporter sequence, e.g ., which may be useful to identify the target antigen or fragment to which the reporter oligonucleotide is bound, and (b) a capture handle sequence. In addition, the reporter oligonucleotide may have a further characteristic in that it may be coupled to a labeling agent. The labeling agent may be coupled to the reporter oligonucleotide via a labeling of the target antigen and/or any fragment thereof, or via a labeling of a nucleotide(s) of the reporter oligonucleotide.

[0087] Nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules, included in the partition with the cell bound to (a) the target antigen and/or fragment of the target antigen or (b) first fragment of the target antigen, may include a partition-specific barcode sequence. A partition-specific barcode sequence may identify the partition in which the nucleic acid barcode molecule is partitioned. Nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules may further include a capture sequence. A capture sequence may be configured to couple to the handle sequence of a reporter oligonucleotide, e.g., by complementary base pairing. A capture sequence may be configured to couple to an mRNA or a DNA analyte. If the capture sequence is configured to couple to an mRNA analyte, it may include a polyT sequence. If the capture sequence is configured to couple to DNA, the DNA may be a cDNA reverse transcribed from the mRNA analyte using primer, e.g., including a polyT sequence, and a reverse transcriptase having terminal transferase activity. The terminal transferase activity of the reverse transcriptase may append nucleotides, e.g., a polyC sequence, to the cDNA to which the capture sequence, e.g., a polyG sequence, of the nucleic acid barcode molecule may couple.

[0088] In any of the methods provided herein barcoded nucleic acid molecules may be generated. In some embodiments, the barcoded nucleic acid molecules, e.g., first and second barcoded nucleic acid molecules, may be generated following (i) coupling of capture sequence(s) of the nucleic acid barcode molecule(s) to the capture handle sequence(s) of the reporter oligonucleotide and/or to mRNA, cDNA or DNA analytes and (ii) pooling of the nucleic acid barcode molecules coupled to the reporter oligonucleotide and/or mRNA, cDNA or DNA analytes from a plurality of partitions, (e.g.. such that the barcoded nucleic acid molecules may be generated in bulk). In other embodiments, the barcoded nucleic acid molecules, including a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule, may be generated in the partition.

[0089] If the barcoded nucleic acid molecules are generated from a partition that had included, or includes, any of (I) a cell bound to the target antigen (coupled to a first reporter oligonucleotide), (II) a cell bound to a fragment of the target antigen (coupled to a second reporter oligonucleotide) or (III) a cell bound to both the target antigen (coupled to the first reporter oligonucleotide) and the fragment of the target antigen (coupled to a second reporter oligonucleotide), then the barcoded nucleic molecules may include:

[0090] (I) (i) a first barcoded nucleic acid molecule including a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; and (ii) a second barcoded nucleic acid molecule including a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof;

[0091] (II) (i) a first barcoded nucleic acid molecule including a sequence of the second reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; and (ii) a second barcoded nucleic acid molecule including a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; or

[0092] (III) (i) a first barcoded nucleic acid molecule including a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof and/or (ii) a third barcoded nucleic acid molecule including a sequence of a second reporter oligonucleotide or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; and (iii) a second barcoded nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. It will be understood that any of the barcoded nucleic acid molecules may further include a unique molecular identifier (UMI). The UMI may be a sequence that originating from a reporter oligonucleotide or a nucleic acid barcode molecule.

[0093] In any of the methods provided herein, if the barcoded nucleic acids are generated from a partition that had included, or includes, any of (I) a cell bound to a first fragment of the target antigen (coupled to a first reporter oligonucleotide), (II) a cell bound to a second fragment of the target antigen non-overlapping with the first fragment of the target antigen (coupled to a second reporter oligonucleotide) or (III) a cell bound to both the first fragment of the target antigen (coupled to the first reporter oligonucleotide) and the second, non-overlapping, fragment of the target antigen (coupled to a second reporter oligonucleotide), then the barcoded nucleic molecules may include:

[0094] (I) (i) a first barcoded nucleic acid molecule including a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; and (ii) a second barcoded nucleic acid molecule including a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof;

[0095] (II) (i) a first barcoded nucleic acid molecule including a sequence of the second reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; and (ii) a second barcoded nucleic acid molecule including a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof;

[0096] (III) (i) (i) a first barcoded nucleic acid molecule including a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof and/or (ii) a third barcoded nucleic acid molecule including a sequence of a second reporter oligonucleotide or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; and (iii) a second barcoded nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. It will be understood that any of the barcoded nucleic acid molecules may further include a UMI. The UMI may be a sequence originating from a reporter oligonucleotide or a nucleic acid barcode molecule. It will be understood that any of the barcoded nucleic acid molecules may further include a functional sequence. Functional sequences are disclosed herein.

[0097] The methods provided herein may, optionally, include subsequent operations following the generation of barcoded nucleic acid molecules in the partition. These subsequent operations may further include amplification of the barcoded nucleic acid molecules. The amplification of the barcoded nucleic acid molecules may optionally be performed using primers that add additional functional sequences to the barcoded nucleic acid molecules. These subsequent operations may include further processing ( e.g ., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations can occur in bulk (e.g, outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. These subsequent operations may include determining sequences of the generated barcoded nucleic acid molecules. In the methods, the determining sequence of the second barcoded nucleic acid molecule may identify the antibody or antigen-binding fragment thereof expressed by the cell in the partition in which the barcoded nucleic was generated. The determining the sequence of the first barcoded nucleic acid molecule may assess the affinity of the antibody or antigen-binding fragment produced by the cell in the partition in which the barcoded nucleic was generated. If a third barcoded nucleic acid molecule is generated in the partition, the third barcoded nucleic acid molecule may further identify or assess the affinity of the antibody or antigen-binding fragment produced by the cell in the partition in which the barcoded nucleic was generated.

[0098] If the methods determine sequences that identify the antibody or antigen-binding fragment thereof expressed by the cell of a partition, the sequences may be nucleic acid sequences encoding the antibody of the antigen-binding fragment thereof. The nucleic acid sequences may encode one or more of a complementarity determining region (CDR), a framework (FWR), a variable heavy chain domain (VH), or a variable light chain domain (VL) of the antibody or antigen-binding fragment thereof. Alternatively, if the methods determine sequences that identify the antibody or antigen-binding fragment thereof expressed by the cell of a partition, the sequences may be amino acid sequences of the antibody or antigen-binding fragment thereof. The amino acid sequences may include a sequence of one or more of a CDR, FWR, VH or VL of the antibody or antigen binding fragment thereof.

[0099] Sequencing may be by performed by any of a variety of approaches, systems, or techniques, including next-generation sequencing (NGS) methods. Sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g, digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR), and/or isothermal amplification. Non limiting examples of nucleic acid sequencing methods include Maxam-Gilbert sequencing and chain-termination methods, de novo sequencing methods including shotgun sequencing and bridge PCR, next-generation methods including Polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD™ sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, and SMRT® sequencing.

[0100] Further, sequence analysis of the nucleic acid molecules can be direct or indirect. Thus, the sequence analysis can be performed on a barcoded nucleic acid molecule or it can be a molecule which is derived therefrom ( e.g ., a complement thereof).

[0101] Other examples of methods for sequencing include, but are not limited to, DNA hybridization methods, restriction enzyme digestion methods, Sanger sequencing methods, ligation methods, and microarray methods. Additional examples of sequencing methods that can be used include targeted sequencing, single molecule real-time sequencing, exon sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, co-amplification at lower denaturation temperature-PCR (COLD-PCR), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, Solexa Genome Analyzer sequencing, MS-PET sequencing, whole transcriptome sequencing, and any combinations thereof.

[0102] If the methods determine binding affinity of an antigen-binding molecule (e.g., antibody or antigen-binding fragment) to a target antigen and/or one or more fragments thereof, and the barcoded nucleic acid molecules include a unique molecular identifier (UMI), the binding affinity can be determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen-binding molecule bound to the target antigen and/or fragments of the target antigen. In embodiments, the binding affinity determined in this manner may be confirmed by other techniques that determine affinity of antigen-binding molecules for target proteins and/or their regions of interest including, for example, competition binning and competition enzyme-linked immunosorbent assay (ELISA), NMR or HDX-MS. [0103] It will be understood that in any of the methods provided herein, the reaction mixture, which includes:

(I) (i) a population of cells expressing antibodies or antigen-binding fragments thereof,

(ii) a target antigen, and (iii) a fragment of the target antigen; or

(II) (i) a population of cells expressing antibodies or antigen-binding fragments thereof, a (ii) plurality of non-overlapping fragments of a target antigen, may further include a further fragment of the target antigen. The further fragment of the target antigen is not meant to necessarily refer one further fragment, as it may refer to one, at least one, two, at least two, three, at least three, four, at least four, five, at least five, six, at least six, seven, at least seven, eight, at least eight, nine, at least nine, ten, at least ten, twenty, at least twenty, thirty, at least thirty, forty, at least forty, fifty, at least fifty, sixty, at least sixty, seventy, at least seventy, eighty, at least eighty, ninety, at least ninety, one hundred, at least one hundred, five hundred, or at least five hundred further fragments. Any further fragment(s) may be coupled to further reporter oligonucleotide(s), which may include sequences of a further reporter sequence (which identifies the further fragment of the target antigen to which it is coupled) and a capture sequence.

[0104] Any further fragment may have one or more substitutions in its sequence relative to the target antigen or any other fragment already present in the reaction mixture. In an example, any further fragment of a length of greater than 200 amino acid residues may have one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 30, or 40 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any further fragment of a length greater than 200 amino acids may have 1-40, 1-30, 1-20, 1- 15, 1-10, or 1-5 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any further fragment of a length of greater than 200 amino acid residues may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding amino acid sequence of the target antigen. In another example, any further fragment of a length of 100 to 200 amino acid residues may have one, two, three, four, five, six, seven, eight, nine,

10, 15, 20, or 30 amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. Any further fragment of a length of 100 to 200 amino acids may have 1-30, 1-20, 1-15, 1-10, or 1-5 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any further fragment of a length of 100 to 200 amino acid residues may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet another example, any further fragment of a length of less than 100 amino acid residues may have one, two, three, four, five, six, seven, eight, nine or 10 amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. Any further fragment of a length of less than 100 amino acids may have 1-10, 1-5, 1-4, or 1-3 amino acid residue substitutions relative to its corresponding amino acid sequence in the target antigen. Any further fragment of a length of less than 100 amino acid residues may have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet another example, any further fragment of a length of less than 40 amino acid residues may have one or two amino acid residue substitutions relative to its corresponding amino acid sequence of the target antigen. Any further fragment of a length of less than 40 amino acids may have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity relative to its corresponding sequence of the target antigen. In yet a further example, any further fragment of a length of less than 20 amino acid residues may have one amino acid residue substitution relative to its corresponding amino acid sequence of the target antigen.

[0105] Also contemplated herein are embodiments wherein the reaction mixture comprises at least one further fragment of the target antigen, and the further fragment of a target antigen and any other fragment of the target antigen in the reaction mixture comprise the same subset of numbered amino acid residues of the target antigen having a set of numbered residues, and the further and/or other fragment(s) comprise different amino acid substitutions. Such embodiments may be useful for positional mutagenesis within a particular region of interest of the target antigen. For example, in some cases, a reaction mixture may include the further and other fragment(s) of the target antigen, and the further and other fragment(s) of the target antigen may substitute different amino acid residues at the same corresponding position of the target antigen amino acid sequence. Alternatively, the further and other fragment(s) of the target antigen may include substitutions at correspondingly different positions of the target antigen amino acid sequence.

[0106] In an embodiment, a reaction mixture may include a set of fragments including further fragments of the same length and from the same length of amino acid residue positions of the target antigen, wherein each fragment includes a different substitution at the same position along the length of the target antigen. For example, the set may include 20 fragments in which each fragment substitutes a different of the 20 naturally-occurring (and/or additionally non-naturally occurring) amino acid residues at the same position along the length. In a different embodiment, a set of fragments including further fragments may include fragments of the same length and from the same length of amino acid residue positions of the target antigen, but include a substitution at each individual amino acid residue position along the length of the target antigen. For example, a set of fragments of 100 amino acid residues in length and of the same length of the target antigen may include fragments in which a substitution is made in each individual different position of the 100 amino acid residue length. Furthermore, a set of fragments including further fragments may include thousands of further fragments of the same length and from the same length of amino acid residue positions of the target antigen, but include a substitution at each individual position along its length, and include more than one substitution at each individual position along its length. For instance, such a set of fragments could include fragments in which each individual amino acid residue along its length is substituted for each of the 20 different naturally-occurring (and/or additionally non-naturally occurring) amino acid residues at each, respective, individual position.

[0107] Amino acid substitutions that may be introduced, generally and as described thoughout the disclosure, may be selected to determine whether antibody or antigen-binding fragment thereof interacts with a particular amino acid residue of the fragment, e.g., if the amino acid residue of the fragment is a part of the epitope for the antibody or antigen-binding fragment thereof. Amino acid substitutions may, but are not necessarily, conservative amino acid substitutions dependent on whether the amino acid substitution would be predicted to result in a protein conformational change. Exemplary conservative amino acids substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256: 1443 45 or in any likelihood matrix, or any PAM matrix. In some embodiments, the substitutions need not be conservative, particularly in light of the fact that further fragments may be included in the reaction mixtures and the further fragments may be usable to introduce more than one different substitution into any given corresponding individual amino acid position in the target antigen sequence. In an embodiment, amino acid substitutions may be incorporated into fragments or further fragments of the target antigen to alanines, or triplets of alanines. Synthetic amino acids or amber codons may also be incorporated as substitutions. Other substitutions that may be incorporated into antigens and fragments thereof are contemplated in the methods, kits, partitions and systems disclosed herein.

[0108] In another example, any of the amino acid substitutions described herein may be introduced to identify antibodies or antigen-binding fragments thereof having affinity for variants, e.g., variants of pathogens such as viruses, that have acquired one or more mutations that improve their binding to or entry into host cells. In some embodiments, the amino acid substitutions are selected to correspond to known disease-associated variants. In some embodiments, the amino acid substitutions are selected to correspond to known naturally occurring or population-associated variants, e.g., of a human antigen.

[0109] In the methods, if the further fragment is included in the reaction mixture, a cell in the reaction mixture may be further bound to the further fragment coupled to the further reporter oligonucleotide. Thus, in one embodiment, a cell in the reaction mixture may be bound to (i) a target antigen coupled to the first reporter oligonucleotide, (ii) a fragment of the target antigen coupled to a second reporter oligonucleotide and (iii) a further fragment coupled to a further reporter oligonucleotide. In yet another embodiment, a cell in the reaction mixture may be bound to (i) a first fragment of the target antigen coupled to a first reporter oligonucleotide, (ii) a second, non-overlapping, fragment of the target antigen coupled to a second reporter oligonucleotide, and (iii) a further, non-overlapping, fragment of the target antigen coupled to the further reporter oligonucleotide. The cell of either of these embodiments may be partitioned as a partitioned cell. The partitioned cell may be in the partition with a plurality of nucleic acid barcode molecules.

[0110] The partitioned cell, which may further be bound to the further fragment coupled to the further reporter oligonucleotide in either of these embodiments, may, in a bulk reaction or in the partition generate: a first barcoded nucleic acid molecule including a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof and/or (ii) a third barcoded nucleic acid molecule including a sequence of a second reporter oligonucleotide or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof; and/or (iii) a further barcoded nucleic acid molecule including a sequence of a further reporter oligonucleotide or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof, and (iv) a second barcoded nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof. It will be understood that any of the barcoded nucleic acid molecules may further include a unique molecular identifier UMI. The UMI may be a sequence that originating from a reporter oligonucleotide or a nucleic acid barcode molecule. It will be understood that any of the barcoded nucleic acid molecules may further comprise a sample index barcode. It will be understood that any of the barcoded nucleic acid molecules may further comprise a barcode that identifies an experimental condition of a cell. Such sample index barcodes or experimental condition barcodes may facilitate multiplexing, where cells of different groups, e.g., different samples or different experimental conditions can be labeled separately and then pooled together for downstream analysis.

[0111] In these methods, the determining sequence of the second barcoded nucleic acid molecule may identify the antibody or antigen-binding fragment thereof expressed by the cell in the partition in which the barcoded nucleic was generated. The determining the sequence of the first and/or third and/or further barcoded nucleic acid molecule may assess the affinity of the antibody or antigen-binding fragment produced by the cell in the partition in which the barcoded nucleic was generated.

[0112] If the methods determine sequences that identify the antibody or antigen-binding fragment thereof expressed by the cell of a partition in these embodiments, the sequences may be nucleic acid sequences encoding the antibody of the antigen-binding fragment thereof. The nucleic acid sequences may encode one or more of a complementarity determining region (CDR), a framework (FWR), a variable heavy chain domain (VH), or a variable light chain domain (VL) of the antibody or antigen-binding fragment thereof. Alternatively, if the methods determine sequences that identify the antibody or antigen binding fragment thereof expressed by the cell of a partition in these embodiments, the sequences may be amino acid sequences of the antibody or antigen-binding fragment thereof. The amino acid sequences may include a sequence of one or more of a CDR, FWR, VH or VL of the antibody or antigen binding fragment thereof.

[0113] In any of the methods described herein, an antibody may be identified or characterized. The identification or characterization may characterize the antibody or antigen-binding fragment thereof as binding a region of interest of the target antigen, or as having binding affinity to the region of interest of the target antigen, or as having it binding affinity mapped to the region of interest of the target antigen if: the antibody or antigen binding fragment is assessed as having binding affinity for the target antigen and/or fragment(s) of the target antigen comprising the region of interest of the target antigen.

[0114] In any of the methods described herein, it will be understood that the partitioning of the reaction mixture may partition more than one cell of the plurality of cells into more than one of a plurality of partitions. The partitioning of the reaction mixture may partition a first cell of the plurality of cells into a first partition, it may further partition a second cell of the plurality of cells into a second partition. Moreover, it may additionally partition a third cell of the plurality of cells into a third partition, a fourth cell of the plurality of cells into a fourth partition, up to hundreds of cells that are each partitioned into a separate, individual, partition. It should be understood that each and every partitioned cell need be bound to one or more in particular of the target antigen or any fragment of the target antigen. However, at least one cell of the population of cells partitioned into a partition will be bound to a target antigen and/or a fragment of target antigen.

[0115] In any of the methods described here, cell of the plurality may be enriched prior to the partitioning. The cells may be enriched for cell type, e.g., B cells, if obtained from a blood sample or may be enriched by sorting, e.g., as cells bound to the target antigen and/or one or more fragments of the target antigen.

PARTITIONS

[0116] In an aspect, the disclosure provides for a partition. It also, in the methods described herein, provides a partition. In a general sense, a “partition,” may, be understood to, and may in embodiments disclosed herein, refer to a space or volume that can be suitable to contain one or more cells, one or more species of features or compounds, or conduct one or more reactions. Examples of partitions include droplets or microwells.

[0117] A partition may include a cell expressing an antigen-binding molecule, such as antibody or an antigen-binding fragment of an antibody. If the antigen-binding molecule is an antibody, the antibody may be an antibody having an immunoglobulin (Ig)A (e.g, IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3 and IgG4) or IgM constant region. If the antigen-binding molecule is a fragment of an antibody, the fragment of the antibody may be any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. An antigen-binding fragment of an antibody may be one of: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) sdAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody ( e.g ., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FWR3-CDR3-FWR4 peptide. Further, an antigen-binding fragment of an antibody may be an engineered molecule, such as a domain-specific antibody, single domain antibody, chimeric antibody, CDR-grafted antibody, diabody, triabody, tetrabody, minibody, nanobody (e.g., monovalent nanobodies, bivalent nanobodies, etc.), a small modular immunopharmaceutical (SMIP), or a shark IgNAR variable domain.

[0118] A cell in the partition, cell expressing the antigen-binding molecule, may be cell of B cell lineage, e.g, a memory B cell, which express an antibody as a cell surface receptor. A cell may also be an engineered cell having been engineered to express antibodies or antigen-binding fragments of antibodies as a cell surface receptor. The cell expressing the antigen-binding molecule, may be a cell obtained from a subject, e.g, a mammal such as a human. If the cell has been obtained from a subject, it may be from a sample of the subject. The sample of the subject may be obtained by biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a plasma or serum sample.

[0119] The sample of the subject, from which the antigen-binding molecule may have obtained, may have been subject to processing steps so as to arrive at the cell for inclusion in the partition. The processing steps may include steps such as filtration, selective precipitation, purification, centrifugation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort cells of different types, e.g, to separate B cells from other cell types. A separation process can be a positive selection process, a negative selection process (e.g, removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g, removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells).

[0120] In embodiments, the partition may include the cell expressing the antigen binding molecule, and the antigen-binding molecule may be bound to an antigen and a fragment of the antigen. In other embodiments, the partition may include the cell expressing the antigen-binding molecule and first and second fragments of an antigen.

[0121] In any of these embodiments, the antigen may be any antigen for which it is desirable to identify an antigen-binding molecule capable of binding, or of having an affinity for binding. Examples of such antigens include antigens associated with an infectious agent, e.g., a viral, bacterial, parasitic, protozoal or prion agent. If the antigen is associated with an infectious agent that is a viral agent, the viral agent may be an influenza virus, a coronavirus, a retrovirus, a rhinovirus, or a sarcoma virus. The viral agent may be severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), a SARS-CoV-2, a Middle East respiratory syndrome coronavirus (MERS-CoV)), or human immunodeficiency virus (HIV), influenza, respiratory syncytial virus, or Ebola virus. If the antigen is associated with an infectious agent that is a viral agent, the target antigen may be corona virus spike (S) protein, e.g., SARS-CoV-2 S protein, an influenza hemagglutinin protein, an HIV envelope protein or any other a viral glycoprotein. Further, the antigen may be associated with a tumor or a cancer. If the antigen is associated with tumors or cancers, it may be, for example, epidermal growth factor receptor (EGFR), CD38, platelet-derived growth factor receptor (PDGFR) alpha, insulin growth factor receptor (IGFR), CD20, CD 19, CD47, or human epidermal growth factor receptor 2 (HER2). In addition, the antigen may be an immune checkpoint molecule that may or may not be associated with tumors or cancers (e.g., CD38, PD-1, CTLA-4,

TIGIT, LAG-3, VISTA, TIM-3), or it may be a cytokine (e.g., a soluble cytokine), a GPCR, a cell-based co-stimulatory molecule, a cell-based co-inhibitory molecule, an ion channel, a glycan, a glycan conjugate, or a growth factor.

[0122] In an embodiment in which the cell expressing the antigen-binding molecule is bound to the antigen and the fragment of the antigen, the antigen to which the cell is bound (as described above) may be in its full-length form, e.g., as expressed by a cell or as expressed the cell following removal of its leader sequence or other similar processing step.

[0123] In embodiments in which (i) the cell expressing the antigen-binding molecule is bound to the antigen and the fragment of the antigen; or (ii) the cell expressing the antigen binding molecule is bound to a first and a second fragment of the antigen, the fragment and/or fragments of the antigen to which the cell is bound may be polypeptides that are shorter in amino acid sequence length than the full-length antigen. For example, the fragment of the antigen may have an amino acid sequence length that is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% that of the antigen. As another example, the fragment of the antigen may have an amino acid sequence length that is 75% or below, 70% or below, 65% or below, 60% or below, 55% or below, 50% or below, 45% or below, 40% or below, 35% or below, 30% or below, 25% or below, 20% or below, 15% or below, 10% or below, or 5% or below that of the antigen. As another example, the fragment of the target antigen may have an amino acid sequence length that is 75% or below, 70% or below, 65% or below, 60% or below, 55% or below, 50% or below, 45% or below, 40% or below, 35% or below, 30% or below, 25% or below, 20% or below, 15% or below, 10% or below, or 5% or below that of the target antigen. The fragment of the antigen may be 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, 20-60, 20-40, 15-20, 40-200, 40-180, 40-160, 40-140, 40-120, 40-100, 40-80, 40-60, 60-200, 60-180, 60- 160, 60-140, 60-120, 60-100, 60-80, 80-200, 80-180, 80-160, 80-140, 80-120, 80-100, 100- 200, 150-100, 25-175, 25-150, 25-125, 25-100, or 25-75 amino acid residues in length, so long as it is shorter in length than the full-length antigen. The fragment of the antigen may include or may be an epitope of the antigen known to be of importance. The fragment of the antigen may include or may be an epitope of the antigen that includes known sequence variations, such as SNVs or amino acid substitutions arising therefrom, deletions, additions, rearrangements, and the like. The fragment of the antigen may include or may be a domain of the antigen known to be of importance. An epitope or domain of importance of the antigen may be an epitope or domain of the antigen that mediates a process, e.g., affects a signaling pathway directly or by costimulation, is critical to host-pathogen interaction, or affects a conformational change. The fragment may include fewer domains and/or epitopes than does the full-length antigen. In some embodiments, the fragment is not complexed with a MHC molecule.

[0124] If the cell in the partition is bound to a first and second fragment of the antigen, the first and the second fragment of the antigen may be non-overlapping. If the first and the second fragment are non-overlapping, they may have completely distinct amino acid sequences and may be from the same different domains or regions or portions the antigen. If the fragments of the antigen are non-overlapping fragments of the antigen, the fragments may have completely distinct amino acid sequences although they are from the same domains or regions or portions the antigen. The non-overlapping fragments need not, however, have completely distinct amino acid sequences along their entire length. The non-overlapping fragments of the antigen may include consecutive amino acid residues that are identical, e.g., at their N- or C-terminus, and consecutive amino acid residue that are completely distinct, i.e., are non-overlapping to an extent. For example, first and second non-overlapping fragments may each be 100 amino acid residues in length, of which the 20 C-terminal amino acid residues of the first and the 20 N-terminal amino acid residues of the second fragment are identical, while the 80 N-terminal amino acid residues of the first and the 80 C-terminal amino acid residues of the second fragment are distinct. It will be understood that non overlapping fragments of the antigen need not be of the same or of similar amino acid residue length. The non-overlapping fragments may share and differ in inclusion of domains and/or epitopes of the target antigen. The non-overlapping fragments may differ in inclusion of domains and/or epitopes of the target antigen. Other characteristics of fragments, “non overlapping”, and amino acid substitutions for introduction in fragments have been described elsewhere herein. By way of example, a fragment of a target antigen may be a fragment of viral antigen, such as a coronavirus antigen, e.g., SARS CoV-2 spike protein. If the fragment of the target antigen is a fragment of a coronavirus antigen, e.g. , SARS Co-V-2 spike protein, it may be or include the receptor binding domain, the N-terminal binding domain, or the extracellular domain of the SARS Co-V-2 spike protein.

[0125] If the partition includes a cell expressing an antigen-binding molecule and the antigen-binding molecule is bound to an antigen and a fragment of the antigen, the antigen may be coupled to a first reporter oligonucleotide and the fragment of the antigen may be bound to a second reporter oligonucleotide. In such an embodiment, the first reporter oligonucleotide may include a first reporter sequence specific to the antigen and a capture handle sequence. The second reporter oligonucleotide may include a second reporter sequence specific to the fragment of the antigen and capture handle sequence.

[0126] If the partition includes a cell expressing an antigen-binding molecule and the antigen-binding molecule is bound to a first and a second fragment of the antigen, the first antigen may be coupled to a first reporter oligonucleotide and the second fragment of the antigen may be bound to a second reporter oligonucleotide. In such an embodiment, the first reporter oligonucleotide may include a first reporter sequence specific to the first fragment of the antigen and a capture handle sequence. The second reporter oligonucleotide may include a second reporter sequence specific to the second fragment of the antigen and a capture handle sequence.

[0127] Any or either of the partitions disclosed herein may further include a plurality of nucleic acid barcode molecules. Nucleic acid barcode molecules of the plurality may include a partition-specific sequence. First nucleic acid barcode molecules of the plurality may further include a capture sequence configured to couple to the capture handle sequence of the first and/or second reporter oligonucleotide of any first and/or second reporter oligonucleotides of the embodiment partitions disclosed herein. Second nucleic acid barcode molecules of the plurality may further include a capture sequence configured to couple to (i) an mRNA or DNA analyte or (ii) a cDNA reverse transcribed from an mRNA analyte by a reverse transcriptase having terminal transferase activity. In embodiments in which the second nucleic acid barcode molecules of the plurality further includes the capture sequence configured to couple to the (ii) cDNA reverse transcribed from the mRNA analyte, the partition may further include a primer (which may couple to the mRNA analyte, e.g ., via complementary base pairing, at a nucleotide sequence of the mRNA analyte, e.g., polyT sequence). In embodiments in which the second nucleic acid barcode molecules of the plurality further include the capture sequence configured to couple to the (ii) cDNA reverse transcribed from the mRNA analyte, the partition may further include a reverse transcriptase with terminal transferase activity.

[0128] Regardless of whether the nucleic acid barcode molecules of the plurality include a capture sequence configured to couple to the capture handle sequence of the first and/or second reporter oligonucleotide or whether the nucleic acid barcode molecules of the plurality include a capture sequence configured to couple to an mRNA, DNA analyte or cDNA of an mRNA analyte, the capture sequence may configured such that its coupling is by complementary base pairing. For example, a capture sequence configured to couple to an mRNA analyte comprises a polyT sequence. In another example, a capture sequence configured to couple to a cDNA reverse transcribed from an mRNA analyte may include a polyG sequence, e.g., for complementary base pairing to a polyC sequence that may be appended to the cDNA during reverse transcription of the mRNA analyte by the reverse transcriptase having terminal transferase activity.

[0129] In a partition in which (i) the cell expressing the antigen-binding molecule is bound to the antigen and the fragment of the antigen; or (ii) the cell expressing the antigen binding molecule is bound to a first and a second fragment of the antigen, the cell expressing the antigen-binding molecule may be bound to a further antigen. If the cell is bound to a further antigen, it may be understood that the cell may be bound to one, at least one, two, at least two, three, at least three, four, at least four, five, at least five, six, at least six, seven, at least seven, eight, at least eight, nine, at least nine, ten, at least ten additional fragments of the target antigen. In the event that the cell is bound to a further fragment of the antigen, the further fragment of the antigen may be coupled to a further reporter oligonucleotide. The further reporter oligonucleotide coupled to the further fragment of the target antigen may include a further reporter sequence which identifies the further fragment to which it is coupled and a capture handle sequence. If the partition includes the further fragment of the antigen coupled to the further reporter oligonucleotide, in which the further reporter oligonucleotide includes a further reporter sequence and a capture handle sequence, then the partition’s included nucleic acid barcode molecules may be capable of coupling to the capture handle sequence of the further reporter oligonucleotide.

[0130] It may be understood that any of the first reporter oligonucleotide and/or second reporter oligonucleotide and/or further reporter oligonucleotide and/or nucleic acid barcode molecules may include a UMI and/or primer sequences. It may also be understood that additional reagents, such as buffers or enzymes, or fluorescent labels may be included in or added to the partitions. Further disclosure related to these reagents can be found in the “ Further Disclosure - Partitions, Partitioning, Reagents and Processing" section, immediately below.

Further Disclosure - Partitions, Partitioning, Reagents and Processing

Systems and methods for partitioning

[0131] In some aspects, such as those that have been described above, the methods provided herein include a step of partitioning, or include a step of generating barcoded nucleic acid molecules, or may include an additional processing step(s). In some aspects, the methods herein provide for a partition. This description sets forth examples, embodiments and characteristics of steps of the methods, of the partitions, and of reagents useful in the methods or as may be provided in the partitions.

[0132] In an aspect, the systems and methods described herein provide for the compartmentalization, depositing, or partitioning of one or more particles (e.g, biological particles, macromolecular constituents of biological particles, beads, reagents, etc.) into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions.

[0133] In some embodiments disclosed herein, the partitioned particle is a labelled cell of B cell lineage, e.g. a memory B cell, which expresses an antigen-binding molecule (e.g. , an immune receptor, an antibody or a functional fragment thereof) on its surface. In other examples, the partitioned particle can be a labelled cell engineered to express antigen-binding molecules ( e.g ., an immune receptors, antibodies or functional fragments thereof).

[0134] The term “partition,” as used herein, generally, refers to a space or volume that can be suitable to contain one or more cells, one or more species of features or compounds, or conduct one or more reactions. A partition can be a physical container, compartment, or vessel, such as a droplet, a flow cell, a reaction chamber, a reaction compartment, a tube, a well, or a microwell. In some embodiments, the compartments or partitions include partitions that are flowable within fluid streams. These partitions can include, for example, micro vesicles that have an outer barrier surrounding an inner fluid center or core, or, in some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials within its matrix. In some aspects, partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase). A variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in detail in, e.g, U.S. Patent Application Publication No. 2010/010511.

[0135] In some embodiments, a partition herein includes a space or volume that can be suitable to contain one or more species or conduct one or more reactions. A partition can be a physical compartment, such as a droplet or well. The partition can be an isolated space or volume from another space or volume. The droplet can be a first phase (e.g, aqueous phase) in a second phase (e.g, oil) immiscible with the first phase. The droplet can be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition can include one or more other (inner) partitions. In some cases, a partition can be a virtual compartment that can be defined and identified by an index (e.g, indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment can include a plurality of virtual compartments.

[0136] In some embodiments, the methods described herein provide for the compartmentalization, depositing or partitioning of individual cells from a sample material containing cells, into discrete partitions, where each partition maintains separation of its own contents from the contents of other partitions. Identifiers including unique identifiers (e.g, UMI) and common or universal tags, e.g, barcodes, can be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments. Further, identifiers including unique identifiers and common or universal tags, e.g., barcodes, can be coupled to labelling agents and previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments. Identifiers including unique identifiers and common or universal tags, e.g, barcodes, can be delivered, for example on an oligonucleotide, to a partition via any suitable mechanism, for example by coupling the barcoded oligonucleotides, e.g., nucleic acid barcode molecules, to a bead. In some embodiments, the barcoded oligonucleotides, e.g., nucleic acid barcode molecules, are reversibly (e.g, releasably) coupled to a bead. The bead suitable for the compositions and methods of the disclosure can have different surface chemistries and/or physical volumes. In some embodiments, the bead includes a polymer gel. In some embodiments, the polymer gel is a polyacrylamide. Additional non-limiting examples of suitable beads include microparticles, nanoparticles, beads, and microbeads. The partition can be a droplet in an emulsion. A partition can include one or more particles. A partition can include one or more types of particles. For example, a partition of the present disclosure can include one or more biological particles, e.g, labelled engineered cells, B cells, or memory B cells, and/or macromolecular constituents thereof. A partition can include one or more gel beads. A partition can include one or more cell beads. A partition can include a single gel bead, a single cell bead, or both a single cell bead and single gel bead. A partition can include one or more reagents. Alternatively, a partition can be unoccupied. For example, a partition cannot comprise a bead. Unique identifiers, such as barcodes, can be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a bead, as described elsewhere herein. Microfluidic channel networks (e.g, on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.

[0137] The partitions can be flowable within fluid streams. The partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core. In some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials (e.g, expressed antibodies or antigen-binding fragments thereof) within its matrix (e.g, via a capture agent configured to couple to both the matrix and the expressed antibody or antigen-binding fragment thereof). The partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible. For example, the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase ( e.g ., oil phase). In another example, the partitions can be droplets of a non-aqueous fluid within an aqueous phase. In some examples, the partitions can be provided in a water- in-oil emulsion or oil-in-water emulsion. A variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in, for example, U.S. Patent Application Publication No. 2010/0105112.

[0138] In the case of droplets in an emulsion, allocating individual particles (e.g., labelled engineered cells) to discrete partitions can, in one non-limiting example, be accomplished by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams. Fluid properties (e.g, fluid flow rates, fluid viscosities, etc.), particle properties (e.g, volume fraction, particle size, particle concentration, etc.), microfluidic architectures (e.g, channel geometry, etc.), and other parameters can be adjusted to control the occupancy of the resulting partitions (e.g, number of biological particles per partition, number of beads per partition, etc.). For example, partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of particles. To generate single biological particle partitions, the relative flow rates of the immiscible fluids can be selected such that, on average, the partitions can contain less than one biological particle per partition in order to ensure that those partitions that are occupied are primarily singly occupied. In some cases, partitions among a plurality of partitions can contain at most one biological particle (e.g, bead, DNA, cell, such as a labelled engineered cells, B cells, or memory B cells, or cellular material). In some embodiments, the various parameters (e.g, fluid properties, particle properties, microfluidic architectures, etc.) can be selected or adjusted such that a majority of partitions are occupied, for example, allowing for only a small percentage of unoccupied partitions. The flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions.

[0139] In some embodiments, the method further includes individually partitioning one or more single cells from a plurality of cells in a partition of a second plurality of partitions.

[0140] In some embodiments, at least one of the first and second plurality of partitions includes a microwell, a flow cell, a reaction chamber, a reaction compartment, or a droplet. In some embodiments, at least one of the first and second plurality of partitions includes individual droplets in emulsion. In some embodiments, the partitions of the first plurality and/or the second plurality of partition have the same reaction volume.

[0141] In the case of droplets in emulsion, allocating individual cells to discrete partitions can generally be accomplished by introducing a flowing stream of cells in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams. By providing the aqueous cell -containing stream at a certain concentration of cells, the occupancy of the resulting partitions ( e.g ., number of cells per partition) can be controlled. For example, where single cell partitions are desired, the relative flow rates of the fluids can be selected such that, on average, the partitions contain less than one cell per partition, in order to ensure that those partitions that are occupied, are primarily singly occupied. In some embodiments, the relative flow rates of the fluids can be selected such that a majority of partitions are occupied, e.g., allowing for only a small percentage of unoccupied partitions. In some embodiments, the flows and channel architectures are controlled as to ensure a desired number of singly occupied partitions, less than a certain level of unoccupied partitions and less than a certain level of multiply occupied partitions.

[0142] In some embodiments, the methods described herein can be performed such that a majority of occupied partitions include no more than one cell per occupied partition. In some embodiments, the partitioning process is performed such that fewer than 25%, fewer than 20%, fewer than 15%, fewer than 10%, fewer than 5%, fewer than 2%, or fewer than 1% the occupied partitions contain more than one cell. In some embodiments, fewer than 20% of the occupied partitions include more than one cell. In some embodiments, fewer than 10% of the occupied partitions include more than one cell per partition. In some embodiments, fewer than 5% of the occupied partitions include more than one cell per partition. In some embodiments, it is desirable to avoid the creation of excessive numbers of empty partitions. For example, from a cost perspective and/or efficiency perspective, it may be desirable to minimize the number of empty partitions. While this can be accomplished by providing sufficient numbers of cells into the partitioning zone, the Poissonian distribution can optionally be used to increase the number of partitions that include multiple cells. As such, in some embodiments described herein, the flow of one or more of the cells, or other fluids directed into the partitioning zone are performed such that no more than 50% of the generated partitions, no more than 25% of the generated partitions, or no more than 10% of the generated partitions are unoccupied. Further, in some aspects, these flows are controlled so as to present non-Poissonian distribution of single occupied partitions while providing lower levels of unoccupied partitions. Restated, in some aspects, the above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in some embodiments, the use of the systems and methods described herein creates resulting partitions that have multiple occupancy rates of less than 25%, less than 20%, less than 15%), less than 10%, and in some embodiments, less than 5%, while having unoccupied partitions of less than 50%), less than 40%, less than 30%, less than 20%, less than 10%, and in some embodiments, less than 5%.

[0143] Although described in terms of providing substantially singly occupied partitions, above, in some embodiments, the methods as described herein include providing multiply occupied partitions, e.g., containing two, three, four or more cells and/or beads comprising nucleic acid barcode molecules within a single partition.

[0144] In some embodiments, the reporter oligonucleotides contained within a partition are distinguishable from the reporter oligonucleotides contained within other partitions of the plurality of partitions.

[0145] In some embodiments, it may be desirable to incorporate multiple different barcode sequences within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known barcode sequences set can provide greater assurance of identification in the subsequent processing, e.g, by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.

Microfluidic channel structures

[0146] Microfluidic channel networks (e.g, on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.

[0147] FIG. 12 shows an example of a microfluidic channel structure 1200 for partitioning individual biological particles. The channel structure 1200 can include channel segments 1202, 1204, 1206 and 1208 communicating at a channel junction 1210. In operation, a first aqueous fluid 1212 that includes suspended biological particles (e.g, cells, for example, labelled engineered cells, B cells, or memory B cells) 1214 can be transported along channel segment 1202 into junction 1210, while a second fluid 1216 that is immiscible with the aqueous fluid 1212 is delivered to the junction 1210 from each of channel segments 1204 and 1206 to create discrete droplets 1218, 1220 of the first aqueous fluid 1212 flowing into channel segment 1208, and flowing away from junction 1210. The channel segment 1208 can be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated can include an individual biological particle 1214 (such as droplets 1218). A discrete droplet generated can include more than one individual biological particle ( e.g ., labelled B cells) 1214 (not shown in FIG. 12). A discrete droplet can contain no biological particle 1214 (such as droplet 1220). Each discrete partition can maintain separation of its own contents (e.g., individual biological particle 1214) from the contents of other partitions.

[0148] The second fluid 1216 can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 1218, 1220. Examples of particularly useful partitioning fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010/0105112.

[0149] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure 1200 can have other geometries. For example, a microfluidic channel structure can have more than one channel junction. For example, a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying particles (e.g, biological particles, cell beads, and/or gel beads) that meet at a channel junction. Fluid can be directed to flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors (e.g, providing positive pressure), pumps (e.g, providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.

[0150] The generated droplets can include two subsets of droplets: (1) occupied droplets 1218, containing one or more biological particles 1214, e.g, labelled engineered cells, B cells, or memory B cells, and (2) unoccupied droplets 1220, not containing any biological particles 1214. Occupied droplets 1218 can include singly occupied droplets (having one biological particle, such as one B cell or memory B cell) and multiply occupied droplets (having more than one biological particle, such as multiple B cells or memory B cells). As described elsewhere herein, in some cases, the majority of occupied partitions can include no more than one biological particle, e.g., labelled engineered cells, B cells, or memory B cells, per occupied partition and some of the generated partitions can be unoccupied (of any biological particle, or labelled engineered cells, B cells, or memory B cells). In some cases, though, some of the occupied partitions can include more than one biological particle, e.g, labelled engineered cells, B cells, or memory B cells. In some cases, the partitioning process can be controlled such that fewer than about 25% of the occupied partitions contain more than one biological particle, and in many cases, fewer than about 20% of the occupied partitions have more than one biological particle, while in some cases, fewer than about 10% or even fewer than about 5% of the occupied partitions include more than one biological particle per partition.

[0151] In some cases, it can be desirable to minimize the creation of excessive numbers of empty partitions, such as to reduce costs and/or increase efficiency. While this minimization can be achieved by providing a sufficient number of biological particles (e.g, biological particles, such as labelled engineered cells, B cells, or memory B cells 1214) at the partitioning junction 1210, such as to ensure that at least one biological particle is encapsulated in a partition, the Poissonian distribution can expectedly increase the number of partitions that include multiple biological particles. As such, where singly occupied partitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions can be unoccupied.

[0152] In some cases, the flow of one or more of the biological particles, such as B cells or memory B cells, (e.g, in channel segment 1202), or other fluids directed into the partitioning junction (e.g, in channel segments 1204, 1206) can be controlled such that, in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled so as to present a non-Poissonian distribution of single-occupied partitions while providing lower levels of unoccupied partitions. The above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, the use of the systems and methods described herein can create resulting partitions that have multiple occupancy rates of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while having unoccupied partitions of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less.

[0153] As will be appreciated, the above-described occupancy rates are also applicable to partitions that include both biological particles ( e.g ., cells) and additional reagents, including, but not limited to, beads (e.g., gel beads) carrying nucleic acid barcode molecules (e.g, barcoded oligonucleotides) (described in relation to FIGS. 12 and 13). The occupied partitions (e.g, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) can include both a bead comprising nucleic acid barcode molecules and a biological particle.

[0154] In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g., cells) may be encapsulated within a particulate material to form a “cell bead”.

[0155] The cell bead can include other reagents. Encapsulation of biological particles, e.g, labelled engineered cells, can be performed by a variety of processes. Such processes can combine an aqueous fluid containing the biological particles with a polymeric precursor material that can be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor. Such stimuli can include, for example, thermal stimuli (e.g, either heating or cooling), photo-stimuli (e.g, through photo-curing), chemical stimuli (e.g, through crosslinking, polymerization initiation of the precursor (e.g, through added initiators)), mechanical stimuli, or a combination thereof.

[0156] Encapsulation of biological particles, e.g, labelled engineered cells, B cells, or memory B cells, can be performed by a variety of methods. For example, air knife droplet or aerosol generators may be used to dispense droplets of precursor fluids into gelling solutions in order to form cell beads that include individual biological particles or small groups of biological particles. Likewise, membrane-based encapsulation systems may be used to generate cell beads comprising encapsulated biological particles as described herein. Microfluidic systems of the present disclosure, such as that shown in FIG. 12, may be readily used in encapsulating biological particles (e.g., cells) as described herein. Exemplary methods for encapsulating biological particles (e.g., cells) are also further described in U.S. Patent Application Pub. No. US 2015/0376609 and PCT/US2018/016019. In particular, and with reference to FIG. 12, the aqueous fluid 1212 comprising (i) the biological particles 1214 and (ii) the polymer precursor material (not shown) is flowed into channel junction 1210, where it is partitioned into droplets 1218, 1220 through the flow of non-aqueous fluid 1216.

In the case of encapsulation methods, non-aqueous fluid 1216 may also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the microcapsule that includes the entrained biological particles. Examples of polymer precursor/initiator pairs include those described in U.S. Patent Application Publication No. 2014/0378345.

[0157] For example, in the case where the polymer precursor material comprises a linear polymer material, such as a linear polyacrylamide, PEG, or other linear polymeric material, the activation agent can include a cross-linking agent, or a chemical that activates a cross-linking agent within the formed droplets. Likewise, for polymer precursors that comprise polymerizable monomers, the activation agent can include a polymerization initiator. For example, in certain cases, where the polymer precursor comprises a mixture of acrylamide monomer with a N,N’-bis-(acryloyl)cystamine (BAC) comonomer, an agent such as tetraethylmethylenediamine (TEMED) can be provided within the second fluid streams 1216 in channel segments 1204 and 1206, which can initiate the copolymerization of the acrylamide and BAC into a cross-linked polymer network, or hydrogel.

[0158] Upon contact of the second fluid stream 1216 with the first fluid stream 1212 at junction 1210, during formation of droplets, the TEMED can diffuse from the second fluid 1216 into the aqueous fluid 1212 comprising the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets 1218, 1220, resulting in the formation of gel ( e.g ., hydrogel) cell beads, as solid or semi-solid beads or particles entraining the cells (e.g., B cells) 1214. Although described in terms of polyacrylamide encapsulation, other “activatable” encapsulation compositions can also be employed in the context of the methods and compositions described herein. For example, formation of alginate droplets followed by exposure to divalent metal ions (e.g, Ca²⁺ ions), can be used as an encapsulation process using the described processes. Likewise, agarose droplets can also be transformed into capsules through temperature based gelling (e.g, upon cooling, etc.).

[0159] In some cases, encapsulated biological particles can be selectively releasable from the cell bead, such as through passage of time or upon application of a particular stimulus, that degrades the encapsulating material sufficiently to allow the biological particles (e.g, labelled B cells), or its other contents to be released from the encapsulating material, such as into a partition (e.g, droplet). For example, in the case of the polyacrylamide polymer described above, degradation of the polymer can be accomplished through the introduction of an appropriate reducing agent, such as DTT or the like, to cleave disulfide bonds that cross link the polymer matrix. See, for example, U.S. Patent Application Publication No. 2014/0378345.

[0160] The biological particle ( e.g ., labelled B cells), can be subjected to other conditions sufficient to polymerize or gel the precursors. The conditions sufficient to polymerize or gel the precursors can include exposure to heating, cooling, electromagnetic radiation, and/or light. The conditions sufficient to polymerize or gel the precursors can include any conditions sufficient to polymerize or gel the precursors. Following polymerization or gelling, a polymer or gel can be formed around the biological particle (e.g., labelled B cells). The polymer or gel can be diffusively permeable to chemical or biochemical reagents. The polymer or gel can be diffusively impermeable to macromolecular constituents (e.g, secreted antibodies or antigen-binding fragments thereof) of the biological particle (e.g, labelled B cells). In this manner, the polymer or gel can act to allow the biological particle (e.g, labelled B cells) to be subjected to chemical or biochemical operations while spatially confining the macromolecular constituents to a region of the droplet defined by the polymer or gel. The polymer or gel can include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel can include any other polymer or gel.

[0161] The polymer or gel can be functionalized (e.g, coupled to a capture agent) to bind to targeted analytes (e.g, secreted antibodies or antigen-binding fragment thereof), such as nucleic acids, proteins, carbohydrates, lipids or other analytes. The polymer or gel can be polymerized or gelled via a passive mechanism. The polymer or gel can be stable in alkaline conditions or at elevated temperature. The polymer or gel can have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel can be of a similar size to the bead. The polymer or gel can have a mechanical strength (e.g, tensile strength) similar to that of the bead. The polymer or gel can be of a lower density than an oil. The polymer or gel can be of a density that is roughly similar to that of a buffer. The polymer or gel can have a tunable pore size. The pore size can be chosen to, for instance, retain denatured nucleic acids. The pore size can be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors. The polymer or gel can be biocompatible. The polymer or gel can maintain or enhance cell viability. The polymer or gel can be biochemically compatible. The polymer or gel can be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically.

[0162] The polymer can include poly(acrylamide-co-acrylic acid) crosslinked with disulfide linkages. The preparation of the polymer can include a two-step reaction. In the first activation step, poly(acrylamide-co-acrylic acid) can be exposed to an acylating agent to convert carboxylic acids to esters. For instance, the poly(acrylamide-co-acrylic acid) can be exposed to 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). The polyacrylamide-co-acrylic acid can be exposed to other salts of 4-(4,6-dimethoxy-l,3,5- triazin-2-yl)-4-methylmorpholinium. In the second cross-linking step, the ester formed in the first step can be exposed to a disulfide crosslinking agent. For instance, the ester can be exposed to cystamine (2,2’-dithiobis(ethylamine)). Following the two steps, the biological particle can be surrounded by polyacrylamide strands linked together by disulfide bridges. In this manner, the biological particle can be encased inside of or comprise a gel or matrix ( e.g ., polymer matrix) to form a “cell bead.” A cell bead can contain biological particles (e.g., labelled B cells) or macromolecular constituents (e.g, RNA, DNA, proteins, secreted antibodies or antigen-binding fragments thereof etc.) of biological particles. A cell bead can include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads. Systems and methods disclosed herein can be applicable to both (i) cell beads (and/or droplets or other partitions) containing biological particles and (ii) cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles.

[0163] Encapsulated biological particles (e.g, labelled B cells) can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it can be desirable to allow biological particles (e.g, labelled B cells) to incubate for a select period of time before analysis, such as in order to characterize changes in such biological particles over time, either in the presence or absence of different stimuli (e.g, cytokines, antigens, etc.). In such cases, encapsulation can allow for longer incubation than partitioning in emulsion droplets, although in some cases, droplet partitioned biological particles can also be incubated for different periods of time, e.g, at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours or more. The encapsulation of biological particles (e.g, labelled B cells) can constitute the partitioning of the biological particles into which other reagents are co-partitioned. Alternatively or in addition, encapsulated biological particles can be readily deposited into other partitions ( e.g ., droplets) as described above.

Microwells

[0164] As described herein, one or more processes can be performed in a partition, which can be a well. The well can be a well of a plurality of wells of a substrate, such as a microwell of a microwell array or plate, or the well can be a microwell or microchamber of a device (e.g., microfluidic device) comprising a substrate. The well can be a well of a well array or plate, or the well can be a well or chamber of a device (e.g, fluidic device). Accordingly, the wells or microwells can assume an “open” configuration, in which the wells or microwells are exposed to the environment (e.g, contain an open surface) and are accessible on one planar face of the substrate, or the wells or microwells can assume a “closed” or “sealed” configuration, in which the microwells are not accessible on a planar face of the substrate. In some instances, the wells or microwells can be configured to toggle between “open” and “closed” configurations. For instance, an “open” microwell or set of microwells can be “closed” or “sealed” using a membrane (e.g, semi-permeable membrane), an oil (e.g, fluorinated oil to cover an aqueous solution), or a lid, as described elsewhere herein. The wells or microwells can be initially provided in a “closed” or “sealed” configuration, wherein they are not accessible on a planar surface of the substrate without an external force. For instance, the “closed” or “sealed” configuration can include a substrate such as a sealing film or foil that is puncturable or pierceable by pipette tip(s). Suitable materials for the substrate include, without limitation, polyester, polypropylene, polyethylene, vinyl, and aluminum foil.

[0165] In some embodiments, the well can have a volume of less than 1 milliliter (mL). For example, the well can be configured to hold a volume of at most 1000 microliters (pL), at most 100 pL, at most 10 pL, at most 1 pL, at most 100 nanoliters (nL), at most 10 nL, at most 1 nL, at most 100 picoliters (pL), at most 10 (pL), or less. The well can be configured to hold a volume of about 1000 pL, about 100 pL, about 10 pL, about 1 pL, about 100 nL, about 10 nL, about 1 nL, about 100 pL, about 10 pL, etc. The well can be configured to hold a volume of at least 10 pL, at least 100 pL, at least 1 nL, at least 10 nL, at least 100 nL, at least 1 pL, at least 10 pL, at least 100 pL, at least 1000 pL, or more. The well can be configured to hold a volume in a range of volumes listed herein, for example, from about 5 nL to about 20 nL, from about 1 nL to about 100 nL, from about 500 pL to about 100 pL, etc. The well can be of a plurality of wells that have varying volumes and can be configured to hold a volume appropriate to accommodate any of the partition volumes described herein.

[0166] In some instances, a microwell array or plate includes a single variety of microwells. In some instances, a microwell array or plate includes a variety of microwells.

For instance, the microwell array or plate can include one or more types of microwells within a single microwell array or plate. The types of microwells can have different dimensions (e.g, length, width, diameter, depth, cross-sectional area, etc.), shapes (e.g, circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, etc.), aspect ratios, or other physical characteristics. The microwell array or plate can include any number of different types of microwells. For example, the microwell array or plate can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more different types of microwells. A well can have any dimension (e.g, length, width, diameter, depth, cross-sectional area, volume, etc.), shape (e.g, circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, other polygonal, etc.), aspect ratios, or other physical characteristics described herein with respect to any well.

[0167] In certain instances, the microwell array or plate includes different types of microwells that are located adjacent to one another within the array or plate. For example, a microwell with one set of dimensions can be located adjacent to and in contact with another microwell with a different set of dimensions. Similarly, microwells of different geometries can be placed adjacent to or in contact with one another. The adjacent microwells can be configured to hold different articles; for example, one microwell can be used to contain a cell, cell bead, or other sample (e.g, cellular components, nucleic acid molecules, etc.) while the adjacent microwell can be used to contain a droplet, bead, or other reagent. In some cases, the adjacent microwells can be configured to merge the contents held within, e.g, upon application of a stimulus, or spontaneously, upon contact of the articles in each microwell.

[0168] As is described elsewhere herein, a plurality of partitions can be used in the systems, compositions, and methods described herein. For example, any suitable number of partitions (e.g, wells or droplets) can be generated or otherwise provided. For example, in the case when wells are used, at least about 1,000 wells, at least about 5,000 wells, at least about 10,000 wells, at least about 50,000 wells, at least about 100,000 wells, at least about 500,000 wells, at least about 1,000,000 wells, at least about 5,000,000 wells at least about 10,000,000 wells, at least about 50,000,000 wells, at least about 100,000,000 wells, at least about 500,000,000 wells, at least about 1,000,000,000 wells, or more wells can be generated or otherwise provided. Moreover, the plurality of wells can include both unoccupied wells ( e.g ., empty wells) and occupied wells.

[0169] A well can include any of the reagents described herein, or combinations thereof. These reagents can include, for example, barcode molecules, enzymes, adapters, and combinations thereof. The reagents can be physically separated from a sample (for example, a cell, cell bead, or cellular components, e.g., proteins, nucleic acid molecules, etc.) that is placed in the well. This physical separation can be accomplished by containing the reagents within, or coupling to, a bead that is placed within a well. The physical separation can also be accomplished by dispensing the reagents in the well and overlaying the reagents with a layer that is, for example, dissolvable, meltable, or permeable prior to introducing the polynucleotide sample into the well. This layer can be, for example, an oil, wax, membrane (e.g, semi-permeable membrane), or the like. The well can be sealed at any point, for example, after addition of the bead, after addition of the reagents, or after addition of either of these components. The sealing of the well can be useful for a variety of purposes, including preventing escape of beads or loaded reagents from the well, permitting select delivery of certain reagents (e.g, via the use of a semi-permeable membrane), for storage of the well prior to or following further processing, etc.

[0170] Once sealed, the well may be subjected to conditions for further processing of a cell (or cells) in the well. For instance, reagents in the well may allow further processing of the cell, e.g., cell lysis, as further described herein. Alternatively, the well (or wells such as those of a well-based array) comprising the cell (or cells) may be subjected to freeze-thaw cycling to process the cell (or cells), e.g., cell lysis. The well containing the cell may be subjected to freezing temperatures (e.g., 0°C, below 0°C, -5°C, -10°C, -15°C, -20°C, -25°C, - 30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -80°C, or -85°C). Freezing may be performed in a suitable manner, e.g., sub-zero freezer or a dry ice/ethanol bath. Following an initial freezing, the well (or wells) comprising the cell (or cells) may be subjected to freeze-thaw cycles to lyse the cell (or cells). In one embodiment, the initially frozen well (or wells) are thawed to a temperature above freezing (e.g., 4°C or above, 8°C or above, 12°C or above, 16°C or above, 20°C or above, room temperature, or 25°C or above).

In another embodiment, the freezing is performed for less than 10 minutes (e.g., 5 minutes or 7 minutes) followed by thawing at room temperature for less than 10 minutes (e.g., 5 minutes or 7 minutes). This freeze-thaw cycle may be repeated a number of times, e.g., 2, 3, 4 or more times, to obtain lysis of the cell (or cells) in the well (or wells). In one embodiment, the freezing, thawing and/or freeze/thaw cycling is performed in the absence of a lysis buffer. Additional disclosure related to freeze-thaw cycling is provided in WO2019165181A1, which is incorporated herein by reference in its entirety.

[0171] A well can include free reagents and/or reagents encapsulated in, or otherwise coupled to or associated with, beads or droplets. In some embodiments, any of the reagents described in this disclosure can be encapsulated in, or otherwise coupled to, a droplet or bead, with any chemicals, particles, and elements suitable for sample processing reactions involving biomolecules, such as, but not limited to, nucleic acid molecules and proteins. For example, a bead or droplet used in a sample preparation reaction for DNA sequencing can include one or more of the following reagents: enzymes, restriction enzymes ( e.g ., multiple cutters), ligase, polymerase, fluorophores, oligonucleotide barcodes, adapters, buffers, nucleotides (e.g., dNTPs, ddNTPs) and the like.

[0172] Additional examples of reagents include, but are not limited to: buffers, acidic solution, basic solution, temperature-sensitive enzymes, pH-sensitive enzymes, light-sensitive enzymes, metals, metal ions, magnesium chloride, sodium chloride, manganese, aqueous buffer, mild buffer, ionic buffer, inhibitor, enzyme, protein, polynucleotide, antibodies, saccharides, lipid, oil, salt, ion, detergents, ionic detergents, non-ionic detergents, oligonucleotides, nucleotides, deoxyribonucleotide triphosphates (dNTPs), dideoxyribonucleotide triphosphates (ddNTPs), DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double stranded DNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA, polymerase, ligase, restriction enzymes, proteases, nucleases, protease inhibitors, nuclease inhibitors, chelating agents, reducing agents, oxidizing agents, fluorophores, probes, chromophores, dyes, organics, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, pharmaceuticals, radioactive molecules, preservatives, antibiotics, aptamers, and pharmaceutical drug compounds. As described herein, one or more reagents in the well can be used to perform one or more reactions, including but not limited to: cell lysis, cell fixation, permeabilization, nucleic acid reactions, e.g, nucleic acid extension reactions, amplification, reverse transcription, transposase reactions (e.g, tagmentation), etc.

[0173] The wells disclosed herein can be provided as a part of a kit. For example, a kit can include instructions for use, a microwell array or device, and reagents ( e.g ., beads). The kit can include any useful reagents for performing the processes described herein, e.g., nucleic acid reactions, barcoding of nucleic acid molecules, sample processing (e.g, for cell lysis, fixation, and/or permeabilization).

[0174] In some cases, a well includes a bead or droplet that includes a set of reagents that has a similar attribute, for example, a set of enzymes, a set of minerals, a set of oligonucleotides, a mixture of different barcode molecules, a mixture of identical barcode molecules. In other cases, a bead or droplet includes a heterogeneous mixture of reagents. In some cases, the heterogeneous mixture of reagents can include all components necessary to perform a reaction. In some cases, such mixture can include all components necessary to perform a reaction, except for 1, 2, 3, 4, 5, or more components necessary to perform a reaction. In some cases, such additional components are contained within, or otherwise coupled to, a different droplet or bead, or within a solution within a partition (e.g, microwell) of the system.

[0175] A non-limiting example of a microwell array in accordance with some embodiments of the disclosure is schematically presented in FIG. 17. In this example, the array can be contained within a substrate 1700. The substrate 1700 includes a plurality of wells 1702. The wells 1702 can be of any size or shape, and the spacing between the wells, the number of wells per substrate, as well as the density of the wells on the substrate 1700 can be modified, depending on the particular application. In one such example application, a sample molecule 1706, which can include a cell or cellular components (e.g, nucleic acid molecules) is co-partitioned with a bead 1704, which can include a nucleic acid barcode molecule coupled thereto. The wells 1702 can be loaded using gravity or other loading technique (e.g, centrifugation, liquid handler, acoustic loading, optoelectronic, etc.). In some instances, at least one of the wells 1702 contains a single sample molecule 1706 (e.g, cell) and a single bead 1704.

[0176] Reagents can be loaded into a well either sequentially or concurrently. In some cases, reagents are introduced to the device either before or after a particular operation. In some cases, reagents (which can be provided, in certain instances, in droplets or beads) are introduced sequentially such that different reactions or operations occur at different steps.

The reagents (or droplets, or beads) can also be loaded at operations interspersed with a reaction or operation step. For example, or droplets or beads including reagents for fragmenting polynucleotides (e.g, restriction enzymes) and/or other enzymes (e.g, transposases, ligases, polymerases, etc.) can be loaded into the well or plurality of wells, followed by loading of droplets or beads including reagents for attaching nucleic acid barcode molecules to a sample nucleic acid molecule. Reagents can be provided concurrently or sequentially with a sample, e.g., a cell or cellular components (e.g, organelles, proteins, nucleic acid molecules, carbohydrates, lipids, etc.). Accordingly, use of wells can be useful in performing multi-step operations or reactions.

[0177] As described elsewhere herein, the nucleic acid barcode molecules and other reagents can be contained within a bead or droplet. These beads or droplets can be loaded into a partition (e.g, a microwell) before, after, or concurrently with the loading of a cell, such that each cell is contacted with a different bead or droplet. This technique can be used to attach a unique nucleic acid barcode molecule to nucleic acid molecules obtained from each cell. Alternatively or in addition, the sample nucleic acid molecules can be attached to a support. For example, the partition (e.g, microwell) can include a bead which has coupled thereto a plurality of nucleic acid barcode molecules. The sample nucleic acid molecules, or derivatives thereof, can couple or attach to the nucleic acid barcode molecules attached on the support. The resulting barcoded nucleic acid molecules can then be removed from the partition, and in some instances, pooled and sequenced. In such cases, the nucleic acid barcode sequences can be used to trace the origin of the sample nucleic acid molecule. For example, polynucleotides with identical barcodes can be determined to originate from the same cell or partition, while polynucleotides with different barcodes can be determined to originate from different cells or partitions.

[0178] The samples or reagents can be loaded in the wells or microwells using a variety of approaches. For example, the samples (e.g, a cell, cell bead, or cellular component) or reagents (as described herein) can be loaded into the well or microwell using an external force, e.g, gravitational force, electrical force, magnetic force, or using mechanisms to drive the sample or reagents into the well, for example, via pressure-driven flow, centrifugation, optoelectronics, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc. In certain cases, a fluid handling system can be used to load the samples or reagents into the well. The loading of the samples or reagents can follow a Poissonian distribution or a non- Poissonian distribution, e.g, super Poisson or sub-Poisson. The geometry, spacing between wells, density, and size of the microwells can be modified to accommodate a useful sample or reagent distribution; for example, the size and spacing of the microwells can be adjusted such that the sample or reagents can be distributed in a super-Poissonian fashion. [0179] In one non-limiting example, the microwell array or plate includes pairs of microwells, in which each pair of microwells is configured to hold a droplet ( e.g ., including a single cell) and a single bead (such as those described herein, which can, in some instances, also be encapsulated in a droplet). The droplet and the bead (or droplet containing the bead) can be loaded simultaneously or sequentially, and the droplet and the bead can be merged, e.g., upon contact of the droplet and the bead, or upon application of a stimulus (e.g, external force, agitation, heat, light, magnetic or electric force, etc.). In some cases, the loading of the droplet and the bead is super-Poissonian. In other examples of pairs of microwells, the wells are configured to hold two droplets including different reagents and/or samples, which are merged upon contact or upon application of a stimulus. In such instances, the droplet of one microwell of the pair can include reagents that can react with an agent in the droplet of the other microwell of the pair. For example, one droplet can include reagents that are configured to release the nucleic acid barcode molecules of a bead contained in another droplet, located in the adjacent microwell. Upon merging of the droplets, the nucleic acid barcode molecules can be released from the bead into the partition (e.g, the microwell or microwell pair that are in contact), and further processing can be performed (e.g, barcoding, nucleic acid reactions, etc.). In cases where intact or live cells are loaded in the microwells, one of the droplets can include lysis reagents for lysing the cell upon droplet merging.

[0180] In some embodiments, a droplet or bead can be partitioned into a well. The droplets can be selected or subjected to pre-processing prior to loading into a well. For instance, the droplets can include cells, and only certain droplets, such as those containing a single cell (or at least one cell), can be selected for use in loading of the wells. Such a pre selection process can be useful in efficient loading of single cells, such as to obtain a non- Poissonian distribution, or to pre-filter cells for a selected characteristic prior to further partitioning in the wells. Additionally, the technique can be useful in obtaining or preventing cell doublet or multiplet formation prior to or during loading of the microwell.

[0181] In some embodiments, the wells can include nucleic acid barcode molecules attached thereto. The nucleic acid barcode molecules can be attached to a surface of the well (e.g, a wall of the well). The nucleic acid barcode molecules may be attached to a droplet or bead that has been partitioned into the well. The nucleic acid barcode molecule (e.g, a partition barcode sequence) of one well can differ from the nucleic acid barcode molecule of another well, which can permit identification of the contents contained with a single partition or well. In some embodiments, the nucleic acid barcode molecule can include a spatial barcode sequence that can identify a spatial coordinate of a well, such as within the well array or well plate. In some embodiments, the nucleic acid barcode molecule can include a unique molecular identifier for individual molecule identification. In some instances, the nucleic acid barcode molecules can be configured to attach to or capture a nucleic acid molecule within a sample or cell distributed in the well. For example, the nucleic acid barcode molecules can include a capture sequence that can be used to capture or hybridize to a nucleic acid molecule (e.g, RNA, DNA) within the sample. In some embodiments, the nucleic acid barcode molecules can be releasable from the microwell. In some instances, the nuelcic acid barcode molecules may be releasable from the bead or droplet. For example, the nucleic acid barcode molecules can include a chemical cross-linker which can be cleaved upon application of a stimulus (e.g, photo-, magnetic, chemical, biological, stimulus). The released nucleic acid barcode molecules, which can be hybridized or configured to hybridize to a sample nucleic acid molecule, can be collected and pooled for further processing, which can include nucleic acid processing (e.g, amplification, extension, reverse transcription, etc.) and/or characterization (e.g, sequencing). In some instances nucleic acid barcode molecules attached to a bead or droplet in a well may be hybridized to sample nucleic acid molecules, and the bead with the sample nucleic acid molecules hybridized thereto may be collected and pooled for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In such cases, the unique partition barcode sequences can be used to identify the cell or partition from which a nucleic acid molecule originated.

[0182] Characterization of samples within a well can be performed. Such characterization can include, in non-limiting examples, imaging of the sample (e.g, cell, cell bead, or cellular components) or derivatives thereof. Characterization techniques such as microscopy or imaging can be useful in measuring sample profiles in fixed spatial locations. For example, when cells are partitioned, optionally with beads, imaging of each microwell and the contents contained therein can provide useful information on cell doublet formation (e.g, frequency, spatial locations, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, expression level of a biomarker (e.g, a surface marker, a fluorescently labeled molecule therein, etc.), cell or bead loading rate, number of cell-bead pairs, etc. In some instances, imaging can be used to characterize live cells in the wells, including, but not limited to: dynamic live-cell tracking, cell-cell interactions (when two or more cells are co partitioned), cell proliferation, etc. Alternatively or in addition to, imaging can be used to characterize a quantity of amplification products in the well.

[0183] In operation, a well can be loaded with a sample and reagents, simultaneously or sequentially. When cells or cell beads are loaded, the well can be subjected to washing, e.g., to remove excess cells from the well, microwell array, or plate. Similarly, washing can be performed to remove excess beads or other reagents from the well, microwell array, or plate. In the instances where live cells are used, the cells can be lysed in the individual partitions to release the intracellular components or cellular analytes. Alternatively, the cells can be fixed or permeabilized in the individual partitions. The intracellular components or cellular analytes can couple to a support, e.g. , on a surface of the microwell, on a solid support (e.g, bead), or they can be collected for further downstream processing. For example, after cell lysis, the intracellular components or cellular analytes can be transferred to individual droplets or other partitions for barcoding. Alternatively, or in addition, the intracellular components or cellular analytes (e.g, nucleic acid molecules) can couple to a bead including a nucleic acid barcode molecule; subsequently, the bead can be collected and further processed, e.g, subjected to nucleic acid reaction such as reverse transcription, amplification, or extension, and the nucleic acid molecules thereon can be further characterized, e.g, via sequencing. Alternatively, or in addition, the intracellular components or cellular analytes can be barcoded in the well (e.g, using a bead including nucleic acid barcode molecules that are releasable or on a surface of the microwell including nucleic acid barcode molecules). The barcoded nucleic acid molecules or analytes can be further processed in the well, or the barcoded nucleic acid molecules or analytes can be collected from the individual partitions and subjected to further processing outside the partition. Further processing can include nucleic acid processing (e.g, performing an amplification, extension) or characterization (e.g, fluorescence monitoring of amplified molecules, sequencing). At any suitable or useful step, the well (or microwell array or plate) can be sealed (e.g, using an oil, membrane, wax, etc.), which enables storage of the assay or selective introduction of additional reagents.

Beads

[0184] In some embodiments of the disclosure, a partition can include one or more unique identifiers, such as barcodes (e.g, a plurality of nucleic acid barcode molecules which can be, for example, a plurality of partition barcode sequences). Barcodes can be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle (e.g, labelled B cells). For example, barcodes can be injected into droplets previous to, subsequent to, or concurrently with droplet generation. In some embodiments, the delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle ( e.g ., labelled B cells) to the particular partition. Barcodes can be delivered, for example on a nucleic acid molecule (e.g., a barcoded oligonucleotide or nucleic acid barcode molecule), to a partition via any suitable mechanism. In some embodiments, nucleic acid barcode molecules can be delivered to a partition via a bead. Beads are described in further detail below.

[0185] In some embodiments, nucleic acid barcode molecules can be initially associated with the bead and then released from the bead. In some embodiments, release of the nucleic acid barcode molecules can be passive (e.g, by diffusion out of the bead). In addition or alternatively, release from the bead can be upon application of a stimulus which allows the nucleic acid barcode molecules to dissociate or to be released from the bead. Such stimulus can disrupt the bead, an interaction that couples the nucleic acid barcode molecules to or within the bead, or both. Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g, change in pH or use of a reducing agent), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g, enzyme), or any combination thereof. Methods and systems for partitioning barcode carrying beads into droplets are provided in US. Patent Publication Nos. 2019/0367997 and 2019/0064173, and International Application Nos. PCT/US20/17785 and PCT/US20/020486.

[0186] Beneficially, a discrete droplet partitioning a biological particle and a barcode carrying bead can effectively allow the attribution of the barcode to macromolecular constituents of the biological particle within the partition. The contents of a partition can remain discrete from the contents of other partitions.

[0187] In operation, the barcoded oligonucleotides can be released (e.g, in a partition), as described elsewhere herein. Alternatively, the nucleic acid molecules bound to the bead (e.g, gel bead) can be used to hybridize and capture analytes (e.g, one or more types of analytes) on the solid phase of the bead.

[0188] In some examples, beads, biological particles (e.g, labelled B cells) and droplets can flow along channels (e.g, the channels of a microfluidic device), in some cases at substantially regular flow profiles (e.g, at regular flow rates). Such regular flow profiles can permit a droplet to include a single bead and a single biological particle. Such regular flow profiles can permit the droplets to have an occupancy (e.g, droplets having beads and biological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flow profiles and devices that can be used to provide such regular flow profiles are provided in, for example, U.S. Patent Publication No. 2015/0292988.

[0189] A bead can be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a bead can be dissolvable, disruptable, and/or degradable. In some cases, a bead cannot be degradable. In some cases, the bead can be a gel bead. A gel bead can be a hydrogel bead. A gel bead can be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid bead can be a liposomal bead. Solid beads can include metals including iron oxide, gold, and silver. In some cases, the bead can be a silica bead. In some cases, the bead can be rigid. In other cases, the bead can be flexible and/or compressible.

[0190] A bead can be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non- spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.

[0191] Beads can be of uniform size or heterogeneous size. In some cases, the diameter of a bead can be at least about 10 nanometers (nm), 100 nm, 500 nm, 1 micrometer (pm), 5pm, 10pm, 20pm, 30pm, 40pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 250pm,

500pm, 1mm, or greater. In some cases, a bead can have a diameter of less than about 10 nm, 100 nm, 500 nm, 1pm, 5pm, 10pm, 20pm, 30pm, 40pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 250pm, 500pm, 1mm, or less. In some cases, a bead can have a diameter in the range of about 40-75pm, 30-75pm, 20-75pm, 40-85pm, 40-95pm, 20-100pm, 10-100pm, 1- 100pm, 20-250pm, or 20-500pm.

[0192] In certain aspects, beads can be provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it may be desirable to provide relatively consistent amounts of reagents within partitions, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency. In some embodiments, the beads described herein can have size distributions that have a coefficient of variation in their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less.

[0193] A bead can include natural and/or synthetic materials. For example, a bead can include a natural polymer, a synthetic polymer or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch ( e.g ., amylose, amylopectin), proteins, enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate, or natural polymers thereof. Examples of synthetic polymers include acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/or combinations ( e.g ., co-polymers) thereof. Beads can also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others.

[0194] In some embodiments, the bead can contain molecular precursors (e.g., monomers or polymers), which can form a polymer network via polymerization of the molecular precursors. In some cases, a precursor can be an already polymerized species capable of undergoing further polymerization via, for example, a chemical cross-linkage. In some embodiments, a precursor can include one or more of an acrylamide or a methacrylamide monomer, oligomer, or polymer. In some cases, the bead can include prepolymers, which are oligomers capable of further polymerization. For example, polyurethane beads can be prepared using prepolymers. In some embodiments, the bead can contain individual polymers that can be further polymerized together. In some cases, beads can be generated via polymerization of different precursors, such that they include mixed polymers, co-polymers, and/or block co-polymers. In some embodiments, the bead can include covalent or ionic bonds between polymeric precursors (e.g, monomers, oligomers, linear polymers), nucleic acid molecules (e.g, oligonucleotides), primers, and other entities.

In some embodiments, the covalent bonds can be carbon-carbon bonds, thioether bonds, or carbon-heteroatom bonds.

[0195] Cross-linking can be permanent or reversible, depending upon the particular cross-linker used. Reversible cross-linking can allow for the polymer to linearize or dissociate under appropriate conditions. In some embodiments, reversible cross-linking can also allow for reversible attachment of a material bound to the surface of a bead. In some embodiments, a cross-linker can form disulfide linkages. In some embodiments, the chemical cross-linker forming disulfide linkages can be cystamine or a modified cystamine.

[0196] In some embodiments, disulfide linkages can be formed between molecular precursor units ( e.g ., monomers, oligomers, or linear polymers) or precursors incorporated into a bead and nucleic acid molecules (e.g., oligonucleotides). Cystamine (including modified cystamines), for example, is an organic agent including a disulfide bond that can be used as a crosslinker agent between individual monomeric or polymeric precursors of a bead. Polyacrylamide can be polymerized in the presence of cystamine or a species including cystamine (e.g, a modified cystamine) to generate polyacrylamide gel beads including disulfide linkages (e.g, chemically degradable beads including chemically-reducible cross linkers). The disulfide linkages can permit the bead to be degraded (or dissolved) upon exposure of the bead to a reducing agent.

[0197] In some embodiments, chitosan, a linear polysaccharide polymer, can be crosslinked with glutaraldehyde via hydrophilic chains to form a bead. Crosslinking of chitosan polymers can be achieved by chemical reactions that are initiated by heat, pressure, change in pH, and/or radiation.

[0198] In some embodiments, a bead can include an acrydite moiety, which in certain aspects can be used to attach one or more nucleic acid molecules (e.g, barcode sequence, barcoded nucleic acid molecule, barcoded oligonucleotide, nucleic acid barcode molecule, primer, or other oligonucleotide) to the bead. In some cases, an acrydite moiety can refer to an acrydite analogue generated from the reaction of acrydite with one or more species, such as, the reaction of acrydite with other monomers and cross-linkers during a polymerization reaction. Acrydite moieties can be modified to form chemical bonds with a species to be attached, such as a nucleic acid molecule (e.g, barcode sequence, barcoded nucleic acid molecule, nucleic acid barcode molecule, barcoded oligonucleotide, primer, or other oligonucleotide). Acrydite moieties can be modified with thiol groups capable of forming a disulfide bond or can be modified with groups already including a disulfide bond. The thiol or disulfide (via disulfide exchange) can be used as an anchor point for a species to be attached or another part of the acrydite moiety can be used for attachment. In some cases, attachment can be reversible, such that when the disulfide bond is broken (e.g, in the presence of a reducing agent), the attached species is released from the bead. In other cases, an acrydite moiety can include a reactive hydroxyl group that can be used for attachment.

[0199] Functionalization of beads for attachment of nucleic acid molecules (e.g, oligonucleotides) can be achieved through a wide range of different approaches, including activation of chemical groups within a polymer, incorporation of active or activatable functional groups in the polymer structure, or attachment at the pre-polymer or monomer stage in bead production.

[0200] For example, precursors ( e.g ., monomers, cross-linkers) that are polymerized to form a bead can include acrydite moieties, such that when a bead is generated, the bead also includes acrydite moieties. The acrydite moieties can be attached to a nucleic acid molecule (e.g., oligonucleotide), which can include a priming sequence (e.g, a primer for amplifying target nucleic acids, random primer, primer sequence for messenger RNA) and/or one or more barcode sequences. The one or more barcode sequences can include sequences that are the same for all nucleic acid molecules coupled to a given bead and/or sequences that are different across all nucleic acid molecules coupled to the given bead. The nucleic acid molecule can be incorporated into the bead.

[0201] In some embodiments, the nucleic acid molecule can include a functional sequence, for example, for attachment to a sequencing flow cell, such as, for example, a P5 sequence for Illumina® sequencing. In some cases, the nucleic acid molecule or derivative thereof (e.g, oligonucleotide or polynucleotide generated from the nucleic acid molecule) can include another functional sequence, such as, for example, a P7 sequence for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid molecule can include a barcode sequence. In some cases, the primer can further include a unique molecular identifier (UMI). In some cases, the primer can include an R1 primer sequence for Illumina sequencing. In some cases, the primer can include an R2 primer sequence for Illumina sequencing. Examples of such nucleic acid molecules (e.g, oligonucleotides, polynucleotides, etc.) and uses thereof, as can be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Pub. Nos. 2014/0378345 and 2015/0376609.

[0202] FIG. 15 illustrates an example of a barcode carrying bead. A nucleic acid molecule 1502, such as an oligonucleotide, can be coupled to a bead 1504 by a releasable linkage 1506, such as, for example, a disulfide linker. The same bead 1504 can be coupled (e.g, via releasable linkage) to one or more other nucleic acid molecules 1518, 1520. The nucleic acid molecule 1502 can be or include a barcode. As noted elsewhere herein, the structure of the barcode can include a number of sequence elements. The nucleic acid molecule 1502 can include a functional sequence 1508 that can be used in subsequent processing. For example, the functional sequence 1508 can include one or more of a sequencer specific flow cell attachment sequence ( e.g ., a P5 sequence for Illumina® sequencing systems) and a sequencing primer sequence (e.g., a R1 primer for Illumina® sequencing systems). The nucleic acid molecule 1502 can include a barcode sequence 1510 for use in barcoding the sample (e.g, DNA, RNA, protein, etc.). In some cases, the barcode sequence 1510 can be bead-specific such that the barcode sequence 1510 is common to all nucleic acid molecules (e.g, including nucleic acid molecule 1502) coupled to the same bead 1504. Alternatively or in addition, the barcode sequence 1510 can be partition-specific such that the barcode sequence 1510 is common to all nucleic acid molecules coupled to one or more beads that are partitioned into the same partition. The nucleic acid molecule 1502 can include a specific priming sequence 1512, such as an mRNA specific priming sequence (e.g, poly-T sequence), a targeted priming sequence, and/or a random priming sequence. The nucleic acid molecule 1502 can include an anchoring sequence 1514 to ensure that the specific priming sequence 1512 hybridizes at the sequence end (e.g, of the mRNA). For example, the anchoring sequence 1514 can include a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longer sequence, which can ensure that a poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA.

[0203] The nucleic acid molecule 1502 can include a unique molecular identifying sequence 1516 (e.g, unique molecular identifier (UMI)). In some cases, the unique molecular identifying sequence 1516 can include from about 5 to about 8 nucleotides. Alternatively, the unique molecular identifying sequence 1516 can compress less than about 5 or more than about 8 nucleotides. The unique molecular identifying sequence 1516 can be a unique sequence that varies across individual nucleic acid molecules (e.g, 1502, 1518, 1520, etc.) coupled to a single bead (e.g, bead 1504). In some cases, the unique molecular identifying sequence 1516 can be a random sequence (e.g, such as a random N-mer sequence). For example, the UMI can provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA. As will be appreciated, although FIG. 15 shows three nucleic acid molecules 1502, 1518, 1520 coupled to the surface of the bead 1504, an individual bead can be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands or even millions of individual nucleic acid molecules. The respective barcodes for the individual nucleic acid molecules can include both common sequence segments or relatively common sequence segments (e.g, 1508, 1510, 1512, etc.) and variable or unique sequence segments (e.g, 1516) between different individual nucleic acid molecules coupled to the same bead. [0204] In operation, a biological particle ( e.g ., cell, DNA, RNA, etc.) can be co partitioned along with a barcode bearing bead 1504. The nucleic acid barcode molecules 1502, 1518, 1520 can be released from the bead 1504 in the partition. By way of example, in the context of analyzing sample RNA, the poly-T segment (e.g., 1512) of one of the released nucleic acid molecules (e.g, 1502) can hybridize to the poly-A tail of a mRNA molecule. Reverse transcription can result in a cDNA transcript of the mRNA, but which transcript includes each of the sequence segments 1508, 1510, 1516 of the nucleic acid molecule 1502. Because the nucleic acid molecule 1502 includes an anchoring sequence 1514, it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA. Within any given partition, all of the cDNA transcripts of the individual mRNA molecules can include a common barcode sequence segment 1510. However, the transcripts made from the different mRNA molecules within a given partition can vary at the unique molecular identifying sequence 1512 segment (e.g, UMI segment). Beneficially, even following any subsequent amplification of the contents of a given partition, the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the biological particle (e.g, cell). As noted above, the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-T primer sequence is described, other targeted or random priming sequences can also be used in priming the reverse transcription reaction. Likewise, although described as releasing the barcoded oligonucleotides into the partition, in some cases, the nucleic acid molecules bound to the bead (e.g, gel bead) can be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents. In such cases, further processing can be performed, in the partitions or outside the partitions (e.g, in bulk). For instance, the RNA molecules on the beads can be subjected to reverse transcription or other nucleic acid processing, additional adapter sequences can be added to the barcoded nucleic acid molecules, or other nucleic acid reactions (e.g, amplification, nucleic acid extension) can be performed. The beads or products thereof (e.g, barcoded nucleic acid molecules) can be collected from the partitions, and/or pooled together and subsequently subjected to clean up and further characterization (e.g, sequencing).

[0205] The operations described herein can be performed at any useful or suitable step. For instance, the beads including nucleic acid barcode molecules can be introduced into a partition (e.g, well or droplet) prior to, during, or following introduction of a sample into the partition. The nucleic acid molecules of a sample can be subjected to barcoding, which can occur on the bead (in cases where the nucleic acid molecules remain coupled to the bead) or following release of the nucleic acid barcode molecules into the partition. In cases where analytes from the sample are captured by the nucleic acid barcode molecules in a partition (e.g., by hybridization), captured analytes from various partitions may be collected, pooled, and subjected to further processing (e.g., reverse transcription, adapter attachment, amplification, clean up, sequencing). For example, in cases wherein the nucleic acid molecules from the sample remain attached to the bead, the beads from various partitions can be collected, pooled, and subjected to further processing (e.g., reverse transcription, adapter attachment, amplification, clean up, and/or sequencing). In other instances, one or more of the processing methods, e.g., reverse transcription, may occur in the partition. For example, conditions sufficient for barcoding, adapter attachment, reverse transcription, or other nucleic acid processing operations can be provided in the partition and performed prior to clean up and sequencing.

[0206] In some instances, a bead can include a capture sequence or binding sequence configured to bind to a corresponding capture sequence or binding sequence. In some instances, a bead can include a plurality of different capture sequences or binding sequences configured to bind to different respective corresponding capture sequences or binding sequences. For example, a bead can include a first subset of one or more capture sequences each configured to bind to a first corresponding capture sequence, a second subset of one or more capture sequences each configured to bind to a second corresponding capture sequence, a third subset of one or more capture sequences each configured to bind to a third corresponding capture sequence, and etc. A bead can include any number of different capture sequences. In some instances, a bead can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences, respectively. Alternatively or in addition, a bead can include at most about 10, 9, 8, 7, 6, 5, 4, 3, or 2 different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences. In some instances, the different capture sequences or binding sequences can be configured to facilitate analysis of a same type of analyte. In some instances, the different capture sequences or binding sequences can be configured to facilitate analysis of different types of analytes (with the same bead). The capture sequence can be designed to attach to a corresponding capture sequence. Beneficially, such corresponding capture sequence can be introduced to, or otherwise induced in, a biological particle ( e.g ., cell, cell bead, etc.) for performing different assays in various formats (e.g., barcoded antibodies including the corresponding capture sequence, barcoded MHC dextramers including the corresponding capture sequence, barcoded guide RNA molecules including the corresponding capture sequence, etc.), such that the corresponding capture sequence can later interact with the capture sequence associated with the bead. In some instances, a capture sequence coupled to a bead (or other support) can be configured to attach to a linker molecule, such as a splint molecule, wherein the linker molecule is configured to couple the bead (or other support) to other molecules through the linker molecule, such as to one or more analytes or one or more other linker molecules.

[0207] FIG. 16 illustrates a non-limiting example of a barcode carrying bead in accordance with some embodiments of the disclosure. A nucleic acid molecule 1605, such as an oligonucleotide, can be coupled to a bead 1604 by a releasable linkage 1606, such as, for example, a disulfide linker. The nucleic acid molecule 1605 can include a first capture sequence 1660. The same bead 1604 can be coupled, e.g, via releasable linkage, to one or more other nucleic acid molecules 1603, 1607 including other capture sequences. The nucleic acid molecule 1605 can be or include a barcode. As described elsewhere herein, the structure of the barcode can include a number of sequence elements, such as a functional sequence 1608 (e.g, flow cell attachment sequence, sequencing primer sequence, etc.), a barcode sequence 1610 (e.g, bead-specific sequence common to bead, partition-specific sequence common to partition, etc.), and a unique molecular identifier 1612 (e.g, unique sequence within different molecules attached to the bead), or partial sequences thereof. The capture sequence 1660 can be configured to attach to a corresponding capture sequence 1665 (e.g, capture handle). In some instances, the corresponding capture sequence 1665 can be coupled to another molecule that can be an analyte or an intermediary carrier. For example, as illustrated in FIG. 16, the corresponding capture sequence 1665 is coupled to a guide RNA molecule 1662 including a target sequence 1664, wherein the target sequence 1664 is configured to attach to the analyte. Another oligonucleotide molecule 1607 attached to the bead 1604 includes a second capture sequence 1680 which is configured to attach to a second corresponding capture sequence (e.g, capture handle) 1685. As illustrated in FIG. 16, the second corresponding capture sequence 1685 is coupled to an antibody 1682. In some cases, the antibody 1682 can have binding specificity to an analyte (e.g, surface protein). Alternatively, the antibody 1682 cannot have binding specificity. Another oligonucleotide molecule 1603 attached to the bead 1604 includes a third capture sequence 470 which is configured to attach to a second corresponding capture sequence 1675. As illustrated in FIG. 16, the third corresponding capture sequence ( e.g ., capture handle) 1675 is coupled to a molecule 1672. The molecule 1672 may or may not be configured to target an analyte. The other oligonucleotide molecules 1603, 1607 can include the other sequences (e.g., functional sequence, barcode sequence, UMI, etc.) described with respect to oligonucleotide molecule 1605. While a single oligonucleotide molecule including each capture sequence is illustrated in FIG. 16, it will be appreciated that, for each capture sequence, the bead can include a set of one or more oligonucleotide molecules each including the capture sequence. For example, the bead can include any number of sets of one or more different capture sequences. Alternatively or in addition, the bead 1604 can include other capture sequences. Alternatively or in addition, the bead 1604 can include fewer types of capture sequences (e.g, two capture sequences). Alternatively or in addition, the bead 1604 can include oligonucleotide molecule(s) including a priming sequence, such as a specific priming sequence such as an mRNA specific priming sequence (e.g, poly-T sequence), a targeted priming sequence, and/or a random priming sequence, for example, to facilitate an assay for gene expression.

[0208] The generation of a barcoded sequence, see, e.g. , FIG. 15, is described herein. [0209] In some embodiments, precursors including a functional group that is reactive or capable of being activated such that it becomes reactive can be polymerized with other precursors to generate gel beads including the activated or activatable functional group. The functional group can then be used to attach additional species (e.g, disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads. For example, some precursors including a carboxylic acid (COOH) group can co-polymerize with other precursors to form a gel bead that also includes a COOH functional group. In some cases, acrylic acid (a species including free COOH groups), acrylamide, and bis(acryloyl)cystamine can be co-polymerized together to generate a gel bead including free COOH groups. The COOH groups of the gel bead can be activated (e.g, via 1 -Ethyl-3 -(3 -dimethylaminopropyl)carbodiimide (EDC) and N- Hydroxysuccinimide (NHS) or 4-(4,6-Dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)) such that they are reactive (e.g, reactive to amine functional groups where EDC/NHS or DMTMM are used for activation). The activated COOH groups can then react with an appropriate species (e.g, a species including an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group) including a moiety to be linked to the bead. [0210] Beads including disulfide linkages in their polymeric network can be functionalized with additional species via reduction of some of the disulfide linkages to free thiols. The disulfide linkages can be reduced via, for example, the action of a reducing agent ( e.g ., DTT, TCEP, etc.) to generate free thiol groups, without dissolution of the bead. Free thiols of the beads can then react with free thiols of a species or a species including another disulfide bond (e.g., via thiol-disulfide exchange) such that the species can be linked to the beads (e.g, via a generated disulfide bond). In some cases, free thiols of the beads can react with any other suitable group. For example, free thiols of the beads can react with species including an acrydite moiety. The free thiol groups of the beads can react with the acrydite via Michael addition chemistry, such that the species including the acrydite is linked to the bead. In some cases, uncontrolled reactions can be prevented by inclusion of a thiol capping agent such as N-ethylmaleimide or iodoacetate.

[0211] Activation of disulfide linkages within a bead can be controlled such that only a small number of disulfide linkages are activated. Control can be exerted, for example, by controlling the concentration of a reducing agent used to generate free thiol groups and/or concentration of reagents used to form disulfide bonds in bead polymerization. In some cases, a low concentration (e.g, molecules of reducing agent: gel bead ratios of less than or equal to about 1:100,000,000,000, less than or equal to about 1:10,000,000,000, less than or equal to about 1:1,000,000,000, less than or equal to about 1:100,000,000, less than or equal to about 1 : 10,000,000, less than or equal to about 1 : 1,000,000, less than or equal to about 1 : 100,000, less than or equal to about 1 : 10,000) of reducing agent can be used for reduction. Controlling the number of disulfide linkages that are reduced to free thiols can be useful in ensuring bead structural integrity during functionalization. In some cases, optically-active agents, such as fluorescent dyes can be coupled to beads via free thiol groups of the beads and used to quantify the number of free thiols present in a bead and/or track a bead.

[0212] In some embodiments, addition of moieties to a gel bead after gel bead formation can be advantageous. For example, addition of an oligonucleotide (e.g, barcoded oligonucleotide, such as a barcoded nucleic acid molecule) after gel bead formation can avoid loss of the species during chain transfer termination that can occur during polymerization. Moreover, smaller precursors (e.g, monomers or cross linkers that do not include side chain groups and linked moieties) can be used for polymerization and can be minimally hindered from growing chain ends due to viscous effects. In some cases, functionalization after gel bead synthesis can minimize exposure of species (e.g, oligonucleotides) to be loaded with potentially damaging agents ( e.g ., free radicals) and/or chemical environments. In some cases, the generated gel can possess an upper critical solution temperature (UCST) that can permit temperature driven swelling and collapse of a bead. Such functionality can aid in oligonucleotide (e.g., a primer) infiltration into the bead during subsequent functionalization of the bead with the oligonucleotide. Post-production functionalization can also be useful in controlling loading ratios of species in beads, such that, for example, the variability in loading ratio is minimized. Species loading can also be performed in a batch process such that a plurality of beads can be functionalized with the species in a single batch.

[0213] A bead injected or otherwise introduced into a partition can include releasably, cleavably, or reversibly attached barcodes (e.g, partition barcode sequences). A bead injected or otherwise introduced into a partition can include activatable barcodes. A bead injected or otherwise introduced into a partition can be degradable, disruptable, or dissolvable beads.

[0214] Barcodes can be releasably, cleavably or reversibly attached to the beads such that barcodes can be released or be releasable through cleavage of a linkage between the barcode molecule and the bead, or released through degradation of the underlying bead itself, allowing the barcodes to be accessed or be accessible by other reagents, or both. In non limiting examples, cleavage can be achieved through reduction of di-sulfide bonds, use of restriction enzymes, photo-activated cleavage, or cleavage via other types of stimuli (e.g, chemical, thermal, pH, enzymatic, etc.) and/or reactions, such as described elsewhere herein. Releasable barcodes can sometimes be referred to as being activatable, in that they are available for reaction once released. Thus, for example, an activatable barcode can be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also envisioned in the context of the described methods and systems.

[0215] In addition to, or as an alternative to the cleavable linkages between the beads and the associated molecules, such as barcode containing nucleic acid molecules (e.g, barcoded oligonucleotides), the beads can be degradable, disruptable, or dissolvable spontaneously or upon exposure to one or more stimuli (e.g, temperature changes, pH changes, exposure to particular chemical species or phase, exposure to light, reducing agent, etc.). In some cases, a bead can be dissolvable, such that material components of the beads are solubilized when exposed to a particular chemical species or an environmental change, such as a change temperature or a change in pH. In some cases, a gel bead can be degraded or dissolved at elevated temperature and/or in basic conditions. In some cases, a bead can be thermally degradable such that when the bead is exposed to an appropriate change in temperature ( e.g ., heat), the bead degrades. Degradation or dissolution of a bead bound to a species (e.g., a nucleic acid molecule, e.g, barcoded oligonucleotide) can result in release of the species from the bead.

[0216] As will be appreciated from the above disclosure, the degradation of a bead can refer to the disassociation of a bound (e.g, capture agent configured to couple to a secreted antibody or antigen-binding fragment thereof) or entrained species (e.g, labelled B cells, or memory B cells, or secreted antibody or antigen-binding fragment thereof) from a bead, both with and without structurally degrading the physical bead itself. For example, the degradation of the bead can involve cleavage of a cleavable linkage via one or more species and/or methods described elsewhere herein. In another example, entrained species can be released from beads through osmotic pressure differences due to, for example, changing chemical environments. By way of example, alteration of bead pore sizes due to osmotic pressure differences can generally occur without structural degradation of the bead itself. In some cases, an increase in pore size due to osmotic swelling of a bead can permit the release of entrained species within the bead. In other cases, osmotic shrinking of a bead can cause a bead to better retain an entrained species due to pore size contraction.

[0217] A degradable bead can be introduced into a partition, such as a droplet of an emulsion or a well, such that the bead degrades within the partition and any associated species (e.g, oligonucleotides) are released within the droplet when the appropriate stimulus is applied. The free species (e.g, oligonucleotides, nucleic acid molecules) can interact with other reagents contained in the partition. For example, a polyacrylamide bead including cystamine and linked, via a disulfide bond, to a barcode sequence, can be combined with a reducing agent within a droplet of a water-in-oil emulsion. Within the droplet, the reducing agent can break the various disulfide bonds, resulting in bead degradation and release of the barcode sequence into the aqueous, inner environment of the droplet. In another example, heating of a droplet including a bead-bound barcode sequence in basic solution can also result in bead degradation and release of the attached barcode sequence into the aqueous, inner environment of the droplet.

[0218] Any suitable number of molecular tag molecules (e.g, primer, barcoded oligonucleotide, nucleic acid barcode molecule) can be associated with a bead such that, upon release from the bead, the molecular tag molecules (e.g, primer, e.g, barcoded oligonucleotide or nucleic acid barcode molecule) are present in the partition at a pre-defmed concentration. Such pre-defmed concentration can be selected to facilitate certain reactions for generating a sequencing library, e.g. , amplification, within the partition. In some cases, the pre-defmed concentration of the primer can be limited by the process of producing nucleic acid molecule (e.g., oligonucleotide) bearing beads.

[0219] In some cases, beads can be non-covalently loaded with one or more reagents. The beads can be non-covalently loaded by, for instance, subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagents to diffuse into the interiors of the beads, and subjecting the beads to conditions sufficient to de-swell the beads. The swelling of the beads can be accomplished, for instance, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ion concentration, and/or subjecting the beads to an electric field. The swelling of the beads can be accomplished by various swelling methods. The de-swelling of the beads can be accomplished, for instance, by transferring the beads in a thermodynamically unfavorable solvent, subjecting the beads to lower or high temperatures, subjecting the beads to a lower or higher ion concentration, and/or removing an electric field. The de-swelling of the beads can be accomplished by various de-swelling methods. Transferring the beads can cause pores in the bead to shrink. The shrinking can then hinder reagents within the beads from diffusing out of the interiors of the beads. The hindrance can be due to steric interactions between the reagents and the interiors of the beads. The transfer can be accomplished microfluidically. For instance, the transfer can be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream. The swellability and/or pore size of the beads can be adjusted by changing the polymer composition of the bead.

[0220] In some cases, an acrydite moiety linked to a precursor, another species linked to a precursor, or a precursor itself can include a labile bond, such as chemically, thermally, or photo-sensitive bond e.g, disulfide bond, UV sensitive bond, or the like. Once acrydite moieties or other moieties including a labile bond are incorporated into a bead, the bead can also include the labile bond. The labile bond can be, for example, useful in reversibly linking (e.g, covalently linking) species (e.g, barcodes, primers, etc.) to a bead. In some cases, a thermally labile bond can include a nucleic acid hybridization based attachment, e.g, where an oligonucleotide is hybridized to a complementary sequence that is attached to the bead, such that thermal melting of the hybrid releases the oligonucleotide, e.g, a barcode containing sequence, from the bead. [0221] The addition of multiple types of labile bonds to a gel bead can result in the generation of a bead capable of responding to varied stimuli. Each type of labile bond can be sensitive to an associated stimulus ( e.g ., chemical stimulus, light, temperature, enzymatic, etc.) such that release of species attached to a bead via each labile bond can be controlled by the application of the appropriate stimulus. Such functionality can be useful in controlled release of species from a gel bead. In some cases, another species including a labile bond can be linked to a gel bead after gel bead formation via, for example, an activated functional group of the gel bead as described above. As will be appreciated, barcodes that are releasably, cleavably or reversibly attached to the beads described herein include barcodes that are released or releasable through cleavage of a linkage between the barcode molecule and the bead, or that are released through degradation of the underlying bead itself, allowing the barcodes to be accessed or accessible by other reagents, or both.

[0222] The barcodes that are releasable as described herein can sometimes be referred to as being activatable, in that they are available for reaction once released. Thus, for example, an activatable barcode can be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also envisioned in the context of the described methods and systems.

[0223] In addition to thermally cleavable bonds, disulfide bonds and UV sensitive bonds, other non-limiting examples of labile bonds that can be coupled to a precursor or bead include an ester linkage (e.g., cleavable with an acid, a base, or hydroxyl amine), a vicinal diol linkage (e.g, cleavable via sodium periodate), a Diels-Alder linkage (e.g, cleavable via heat), a sulfone linkage (e.g, cleavable via a base), a silyl ether linkage (e.g, cleavable via an acid), a glycosidic linkage (e.g, cleavable via an amylase), a peptide linkage (e.g, cleavable via a protease), or a phosphodi ester linkage (e.g, cleavable via a nuclease (e.g, DNAase)). A bond can be cleavable via other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g, restriction endonucleases), as described further below.

[0224] Species can be encapsulated in beads (e.g, capture agent) during bead generation (e.g, during polymerization of precursors). Such species may or may not participate in polymerization. Such species can be entered into polymerization reaction mixtures such that generated beads include the species upon bead formation. In some cases, such species can be added to the gel beads after formation. Such species can include, for example, nucleic acid molecules (e.g, oligonucleotides), reagents for a nucleic acid amplification reaction (e.g, primers, polymerases, dNTPs, co-factors (e.g, ionic co-factors, buffers) including those described herein, reagents for enzymatic reactions ( e.g ., enzymes, co-factors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and/or reagents for template preparation (e.g., tagmentation) for one or more sequencing platforms (e.g, Nextera® for Illumina®). Such species can include one or more enzymes described herein, including without limitation, polymerase, reverse transcriptase, restriction enzymes (e.g, endonuclease), transposase, ligase, proteinase K, DNAse, etc. Such species can include one or more reagents described elsewhere herein (e.g, lysis agents, inhibitors, inactivating agents, chelating agents, stimulus). Trapping of such species can be controlled by the polymer network density generated during polymerization of precursors, control of ionic charge within the gel bead (e.g, via ionic species linked to polymerized species), or by the release of other species. Encapsulated species can be released from a bead upon bead degradation and/or by application of a stimulus capable of releasing the species from the bead. Alternatively or in addition, species can be partitioned in a partition (e.g, droplet) during or subsequent to partition formation. Such species can include, without limitation, the abovementioned species that can also be encapsulated in a bead.

[0225] A degradable bead can include one or more species with a labile bond such that, when the bead/species is exposed to the appropriate stimuli, the bond is broken and the bead degrades. The labile bond can be a chemical bond (e.g, covalent bond, ionic bond) or can be another type of physical interaction (e.g, van der Waals interactions, dipole-dipole interactions, etc.). In some cases, a crosslinker used to generate a bead can include a labile bond. Upon exposure to the appropriate conditions, the labile bond can be broken and the bead degraded. For example, upon exposure of a polyacrylamide gel bead including cystamine crosslinkers to a reducing agent, the disulfide bonds of the cystamine can be broken and the bead degraded.

[0226] A degradable bead can be useful in more quickly releasing an attached species (e.g, a nucleic acid molecule, a barcode sequence, a primer, etc.) from the bead when the appropriate stimulus is applied to the bead as compared to a bead that does not degrade. For example, for a species bound to an inner surface of a porous bead or in the case of an encapsulated species, the species can have greater mobility and accessibility to other species in solution upon degradation of the bead. In some cases, a species can also be attached to a degradable bead via a degradable linker (e.g, disulfide linker). The degradable linker can respond to the same stimuli as the degradable bead or the two degradable species can respond to different stimuli. For example, a barcode sequence can be attached, via a disulfide bond, to a polyacrylamide bead including cystamine. Upon exposure of the barcoded-bead to a reducing agent, the bead degrades and the barcode sequence is released upon breakage of both the disulfide linkage between the barcode sequence and the bead and the disulfide linkages of the cystamine in the bead.

[0227] As will be appreciated from the above disclosure, while referred to as degradation of a bead, in many instances as noted above, that degradation can refer to the disassociation of a bound or entrained species from a bead, both with and without structurally degrading the physical bead itself. For example, entrained species can be released from beads through osmotic pressure differences due to, for example, changing chemical environments. By way of example, alteration of bead pore sizes due to osmotic pressure differences can generally occur without structural degradation of the bead itself. In some cases, an increase in pore size due to osmotic swelling of a bead can permit the release of entrained species within the bead. In other cases, osmotic shrinking of a bead can cause a bead to better retain an entrained species due to pore size contraction.

[0228] Where degradable beads are provided, it can be beneficial to avoid exposing such beads to the stimulus or stimuli that cause such degradation prior to a given time, in order to, for example, avoid premature bead degradation and issues that arise from such degradation, including for example poor flow characteristics and aggregation. By way of example, where beads include reducible cross-linking groups, such as disulfide groups, it will be desirable to avoid contacting such beads with reducing agents, e.g ., DTT or other disulfide cleaving reagents. In such cases, treatment to the beads described herein will, in some cases be provided free of reducing agents, such as DTT. Because reducing agents are often provided in commercial enzyme preparations, it can be desirable to provide reducing agent free (or DTT free) enzyme preparations in treating the beads described herein. Examples of such enzymes include, e.g. , polymerase enzyme preparations, reverse transcriptase enzyme preparations, ligase enzyme preparations, as well as many other enzyme preparations that can be used to treat the beads described herein. The terms “reducing agent free” or “DTT free” preparations can refer to a preparation having less than about 1/10th, less than about 1/50th, or even less than about 1/lOOth of the lower ranges for such materials used in degrading the beads. For example, for DTT, the reducing agent free preparation can have less than about 0.01 millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even less than about 0.0001 mM DTT. In many cases, the amount of DTT can be undetectable. [0229] Numerous chemical triggers can be used to trigger the degradation of beads. Examples of these chemical changes can include, but are not limited to pH-mediated changes to the integrity of a component within the bead, degradation of a component of a bead via cleavage of cross-linked bonds, and depolymerization of a component of a bead.

[0230] In some embodiments, a bead can be formed from materials that include degradable chemical crosslinkers, such as BAC or cystamine. Degradation of such degradable crosslinkers can be accomplished through a number of mechanisms. In some examples, a bead can be contacted with a chemical degrading agent that can induce oxidation, reduction or other chemical changes. For example, a chemical degrading agent can be a reducing agent, such as dithiothreitol (DTT). Additional examples of reducing agents can include b-mercaptoethanol, (2S)-2-amino-l,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof. A reducing agent can degrade the disulfide bonds formed between gel precursors forming the bead, and thus, degrade the bead. In other cases, a change in pH of a solution, such as an increase in pH, can trigger degradation of a bead. In other cases, exposure to an aqueous solution, such as water, can trigger hydrolytic degradation, and thus degradation of the bead. In some cases, any combination of stimuli can trigger degradation of a bead. For example, a change in pH can enable a chemical agent ( e.g ., DTT) to become an effective reducing agent.

[0231] Beads can also be induced to release their contents upon the application of a thermal stimulus. A change in temperature can cause a variety of changes to a bead. For example, heat can cause a solid bead to liquefy. A change in heat can cause melting of a bead such that a portion of the bead degrades. In other cases, heat can increase the internal pressure of the bead components such that the bead ruptures or explodes. Heat can also act upon heat- sensitive polymers used as materials to construct beads.

[0232] Any suitable agent can degrade beads. In some embodiments, changes in temperature or pH can be used to degrade thermo-sensitive or pH-sensitive bonds within beads. In some embodiments, chemical degrading agents can be used to degrade chemical bonds within beads by oxidation, reduction or other chemical changes. For example, a chemical degrading agent can be a reducing agent, such as DTT, wherein DTT can degrade the disulfide bonds formed between a crosslinker and gel precursors, thus degrading the bead. In some embodiments, a reducing agent can be added to degrade the bead, which may or may not cause the bead to release its contents. Examples of reducing agents can include dithiothreitol (DTT), b-mercaptoethanol, (2S)-2-amino-l,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof. The reducing agent can be present at a concentration of about 0. ImM, 0.5mM, ImM, 5mM, or lOmM. The reducing agent can be present at a concentration of at least about 0. ImM, 0.5mM, ImM, 5mM, lOmM, or greater than 10 mM. The reducing agent can be present at concentration of at most about lOmM, 5mM, ImM, 0.5mM, O.lmM, or less.

[0233] Any suitable number of molecular tag molecules ( e.g ., primer, barcoded oligonucleotide, nucleic acid barcode molecule) can be associated with a bead such that, upon release from the bead, the molecular tag molecules (e.g., primer, e.g, barcoded oligonucleotide, nucleic acid barcode molecule) are present in the partition at a pre-defmed concentration. Such pre-defmed concentration can be selected to facilitate certain reactions for generating a sequencing library, e.g, amplification, within the partition. In some cases, the pre-defmed concentration of the primer can be limited by the process of producing oligonucleotide bearing beads.

[0234] Although FIG. 12 and FIG. 13 have been described in terms of providing substantially singly occupied partitions, above, in certain cases, it may be desirable to provide multiply occupied partitions, e.g, containing two, three, four or more cells and/or beads including nucleic acid barcode molecules (e.g, oligonucleotides) within a single partition (e.g, multi-omics method described elsewhere, herein). Accordingly, as noted above, the flow characteristics of the biological particle and/or bead containing fluids and partitioning fluids can be controlled to provide for such multiply occupied partitions. In particular, the flow parameters can be controlled to provide a given occupancy rate at greater than about 50% of the partitions, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or higher.

[0235] In some cases, additional beads can be used to deliver additional reagents to a partition. In such cases, it can be advantageous to introduce different beads into a common channel or droplet generation junction, from different bead sources (e.g, containing different associated reagents) through different channel inlets into such common channel or droplet generation junction (e.g, junction 1210). In such cases, the flow and frequency of the different beads into the channel or junction can be controlled to provide for a certain ratio of beads from each source, while ensuring a given pairing or combination of such beads into a partition with a given number of biological particles (e.g, one biological particle and one bead per partition).

[0236] The partitions described herein can include small volumes, for example, less than about 10 microliters (pL), 5pL, lpL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

[0237] For example, in the case of droplet based partitions, the droplets can have overall volumes that are less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, or less. Where co-partitioned with beads, it will be appreciated that the sample fluid volume, e.g ., including co-partitioned biological particles and/or beads, within the partitions can be less than about 90% of the above described volumes, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the above described volumes.

[0238] As is described elsewhere herein, partitioning species can generate a population or plurality of partitions. In such cases, any suitable number of partitions can be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000,000 partitions, or more partitions can be generated or otherwise provided. Moreover, the plurality of partitions can include both unoccupied partitions (e.g, empty partitions) and occupied partitions.

Reagents

[0239] In accordance with certain aspects, biological particles can be partitioned along with lysis reagents in order to release the contents of the biological particles within the partition. See, e.g., U.S. Pat. Pub. 2018/0216162 (nowU.S. Pat. 10,428,326), U.S. Pat. Pub. 2019/0100632 (now U.S. Pat. 10,590,244), and U.S. Pat. Pub. 2019/0233878. Biological particles (e.g, cells, cell beads, cell nuclei, organelles, and the like) can be partitioned together with nucleic acid barcode molecules and the nucleic acid molecules of or derived from the biological particle (e.g, mRNA, cDNA, gDNA, etc.,) can be barcoded as described elsewhere herein. In some embodiments, biological particles are co-partitioned with barcode carrying beads (e.g, gel beads) and the nucleic acid molecules of or derived from the biological particle are barcoded as described elsewhere herein. In such cases, the lysis agents can be contacted with the biological particle suspension concurrently with, or immediately prior to, the introduction of the biological particles into the partitioning junction/droplet generation zone ( e.g ., junction 1210), such as through an additional channel or channels upstream of the channel junction. In accordance with other aspects, additionally or alternatively, biological particles can be partitioned along with other reagents, as will be described further below.

[0240] Beneficially, when lysis reagents and biological particles are co-partitioned, the lysis reagents can facilitate the release of the contents of the biological particles within the partition. The contents released in a partition can remain discrete from the contents of other partitions.

[0241] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structures can have other geometries and/or configurations. For example, a microfluidic channel structure can have more than two channel junctions. For example, a microfluidic channel structure can have 2, 3, 4, 5 channel segments or more each carrying the same or different types of beads, reagents, and/or biological particles that meet at a channel junction. Fluid flow in each channel segment can be controlled to control the partitioning of the different elements into droplets. Fluid can be directed flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can include compressors (e.g., providing positive pressure), pumps (e.g, providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.

[0242] Examples of lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g, gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes available from, e.g, Sigma-Aldrich, Inc. (St Louis, MO), as well as other commercially available lysis enzymes. Other lysis agents can additionally or alternatively be co-partitioned with the biological particles to cause the release of the biological particle’s contents into the partitions. For example, in some cases, surfactant-based lysis solutions can be used to lyse cells (e.g, labelled engineered cells), although these can be less desirable for emulsion based systems where the surfactants can interfere with stable emulsions. In some cases, lysis solutions can include non-ionic surfactants such as, for example, Triton X-100 and Tween 20. In some cases, lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical cellular disruption can also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of biological particles that can be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption.

[0243] Alternatively or in addition to the lysis agents co-partitioned with the biological particles (e.g, labelled engineered cells) described above, other reagents can also be co partitioned with the biological particles, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids. In addition, in the case of encapsulated biological particles (e.g, cell beads comprising labelled engineered cells), the biological particles can be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned cell bead. For example, in some cases, a chemical stimulus can be co-partitioned along with an encapsulated biological particle to allow for the degradation of the encapsulating material and release of the cell or its contents into the larger partition. In some cases, this stimulus can be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g, oligonucleotides) from their respective bead. In alternative aspects, this can be a different and non-overlapping stimulus, in order to allow an encapsulated biological particle to be released into a partition at a different time from the release of nucleic acid molecules into the same partition.

[0244] Additional reagents can also be co-partitioned with the biological particles (e.g, labelled engineered cells), such as endonucleases to fragment a biological particle’s DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle’s nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments. Other enzymes can be co-partitioned, including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. In an example of template switching, cDNA can be generated from reverse transcription of a template, e.g ., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g. , polyC, to the cDNA in a template independent manner. Switch oligos can include sequences complementary to the additional nucleotides, e.g. , polyG. The additional nucleotides (e.g, polyC) on the cDNA can hybridize to the additional nucleotides (e.g, polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Template switching oligonucleotides can include a hybridization region and a template region. The hybridization region can include any sequence capable of hybridizing to the target. In some cases, as previously described, the hybridization region includes a series of G bases to complement the overhanging C bases at the 3’ end of a cDNA molecule. The series of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The template sequence can include any sequence to be incorporated into the cDNA. In some cases, the template region includes at least 1 (e.g, at least 2, 3, 4, 5 or more) tag sequences and/or functional sequences. Switch oligos can include deoxyribonucleic acids; ribonucleic acids; modified nucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC, 2’-deoxyInosine, Super T (5-hydroxybutynl-2’-deoxyuridine), Super G (8- aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g, UNA- A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, T Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), or any combination.

[0245] In some cases, the length of a switch oligo can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,

196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides or longer.

[0246] In some cases, the length of a switch oligo can be at most about 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

196, 197 , 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or

250 nucleotides.

[0247] Once the contents of the cells ( e.g ., B cells) are released into their respective partitions, the macromolecular components (e.g. , macromolecular constituents of biological particles, such as RNA, DNA, proteins, or secreted antibodies or antigen-binding fragments thereof) contained therein can be further processed within the partitions. In accordance with the methods and systems described herein, the macromolecular component contents of individual biological particles (e.g., B cells) can be provided with unique identifiers such that, upon characterization of those macromolecular components they can be attributed as having been derived from the same biological particle or particles. The ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles. Unique identifiers, e.g, in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles.

[0248] In some aspects, this is performed by co-partitioning the individual biological particle ( e.g ., B cells) or groups of biological particles (e.g. , B cells) with the unique identifiers, such as described above (with reference to FIGS. 12 and 13). In some aspects, the unique identifiers are provided in the form of nucleic acid molecules (e.g., oligonucleotides) that include nucleic acid barcode sequences that can be attached to or otherwise associated with the nucleic acid contents of individual biological particle, or to other components of the biological particle, and particularly to fragments of those nucleic acids. The nucleic acid molecules are partitioned such that as between nucleic acid molecules in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the nucleic acid molecule can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences can be present.

[0249] The nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g, oligonucleotides). The nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of a barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by 1 or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence can be about 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0250] The co-partitioned nucleic acid molecules can also include other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles (e.g, labelled B cells). These sequences include, e.g, targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g ., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co-partitioning oligonucleotides can also be employed, including, e.g. , coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides into partitions, e.g. , droplets within microfluidic systems.

[0251] In an example, beads are provided that each include large numbers of the above described nucleic acid barcode molecules (e.g, barcoded oligonucleotides) releasably attached to the beads, where all of the nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but where a large number of diverse barcode sequences are represented across the population of beads used. In some embodiments, hydrogel beads, e.g, including polyacrylamide polymer matrices, are used as a solid support and delivery vehicle for the nucleic acid molecules into the partitions, as they are capable of carrying large numbers of nucleic acid molecules, and can be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein. In some cases, the population of beads provides a diverse barcode sequence library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences, or more. In some cases, the population of beads provides a diverse barcode sequence library that includes about 1,000 to about 10,000 different barcode sequences, about 5,000 to about 50,000 different barcode sequences, about 10,000 to about 100,000 different barcode sequences, about 50,000 to about 1,000,000 different barcode sequences, or about 100,000 to about 10,000,000 different barcode sequences.

[0252] Additionally, each bead can be provided with large numbers of nucleic acid (e.g, oligonucleotide) molecules attached. In particular, the number of molecules of nucleic acid molecules including the barcode sequence on an individual bead can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules, or more. In some embodiments, the number of nucleic acid molecules including the barcode sequence on an individual bead is between about 1,000 to about 10,000 nucleic acid molecules, about 5,000 to about 50,000 nucleic acid molecules, about 10,000 to about 100,000 nucleic acid molecules, about 50,000 to about 1,000,000 nucleic acid molecules, about 100,000 to about 10,000,000 nucleic acid molecules, about 1,000,000 to about 1 billion nucleic acid molecules. Nucleic acid molecules of a given bead can include identical (or common) barcode sequences, different barcode sequences, or a combination of both. Nucleic acid molecules of a given bead can include multiple sets of nucleic acid molecules. Nucleic acid molecules of a given set can include identical barcode sequences. The identical barcode sequences can be different from barcode sequences of nucleic acid molecules of another set. In some embodiments, such different barcode sequences can be associated with a given bead.

[0253] Moreover, when the population of beads is partitioned, the resulting population of partitions can also include a diverse barcode library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. Additionally, each partition of the population can include at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules.

[0254] In some cases, the resulting population of partitions provides a diverse barcode sequence library that includes about 1,000 to about 10,000 different barcode sequences, about 5,000 to about 50,000 different barcode sequences, about 10,000 to about 100,000 different barcode sequences, about 50,000 to about 1,000,000 different barcode sequences, or about 100,000 to about 10,000,000 different barcode sequences. Additionally, each partition of the population can include between about 1,000 to about 10,000 nucleic acid barcode molecules, about 5,000 to about 50,000 nucleic acid barcode molecules, about 10,000 to about 100,000 nucleic acid barcode molecules, about 50,000 to about 1,000,000 nucleic acid barcode molecules, about 100,000 to about 10,000,000 nucleic acid barcode molecules, about 1,000,000 to about 1 billion nucleic acid barcode molecules.

[0255] In some cases, it may be desirable to incorporate multiple different barcodes within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known set of barcode sequences can provide greater assurance of identification in the subsequent processing, e.g ., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.

[0256] The nucleic acid molecules (e.g, oligonucleotides) are releasable from the beads upon the application of a particular stimulus to the beads. In some cases, the stimulus can be a photo-stimulus, e.g, through cleavage of a photo-labile linkage that releases the nucleic acid molecules. In other cases, a thermal stimulus can be used, where elevation of the temperature of the beads environment will result in cleavage of a linkage or other release of the nucleic acid molecules from the beads. In still other cases, a chemical stimulus can be used that cleaves a linkage of the nucleic acid molecules to the beads, or otherwise results in release of the nucleic acid molecules from the beads. In one case, such compositions include the polyacrylamide matrices described above for encapsulation of biological particles, and can be degraded for release of the attached nucleic acid molecules through exposure to a reducing agent, such as DTT.

Systems and methods for controlled partitioning

[0257] In some aspects, provided are systems and methods for controlled partitioning. Droplet size can be controlled by adjusting certain geometric features in channel architecture (e.g, microfluidics channel architecture). For example, an expansion angle, width, and/or length of a channel can be adjusted to control droplet size.

[0258] FIG. 14 shows an example of a microfluidic channel structure 1400 for delivering barcode carrying beads to droplets. The channel structure 1400 can include channel segments 1401, 1416, 1404, 1406 and 1408 communicating at a channel junction 1410. In operation, the channel segment 1401 may transport an aqueous fluid 1412 that includes a plurality of beads 1414 (e.g., with nucleic acid molecules, oligonucleotides, molecular tags) along the channel segment 1401 into junction 1410. The plurality of beads 1414 may be sourced from a suspension of beads. For example, the channel segment 1401 may be connected to a reservoir comprising an aqueous suspension of beads 1414. The channel segment 1402 may transport the aqueous fluid 1412 that includes a plurality of biological particles 1416 along the channel segment 1402 into junction 1410. The plurality of biological particles 1416 may be sourced from a suspension of biological particles. For example, the channel segment 1402 may be connected to a reservoir comprising an aqueous suspension of biological particles 1416. In some instances, the aqueous fluid 1412 in either the first channel segment 1401 or the second channel segment 1402, or in both segments, can include one or more reagents, as further described below. A second fluid 1418 that is immiscible with the aqueous fluid 1412 (e.g., oil) can be delivered to the junction 1410 from each of channel segments 1404 and 1406. Upon meeting of the aqueous fluid 1412 from each of channel segments 1401 and 1402 and the second fluid 1418 from each of channel segments 1404 and 1406 at the channel junction 1410, the aqueous fluid 1412 can be partitioned as discrete droplets 1420 in the second fluid 1418 and flow away from the junction 1410 along channel segment 1408. The channel segment 1408 may deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 1408, where they may be harvested. As an alternative, the channel segments 1401 and 1402 may meet at another junction upstream of the junction 1410. At such junction, beads and biological particles may form a mixture that is directed along another channel to the junction 1410 to yield droplets 1420. The mixture may provide the beads and biological particles in an alternating fashion, such that, for example, a droplet comprises a single bead and a single biological particle.

[0259] FIG. 13 shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets. A channel structure 1300 can include a channel segment 1302 communicating at a channel junction 1306 (or intersection) with a reservoir 1304. The reservoir 1304 can be a chamber. Any reference to “reservoir,” as used herein, can also refer to a “chamber.” In operation, an aqueous fluid 1308 that includes suspended beads 1312 can be transported along the channel segment 1302 into the junction 1306 to meet a second fluid 1310 that is immiscible with the aqueous fluid 1308 in the reservoir 1304 to create droplets 1316, 1318 of the aqueous fluid 1308 flowing into the reservoir 1304. At the junction 1306 where the aqueous fluid 1308 and the second fluid 1310 meet, droplets can form based on factors such as the hydrodynamic forces at the junction 1306, flow rates of the two fluids 1308, 1310, fluid properties, and certain geometric parameters ( e.g ., w, ho, a, etc.) of the channel structure 1300. A plurality of droplets can be collected in the reservoir 1304 by continuously injecting the aqueous fluid 1308 from the channel segment 1302 through the junction 1306.

[0260] A discrete droplet generated can include a bead (e.g., as in occupied droplets 1316). Alternatively, a discrete droplet generated can include more than one bead. Alternatively, a discrete droplet generated cannot include any beads (e.g, as in unoccupied droplet 1318). In some instances, a discrete droplet generated can contain one or more biological particles, as described elsewhere herein. In some instances, a discrete droplet generated can include one or more reagents, as described elsewhere herein.

[0261] In some instances, the aqueous fluid 1308 can have a substantially uniform concentration or frequency of beads 1312. The beads 1312 can be introduced into the channel segment 1302 from a separate channel (not shown in FIG. 13). The frequency of beads 1312 in the channel segment 1302 can be controlled by controlling the frequency in which the beads 1312 are introduced into the channel segment 1302 and/or the relative flow rates of the fluids in the channel segment 1302 and the separate channel. In some instances, the beads can be introduced into the channel segment 1302 from a plurality of different channels, and the frequency controlled accordingly.

[0262] In some instances, the aqueous fluid 1308 in the channel segment 1302 can include biological particles (e.g, described with reference to FIG. 12). In some instances, the aqueous fluid 1308 can have a substantially uniform concentration or frequency of biological particles. As with the beads, the biological particles (e.g, labelled engineered cells) can be introduced into the channel segment 1302 from a separate channel. The frequency or concentration of the biological particles in the aqueous fluid 1308 in the channel segment 1302 can be controlled by controlling the frequency in which the biological particles are introduced into the channel segment 1302 and/or the relative flow rates of the fluids in the channel segment 1302 and the separate channel. In some instances, the biological particles can be introduced into the channel segment 1302 from a plurality of different channels, and the frequency controlled accordingly. In some instances, a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment 1302. The first separate channel introducing the beads can be upstream or downstream of the second separate channel introducing the biological particles.

[0263] The second fluid 1310 can include an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets.

[0264] In some instances, the second fluid 1310 cannot be subjected to and/or directed to any flow in or out of the reservoir 1304. For example, the second fluid 1310 can be substantially stationary in the reservoir 1304. In some instances, the second fluid 1310 can be subjected to flow within the reservoir 1304, but not in or out of the reservoir 1304, such as via application of pressure to the reservoir 1304 and/or as affected by the incoming flow of the aqueous fluid 1308 at the junction 1306. Alternatively, the second fluid 1310 can be subjected and/or directed to flow in or out of the reservoir 1304. For example, the reservoir 1304 can be a channel directing the second fluid 1310 from upstream to downstream, transporting the generated droplets.

[0265] The channel structure 1300 at or near the junction 1306 can have certain geometric features that at least partly determine the sizes of the droplets formed by the channel structure 1300. The channel segment 1302 can have a height, ho and width, w, at or near the junction 1306. By way of example, the channel segment 1302 can include a rectangular cross-section that leads to a reservoir 1304 having a wider cross-section (such as in width or diameter). Alternatively, the cross-section of the channel segment 1302 can be other shapes, such as a circular shape, trapezoidal shape, polygonal shape, or any other shapes. The top and bottom walls of the reservoir 1304 at or near the junction 1306 can be inclined at an expansion angle, a. The expansion angle, a, allows the tongue (portion of the aqueous fluid 1308 leaving channel segment 1302 at junction 1306 and entering the reservoir 1304 before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet. Droplet size can decrease with increasing expansion angle. The resulting droplet radius, R_d , can be predicted by the following equation for the aforementioned geometric parameters of ho , w, and a:

R_d * 0.44 ( 1 + 2.2Vten a^- ) _, ^k°

\ h_{0 )} Vtan a

[0266] By way of example, for a channel structure with w = 21 pm, h = 21 pm, and a = 3°, the predicted droplet size is 121 pm. In another example, for a channel structure with w = 25 pm, h = 25 pm, and a = 5°, the predicted droplet size is 123 pm. In another example, for a channel structure with w = 28 pm, h = 28 pm, and a = 7°, the predicted droplet size is 124 pm.

[0267] In some instances, the expansion angle, a, can be between a range of from about 0.5° to about 4°, from about 0.1° to about 10°, or from about 0° to about 90°. For example, the expansion angle can be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, the expansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.G, 0.0P, or less. In some instances, the width, w, can be between a range of from about 100 micrometers (pm) to about 500 pm. In some instances, the width, w, can be between a range of from about 10 pm to about 200 pm. Alternatively, the width can be less than about 10 pm. Alternatively, the width can be greater than about 500 pm. In some instances, the flow rate of the aqueous fluid 1308 entering the junction 1306 can be between about 0.04 microliters (pL)/minute (min) and about 40 pL/min. In some instances, the flow rate of the aqueous fluid 1308 entering the junction 1306 can be between about 0.01 microliters (pL)/minute (min) and about 100 pL/min. Alternatively, the flow rate of the aqueous fluid 1308 entering the junction 1306 can be less than about 0.01 pL/min. Alternatively, the flow rate of the aqueous fluid 1308 entering the junction 1306 can be greater than about 40 pL/min, such as 45 pL/min, 50 pL/min, 55 pL/min, 60 pL/min, 65 pL/min, 70 pL/min, 75 pL/min, 80 pL/min, 85 pL/min, 90 pL/min, 95 pL/min, 100 pL/min, 110 pL/min , 120 pL/min , 130 pL/min , 140 pL/min , 150 pL/min, or greater. At lower flow rates, such as flow rates of about less than or equal to 10 microliters/minute, the droplet radius cannot be dependent on the flow rate of the aqueous fluid 1308 entering the junction 1306.

[0268] In some instances, at least about 50% of the droplets generated can have uniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the droplets generated can have uniform size. Alternatively, less than about 50% of the droplets generated can have uniform size.

[0269] The throughput of droplet generation can be increased by increasing the points of generation, such as increasing the number of junctions ( e.g ., junction 1306) between aqueous fluid 1308 channel segments (e.g., channel segment 1302) and the reservoir 1304. Alternatively or in addition, the throughput of droplet generation can be increased by increasing the flow rate of the aqueous fluid 1308 in the channel segment 1302.

[0270] The methods and systems described herein can be used to greatly increase the efficiency of single cell applications and/or other applications receiving droplet-based input.

[0271] Subsequent operations that can be performed can include generation of amplification products, purification ( e.g ., via solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, ligation of functional sequences, and subsequent amplification (e.g, via PCR)). These operations can occur in bulk (e.g, outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. Additional reagents that can be co partitioned along with the barcode bearing bead can include oligonucleotides to block ribosomal RNA (rRNA) and nucleases to digest genomic DNA from cells. Alternatively, rRNA removal agents can be applied during additional processing operations. The configuration of the constructs generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing and/or sequence the 5’ end of a polynucleotide sequence. The amplification products, for example, first amplification products and/or second amplification products, can be subject to sequencing for sequence analysis. In some cases, amplification can be performed using the Partial Hairpin Amplification for Sequencing (PHASE) method.

[0272] A variety of applications require the evaluation of the presence and quantification of different biological particle or organism types within a population of biological particles, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g. , in tracing contamination or the like.

[0273] Partitions including a barcode bead (e.g, a gel bead) associated with barcode molecules and a bead encapsulating cellular constituents (e.g, a cell bead) such as cellular nucleic acids can be useful in constituent analysis as is described in U.S. Patent Publication No. 2018/0216162.

Sample and cell processing

[0274] A sample can be derived from any useful source including any subject, such as a human subject. A sample can include material (e.g, one or more cells) from one or more different sources, such as one or more different subjects. Multiple samples, such as multiple samples from a single subject (e.g, multiple samples obtained in the same or different manners from the same or different bodily locations, and/or obtained at the same or different times (e.g, seconds, minutes, hours, days, weeks, months, or years apparat)), or multiple samples from different subjects, can be obtained for analysis as described herein. For example, a first sample can be obtained from a subject at a first time and a second sample can be obtained from the subject at a second time later than the first time. The first time can be before a subject undergoes a treatment regimen or procedure ( e.g ., to address a disease or condition), and the second time can be during or after the subject undergoes the treatment regimen or procedure. In another example, a first sample can be obtained from a first bodily location or system of a subject (e.g., using a first collection technique) and a second sample can be obtained from a second bodily location or system of the subject (e.g, using a second collection technique), which second bodily location or system can be different than the first bodily location or system. In another example, multiple samples can be obtained from a subject at a same time from the same or different bodily locations. Different samples, such as different samples collected from different bodily locations of a same subject, at different times, from multiple different subjects, and/or using different collection techniques, can undergo the same or different processing (e.g, as described herein). For example, a first sample can undergo a first processing protocol and a second sample can undergo a second processing protocol.

[0275] A sample can be a biological sample, such as a cell sample (e.g, as described herein). A sample can include one or more biological particles, such as one or more cells and/or cellular constituents, such as one or more cell nuclei. For example, a sample can include a plurality of cells and/or cellular constituents. Components (e.g, cells or cellular constituents, such as cell nuclei) of a sample can be of a single type or a plurality of different types. For example, cells of a sample can include one or more different types of blood cells.

[0276] A biological sample can include a plurality of cells having different dimensions and features. In some cases, processing of the biological sample, such as cell separation and sorting (e.g, as described herein), can affect the distribution of dimensions and cellular features included in the sample by depleting cells having certain features and dimensions and/or isolating cells having certain features and dimensions.

[0277] A sample may undergo one or more processes in preparation for analysis (e.g, as described herein), including, but not limited to, filtration, selective precipitation, purification, centrifugation, permeabilization, isolation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In an example, a filtration process can include the use of microfluidics (e.g, to separate biological particles of different sizes, types, charges, or other features).

[0278] In an example, a sample including one or more cells can be processed to separate the one or more cells from other materials in the sample (e.g, using centrifugation and/or another process). In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort groups of cells and/or cellular constituents, such as to separate and/or sort cells and/or cellular constituents of different types. Examples of cell separation include, but are not limited to, separation of white blood cells or immune cells from other blood cells and components, separation of circulating tumor cells from blood, and separation of bacteria from bodily cells and/or environmental materials. A separation process can include a positive selection process ( e.g ., targeting of a cell type of interest for retention for subsequent downstream analysis, such as by use of a monoclonal antibody that targets a surface marker of the cell type of interest), a negative selection process (e.g., removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g, removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells).

[0279] Separation of one or more different types of cells can include, for example, centrifugation, filtration, microfluidic-based sorting, flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), buoyancy-activated cell sorting (BACS), or any other useful method. For example, a flow cytometry method can be used to detect cells and/or cellular constituents based on a parameter such as a size, morphology, or protein expression. Flow cytometry-based cell sorting can include injecting a sample into a sheath fluid that conveys the cells and/or cellular constituents of the sample into a measurement region one at a time. In the measurement region, a light source such as a laser can interrogate the cells and/or cellular constituents and scattered light and/or fluorescence can be detected and converted into digital signals. A nozzle system (e.g, a vibrating nozzle system) can be used to generate droplets (e.g, aqueous droplets) including individual cells and/or cellular constituents. Droplets including cells and/or cellular constituents of interest (e.g, as determined via optical detection) can be labeled with an electric charge (e.g, using an electrical charging ring), which charge can be used to separate such droplets from droplets including other cells and/or cellular constituents. For example, FACS can include labeling cells and/or cellular constituents with fluorescent markers (e.g, using internal and/or external biomarkers). Cells and/or cellular constituents can then be measured and identified one by one and sorted based on the emitted fluorescence of the marker or absence thereof. MACS can use micro- or nano-scale magnetic particles to bind to cells and/or cellular constituents (e.g, via an antibody interaction with cell surface markers) to facilitate magnetic isolation of cells and/or cellular constituents of interest from other components of a sample (e.g, using a column-based analysis). BACS can use microbubbles (e.g, glass microbubbles) labeled with antibodies to target cells of interest. Cells and/or cellular components coupled to microbubbles can float to a surface of a solution, thereby separating target cells and/or cellular components from other components of a sample. Cell separation techniques can be used to enrich for populations of cells of interest ( e.g ., prior to partitioning, as described herein). For example, a sample including a plurality of cells including a plurality of cells of a given type can be subjected to a positive separation process. The plurality of cells of the given type can be labeled with a fluorescent marker (e.g., based on an expressed cell surface marker or another marker) and subjected to a FACS process to separate these cells from other cells of the plurality of cells. The selected cells can then be subjected to subsequent partition- based analysis (e.g, as described herein) or other downstream analysis. The fluorescent marker can be removed prior to such analysis or can be retained. The fluorescent marker can include an identifying feature, such as a nucleic acid barcode sequence and/or unique molecular identifier.

[0280] In another example, a first sample including a first plurality of cells including a first plurality of cells of a given type (e.g, immune cells expressing a particular marker or combination of markers) and a second sample including a second plurality of cells including a second plurality of cells of the given type can be subjected to a positive separation process. The first and second samples can be collected from the same or different subjects, at the same or different types, from the same or different bodily locations or systems, using the same or different collection techniques. For example, the first sample can be from a first subject and the second sample can be from a second subject different than the first subject. The first plurality of cells of the first sample can be provided a first plurality of fluorescent markers configured to label the first plurality of cells of the given type. The second plurality of cells of the second sample can be provided a second plurality of fluorescent markers configured to label the second plurality of cells of the given type. The first plurality of fluorescent markers can include a first identifying feature, such as a first barcode, while the second plurality of fluorescent markers can include a second identifying feature, such as a second barcode, that is different than the first identifying feature. The first plurality of fluorescent markers and the second plurality of fluorescent markers can fluoresce at the same intensities and over the same range of wavelengths upon excitation with a same excitation source (e.g, light source, such as a laser). The first and second samples can then be combined and subjected to a FACS process to separate cells of the given type from other cells based on the first plurality of fluorescent markers labeling the first plurality of cells of the given type and the second plurality of fluorescent markers labeling the second plurality of cells of the given type. Alternatively, the first and second samples can undergo separate FACS processes and the positively selected cells of the given type from the first sample and the positively selected cells of the given type from the second sample can then be combined for subsequent analysis. The encoded identifying features of the different fluorescent markers can be used to identify cells originating from the first sample and cells originating from the second sample. For example, the first and second identifying features can be configured to interact (e.g, in partitions, as described herein) with nucleic acid barcode molecules (e.g, as described herein) to generate barcoded nucleic acid products detectable using, e.g, nucleic acid sequencing.

[0281] FIG. 18 schematically shows an example workflow for processing nucleic acid molecules within a sample. A substrate 1800 including a plurality of microwells 1802 can be provided. A sample 1806 which can include a cell, cell bead, cellular components or analytes (e.g, proteins and/or nucleic acid molecules) can be co-partitioned, in a plurality of microwells 1802, with a plurality of beads 1804 including nucleic acid barcode molecules. During a partitioning process, the sample 1806 can be processed within the partition. For instance, in the case of live cells, the cell can be subjected to conditions sufficient to lyse the cells and release the analytes contained therein. In process 1820, the bead 1804 can be further processed. By way of example, processes 1820a and 1820b schematically illustrate different workflows, depending on the properties of the bead 1804.

[0282] In 1820a, the bead includes nucleic acid barcode molecules that are attached thereto, and sample nucleic acid molecules (e.g, RNA, DNA) can attach, e.g, via hybridization of ligation, to the nucleic acid barcode molecules. Such attachment can occur on the bead. In process 1830, the beads 1804 from multiple wells 1802 can be collected and pooled. Further processing can be performed in process 1840. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 1850, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g, in a plot.

[0283] In 1820b, the bead includes nucleic acid barcode molecules that are releasably attached thereto, as described below. The bead can degrade or otherwise release the nucleic acid barcode molecules into the well 1802; the nucleic acid barcode molecules can then be used to barcode nucleic acid molecules within the well 1802. Further processing can be performed either inside the partition or outside the partition. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 1850, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g., in a plot.

Multiplexing methods

[0284] In some embodiments of the disclosure, steps (a) and (b) of the methods described herein are performed in multiplex format. For example, in some embodiments, step (a) of the methods disclosed herein can include individually partitioning additional single cells (e.g, B cells) of the plurality of cells in additional partitions of the plurality of partitions, and step (b) can further include determining all or a part of the nucleic acid sequences encoding antibodies or antigen-binding fragments thereof produced by the additional cells (e.g, B cells).

[0285] Accordingly, in some embodiments, the present disclosure provides methods and systems for multiplexing, and otherwise increasing throughput of samples for analysis. For example, a single or integrated process workflow may permit the processing, identification, and/or analysis of more or multiple analytes, more or multiple types of analytes, and/or more or multiple types of analyte characterizations. For example, in the methods and systems described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more cells or cell features can be used to characterize cells and/or cell features. In some instances, cell features include cell surface features. Cell surface features can include, but are not limited to, a receptor, an antigen or antigen fragment (e.g., an antigen or antigen fragment that binds to an antigen-binding molecule located on a cell surface), a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features can include intracellular analytes, such as proteins, protein modifications ( e.g ., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof. A labelling agent can include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), an antigen, an antigen fragment, a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a Darpin, and a protein scaffold, or any combination thereof. The labelling agents can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide can include a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g, a first cell surface feature) can have a first reporter oligonucleotide coupled thereto, while a labelling agent that is specific to a different cell feature (e.g, a second cell surface feature) can have a different reporter oligonucleotide coupled thereto. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g, U.S. Pat.

10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969.

[0286] In a particular example, a library of potential cell feature labelling agents can be provided, where the respective cell feature labelling agents are associated with nucleic acid reporter molecules, such that a different reporter oligonucleotide sequence is associated with each labelling agent capable of binding to a specific cell feature. In some embodiments, the cell feature labelling agents comprise a target antigen and a fragment of the target antigen, as disclosed herein. In some embodiments, the cell feature labelling agents comprise a plurality of non-overlapping fragments of a target antigen. In other aspects, different members of the library can be characterized by the presence of a different oligonucleotide sequence label. For example, an antibody capable of binding to a target protein can have associated with it a first reporter oligonucleotide sequence, while an antibody, (which may be the same antibody), capable of binding to a fragment or fragments of the target antigen can have a different, (or additional if the same antibody), reporter oligonucleotide sequence(s) associated with it. The presence of the particular oligonucleotide sequence(s) can be indicative of the presence of a particular antibody or cell feature which can be recognized or bound by the particular antibody.

[0287] Labelling agents capable of binding to or otherwise coupling to one or more cells can be used to characterize a cell as belonging to a particular set of cells. For example, labellinging agents can be used to label a sample of cells, e.g., to provide a sample index. For other example, labelling agents can be used to label a group of cells belonging to a particular experimental condition. In this way, a group of cells can be labeled as different from another group of cells. In an example, a first group of cells can originate from a first sample and a second group of cells can originate from a second sample. Labelling agents can allow the first group and second group to have a different labeling agent (or reporter oligonucleotide associated with the labeling agent). This can, for example, facilitate multiplexing, where cells of the first group and cells of the second group can be labeled separately and then pooled together for downstream analysis. The downstream detection of a label can indicate analytes as belonging to a particular group.

[0288] For example, a reporter oligonucleotide can be linked to an antibody or an epitope binding fragment thereof, and labeling a cell can include subjecting the antibody- linked barcode molecule or the epitope binding fragment-linked barcode molecule to conditions suitable for binding the antibody to a molecule present on a surface of the cell.

The binding affinity between the antibody or the epitope binding fragment thereof and the molecule present on the surface can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule. For example, the binding affinity can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule during various sample processing steps, such as partitioning and/or nucleic acid amplification or extension. A dissociation constant (Kd) between the antibody or an epitope binding fragment thereof and the molecule to which it binds can be less than about 100 mM, 90 mM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM,

7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, or 1 pM. For example, the dissociation constant can be less than about 10 mM. In some embodiments, the antibody or epitope binding fragment thereof has a desired off rate (koff), such that the antibody or antigen binding fragment thereof remains bound to the target antigen or antigen fragment during various sample processing steps.

[0289] In another example, a reporter oligonucleotide can be coupled to a cell- penetrating peptide (CPP), and labeling cells can include delivering the CPP coupled reporter oligonucleotide into a biological particle. Labeling biological particles can include delivering the CPP conjugated oligonucleotide into a cell and/or cell bead by the cell-penetrating peptide. A CPP that can be used in the methods provided herein can include at least one non functional cysteine residue, which can be either free or derivatized to form a disulfide link with an oligonucleotide that has been modified for such linkage. Non-limiting examples of CPPs that can be used in embodiments herein include penetratin, transportan, plsl, TAT(48- 60), pVEC, MTS, and MAP. Cell-penetrating peptides useful in the methods provided herein can have the capability of inducing cell penetration for at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of cells of a cell population. The CPP can be an arginine-rich peptide transporter. The CPP can be Penetratin or the Tat peptide. In another example, a reporter oligonucleotide can be coupled to a fluorophore or dye, and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions suitable for binding the fluorophore to the surface of the cell. In some instances, fluorophores can interact strongly with lipid bilayers and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions such that the fluorophore binds to or is inserted into a membrane of the cell. In some cases, the fluorophore is a water-soluble, organic fluorophore. In some instances, the fluorophore is Alexa 532 maleimide, tetramethylrhodamine-5-maleimide (TMR maleimide), BODIPY-TMR maleimide, Sulfo- Cy3 maleimide, Alexa 546 carboxylic acid/succinimidyl ester, Atto 550 maleimide, Cy3 carboxylic acid/succinimidyl ester, Cy3B carboxylic acid/succinimidyl ester, Atto 565 biotin, Sulforhodamine B, Alexa 594 maleimide, Texas Red maleimide, Alexa 633 maleimide, Abberior STAR 635P azide, Atto 647N maleimide, Atto 647 SE, or Sulfo-Cy5 maleimide. See, e.g, Hughes L D, et al. PLoS One. 2014 Feb. 4; 9(2):e87649 for a description of organic fluorophores.

[0290] A reporter oligonucleotide can be coupled to a lipophilic molecule, and labeling cells can include delivering the nucleic acid barcode molecule to a membrane of a cell or a nuclear membrane by the lipophilic molecule. Lipophilic molecules can associate with and/or insert into lipid membranes such as cell membranes and nuclear membranes. In some cases, the insertion can be reversible. In some cases, the association between the lipophilic molecule and the cell or nuclear membrane can be such that the membrane retains the lipophilic molecule (e.g, and associated components, such as nucleic acid barcode molecules, thereof) during subsequent processing (e.g, partitioning, cell permeabilization, amplification, pooling, etc.). The reporter nucleotide can enter into the intracellular space and/or a cell nucleus. In some embodiments, a reporter oligonucleotide coupled to a lipophilic molecule will remain associated with and/or inserted into lipid membrane (as described herein) via the lipophilic molecule until lysis of the cell occurs, e.g ., inside a partition. Exemplary embodiments of lipophilic molecules coupled to reporter oligonucleotides are described in PCT/US2018/064600.

[0291] A reporter oligonucleotide can be part of a nucleic acid molecule including any number of functional sequences, as described elsewhere herein, such as a target capture sequence, a random primer sequence, and the like, and coupled to another nucleic acid molecule that is, or is derived from, the analyte.

[0292] Prior to partitioning, the cells can be incubated with the library of labelling agents, that can be labelling agents to a broad panel of different cell features, e.g. , receptors, proteins, etc., and which include their associated reporter oligonucleotides. Unbound labelling agents can be washed from the cells, and the cells can then be co-partitioned (e.g, into droplets or wells) along with partition-specific barcode oligonucleotides (e.g, attached to a support, such as a bead or gel bead) as described elsewhere herein. As a result, the partitions can include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides.

[0293] In other instances, e.g, to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature can have a first plurality of the labelling agent (e.g, an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent coupled to a second reporter oligonucleotide. For example, the first plurality of the labeling agent and second plurality of the labeling agent can interact with different cells, cell populations or samples, allowing a particular report oligonucleotide to indicate a particular cell population (or cell or sample) and cell feature. In this way, different samples or groups can be independently processed and subsequently combined together for pooled analysis (e.g, partition-based barcoding as described elsewhere herein). See, e.g,

U.S. Pat. Pub. 20190323088.

[0294] In some embodiments, to facilitate sample multiplexing, individual samples can be stained with lipid tags, such as cholesterol -modified oligonucleotides (CMOs, see, e.g, FIG. 7), anti-calcium channel antibodies, or anti-ACTB antibodies. Non-limiting examples of anti-calcium channel antibodies include anti-KCNN4 antibodies, anti-BK channel beta 3 antibodies, anti-alB calcium channel antibodies, and anti-CACNAl A antibodies. Examples of anti-ACTB antibodies suitable for the methods of the disclosure include, but are not limited to, mAbGEa, ACTN05, AC- 15, 15G5A11/E2, BA3R, and HHF35.

[0295] As described elsewhere herein, libraries of labelling agents can be associated with a particular cell feature as well as be used to identify analytes as originating from a particular cell population, or sample. Cell populations can be incubated with a plurality of libraries such that a cell or cells include multiple labelling agents. For example, a cell can include coupled thereto a lipophilic labeling agent and an antibody. The lipophilic labeling agent can indicate that the cell is a member of a particular cell sample, whereas the antibody can indicate that the cell includes a particular analyte. In this manner, the reporter oligonucleotides and labelling agents can allow multi-analyte, multiplexed analyses to be performed.

[0296] In some instances, these reporter oligonucleotides can include nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The use of oligonucleotides as the reporter can provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g, using sequencing or array technologies.

[0297] Attachment (coupling) of the reporter oligonucleotides to the labelling agents can be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, reporter oligonucleotides can be covalently attached to a portion of a labelling agent (such a protein, e.g, an antigen or antigen fragment, an antibody or antibody fragment) using chemical conjugation techniques (e.g, Lightning- Link® antibody labelling kits available from Innova Biosciences), as well as other non- covalent attachment mechanisms, e.g, using biotinylated antibodies (or biotinylated antigens, or biotinylated antigen fragments) and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g, Fang, etal., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708- 715. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g, U.S. Pat. No. 6,265,552. Furthermore, click reaction chemistry such as a Methyl tetrazine-PEG5-NHS Ester reaction, a TCO-PEG4-NHS Ester reaction, or the like, can be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art can be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly ( e.g ., via hybridization) coupled to a reporter oligonucleotide including a barcode sequence that identifies the label agent. For instance, the labelling agent can be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that includes a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide can be attached to the labeling agent through a labile bond (e.g, chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. In some instances, the reporter oligonucleotides described herein can include one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0298] In some cases, the labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a monomer. In some cases, the labelling agent is presented as a multimer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a dimer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a trimer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a tetramer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a pentamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a hexamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a heptamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as an octamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a nonamer.

In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a decamer. In some cases, a labellign agent (.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a 10+-mer.

[0299] In some cases, the labelling agent can include a reporter oligonucleotide and a label. A label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly ( e.g ., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to an oligonucleotide that is complementary to a sequence of the reporter oligonucleotide, and the oligonucleotide can be allowed to hybridize to the reporter oligonucleotide.

[0300] FIG. 19 describes exemplary labelling agents (1910, 1920, 1930) including reporter oligonucleotides (1940) attached thereto. Labelling agent 1910 (e.g., any of the labelling agents described herein) is attached (either directly, e.g, covalently attached, or indirectly) to reporter oligonucleotide 1940. Reporter oligonucleotide 1940 can include barcode sequence 1942 that identifies labelling agent 1910. Reporter oligonucleotide 1940 can also include one or more functional sequences 1943 that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, or a sequencing primer or primer binding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0301] Referring to FIG. 19, in some instances, reporter oligonucleotide 1940 conjugated to a labelling agent (e.g, 1910, 1920, 1930) includes a functional sequence 1941, a reporter barcode sequence 1942 that identifies the labelling agent (e.g, 1910, 1920, 1930), and reporter capture handle 1943. Reporter capture handle sequence 1943 can be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule 1990 (not shown), such as those described elsewhere herein.

In some instances, nucleic acid barcode molecule 1990 is attached to a support (e.g, a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1990 can be attached to the support via a releasable linkage (e.g, including a labile bond), such as those described elsewhere herein. In some instances, reporter oligonucleotide 1940 includes one or more additional functional sequences, such as those described above.

[0302] In some instances, the labelling agent 1910 is a protein or polypeptide (e.g, an antigen or prospective antigen, or a fragment of an antigen or prospective antigen) including reporter oligonucleotide 1940. Reporter oligonucleotide 1940 includes reporter barcode sequence 1942 that identifies polypeptide 1910 and can be used to infer the presence of an analyte, e.g., a binding partner of polypeptide 1910 (i.e., a molecule or compound to which polypeptide 1910 can bind). In some instances, the labelling agent 1910 is a lipophilic moiety (e.g, cholesterol) including reporter oligonucleotide 1940, where the lipophilic moiety is selected such that labelling agent 710 integrates into a membrane of a cell or nucleus. Reporter oligonucleotide 740 includes reporter barcode sequence 742 that identifies lipophilic moiety 1910 which in some instances is used to tag cells (e.g, groups of cells, cell samples, etc.) and can be used for multiplex analyses as described elsewhere herein. In some instances, the labelling agent is an antibody 1920 (or an epitope binding fragment thereof) including reporter oligonucleotide 1940. Reporter oligonucleotide 1940 includes reporter barcode sequence 1942 that identifies antibody 1920 and can be used to infer the presence of, e.g, a target of antibody 1920 (i.e., a molecule or compound to which antibody 1920 binds). In other embodiments, labelling agent 1930 includes an MHC molecule 1931 including peptide 1932 and reporter oligonucleotide 1940 that identifies peptide 1932. In some instances, the MHC molecule is coupled to a support 1933. In some instances, support 1933 can be a polypeptide, such as streptavidin, or a polysaccharide, such as dextran. In some instances, reporter oligonucleotide 1940 can be directly or indirectly coupled to MHC labelling agent 1930 in any suitable manner. For example, reporter oligonucleotide 1940 can be coupled to MHC molecule 1931, support 1933, or peptide 1932. In some embodiments, labelling agent 1930 includes a plurality of MHC molecules, (e.g. is an MHC multimer, which can be coupled to a support (e.g, 1933)). There are many possible configurations of Class I and/or Class II MHC multimers that can be utilized with the compositions, methods, and systems disclosed herein, e.g, MHC tetramers, MHC pentamers (MHC assembled via a coiled-coil domain, e.g, Pro5® MHC Class I Pentamers, (Prolmmune, Ltd.), MHC octamers, MHC dodecamers, MHC decorated dextran molecules (e.g, MHC Dextramer® (Immudex)), etc.

For a description of exemplary labelling agents, including antibody and MHC -based labelling agents, reporter oligonucleotides, and methods of use, see, e.g, U.S. Pat. 10,550,429 and U.S. Pat. Pub. 20190367969.

[0303] Exemplary barcode molecules attached to a support (e.g, a bead) is shown in FIG. 20. In some embodiments, analysis of multiple analytes (e.g, RNA and one or more analytes using labelling agents described herein) can include nucleic acid barcode molecules as generally depicted in FIG. 20. In some embodiments, nucleic acid barcode molecules 2010 and 2020 are attached to support 2030 via a releasable linkage 2040 ( e.g ., including a labile bond) as described elsewhere herein. Nucleic acid barcode molecule 2010 can include functional sequence 2011, barcode sequence 2012 and capture sequence 2013. Nucleic acid barcode molecule 2020 can include adapter sequence 2021, barcode sequence 2012, and capture sequence 2023, wherein capture sequence 2023 includes a different sequence than capture sequence 2013. In some instances, adapter 2011 and adapter 2021 include the same sequence. In some instances, adapter 2011 and adapter 2021 include different sequences. Although support 2030 is shown including nucleic acid barcode molecules 2010 and 2020, any suitable number of barcode molecules including common barcode sequence 2012 are contemplated herein. For example, in some embodiments, support 2030 further includes nucleic acid barcode molecule 2050. Nucleic acid barcode molecule 2050 can include adapter sequence 2051, barcode sequence 2012 and capture sequence 2053, wherein capture sequence 2053 includes a different sequence than capture sequence 2013 and 2023. In some instances, nucleic acid barcode molecules (e.g., 2010, 2020, 2050) include one or more additional functional sequences, such as a UMI or other sequences described herein. The nucleic acid barcode molecules 2010, 2020 or 2050 can interact with analytes as described elsewhere herein, for example, as depicted in FIGS. 21A-21C.

[0304] Referring to FIG. 21A, in an instance where cells are labelled with labeling agents, capture sequence 2123 can be complementary to an adapter sequence of a reporter oligonucleotide. Cells can be contacted with one or more reporter oligonucleotide 2120 conjugated labelling agents 2110 (e.g, polypeptide such as an antigen or fragment of an antigen, antibody, or others described elsewhere herein). In some cases, the cells can be further processed prior to barcoding. For example, such processing steps can include one or more washing and/or cell sorting steps. In some instances, a cell that is bound to labelling agent 2110 which is conjugated to oligonucleotide 2120 and support 2130 (e.g, a bead, such as a gel bead) including nucleic acid barcode molecule 2190 is partitioned into a partition amongst a plurality of partitions (e.g, a droplet of a droplet emulsion or a well of a microwell array). In some instances, the partition includes at most a single cell bound to labelling agent 2110. In some instances, reporter oligonucleotide 2120 conjugated to labelling agent 2110 (e.g, polypeptide such as an antigen or fragment of an antigen, an antibody, pMHC molecule such as an MHC multimer, etc.) includes a first functional sequence 2111 (e.g, a primer sequence), a barcode sequence 2112 that identifies the labelling agent 2110 (e.g, the polypeptide such as an antigen or fragment of an antigen, antibody, or peptide of a pMHC molecule or complex), and a capture handle sequence 2113. Capture handle sequence 2113 can be configured to hybridize to a complementary sequence, such as capture sequence 2123 present on a nucleic acid barcode molecule 2190 ( e.g ., partition-specific barcode molecule).

In some instances, oligonucleotide 2110 includes one or more additional functional sequences, such as those described elsewhere herein.

[0305] Barcoded nucleic acid molecules can be generated (e.g., via a nucleic acid reaction, such as nucleic acid extension, reverse transcription, or ligation) from the constructs described in FIGS. 21A-21C. For example, capture handle sequence 2113 can then be hybridized to complementary capture sequence 2123 to generate (e.g, via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule including cell barcode (e.g, common barcode or partition-specific barcode) sequence 2122 (or a reverse complement thereof) and reporter barcode sequence 2112 (or a reverse complement thereof). In some embodiments, the nucleic acid barcode molecule 2190 (e.g, partition-specific barcode molecule) further includes a UMI. Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g, to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g, U.S. Pat. Pub. 2018/0105808. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform.

[0306] In some instances, analysis of multiple analytes (e.g, nucleic acids and one or more analytes using labelling agents described herein) can be performed. For example, the workflow can include a workflow as generally depicted in any of FIGS. 21A-21C, or a combination of workflows for an individual analyte, as described elsewhere herein. For example, by using a combination of the workflows as generally depicted in FIGS. 21A-21C, multiple analytes can be analyzed.

[0307] In some instances, analysis of an analyte (e.g. a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.) includes a workflow as generally depicted in FIG. 21 A. A nucleic acid barcode molecule 2190 can be co-partitioned with the one or more analytes. In some instances, nucleic acid barcode molecule 2190 is attached to a support 2130 (e.g, a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 2190 can be attached to support 2130 via a releasable linkage 2140 (e.g, including a labile bond), such as those described elsewhere herein. Nucleic acid barcode molecule 2190 can include a functional sequence 2121 and optionally include other additional sequences, for example, a barcode sequence 2122 ( e.g ., common barcode, partition-specific barcode, or other functional sequences described elsewhere herein), and/or a UMI sequence 2125. The nucleic acid barcode molecule 2190 can include a capture sequence 2123 that can be complementary to another nucleic acid sequence, such that it can hybridize to a particular sequence.

[0308] For example, capture sequence 2123 can include a poly-T sequence and can be used to hybridize to mRNA. Referring to FIG. 21C, in some embodiments, nucleic acid barcode molecule 2190 includes capture sequence 2123 complementary to a sequence of RNA molecule 2160 from a cell. In some instances, capture sequence 2123 includes a sequence specific for an RNA molecule. Capture sequence 2123 can include a known or targeted sequence or a random sequence. In some instances, a nucleic acid extension reaction can be performed, thereby generating a barcoded nucleic acid product including capture sequence 2123, the functional sequence 2121, UMI sequence 2125, any other functional sequence, and a sequence corresponding to the RNA molecule 2160.

[0309] In another example, capture sequence 2123 can be complementary to an overhang sequence or an adapter sequence that has been appended to an analyte. For example, referring to FIG. 21B, in some embodiments, primer 2150 includes a sequence complementary to a sequence of nucleic acid molecule 2160 (such as an RNA encoding for a BCR sequence) from a biological particle. In some instances, primer 2150 includes one or more sequences 2151 that are not complementary to RNA molecule 2160. Sequence 2151 can be a functional sequence as described elsewhere herein, for example, an adapter sequence, a sequencing primer sequence, or a sequence the facilitates coupling to a flow cell of a sequencer. In some instances, primer 2150 includes a poly-T sequence. In some instances, primer 2150 includes a sequence complementary to a target sequence in an RNA molecule. In some instances, primer 2150 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Primer 2150 is hybridized to nucleic acid molecule 2160 and complementary molecule 2170 is generated. For example, complementary molecule 2170 can be cDNA generated in a reverse transcription reaction. In some instances, an additional sequence can be appended to complementary molecule 2170. For example, the reverse transcriptase enzyme can be selected such that several non- templated bases 2180 (e.g., a poly-C sequence) are appended to the cDNA. In another example, a terminal transferase can also be used to append the additional sequence. Nucleic acid barcode molecule 2190 includes a sequence 2124 complementary to the non-templated bases, and the reverse transcriptase performs a template switching reaction onto nucleic acid barcode molecule 2190 to generate a barcoded nucleic acid molecule including cell ( e.g ., partition specific) barcode sequence 2122 (or a reverse complement thereof) and a sequence of complementary molecule 2170 (or a portion thereof). In some instances, capture sequence 2123 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Capture sequence 2123 is hybridized to nucleic acid molecule 2160 and a complementary molecule 2170 is generated. For example, complementary molecule 2170 can be generated in a reverse transcription reaction generating a barcoded nucleic acid molecule including cell barcode (e.g., common barcode or partition- specific barcode) sequence 2122 (or a reverse complement thereof) and a sequence of complementary molecule 2170 (or a portion thereof). Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D)J regions of an immune cell receptor and/or barcoding methods and composition including a template switch oligonucleotide are described in International Patent Application WO2018/075693, U.S. Patent Publication No. 2018/0105808, U.S. Patent Publication No. 2015/0376609, filed June 26, 2015, and U.S. Patent Publication No. 2019/0367969.

KITS

[0310] Further provided herein are kits, which may be useful for the practice of a method described herein. In some embodiments, the kits are for (i) identification of an antibody, or an antigen-binding fragment thereof, to a region of interest of a target antigen,

(ii) mapping binding affinity for at least one region of interest of a target antigen by an antibody, or antigen-binding fragment thereof, or (iii) characterizing an antibody or antigen- fragment thereof. In one embodiment, the kit may include: (a) instructions for use and (b) a target antigen and a fragment of the target antigen. In another embodiment, the kit may include: (a) instructions for use and (b) a plurality of fragments of the target antigen.

[0311] The antibody, which may be characterized, identified or whose binding affinity may be mapped by use of the kits may be an antibody having an Immunoglobulin (Ig)A (e.g, IgAl or IgA2), IgD, IgE, IgG (e.g, IgGl, IgG2, IgG3 and IgG4) or IgM constant region. The antigen-binding fragment of the antibody, which may be characterized, identified or whose binding affinity may be mapped by use of the kits herein may be any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. For example, an antigen-binding fragment of an antibody may be any one of: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) sdAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody ( e.g ., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FWR3-CDR3-FWR4 peptide. Further, an antigen-binding fragment of an antibody may be an engineered molecule, such as a domain-specific antibody, single domain antibody, chimeric antibody, CDR-grafted antibody, diabody, triabody, tetrabody, minibody, nanobody (e.g., monovalent nanobodies, bivalent nanobodies, etc.), a small modular immunopharmaceutical (SMIP), or a shark variable IgNAR domain.

[0312] The target antigen, which may be included in the kits, and to which the antibody or antigen-binding fragment thereof may having binding affinity for a region of interest, may be any antigen for which characterization and/or identification of binders thereto, is desirable. The target antigen may be an antigen associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal or prion agent. If the target antigen is associated with an infectious agent that is a viral agent, the viral agent may be an influenza virus, a coronavirus, a retrovirus, a rhinovirus, or a sarcoma virus. The viral agent may be severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), a SARS-CoV-2, a Middle East respiratory syndrome coronavirus (MERS-CoV)), or human immunodeficiency virus (HIV), influenza, respiratory syncytial virus, or Ebola virus. If the target antigen is associated with an infectious agent that is a viral agent, the target antigen may be corona virus spike (S) protein, e.g., a SARS-CoV-2 spike protein, an influenza hemagglutinin protein, an HIV envelope protein or any other a viral glycoprotein. Further, the target antigen may be associated with a tumor or a cancer. If the target agent is associated with a tumor, the target agent may be associated tumors or cancers. If the target antigen is associated with tumors or cancers, it may be, for example, epidermal growth factor receptor (EGFR), CD38, platelet-derived growth factor receptor (PDGFR) alpha, insulin growth factor receptor (IGFR), CD20, CD 19, CD47, or human epidermal growth factor receptor 2 (HER2). In addition, the target antigen may be an immune checkpoint molecule that may or may not be associated with tumors or cancers (e.g, CD38, PD-1, CTLA-4, TIGIT, LAG-3, VISTA, TIM-3), or it may be a cytokine (e.g, soluble cytokine), a GPCR, a cell-based co-stimulatory molecule, a cell-based co- inhibitory molecule, an ion channel, a glycan, a glycan conjugate, or a growth factor.

[0313] The region of interest of the target antigen, e.g., for which antigen-binding molecules such as antibodies or antigen-binding fragments thereof may be characterized as having binding affinity to or to which they may be mapped, may be a less than the full-length target antigen. The region of interest of the target antigen may include or may be an epitope of the target antigen, e.g ., a linear or conformational or cryptic epitope. The region of interest of the target antigen may include or may be a domain of the target antigen. A domain of a target antigen may also be referred to as a unit or portion an antigen that is self-stabilizing and folds independently of the remainder of the antigen. Domains of antigens may be determined by Hydrophobicity/Kyte-Doolittle plots, which can identify extracellular vs. intracellular domains of proteins. Domains of antigens may also be determined using tools such as InterPro or PROSITE (https://www.ebi.ac.uk/interpro/) or protein BLAST; each of which is capable of identifying protein domains via sequence similarities shared by other proteins having similar structures and/or functions. The region of interest of the target antigen may be a 20-200, a 20-180, a 20-160, a 20-140, a 20-120, a 20-100, a 20-80, a 20-60, a 20-40, a 40-200, a 40-180, a 40-160, a 40-140, a 40-120, a 40-100, a 40-80, a 40-60, 60- 200, a 60-180, a 60-160, a 60-140, a 60-120, a 60-100, a 60-80, a 80-200, a 80-180, a 80-160, a 80-140, a 80-120, a 80-100, a 100-200, a 150-100, or a 25-175 amino acid residue peptide of the full-length fragment of the target antigen. The region of interest may be selected as it may be involved in a signaling pathway, interact with other proteins or peptides, or result in or prevent a conformational change in the antigen.

[0314] If the kit includes the target antigen, then the target antigen may be a full-length version of the polypeptide as discussed throughout the disclosure herein. The kit may be understood to accommodate any target antigen of any amino acid length, including those that are at least 20 amino acid residues, at least 40 amino acid residues, at least 60 amino acid residues, at least 80 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, at least 700 amino acids, at least 800 amino acid residues, at least 900 amino acid residues, at least 1000 amino acid residues, at least 1100 amino acid residues, at least 1200 amino acid residues, at least 1300 amino acid residues, up to 40 amino acid residues, up to 60 amino acid residues, up to 80 amino acid residues, up to 100 amino acid residues, up to 200 amino acid residues, up to 300 amino acid residues, up to 400 amino acid residues, up to 500 amino acid residues, up to 600 amino acid residues, up to 700 amino acids, up to 800 amino acid residues, up to 900 amino acid residues, up to 1000 amino acid residues, up to 1100 amino acid residues, up to 1200 amino acid residues or up to 1300 amino acid residues. The target antigen, including those referenced above, e.g., of an infectious agent, may be any polypeptide having any number of domains, e.g., one domain, at least one domain, two domains, at least two domains, three domains, at least three domains, four domains, at least four domains, five domains, at least five domains, six domains, at least six domains, seven domains, at least seven domains, eight domains, at least eight domains, nine domains, at least nine domains, ten domains, at least ten domains, at least thirty domains, at least forty domains, at least fifty domains, at least sixty domains, at least seventy domains, at least eighty domains, at least ninety domains, at least one hundred domains, at most two hundred domains, at most 175 domains, at most 150 domains, at most 125 domains, at most 100 domains, at most 75 domains, at most 50 domains, at most 25 domains, at most 20 domains, at most 15 domains, at most 10 domains, or at most 5 domains.

[0315] If the kit includes a fragment of the target antigen, then the fragment of the target antigen may be of any amino acid residue length such that it is less than the length of the target antigen. The fragment of the target antigen may have an amino acid length that is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% that of the target antigen. As another example, the fragment of the target antigen may have an amino acid sequence length that is 75% or below, 70% or below, 65% or below, 60% or below, 55% or below, 50% or below, 45% or below, 40% or below, 35% or below, 30% or below, 25% or below, 20% or below, 15% or below, 10% or below, or 5% or below that of the target antigen. The fragment of the target antigen may be 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, 20-60, 20-40, 15-20, 40-200, 40-180, 40-160, 40- 140, 40-120, 40-100, 40-80, 40-60, 60-200, 60-180, 60-160, 60-140, 60-120, 60-100, 60-80, 80-200, 80-180, 80-160, 80-140, 80-120, 80-100, 100-200, 150-100, 25-175, 25-150, 25-125, 25-100, or 25-75 amino acid residues in length, so long as its length is shorter than the length than the full-length target antigen. The fragment of the target antigen may include or may be an epitope of the target antigen known to be of importance. The fragment of the target antigen may include or may be a domain of the target antigen known to be of importance. An epitope or domain of importance of the target antigen may be an epitope or domain of the target antigen that mediates a process, e.g., affects a signaling pathway directly or by costimulation, is critical to host-pathogen interaction, or affects a conformational change. Other characteristics of fragments, fragments that may be “non-overlapping”, and amino acid substitutions for introduction in fragments have been described elsewhere herein. By way of example, a fragment of a target antigen may be a fragment of viral antigen, such as a coronavirus antigen, e.g., SARS CoV-2 spike protein. If the fragment of the target antigen is a fragment of a coronavirus antigen, e.g. , SARS Co-V-2 spike protein, it may be or include the receptor binding domain, the N-terminal binding domain, or the extracellular domain of the SARS Co-V-2 spike protein.

[0316] If the kit includes the plurality of fragments of the target antigen, the kit may include a first and a second fragment of the target antigen. The first and the second fragment of the target antigen may each be of any amino acid residue length so long as it is less than the length of the full-length target antigen. The first and the second fragment of the target antigen need not be of the same or of similar amino acid length.

[0317] The first and second fragments of the target antigen may be non-overlapping fragments. If the fragments of the target antigen are non-overlapping, the fragments may have completely distinct amino acid sequences and may be from the same different domains or regions or portions the target antigen. If the fragments of the target antigen are non overlapping fragments, the fragments may have completely distinct amino acid sequences although they are from the same domains or regions or portions the target antigen. The non overlapping fragments need not, however, have completely distinct amino acid sequences along their entire length. The non-overlapping fragments of the target antigen may include consecutive amino acid residues that are identical, e.g., at their N- or C-terminus, and consecutive amino acid residue that are completely distinct, i.e., are non-overlapping to an extent. For example, first and second non-overlapping fragments may each be 100 amino acid residues in length, of which the 20 C-terminal amino acid residues of the first and the 20 N-terminal amino acid residues of the second fragment are identical, while the 80 N-terminal amino acid residues of the first and the 80 C-terminal amino acid residues of the second fragment are distinct. It will be understood that the non-overlapping fragments, that are non overlapping to an extent, of the target antigen may include one or more of the same, but one or more different epitopes and/or domains of the target antigen.

[0318] If the kit includes the target antigen and the fragment of the target antigen, the target antigen and the fragment of the target antigen may each be coupled to a reporter oligonucleotide. If the kit includes a plurality of fragments, each of the plurality of fragments may be coupled to a reporter oligonucleotide.

[0319] If the kit includes the target antigen and the fragment of the target antigen, and the target antigen and the fragment of the target antigen are coupled to reporter oligonucleotides, then the target antigen may be coupled to a first reporter oligonucleotide and the fragment of the target antigen may be coupled to a second reporter oligonucleotide. The first reporter oligonucleotide, coupled to the target antigen, may include a first reporter sequence and a capture sequence. The first reporter sequence may be specific to the target antigen to which the first reporter oligonucleotide is coupled. The second reporter oligonucleotide, coupled to the fragment of the target antigen, may include a second reporter sequence and a capture sequence. The second reporter sequence may be specific to the fragment of the target antigen to which the second reporter oligonucleotide is coupled.

[0320] If the kit includes a plurality of fragments of the target antigen, and each of the plurality of fragments is coupled to a reporter oligonucleotide, then a first of the plurality of fragments may be coupled to a first reporter oligonucleotide and a second of the plurality of fragments may be coupled to a second reporter oligonucleotide. The first reporter oligonucleotide, coupled to the first fragment of the target antigen, may include a first reporter sequence and a capture sequence. The first reporter sequence may be specific to the fragment of the target antigen to which the first reporter oligonucleotide is coupled. The second reporter oligonucleotide, coupled to the second fragment of the target antigen, may include a second reporter sequence and a capture sequence. The second reporter sequence may be specific to the second fragment of the target antigen to which the second reporter oligonucleotide is coupled.

[0321] In any of the kits described herein, a plurality of nucleic acid barcode molecules may also be included therein. The plurality of nucleic acid barcode molecules may include a capture sequence, which may be complementary to a capture handle sequence to any of the first and/or second reporter oligonucleotides. In some aspects, one of either the reporter oligonucleotides or the plurality of nucleic acid barcode molecules may include a unique molecular identifier (UMI).

[0322] Any of the kits provided herein, whether they include: (a) the target antigen and the fragment of the target antigen; or (b) the plurality of fragments of the target antigen, may include a further fragment of the target antigen. By a further fragment of the target antigen, it may be understood that the kits include any of an additional number of fragments of the target antigen. For example, the kits may include one, at least one, two, at least two, three, at least three, four, at least four, five, at least five, six, at least six, seven, at least seven, eight, at least eight, nine, at least nine, ten, at least ten, twenty, at least twenty, thirty, at least thirty, forty, at least forty, fifty, at least fifty, sixty, at least sixty, seventy, at least seventy, eighty, at least eighty, ninety, at least ninety, 100, at least 100, 200, at least 200, 300, at least 300, 400, at least 400, 500, at least 500, 600, at least 600, 700, at least 700, 800, at least 800, 900, at least 900, 1000, at least 1000, 1500, at least 1500, 2000, at least 2000, 2500, at least 2500 additional fragments of the target antigen. The further fragment of the target antigen may be non-overlapping with all or a portion of one or more other fragment(s) in the kit. In the event that the kit further includes a further fragment of the target antigen, the further fragment of the target antigen may be coupled to a further reporter oligonucleotide. The further reporter oligonucleotide coupled to the further fragment of the target antigen may include a further reporter sequence which identifies the further fragment to which it is coupled and a capture handle sequence.

[0323] Any of the kits provided herein may further include a non-target antigen or a fragment of a non-target antigen, e.g., a peptide control. If any kit provided herein includes the non-target antigen or fragment of the non-target antigen, the non-target antigen or fragment of the non-target antigen may be coupled to a non-target reporter oligonucleotide. The non-target reporter oligonucleotide may include a non-target reporter sequence, which may be specific to the non-target antigen or fragment thereof to which the non-target reporter oligonucleotide is coupled. It will be understood that the kits may further include other control reagents or other reagents as may be needed to processing of samples.

[0324] In some embodiments, the kits may further include enzymes, aqueous or frozen solutions, primers or other reagents, e.g., labeling reagents, as may be desirable for using the kit for its intended purpose. Some of the reagents are described in the “ Further Disclosure - Partitions, Partitioning, Reagents and Processing" section, above. The various components of the kit can each be in separate containers, combined in single container, or combined in various container as appropriate.

[0325] The kit, whether it includes the (i) target antigen and the fragment of the target antigen, or (ii) plurality of fragments of the target antigen, may further include instructions for use thereof. The instructions for use may be included a package insert including information concerning the components of the kit by the user and/or informational aids. Generally, informational aids may include proper storage conditions, references, manufacturer/distributor information, compatible systems for use and intellectual property information.

[0326] The instructions for may be provided in any format, e.g., they may be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions may, alternatively, be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the instructions are not physically present in the kit, but means for obtaining the instructions from a remote source (e.g, via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0327] The kits may be included, or employed, in a system. Such a system may further include reagents for generating barcoded nucleic acid molecules, e.g., barcoded nucleic acid molecules that may be formed by complementary base pairing of (i) the capture sequence of a nucleic acid barcode of the plurality of nucleic acid barcode molecules and (ii) the capture handle sequence of the first and/or second reporter oligonucleotide, (the first and/or second reporter oligonucleotide coupled or having been coupled to the target antigen and the fragment of the target antigen, or coupled or having been coupled to each of fragments of the target antigen). Further yet, the system may include reagents for determining sequences from the generated barcoded nucleic acid molecules. Reagents for determining sequences from the generated barcoded nucleic acid molecules may include a sequencer or sequencing system. The sequencing reagents, e.g., sequencer or sequencing system, may determine sequence or sequences of barcoded nucleic acid molecules encoding an antigen binding molecule. Any system described herein may further include reagents for determining affinity of the antigen binding molecule, e.g, from a barcoded nucleic acid molecule formed by complementary base pairing of (i) the capture sequence of a nucleic acid barcode of the plurality of nucleic acid barcode molecules and (ii) the capture handle sequence of the first and/or second reporter oligonucleotide, (the first and/or second reporter oligonucleotide being or having been coupled to the target antigen and the fragment of the target antigen or being or having been coupled to each of fragments of the target antigen). Furthermore, any of the systems that include, or employ, any of the kits disclosed herein may further include an analysis engine, and/or a network. Moreover, any system including, or employing, any of the kits provided herein, may implement the methods provided in the disclosure or as set forth in the appended claims.

[0328] FIG. 22 depicts a block diagram illustrating an example of a computer system 201, in accordance with some example embodiments. Referring to FIG. 22, the computer system 201 may be configured to implement one or more of the analysis engine 2202, the sequencing platform 2204, and the client device 2206. The computer system 201 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0329] The computer system 201 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 205, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 201 also includes memory or memory location 210 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 215 (e.g., hard disk), communication interface 220 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 225, such as cache, other memory, data storage and/or electronic display adapters.

The memory 210, storage unit 215, interface 220 and peripheral devices 225 are in communication with the CPU 205 through a communication bus (solid lines), such as a motherboard. The storage unit 215 can be a data storage unit (or data repository) for storing data. The computer system 201 can be operatively coupled to a computer network (“network”) 230 with the aid of the communication interface 220. The network 230 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 230 in some cases is a telecommunication and/or data network. The network 230 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 230, in some cases with the aid of the computer system 201, can implement a peer-to-peer network, which may enable devices coupled to the computer system 201 to behave as a client or a server.

[0330] The CPU 205 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 210. The instructions can be directed to the CPU 205, which can subsequently program or otherwise configure the CPU 205 to implement methods of the present disclosure. Examples of operations performed by the CPU 205 can include fetch, decode, execute, and writeback.

[0331] The CPU 205 can be part of a circuit, such as an integrated circuit. One or more other components of the system 201 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0332] The storage unit 215 can store files, such as drivers, libraries and saved programs. The storage unit 215 can store user data, e.g., user preferences and user programs. The computer system 201 in some cases can include one or more additional data storage units that are external to the computer system 201, such as located on a remote server that is in communication with the computer system 201 through an intranet or the Internet.

[0333] The computer system 201 can communicate with one or more remote computer systems through the network 230. For instance, the computer system 201 can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android- enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 201 via the network 230.

[0334] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 201, such as, for example, on the memory 210 or electronic storage unit 215. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 205. In some cases, the code can be retrieved from the storage unit 215 and stored on the memory 210 for ready access by the processor 205. In some situations, the electronic storage unit 215 can be precluded, and machine-executable instructions are stored on memory 210.

[0335] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0336] Aspects of the systems and methods provided herein, such as the computer system 201, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non- transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0337] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform.

Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.

Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0338] The computer system 201 can include or be in communication with an electronic display 235 that comprises a user interface (EΊ) 240 for providing, for example, results of the assay, such as a summary of one or more antigen binding molecules that bind the antigen/antigen fragment(s), a summary of one or more antigens not bound by an antigen binding molecule in the composition, a site on the antigen/antigen fragment(s) that binds to the antigen binding molecule, or proposed modifications to the ntigen/antigen fragment(s)that can reduce affinity of the antigen binding fragment for the ntigen/antigen fragment(s). Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0339] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 205. The algorithm can, for example, contact an anti gen/anti gen fragment with an antigen binding molecule, isolate the antigen binding molecule, or identify the antigen binding molecule as described herein. In some embodiments, an algorithm can determine a relative dissociation constant for an antigen binding molecule such as an immune cell receptor (e.g., a B-cell receptor, and/or the like).

The algorithm can further identify, based at least on the relative dissociation constant, the antigen binding molecule as binding specifically to a target antigen/antigen fragment(s)such as, for example, a spike (S) protein of a coronavirus (CoV-S), e.g., a severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), a SARS-CoV-2, and/or a Middle East respiratory syndrome coronavirus (MERS-CoV). The algorithm using the relative dissociation constant may also be applied to detect antigen binding molecules that bind specifically to any target antigen of interest. Exemplary target antigens include, but are not limited to, an immune checkpoint molecule (e.g, CD38, PD-1, CTLA-4, TIGIT, LAG-3, VISTA, TIM-3), an influenza hemagglutinin, an HIV envelope protein, a cytokine, a viral glycoprotein, and/or the like.

[0340] Devices, systems, compositions and methods of the present disclosure may be used for various applications, such as, for example, processing a single analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein) from a single cell. For example, a biological particle (e.g., a cell or cell bead) is partitioned in a partition (e.g., droplet), and multiple analytes from the biological particle are processed for subsequent processing. The multiple analytes may be from the single cell. This may enable, for example, simultaneous proteomic, transcriptomic and genomic analysis of the cell

ANTIBODIES AND ANTIBODY FRAGMENTS

[0341] As described in greater detail below, one aspect of the present disclosure relates to anti-CoV-S antigen-binding polypeptides, such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or a region of interest thereof. Also provided, in other related aspects of the disclosure, are nucleic acids encoding the antibodies and antigen-binding fragments as disclosed herein, recombinant cells and transgenic animals engineered to produce the antibodies and antigen-binding fragments as disclosed herein, pharmaceutical compositions containing one or more of the nucleic acids, recombinant cells, and antibodies and antigen-binding fragments as disclosed herein.

Antigen-binding proteins

[0342] One aspect of the present disclosure relates to antigen-binding polypeptides, such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or a region of interest of the CoV spike protein. The region of interest of the CoV spike protein, may be or may include one or more domains of the CoV spike protein, such as the RBD, ECD or NTD domain.

[0343] An antibody may be an immunoglobulin molecule including four polypeptide chains, two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds (i.e., “full antibody molecules”), as well as multimers thereof (e.g. IgM). Exemplary antibodies may include, for example, those listed in Table 1. Each heavy chain may include a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (which is comprised of domains CHI, CH2 and CH3). Each light chain may be comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FWR). Each VH and VL includes three CDRs and four FWRs, arranged from amino-terminus to carboxy-terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. Heavy chain CDRs can also be referred to as HCDRs, and numbered as described above (e.g, HCDR1, HCDR2, and HCDR3). Likewise, light chain CDRs can be referred to as LCDRs, and numbered LCDR1, LCDR2, and LCDR3. In some embodiments of the disclosure, the FWRs of the antibodies or antigen binding fragments thereof are identical to the human germline sequences, or are naturally or artificially modified. Thus, the present disclosure provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g, anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) including HCDR and LCDR sequences of Table 1 within a variable heavy chain or light chain region of human germline immunoglobulin sequences.

[0344] In some embodiments of the disclosure, the assignment of amino acids to each domain is in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, etal. National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al. , (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al. , (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

Table 1: Exemplary antigen-binding polypeptides, e.g ., antibodies, of the disclosure.

[0345] An “antigen-binding fragment” of an antibody or antigen-binding polypeptide, as used herein, may include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments may include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) sdAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody ( e.g ., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FWR3-CDR3-FWR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein. The antigen-binding fragment may include three or more CDRs of an antibody of Table 1 (e.g, HCDR1, HCDR2 and HCDR3; or LCDR1, CDR2 and LCDR3).

[0346] An antigen-binding fragment of an antibody, in some embodiment of the disclosure, may include at least one variable domain. The variable domain can be of any size or amino acid composition and will generally include at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be situated relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0347] In some embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that can be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) V_H-C_H1; (ii) V_H-C_H2; (iii) V_H-C_H3; (iv) V_H-C_H1-C_H2; (V) V_H-C_H1-C_H2-C_H3; (vi) V_H-C_H2-C_H3; (vii) V_H- C_L; (viii) V_L-C_H1; (ix) V_L-C_H2; (X) V_L-C_H3; (xi) V_L-C_H1-C_H2; (xii) V_L-C_H1-C_H2-C_H3; (xiii) V_L-C_H2-C_H3; and (xiv) V_L-C_L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 ( e.g ., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen binding fragment of an antibody of the present disclosure may include a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). Antigen-binding proteins (e.g, antibodies and antigen-binding fragments) can be mono-specific or multi-specific (e.g, bi- specific).

[0348] In some embodiments, the binding affinity between the antibody or the antigen binding fragment thereof and the target antigen or fragment of the target antigen can be within a desired range to ensure that the antibody or the antigen binding fragment thereof remains bound to its target antigen or fragment of the target antigen. For example, the binding affinity can be within a desired range to ensure that the antibody or the antigen binding fragment thereof remains bound to the target antigen or fragment of the target antigen during various sample processing steps, such as partitioning and/or nucleic acid amplification or extension. A dissociation constant (Kd) between the antibody or the antigen binding fragment thereof and the target antigen or fragment of the target antigen to which it binds can be less than about 100 mM, 90 mM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM,

900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, or 1 pM. For example, the dissociation constant can be less than about 10 mM. In some embodiments, the antibody or the antigen binding fragment thereof has a desired off rate (koff), such that the antibody or antigen binding fragment thereof remains bound to the target antigen or antigen fragment during various sample processing steps.

[0349] In one aspect of the disclosure, provided herein are isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the antibodies or antigen binding fragments include: (a) a heavy chain complementary determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 861- 1075; (b) a HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1076-1290 and c) a HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1291-1505. The isolated antibodies or antigen-binding fragments thereof, which specifically bind to a spike (S) protein SARS-CoV-2, may alternatively include the HCDR1, HCDR2 and HCDR3 amino acid sequences as provided in any row of Table 1. In some embodiments, the isolated antibodies, or antigen-binding fragments thereof, having the HCDR1, HCDR2, and HCDR3 that include the amino acid sequences as shown in SEQ ID NOS: 861-1075; SEQ ID NOS: 1076-1290 and SEQ ID NOS: 1291-1505, respectively, may have a VH that includes an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 216-430 or a VH that includes the amino acid sequence of any of SEQ ID NOS: 216-430. In other embodiments, the isolated antibodies, or antigen-binding fragments thereof, that include the HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in any row of Table 1, may have a VH that includes an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 216-430 or have a VH including the amino acid sequence of any of SEQ ID NOS: 216-430. Any of these isolated antibodies, antibodies, or antigen-binding fragments thereof, that specifically bind to a spike (S) protein of SARS-CoV-2 and have the aforementioned VH CDR or VH domain sequences, may further include a) a light chain complementary determining region 1 (LCDR1) comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1506-1720; (b) a LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1721-1935; and (c) a LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1936-2150, or they may further include LCDR1, LCDR2 and LCDR3 amino acid sequences as provided in any row of Table 1. Any of these isolated antibodies, antibodies, or antigen-binding fragments thereof, that specifically bind to a spike (S) protein of SARS-CoV- 2 and have the aforementioned VH CDR or VH domain sequences, may further include a VL that includes an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 646-860 (while having aVL CDR1, VL CDR3 and VL CDR3 having the amino acid sequences of SEQ ID NOS: 1506-1720, SEQ ID NOS: 1721-1935 and SEQ ID NOS: 1936-2150, respectively or as provided in any row of Table 1) or a VL that includes the amino acid sequence of any of SEQ ID NOS: 646-860.

[0350] In one aspect, some embodiments of the disclosure provide isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the antibodies or antigen binding fragments include: (a) a light chain complementary determining region 1 (LCDR1) comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1506-1720; (b) a LCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1721-1935; and (c) a LCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1936-2150. The isolated antibodies or antigen-binding fragments thereof, which specifically bind to a spike (S) protein of SARS-CoV-2, may include LCDR1, LCDR2 and LCDR3 amino acid sequences as provided in any row of Table 1. In some embodiments, the isolated antibodies, or antigen binding fragments thereof, having the LCDR1, LCDR2, and LCDR3 that includes the amino acid sequences as shown in SEQ ID NOS: 1506-1720; SEQ ID NOS: 1721-1935 and SEQ ID NOS: 1936-2150, respectively, may have a VL that includes an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 646-860 or a VL that includes the amino acid sequence of any of SEQ ID NOS: 646-860. In other embodiments, the isolated antibodies, or antigen-binding fragments thereof, that have the LCDR1, LCDR2, and LCDR3 that includes the amino acid sequences as shown in any row of Table 1, may have a VL that includes an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 646-860 or a VL that includes the amino acid sequence of any of SEQ ID NOS: 646-860. Any of these isolated antibodies, antibodies, or antigen binding fragments thereof, that specifically bind to a spike (S) protein of SARS-CoV-2 and have the aforementioned VL CDR or VL domain sequences, may further include (a) a heavy chain complementary determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 861-1075; (b) a HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1076-1290 and c) a HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1291-1505. Any of these isolated antibodies, antibodies, or antigen-binding fragments thereof, that specifically bind to a spike (S) protein of SARS-CoV-2 and have the aforementioned VL CDR or VL domain sequences, may further include HCDR1, HCDR2 and HCDR3 amino acid sequences as provided in any row of Table 1. Any of these isolated antibodies, antibodies, or antigen-binding fragments thereof, that specifically bind to a spike (S) protein of SARS-CoV-2 may further include a VH that includes an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 216-430 (while having aVH CDR1, VH CDR3 and VH CDR3 having the amino acid sequences of SEQ ID NOS: 861-1075; SEQ ID NOS: 1076-1290 and SEQ ID NOS: 1291-1505, respectively, or as provided in any row of Table 1) or a VH that includes the amino acid sequence of any of SEQ ID NOS: 216-430.

[0351] In another aspect, provided herein are isolated antibodies or antigen-binding fragments thereof, that bind specifically to a spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the antibodies or antigen-binding fragments include: (a) a HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 861-1075; (b) a HCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1076-1290; c) a HCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1291-1505; (d) a LCDR1 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1506-1720; (e) a LCDR2 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1721-1935; and (f) a LCDR3 comprising an amino acid sequence is selected from the group consisting of SEQ ID NOS: 1936-2150.

[0352] In some embodiments, the antibody or antigen-binding fragment comprises: (a) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in any row of Table 1. In some embodiments, the isolated antibodies, or antigen binding fragments thereof, having the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in any row of Table 1 may further include a VH including an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 216-430 or may include a VH including the amino acid sequence of any of SEQ ID NOS: 216-430. In other embodiments, the isolated antibodies, or antigen-binding fragments thereof, having the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in any row of Table 1 may further include a VL including an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 646-860 or may include a VL including the amino acid sequence of any of SEQ ID NOS: 646-860. In yet other embodiments, the isolated antibodies, or antigen-binding fragments thereof, having the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in any row of Table 1 may further include a VH having amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 216-430 and a VL having an amino acid sequence at least 90% identical to the amino acid sequence of any of SEQ ID NOS: 646-860. In further embodiments, the isolated antibodies, or antigen-binding fragments thereof, having the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as set forth in any row of Table 1 may include a VH having the amino acid sequence of any of SEQ ID NOS: 216-430 and a VL having the amino acid sequence of any of SEQ ID NOS: 646-860. The isolated antibody, or antigen-binding fragment thereof, may include a VH and a VL having the amino acid sequence as provided in any row of Table 1.

[0353] Non-limiting exemplary embodiments of the antibodies and antigen-binding fragments thereof of the disclosure can include one or more of the following features. In some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure can include a polypeptide including an amino acid sequence that is set forth herein except for one or more ( e.g ., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, missense mutations (e.g., conservative substitutions), non-sense mutations, deletions, or insertions. For example, the present disclosure includes antigen-binding polypeptides which include an immunoglobulin light chain variant comprising an LCVR amino acid sequence set forth in Table 1 and Sequence Listing but having one or more of such mutations and/or an immunoglobulin heavy chain variant comprising an HCVR amino acid sequence set forth in Table 1 and Sequence Listing but having one or more of such mutations. As described in greater detail below, in some embodiments, an anti-CoV-S antibody or antigen-binding fragment of the disclosure can include an immunoglobulin light chain variant comprising LCDR1, LCDR2 and LCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g, conservative substitutions) and/or an immunoglobulin heavy chain variant comprising HCDR1, HCDR2 and HCDR3 wherein one or more (e.g., 1 or 2 or 3) of such CDRs has one or more of such mutations (e.g, conservative substitutions). Such substitutions can be in a CDR, framework, and/or constant region of an antibody or antigen binding fragment.

[0354] Accordingly, in some embodiments, the antibodies and antigen-binding fragments thereof of the disclosure can include one or more variant CDRs (e.g, any one or more of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3) that are set forth herein with at least 90%, e.g, at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to, e.g, the heavy chain and light chain CDRs of any row of Table 1 and Sequence Listing.

[0355] In some embodiments, the HCDR1 amino acid sequence is at least 90%, e.g. , at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 861-1075. In some embodiments, the HCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 861-1075, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 861- 1075. In some embodiments, the HCDR2 amino acid sequence is at least 90%, e.g. , at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1076-1290. In some embodiments, the HCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1076-1290, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1076-1290. In some embodiments, the HCDR3 amino acid sequence is at least 90%, e.g. , at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1291-1505. In some embodiments, the HCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1291-1505 and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the HCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1291-1505.

[0356] In some embodiments, the LCDR1 amino acid sequence is at least 90%, e.g. , at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1506-1720. In some embodiments, the LCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1506-1720, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR1 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1506-1720. In some embodiments, the LCDR2 amino acid sequence is at least 90%, e.g, at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1721-1935. In some embodiments, the LCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1721- 1935, and further wherein one, two, or three amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR2 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1721-1935. In some embodiments, the LCDR3 amino acid sequence is at least 90%, e.g, at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1936-2150. In some embodiments, the LCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOS: 1936- 2150, and further wherein one, two, three, four, or five amino acids in the amino acid sequence is substituted by a different amino acid. In some embodiments, the LCDR3 amino acid sequence is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1936-2150.

[0357] In some embodiments, an antibody or antigen-binding fragment of the disclosure includes: (a) a HCDR1 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 861-1075; (b) a HCDR2 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 1076-1290; c) a HCDR3 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 1291-1505; (d) a LCDR1 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 1506- 1720 (e) a LCDR2 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 1721-1935; and (f) a LCDR3 comprising an amino acid sequence having 100% sequence identity to SEQ ID NOS: 1936-2150.

[0358] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes three heavy chain CDRs (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein the heavy chain CDRs and light chain CDRs are independently selected from the HCDRs and LCDRs of the antibodies, are selected from antibodies that bind to the same region of interest, RBD, ECD, or NTD of the S protein. [0359] Conservatively modified variant anti-CoV-S antibodies and antigen-binding fragments thereof are also contemplated as part of the present disclosure. A “conservatively modified variant” or a “conservative substitution” refers to a variant wherein there is one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics ( e.g . charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.). Such changes can frequently be made without significantly disrupting the biological activity of the antibody or fragment. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.

[0360] Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan;

5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Exemplary conservative amino acids substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine- glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256: 1443 45.

[0361] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes a framework region having at least 90%, e.g. , at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2151-3870.

[0362] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a heavy chain framework region 1 (HFWR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2151-2365; (b) a heavy chain framework region 2 (HFWR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2366-2580; (c) a heavy chain framework region 3 (HFWR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2581-2795; and (d) a heavy chain framework region 4 (HFWR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2796-3010.

[0363] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a light chain framework region 1 (LFWR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3011-3225; (b) a light chain framework region 2 (LFWR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3226-3440; (c) a light chain framework region 3 (LFWR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3441-3655; and (d) a light chain framework region 4 (LFWR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3656-3870

[0364] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes the heavy chain framework regions HFWR1, HFWR2, HFWR3, and HFWR4 of the same antibody or antigen-binding fragment as shown in Table 2. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes the light chain framework regions LFWR1, LFWR2, LFWR3, and LFWR4 of the same antibody or antigen-binding fragment as shown in Table 2.

Table 2: Exemplary antibodies of the disclosure and corresponding framework regions.

[0365] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 90%, e.g, at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 216-430. In some embodiments, the HCVR comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 216-430. In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region (LCVR) comprising an amino acid sequence having at least 90%, e.g. , at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 646-860. In some embodiments, the LCVR comprises an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 646-860.

[0366] In some embodiments, the antibody or antigen-binding fragment of the disclosure includes: (a) a HCVR comprising an amino acid sequence having at least 90%, e.g, at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 861-1075; and b) a LCVR comprising an amino acid sequence having at least 90%, e.g ., at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 646-860. In some embodiments, the antibody or antigen-binding fragment includes: a HCVR and a LCVR which respectively are 90%, e.g, at least 91%, at least 92%, at least 93%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to the HCVR and LCVR of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment includes the HCVR and LCVR of an antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment of the disclosure is selected from Table 1.

[0367] In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a constant region. In some embodiments, the constant region is an IgA, IgD, IgE, IgG, or IgM heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment of the disclosure includes a constant region of the type IgA (e.g, IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3 and IgG4) or IgM. In some embodiments, the constant region is an IgG constant region.

[0368] In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a kappa type light chain constant region. In some embodiments, the antibody or antigen-binding fragment of the disclosure further includes a lambda type light chain constant region.

[0369] In some embodiments, the antibody or antigen-binding fragment of the disclosure is a human antibody or antigen-binding fragment. One of ordinary skill in the art will understand that the term “human” antibody includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g, a mouse cell. The human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, such as CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g, mouse) have been grafted onto human FWR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal or derived from transgenic animals expressing human antibodies.

[0370] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody, a chimeric antibody, or a hybrid antibody. The term “humanized antibody” as used herein encompasses antibodies comprising heavy and light chain variable region sequences from a non-human species ( e.g ., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Another type of humanized antibody is a FWR- grafted antibody in which human FWR sequences are introduced into non-human VH and VL sequences to replace corresponding non-human FWR sequences. In some embodiments, the antibodies or antigen-binding fragments of the disclosure include a murine antibody, phage display antibody, or nanobody / VHH containing the frameworks and/or CDRs described in this disclosure (e.g., Table 1 and Sequence Listing). As used herein, the term “chimeric antibody” encompasses antibodies having the variable domain from a first antibody and the constant domain from a second antibody, wherein the first and second antibodies are from different species. As used herein, the term “hybrid antibody” encompasses antibodies having the variable domain from a first antibody and the constant domain from a second antibody, wherein the first and second antibodies are from different animals, or wherein the variable domain, but not the constant region, is from a first animal. For example, a variable domain can be taken from an antibody isolated from a human and expressed with a fixed constant region not isolated from that antibody. Hybrid antibodies are synthetic and non-naturally occurring because the variable and constant regions they contain are not isolated from a single natural source. In some embodiments, the hybrid antibodies of the disclosure includes a light chain from a first antibody and a heavy chain from a second antibody, wherein the first and second antibodies are from different species. In some embodiments, the chimeric antibodies of the disclosure includes a non-human light chain which is combined with a heavy chain or set of heavy chain CDRs disclosed in this application.

[0371] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment (scFv), a Fab, a Fab', a Fab'-SH, a F(ab')2, or a Fv fragment.

[0372] In some embodiments, the antibody or antigen-binding fragment has a binding affinity to an epitope in a domain of the S protein of SARS-CoV-2. The epitope may be in any one or more the S protein’s NTD, RBD or ECD domains.

[0373] Generally, binding affinity can be used as a measure of the strength of a non- covalent interaction between two molecules, e.g ., an antibody or antigen-binding fragment thereof and an antigen (e.g, coronavirus S protein antigen). In some cases, binding affinity can be used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules can be quantified by determination of the equilibrium dissociation constant (K_D). In turn, K_D can be determined by measurement of the kinetics of complex formation and dissociation using, e.g, the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants k_a (or k_on) and dissociation rate constant k_d (or k_off), respectively. K_D is related to k_a and k_d through the equation K_D = k_d / k_a. The value of the dissociation constant can be determined directly by various methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al.

(1984, Byte 9: 340-362). For example, the K_D can be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432). Other standard assays to evaluate the binding ability of the antibodies and antigen-binding fragments of the present disclosure towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA. In some embodiments, the binding affinity of an antibody or an antigen-binding fragment for a target antigen (e.g, coronavirus S protein antigen) can be calculated by the Scatchard method described by Frankel et aI.,MoI.

Immunol, 16: 101-106, 1979. It will be understood that an antibody or antigen-binding fragment that “specifically binds” a target antigen (such as S protein) is an antigen-binding fragment that does not significantly bind other antigens but binds the target antigen with high affinity, e.g, with an equilibrium dissociation constant (K_D) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less. In some embodiments, the antibodies or antigen-binding fragments of the disclosure that specifically bind a target antigen, such as a CoV-S protein (e.g, SARS- CoV-2 S protein), have a binding affinity to the target antigen expressed as K_D, of at least about 10^-8 M, as measured by real-time, label free bio-layer interferometry assay, for example, at 25° C. or 37°C, e.g, an Octet® HTX biosensor, or by surface plasmon resonance, e.g. , BIACORE™, or by solution-affinity ELISA.

[0374] In some embodiments, the binding affinity of an antigen-binding molecule (e.g, antibody or antigen-binding fragment) to a target antigen (such as S protein and/or one or more of its ECD, RBD or NTD domains) can be determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen-binding molecule bound to the antigen (see, e.g, Example 6 and Table 3 below).

[0375] In some embodiments, the antibodies and antigen-binding fragments of the disclosure bind to a target antigen, such as a CoV-S protein (e.g, SARS-CoV-2 S protein), and compete for binding with another antigen-binding polypeptide (e.g, antibody or antigen binding fragment thereof) to the target antigen. Accordingly, also provided herein are antibodies or antigen-binding fragments thereof that compete for binding with an antibody disclosed herein, e.g., in Table 1.

[0376] The term “competes” as used herein, refers to an antibody or antigen-binding fragment that binds to a target antigen, and inhibits or blocks the binding of another antigen binding polypeptide (e.g, antibody or antigen-binding fragment thereof) to the target antigen. The term also includes competition between two antigen-binding polypeptides e.g, antibodies, in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice versa. In some embodiments, the first antigen-binding polypeptide (e.g, antibody or antigen-binding fragment) and second antigen-binding polypeptide (e.g, antibody or antigen-binding fragment thereof) may bind to the same epitope. Alternatively, the first and second antigen-binding polypeptides (e.g, antibodies or antigen-binding fragments) may bind to different, but, for example, overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g, via steric hindrance. Competition between antigen-binding polypeptides (e.g, antibodies or anti gen -binding fragments) may be measured by methods known in the art, for example, by a real-time, label- free bio-layer interferometry assay. Epitope mapping (e.g, via alanine scanning or hydrogen- deuterium exchange (HDX)) can be used to determine whether two or more antibodies are non-competing (e.g, on a spike protein receptor binding domain (RBD) monomer), competing for the same epitope, or competing but with diverse micro-epitopes (e.g, identified through HDX). In some embodiments, competition between a first and second anti- CoV-S antigen-binding polypeptide (e.g, antibody or antigen-binding fragment thereof) is determined by measuring the ability of an immobilized first anti-CoV-S antigen-binding polypeptide ( e.g ., antibody) (not initially complexed with CoV-S protein) to bind to soluble CoV-S protein complexed with a second anti -CoV-S antigen-binding polypeptide (e.g., antibody or antigen-binding fragment thereof). A reduction in the ability of the first anti- CoV-S antigen-binding polypeptide (e.g, antibody or antigen-binding fragment thereof) to bind to the complexed CoV-S protein, relative to uncomplexed CoV-S protein, indicates that the first and second anti -CoV-S antigen-binding polypeptides (e.g, antibodies or antigen binding fragments thereof) compete. The degree of competition can be expressed as a percentage of the reduction in binding. Such competition can be measured using a real time, label -free bio-layer interferometry assay, e.g, on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance).

[0377] In some embodiments, the antibodies and antigen-binding fragments of the disclosure have a neutralizing activity (e.g, antagonistic activity) against SARS-CoV-2, e.g, able to bind to and neutralize the activity of SARS-CoV-S, as determined by in vitro or in vivo assays. The ability of the antibodies of the disclosure to bind to, block and/or neutralize the activity of SARS-CoV-2 may be measured using any standard method known to those skilled in the art, including binding assays, or activity assays, as described herein. For example, the binding affinity and dissociation constants of anti-SARS-CoV-2 antigen-binding polypeptides for SARS-CoV-2 can be determined by surface plasmon resonance assay. Alternatively, neutralization assays were used to determine infectivity of SARS-CoV-2 S protein-containing virus-like particles. One of ordinary skill in the art will understand that a neutralizing or antagonistic CoV-S antigen-binding polypeptide, e.g, antibody or antigen binding fragment, generally refers to a molecule that inhibits an activity of CoV-S to any detectable degree, e.g, inhibits or reduces the ability of CoV-S to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into a host cell or mediate viral reproduction in a host cell. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have a neutralization activity IC50 value of less than 150 ng/ml for viral neutralization, as determined a quantitative focus reduction neutralization test (FRNT) described previously by Zost et al. (Nature, 584:443-449, 2020).

In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 150 ng/ml for blocking ACE2. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 10 ng/ml for S2P ectodomain binding. In some embodiments, the antibodies and antigen-binding fragments of the disclosure have blocking activity IC50 value of less than 10 ng/ml for RBD ectodomain binding. In some embodiments, the antibody or antigen-binding fragment neutralizes at least 50% of 200 times the tissue culture infectious dose (200xTCID50) of the coronavirus at an antibody concentration of 12.5 pg/ml or less. Here, TCID50 represents the viral load at which 50% of cells are infected when a solution containing the virus is added to cell culture. In some embodiments, neutralizing antibodies are effective at antibody concentrations of <3.125 pg/ml, <.8 pg/ml, <.2 pg/ml, or <1 pg/ml

[0378] In some embodiments, an anti-SARS-CoV-S antigen-binding polypeptide (e.g, antibody or antigen-binding fragment) described herein is not an antibody or antigen-binding fragment described in the following patent publications CN111620946A, CN111690059A, US10787501, and WO2015179535. In some embodiments, the an anti-SARS-CoV-S antigen binding polypeptide (e.g, antibody or antigen-binding fragment) described herein is not an antibody or antigen-binding fragment described in the following documents Jakob Kreye et al., 2020; Seth Zost et al., (Nature Medicine, July 10, 2020); Xiaojian Han et al., (BioRxiv, Aug 21, 2020); Tal Noy-Porat et al., (Nature Comm., Aug. 27, 2020); Edurne Rujas et al. (BioRxiv, Oct 16, 2020), 2020; Renhong Yan et al., (BioRxiv, 2020); Christoph Kreer et al., (Cell, Aug 20, 2020; Vol. 182, Issue 4, pp. 843-854); Yunlong Cao et al., (Cell, July 9, Vol. 182, Issue 1, 2020); and Thomas Rogers et al., (Science Aug 21, 2020: Vol. 369, Issue 6506, pp. 956-963).

Nucleic acids

[0379] In discussed above, one aspect of the disclosure relates to recombinant nucleic acids including a nucleic acid sequence that encodes an antibody of the disclosure or an antigen-binding fragment thereof. In some embodiments, the recombinant nucleic acids of the disclosure can be configured as expression cassettes or vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which allow in vivo expression of the receptor in a host cell.

[0380] Nucleic acid molecules of the present disclosure can be of any length, including for example, between about 1 Kb and about 50 Kb, e.g, between about 1.2 Kb and about 10 Kb, between about 2 Kb and about 15 Kb, between about 5 Kb and about 20 Kb, between about 10 Kb and about 20 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0381] Accordingly, in some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding an antibody of the disclosure or an antigen-binding fragment thereof. In some embodiments, the nucleotide sequence is incorporated into an expression cassette, a vector, or an expression vector. It will be understood by the skilled artisan that an expression cassette generally includes a construct of genetic material that contains coding sequences of the antibody or antigen-binding fragment thereof and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. Generally, the expression cassette can be inserted into a vector for targeting to a desired host cell and/or into an individual. As such, in some embodiments, an expression cassette of the disclosure include a coding sequence for an antibody of the disclosure or an antigen-binding fragment thereof, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0382] An expression cassette can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, as a linear or circular, single- stranded or double-stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g ., operably linked.

[0383] In some embodiments, the nucleic acid molecule of the disclosure is incorporated into an expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that can be used for the purpose of transformation, e.g, the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0384] In some embodiments, the expression vector can be a viral vector. As will be appreciated by one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g, a transfer plasmid) that includes virus-derived nucleic acid elements that facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will generally include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector can refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.

[0385] The nucleic acid sequences encoding the antibodies and antigen-binding fragments as disclosed herein can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon usage optimization are known in the art. Codon usages within the coding sequence of the antibodies and antigen-binding fragment disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0386] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any antibody or an antigen-binding fragment thereof as disclosed herein. The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed/transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. etal. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. etal. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications.

San Diego, CA: Academic Press; Lefkovits, T (1997). The Immunology Methods Manual:

The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. etal. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY : Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference).

[0387] DNA vectors can be introduced into cells, e.g. , eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.

[0388] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example,

Gluzman (Ed.), Eukaryotic Viral Vectors , CSH Laboratory Press, Cold Spring Harbor, N. Y.).

[0389] For example, an antibody or an antigen-binding fragment thereof as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells (e.g, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans can consult P. Jones, “Vectors: Cloning Applications”, John Wiley and Sons, New York, N.Y., 2009).

[0390] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g, antibody. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded ( e.g ., either a sense or an antisense strand).

[0391] The nucleic acid molecules are not limited to sequences that encode polypeptides (e.g., antibodies); some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g, the coding sequence of an antibody) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription. Recombinant cell and cell cultures

[0392] The nucleic acid of the present disclosure can be introduced into a host cell, such as, for example, a Chinese hamster ovary (CHO) cell, to produce a recombinant cell containing the nucleic acid molecule. Introduction of the nucleic acid molecules (e.g, DNA or RNA, including mRNA) or vectors of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery. For example, methods for introduction of heterologous nucleic acid molecules into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the nucleic acid molecule(s) in liposomes, lipid nanoparticle technology, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus. As discussed in greater detail below, in some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be introduced to a subject in nucleic acid form (e.g, DNA or RNA, including mRNA), such that the subject's own cells produce the antibody. The present disclosure further provides modifications to nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g, mRNA) stability, or improved affinity or specificity of the antibodies for the CoV spike protein.

[0393] Accordingly, in some embodiments, the nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas genome editing, or DNA- guided endonuclease genome editing with NgAgo (Nalronobacleriiim gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini circle expression vector for transient expression.

[0394] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0395] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0396] In some embodiments, host cells can be genetically engineered ( e.g. , transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0397] In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the animal cell is a non-human animal cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the recombinant cell is selected from the group consisting of a baby hamster kidney (BHK) cell, a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, a NSO murine myeloma cell, a human epidermoid larynx cell, a human fibroblast cell, a human HEK-293 cell, a human HeLa cell, a human HepG2 cell, a human HUH-7 cell, a human MRC-5 cell, a human muscle cell, a mouse 3T3 cell, a mouse connective tissue cell, a mouse muscle cell, and a rabbit kidney cell. In some embodiments, the recombinant cell is a Pichia pastoris cell or a Saccharomyces cerevisiae cell, both of which are also suitable for production of scFv, scFv- Fc, Fab, and F(ab’)2.

[0398] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0399] Also provided, in another aspect, are animals including a recombinant nucleic acid or a vector as disclosed herein. In some embodiments, the disclosure provides a transgenic animal that is a non-human animal. In some embodiments, the transgenic animal produces an antibody or antigen-binding fragment as disclosed herein.

[0400] The transgenic non-human host animals of the disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal. In some embodiments, the non-human animals of the disclosure are mice. Other animal species suitable for the compositions and methods of the disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include but are not limited to rats, rabbits, chickens, goats, pigs, sheep and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm ( e.g ., in pig), retroviral vectors (e.g, avian species), somatic cell nuclear transfer (e.g, in goats). The state of the art in the preparation of transgenic domestic farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0401] In some embodiments, the animal is a vertebrate animal or an invertebrate animal. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammalian animal is a non-human animal. In some embodiments, the transgenic animals of the disclosure can be made using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas genome editing, or DNA- guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). IN some embodiments, the transgenic animals of the disclosure can be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination.

[0402] In another aspect, provided herein are methods for producing an antibody or antigen-binding fragment thereof, wherein the methods include growing (i) a transgenic animal as disclosed herein, or (ii) a recombinant cell as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced.

[0403] In some embodiments, the methods for producing an antibody or antigen binding fragment thereof as described herein further include isolating the produced antibody or antigen-binding fragment from (i) the transgenic animal or (ii) recombinant cell and/or the medium in which the recombinant cell is cultured. In some embodiments, the mammalian animal is a non-human primate. Accordingly, the antibodies or antigen-binding fragments produced by the methods disclosed herein are also within the scope of the disclosure.

[0404] In some embodiments, antibodies and antigen-binding fragments of the present disclosure include immunoglobulin chains having the amino acid sequences set forth herein as well as cellular modifications and in vitro post-translational modifications to the antibody and antigen-binding fragment. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to CoV-S comprising heavy and/or light chain amino acid sequences set forth herein ( e.g ., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and/or LCDR3) as well as antibodies and fragments wherein one or more amino acid residues is glycosylated, one or more Asn residues is deamidated, one or more residues (e.g., Met, Trp and/or His) is oxidized, the N-terminal Gin is pyroglutamate (pyroE) and/or the C- terminal Lysine is missing.

Pharmaceutical compositions

[0405] The antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures of the disclosure can be incorporated into compositions, including pharmaceutical compositions.

[0406] In another aspect, the antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures of the disclosure can be incorporated into compositions suitable for various downstream applications, for example, pharmaceutical compositions. Exemplary compositions of the disclosure include pharmaceutical compositions which generally include one or more of the antibodies, antigen-binding fragments, nucleic acids, recombinant cells, and/or cell cultures as described herein and a pharmaceutically acceptable excipient, e.g, carrier. In some embodiments, the composition is a sterile composition. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the composition further includes an adjuvant.

[0407] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to an individual. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. The scope of the present disclosure includes desiccated, e.g, freeze-dried, compositions comprising an anti-CoV-S antigen-binding polypeptides, e.g, antibody or antigen-binding fragment thereof (e.g, of Table 1), or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into an individual or an animal (some non-limiting examples include a human, or a mammal). In some embodiments, the individual is a human.

[0408] The terms “administration” and “administering”, as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, oral, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0409] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to an individual. For example, the pharmaceutical composition can be formulated to be suitable for parenteral, intraperitoneal, colorectal, intraperitoneal, and intratumoral administration. In some embodiments, the pharmaceutical composition can be formulated for oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial administration. One of ordinary skilled in the art will appreciate that the formulation should suit the mode of administration.

[0410] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In some embodiments, the composition should be sterile and should be fluid to the extent that easy syringability exists. It can be stabilized under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0411] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0412] In some embodiments, the pharmaceutical composition of the disclosure further includes a further therapeutic agent. Non-limiting examples of further therapeutic agents include (i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. Accordingly, in some embodiments, the pharmaceutical composition of the disclosure further includes a further therapeutic agent selected from the group consisting of:

(i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds the serine protease TMPRSS2 of a target cell, and (iv) a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. In some embodiments, the further therapeutic agent is a second antibody or antigen binding fragment disclosed herein, e.g. , of Table 1. In some embodiments, one, two, three, four, or more antibodies, or antigen-binding fragments thereof, of Table 1 can be used in combination. [0413] In some embodiments, the one or more further therapeutic agents includes an antiviral drug or a vaccine. One of ordinary skill in the art will understand that the antiviral drug of the disclosure can include any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. In some embodiments, the antiviral drug includes, but is not limited to a cationic steroid antimicrobial, leupeptin, aprotinin, ribavirin, or interferon-alpha2b. Methods for treating or preventing virus (e.g, coronavirus) infection in a subject in need of said treatment or prevention by administering an antibody or antigen binding fragment of Table 1 in association with a further therapeutic agent are part of the present disclosure.

[0414] For example, in some embodiments of the disclosure, the further therapeutic agent is a vaccine, e.g. , a coronavirus vaccine. In some embodiments, a vaccine is an inactivated/killed virus vaccine, a live attenuated virus vaccine or a virus subunit vaccine.

Methods for detecting SARS-CoV-2 S protein and/or SARS-CoV-2

[0415] As discussed above, one aspect of the present disclosure relates to methods for detecting the presence of SARS-CoV-2 S protein and/or SARS-CoV-2 in a biological sample, the methods including contacting an antibody or antigen-binding fragment as disclosed herein with a biological sample from an individual infected with or suspected of being infected with SARS-CoV-2. For example, the anti- SARS-CoV-2 S antibodies and antigen-binding fragments of the present disclosure can be used to detect and/or measure SARS-CoV in a sample, e.g. , for diagnostic purposes.

[0416] In some embodiments, the methods include (i) contacting an antibody or antigen-binding fragment as disclosed herein with a biological sample from an individual infected with or suspected of being infected with SARS-CoV-2, (ii) detecting the formation of an antigen-antibody complex between the antibody or antigen-binding fragment and a SARS-CoV-2 S protein present in the biological sample. The formation of the antigen- antibody complex can be detected by one or more techniques known in the art, such as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (IF A), dot blot or western blot. In some embodiments, the formation of the antigen-antibody complex can be detected by ELISA, dot blot or western blot.

[0417] For example, the anti-CoV-S antigen-binding polypeptides, e.g. , antibodies or antigen-binding fragments thereof of the present disclosure (e.g, of Table 1), can be used to detect and/or measure CoV-S in a sample. Exemplary assays for CoV-S include, but are not limited to, contacting a biological sample with an anti-CoV-S antigen-binding polypeptide ( e.g ., antibody or antigen-binding fragment thereof) of the disclosure, wherein the anti-CoV- S antigen-binding polypeptide is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CoV-S from biological samples. The presence of an anti-CoV-S antigen-binding polypeptide complexed with CoV-S indicates the presence of CoV-S in the sample. Alternatively, an unlabeled anti-CoV-S antibody can be used in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as ¾, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, b-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CoV-S in a sample include neutralization assays, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (MA), and fluorescence-activated cell sorting (FACS). Thus, the present disclosure includes a method for detecting the presence of spike protein polypeptide in a sample comprising contacting the sample with an anti-CoV-S antigen-binding polypeptide and detecting the presence of a CoV-S/anti-CoV-S antigen-binding polypeptide wherein the presence of the complex indicates the presence of CoV-S.

[0418] In principle, there are no particular restrictions in regard to the types of biological samples suitable for use in the methods described herein. For example, samples that can be suitably used in SARS-CoV diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a subject, which contains detectable quantities of either SARS-CoV spike protein, or fragments thereof, under normal or pathological conditions. In some embodiments, the biological sample includes sputum, bronchoalveolar lavage, pleural effusion, tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, circulating tumor cells, circulating nucleic acids, bone marrow, or any combination thereof. In some embodiments, the biological sample includes cells or tissue. For example, the biological sample can be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. In some embodiments, the biological sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. In some embodiments, the biological sample can be a skin sample. In some embodiments, the biological sample can be a cheek swab. In some embodiments, the biological sample includes whole blood and blood components.

[0419] Generally, levels of SARS-CoV spike protein in a particular sample obtained from a healthy subject ( e.g ., a subject not afflicted with a disease associated with SARS- CoV) are measured to initially establish a baseline, or standard, level of SARS-CoV. This baseline level of MERS-CoV can then be compared against the levels of SARS-CoV measured in samples obtained from individuals suspected of having a SARS-CoV-associated condition, or symptoms associated with such condition.

[0420] The antibodies and antigen-binding fragments specific for SARS-CoV spike protein may contain no additional labels or moieties, or they may contain an N-terminal or C- terminal label or moiety. In some embodiments the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.

Methods of treatment

[0421] As discussed above, one aspect of the disclosure relate to methods for treating, ameliorating or preventing viral infection (e.g., reducing the likelihood of a viral infection such as coronavirus, e.g., SARS-CoV-2, infection) or a health condition associated with a viral (such as coronavirus, e.g., SARS-CoV-2) infection by administering a composition comprising therapeutically effective amount of an anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment, (e.g, of Table 1) to a subject in need of such treatment or prevention. In a related aspect, some embodiments of the disclosure relate methods for reducing binding of SARS-Co-2V S protein to and/or reducing SARS-CoV-2 entry into a cell of a subject, the method including administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment as disclosed herein. The composition may be administered subcutaneously, intravenously, and/or intramuscularly.

[0422] The treatment methods of the disclosure involve administering an anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment of the present disclosure (e.g, of Table 1), to a subject having one or more signs or symptoms of a disease or infection, e.g, viral infection, for which the antigen-binding polypeptide is effective when administered to the subject at an effective or therapeutically effective amount or dose. An effective or therapeutically effective dose of anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment (e.g, of Table 1), for treating or preventing a viral infection refers to the amount of the antibody or fragment sufficient to alleviate one or more signs and/or symptoms of the infection in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms. Health conditions and symptoms associated with SARS-CoV-2 infection include respiratory tract infections, often in the lower respiratory tract. Accordingly, some embodiments of the disclosure relate to methods of for reducing one or more signs or symptoms associated with coronavirus infection, such as high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure and renal dysfunction), septic shock, and death in severe cases. In some embodiments, a sign or symptom of a coronavirus infection in a subject is survival or proliferation of virus in the body of the subject, e.g ., as determined by viral titer assay (e.g. , coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay). Other signs and symptoms of viral infection include, but are not limited to fever or feeling feverish/chills, cough, sore throat, runny or stuffy nose, sneezing, muscle or body aches, headaches, fatigue (tiredness), vomiting, diarrhea, respiratory tract infection, chest discomfort, shortness of breath, bronchitis, and pneumonia.

[0423] The present disclosure also encompasses prophylactically administering an anti- CoV-S antigen-binding polypeptide, e.g. , antibody or antigen-binding fragment thereof of the present disclosure (e.g, of Table 1), to a subject who is at risk of viral infection so as to prevent such infection (e.g, reducing the likelihood of a viral infection). Passive antibody- based immunoprophylaxis has proven an effective strategy for preventing subject from viral infection. The preventive methods of the disclosure involve administering a composition comprising an anti-CoV-S antigen-binding polypeptide, e.g, antibody or antigen-binding fragment of the present disclosure (e.g, of Table 1), to a subject to inhibit the manifestation of a disease or infection (e.g, viral infection) in the body of a subject, for which the antigen binding polypeptide is effective when administered to the subject at an effective or therapeutically effective amount or dose.

[0424] The dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. In some embodiments, an effective or therapeutically effective dose of antibody or antigen-binding fragment thereof of the present disclosure, for treating or preventing viral infection, e.g, in an adult human subject, is about 0.01 to about 200 mg/kg, e.g, up to about 150 mg/kg. In some embodiments, the dosage is up to about 10.8 or 11 grams (e.g, about 1, 2, 3, 4, 5, 6, 7, 8, 9,

10 or 11 grams). Depending on the severity of the infection, the frequency and the duration of the treatment can be adjusted. In some embodiments, the antigen-binding polypeptide of the present disclosure can be administered at an initial dose, followed by one or more secondary doses. In some embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0425] In some embodiments, the administered composition reduces the likelihood of a coronavirus infection by at least 50%, e.g, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a subject that has not been administered with the composition.

[0426] In some embodiments, the administered composition reduces binding of SARS- Co-2V S protein to and/or reducing SARS-CoV-2 entry into a cell of a subject by at least 50%, e.g, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a subject that has not been administered with the composition.

[0427] In a related aspect, some embodiments of the disclosure relate methods for reducing binding of SARS-Co-2V S protein to a cell of a subject and/or reducing SARS- CoV-2 entry into a cell of a subject, the method including administering to the subject a composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment as disclosed herein.

[0428] Non-limiting exemplary embodiments of the methods as described herein can include one or more of the following features. In some embodiments, the antibody or antigen binding fragment is administered in combination with a SARS-Co-2V S protein conjugated to a therapeutic agent. In some embodiments, the subject is administered one or more further therapeutic agents.

[0429] In some embodiments, the one or more further therapeutic agents is selected from the group consisting of: (i) an antiviral agent, (ii) an anti-inflammatory agent, (iii) an antibody or antigen-binding fragment thereof that specifically binds TMPRSS2, and (iv) an antibody or antigen-binding fragment thereof that specifically binds to CoV-S protein. In some embodiments, the further therapeutic agent is a second antibody or antigen-binding fragment disclosed herein, e.g, of Table 1. In some embodiments, one, two, three, four, or more antibodies, or antigen-binding fragments thereof, of Table 1 can be administered in combination ( e.g ., concurrently or sequentially).

[0430] In some embodiments, the one or more further therapeutic agents includes an antiviral drug or a vaccine. One of ordinary skill in the art will understand that the antiviral drug of the disclosure can include any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. In some embodiments, the antiviral drug includes, but is not limited to a cationic steroid antimicrobial, leupeptin, aprotinin, ribavirin, or interferon-alpha2b. Methods for treating or preventing virus (e.g., coronavirus) infection in a subject in need of said treatment or prevention by administering an antibody or antigen binding fragment of Table 1 in association with a further therapeutic agent are part of the present disclosure.

[0431] For example, in some embodiments of the disclosure, the further therapeutic agent is a vaccine, e.g, a coronavirus vaccine. In some embodiments, a vaccine is an inactivated/killed virus vaccine, a live attenuated virus vaccine or a virus subunit vaccine.

[0432] In some embodiments, the therapeutic composition is formulated to be compatible with its intended route of administration. For example, the antibodies and antigen binding fragments of the disclosure may be given orally or by inhalation, but it is more likely that they will be administered through a parenteral route. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), and transmucosal administration. In some embodiments, the antibody or antigen binding fragment is administered to the subject subcutaneously, intravenously, and/or intramuscularly.

[0433] Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and/or di basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g, to a pH of about 7.2- 7.8, e.g, 7.5). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0434] Dosage, toxicity and therapeutic efficacy of such subject antibodies and antigen- binding fragments of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are generally suitable. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0435] For example, the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 {e-g·, the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Combination therapies

[0436] As discussed above, according to some specific examples, the methods of the disclosure include administering to a subject one or more additional therapies in combination with an anti-CoV-S protein antibody or antigen-binding fragment as disclosed herein.

[0437] Administration “in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order. Accordingly, the additional therapies can be administered before, after, or at the same time as a pharmaceutical composition comprising an anti-CoV-S protein antibody or antigen-binding fragment of the disclosure. Administration “in combination with” also includes the continuous or simultaneous administration of an anti-CoV-S antibody or antigen-binding fragment and a second therapy.

[0438] For example, when the pharmaceutical composition containing the anti-CoV-S antibody or antigen-binding fragment of the disclosure is administered “before”, the additional therapy can be administered for about 72 hours, about 60 hours, or about 48 hours, to the pharmaceutical composition containing the anti-CoV-S antibody or antigen-binding fragment thereof. About 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before. When the pharmaceutical composition containing the anti-CoV-S antibody or antigen-binding fragment is administered “after”, the additional therapy can be administered for about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after.

[0439] The combination therapy may include an anti-CoV-S antibody or antigen binding fragment of the disclosure and any additional therapeutic agent that can be advantageously combined with an anti-CoV-S antibody or antigen-binding fragment of the disclosure.

[0440] For example, a second or third therapeutic agent may be used to help reduce viral load in the lungs, such as an antiviral agent ( e.g ., ribavirin). Antibodies can also be used in combination with other therapies as described above, including vaccines specific to CoV, secondary antibodies specific to CoV, antiviral agents, anti-inflammatory agents, antibodies specifically binds the serine protease TMPRSS2 of a target cell, and additional antibodies or antigen-binding fragment thereof that specifically bind to CoV-S protein.

[0441] As noted above, in some embodiments of the disclosure, the subject may be a non-human animal, and the antigen-binding polypeptides (e.g., antibodies and antigen binding fragments) discussed herein may be used in a veterinary context to treat and/or prevent disease associated with coronavirus in the non-human animals (e.g, cats, dogs, pigs, cows, horses, goats, rabbits, sheep, etc.).

[0442] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0443] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0444] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0445] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

EXAMPLES

EXAMPLE 1 Biological samples

[0446] Sample procurement. The experiments described in the below Examples were performed with peripheral blood mononuclear cells (PBMCs) collected from convalescent human survivors of natural infection with SARS-CoV2. Specifically, Donor 531 PBMCs were purchased from Cellero (~112m/vial product, Cat. # 1146-4785JY20) and used in these experiments.

[0447] Sample background/timeline. The donor tested positive via nasopharyngeal swab while presenting asymptomatic/presymptomatic on Day 0. Hospitalization was not required. The donor tested negative for SARS-2 on Day 23. Plasma and apheresis sample collection were performed on Day 104. The donor is also seropositive for cytomegalovirus, a ubiquitous human herpesvirus.

EXAMPLE 2 Enrichment of B cells

[0448] A vial of approximately 100-120 million frozen peripheral blood mononuclear cells (PBMCs) was thawed for 1-2 min in a water bath, then transferred into 8-10 mL of 10% Fetal Bovine Serum (FBS) in Phosphate buffered saline (PBS), and centrifuged for 5 min at 350g. The cell pellet was washed three times by resuspending in 0.04% Bovine Serum Albumin (BSA) in PBS and centrifuging at room temperature (RT) at 350g for 5 min each wash, with the final pellet resuspended to a concentration of ~20 million cells per mL in a total volume of 5 mL (-100 million cells total). B cells were enriched using the B Cell Isolation Kit II (human; MACS™ Miltenyi) according to manufacturer’s instructions, and approximately 50 million cells were applied to each of two LS columns designed for positive selection of cells. The effluent was concentrated and prepared for cell labeling.

EXAMPLE 3

Antigen sourcing, preparation, and conjugation

[0449] Biotinylated antigens were sourced from suppliers as follows:

[0450] 1) Biotinylated trimerized S (SARS-2) was sourced from ACRO Biosystems, catalog # SPN-C82E9-25 (https://www.acrobiosystems.com/P3345-Biotinylated-SARS-CoV- 2-S-protein-HisAvitag™-Superstable-trimer-%28MALS-verified%29.html). This protein carries a polyhistidine tag at the C-terminus, followed by an Avi tag. Biotinylation of this product is performed using Avitag™ technology. Briefly, the single lysine residue in the Avitag is enzymatically labeled with biotin.

[0451] 2) Biotinylated trimerized S D614G (SARS-2), from ACRO Biosystems, catalog # SPN-C82E3-25 (https://www.acrobiosystems.com/P3431-Biotinylated-SARS-CoV- 2-S-protein-%28D614G%29-HisAvitag™-Super-stable-trimer-%28MALS- verified%29.html). This protein contains D614G mutation, which has become increasingly common in SARS-CoV-2 viruses from around the world. This protein also carries a polyhistidine tag at the C-terminus, followed by an Avi tag. Biotinylation of this product is performed using Avitag™ technology. Briefly, the single lysine residue in the Avitag is enzymatically labeled with biotin.

[0452] 3) Biotinylated Human Serum Albumin (HSA) HSA-H82E3, from Sapphire, catalog # (https: // sapphireusa.com/productj sp?q=ns%3ANS0000368507).

[0453] 4) Biotinylated-SARS-CoV-2 (2019-nCoV) Spike SI NTD-His & AVI recombinant protein, was supplied by Sino Biological (https: // www.sinobiologicalcdn.com/reagent/40591-V49H-B.pdf).

[0454] 5) Biotinylated SARS-CoV-2 (2019-nCoV) Spike S2 ECD-His Recombinant

Protein was supplied by Sino Biological (https: // www.sinobiologicalcdn.com/reagent/40590-V08B-B. pdf).

[0455] 6) Biotinylated SARS-CoV-2 (2019-nCoV) Spike RBD-AVI and His recombinant protein, (Lot #MB14MY1292) was supplied by Sino Biological (https://www.sinobiologicalcdn.com/reagent/40592-V27H-B.pdf).

[0456] For biotinylated antigens requiring resuspension, each was solubilized per manufacturer’s instructions. In each case they were thawed and dissolved in sterile deionized water for 30-60 minutes at room temperature with occasional gentle mixing for a final concentration of 100 microgram/mL (for HSA) or 200 microgram/mL for both of the trimerized S antigens.

[0457] Solubilized antigens were each conjugated to one of the following TotalSeqC reagents, supplied by BioLegend, which each contain a unique barcoded DNA oligonucleotide supplied by the vendor as follows:

[0458] 1) TotalSeq-C0951 PE Streptavidin was conjugated to biotinylated SARS-

CoV-2 trimerized S protein.

[0459] 2) TotalSeq-C0956 APC Streptavidin was conjugated to biotinylated SARS-

CoV2 trimerized S protein (D614G).

[0460] 3) TotalSeq-C0952 PE Streptavidin was conjugated to biotinylated human serum albumin.

[0461] 4) TotalSeq-C0953 PE Streptavidin was conjugated to biotinylated SARS-

CoV-2 (2019-nCoV) Spike SI NTD-His and AVI.

[0462] 5) TotalSeq-C0958 APC Streptavidin was conjugated to biotinylated SARS-

CoV-2 (2019nCoV) Spike S2 ECD-His recombinant protein.

[0463] 6) TotalSeq-C0954 PE Streptavidin was conjugated to biotinylated SARS-

CoV-2 (2019-nCoV) Spike RBD-AVI and His recombinant proteins.

[0464] 7) TotalSeq-C0957 APC Streptavidin was conjugated to biotinylated human serum albumin.

[0465] Briefly, each Total Seq-C barcoded streptavidin PE or APC reagent was diluted to 0.1 mg/mL and then mixed with biotinylated antigen at a 5X molar excess of antigen to streptavidin, based on a fixed amount of 0.5 pg PE-SA. One fifth of the streptavidin-oligo PE or APC conjugate was added to the antigen every 20 minutes at 4°C. The reaction was then quenched with 5 pi 4mM biotin (Pierce, Thermo Fisher) for 30 minutes for a total probe volume of 20 pL. The final conjugated antigen probes were then centrifuged for 5 minutes at 5000g at 4°C and used immediately for cell labeling at a dilution of 1 : 100 as outlined in Example 4.

EXAMPLE 4 Cell labeling

[0466] This Example describes experiments performed to stain B cells with a number of barcoded antibodies and conjugated antigens. In these experiments, approximately 3.0-4.0 million enriched B cells were first resuspended in 50 pL labeling buffer (1% BSA in PBS) and then Fc blocked for 10 minutes on ice using 5 mΐ. Human TruStain FcX (BioLegend).

[0467] Next, cells were stained with the following cocktail of antibodies, antigens and dyes:

[0468] 1) B cells labeling

[0469] -CD 19 PE-Cy7 FITC* (clone SJ25C1, BD Pharmingen) for verification of CD 19+ cells by FACS.

[0470] -Barcoded Antibodies for lOx Single Cell Immune profiling, which included the following TotalSeq-C oligo barcoded antibodies:

[0471] — Total Seq-C0389 anti-human CD38.

[0472] — ' Total Seq-CO 154 anti-human CD27.

[0473] —Total Seq-CO 189 anti-human CD24.

[0474] — Total Seq-C0384 anti-human IgD.

[0475] — Total Seq-CO 100 anti-human CD20.

[0476] — TotalSeq-C0050 anti-human CD19 (clone HIB19, to distinguish it from the flow clone).

[0477] — Total Seq-C0049 anti-human CD3E.

[0478] — Total Seq-C0045 anti-human CD4.

[0479] — Total Seq-C0046 anti-human CD8A.

[0480] — Total Seq-C0051 anti-human CD14.

[0481] — Total Seq-C0083 anti-human CD16.

[0482] — Total Seq-C0090 mouse IgGl K isotype control.

[0483] — Total Seq-C0091 mouse IgG2a K isotype control.

[0484] — Total Seq-C0092 mouse IgG2b K isotype control.

[0485] Final conjugated antigens:

[0486] — ' Total Seq-C0951 PE trimerized S (SARS-2).

[0487] — Total Seq-C0952 PE Human Serum Albumin.

[0488] — TotalSeq-C0956 APC trimerized S D614G (SARS-2).

[0489] — TotalSeq-C0957 APC Human Serum Albumin.

[0490] — TotalSeq-C0953 PE SI NTD

[0491] — TotalSeq-C0958 APC S2 ECD

[0492] — ' Total Seq-C0954 PE Spike RBD

[0493] 7AAD for live/dead cell discrimination.

[0494] Cells were stained in labeling buffer (1% BSA in PBS) in the dark for 30 minutes on ice, then cells were washed 3 times with 2 mL of cold labeling buffer at 350*g for 5 minutes at 4°C, resuspended in cold labeling buffer and a 1:200 addition of live/dead cell discriminating agent 7AAD for 10 minutes on ice in the dark, then washed one more time with labeling buffer at 350*g for 5 minutes at 4C, then resuspended in labeling buffer and loaded into a Sony MA900 Cell Sorter using a 70 microM sorting chip.

EXAMPLE 5

Antigen-specific enrichment via FACS

[0495] Sorting was performed on a Sony MA900 cell sorter and that software was used to set determine compensation settings using a combination of unlabeled, and single color controls. Cells were then gated on being single, live (7AAD-negative) and sorted based on their PE and/or APC status directly into a mixture of master mix and water based on one of four criteria:

[0496] 1) PE+, representing some combination of trimerized S (SARS-2) wt+Sl

NTD+RBD antigen+ and/or HSA+ control cells (see FIG. 1 at Quadrant (Q)l; 6733 events/cells);

[0497] 2) APC+, representing trimerized S D614G (SARS-2), S2 ECD and/or HSA control antigen+ cells (see FIG. 1 at Q3; 6652 events/cells);

[0498] 3) Dual PE+ and APC+, representing a combination of dual trimerized S

(SARS-2) antigen+, trimerized S D614G (SARS-2), SI (NTD), S2 (ECD), RBD+ and/or HSA control antigen-positive cells (FIG. 1 at Q2; 11,900 events/cells);

[0499] 4) PE and APC negative cells, representing cells that are not binding any antigen, or at a level below the thresholding/gating/detection we set on FACS (see FIG. 1 at Q4; 6200 events/cells).

[0500] In FIG. 1, the Y axis represents PE (some combination of trimerized S (SARS-2) wt+Sl NTD+RBD antigen+ and/or HSA+ control cells) signal. The X axis represents APC trimerized S D614G (SARS-2), S2 ECD and/or HSA control antigen+ cells. The numbers beneath each Quadrant represent the fraction of events of the parent population (single, live, CD19+ cells) for that Quadrant. FACS data were analyzed with FlowJo.

[0501] The events/cells were sorted into a mixture of enzyme, buffer, primer and water for loading the lOx 5Ύ2 Single Cell Immune Profiling V2 kit. Standard gene expression, V(D)J, and barcoded antigen libraries were constructed using the 10x 5’ V2 Single Cell Immune Profiling kit per manufacturer's instructions. Additional information in this regard can be found at “support.10xgenomics.com/permalink/getting-started-immune- profiling-feature-barcoding.” The system can typically capture 65% of cells loaded in system.

EXAMPLE 6 Sequencing analysis

[0502] The libraries resulting from the experiments described in Example 5 above were sequenced on a NovaSeq 3 using a NovaSeq S4200 cycles 2020 vl.5 kit, targeting using read 28, 10, 10, and 90 cycles targeting 20,000, 30,000, or 6000 reads per cell for gene expression, barcoded antigen, or Ig libraries, respectively.

[0503] Data was analyzed using lOx Genomics “Enel one” (available at https://bit.ly/enclone), which is a computational tool developed for clonal grouping to study the adaptive immune system. In this computational tool, the lOx Genomics Chromium Single Cell V(D)J data containing B cell receptor (BCR) and T cell receptor (TCR) RNA sequences are provided as input data to Enclone. Based on the input, Enclone finds and organizes cells arising from the same progenitors into groups ( e.g ., clonotype families) and compactly displays each clonotype along with its salient features, including mutated amino acids. Antibodies in the dataset were classified via a process termed “barcode-enabled antigen mapping by sequencing” (BEAM-seq).

[0504] In a dataset where there is considerable enrichment for genuine antigen binding cells, the binding affinity of an antigen-binding molecule (e.g., antibody or antigen binding fragment) to a target antigen (such as S protein or a fragment thereof) were determined based on a quantity/number of unique molecular identifiers (UMIs) associated with the antigen bound to each cell.

[0505] Over 4000 antibodies were identified that had binding affinity for one or more of the spike antigens or antigen fragments listed in Example 3.

Table 3: Binding affinity of exemplary antibodies. The integer values displayed in the table below represent median antigen UMI counts for each of the individual on-target (Wildtype Spike (S), Mutant S, S N-terminal binding domain, S receptor binding domain, and S extracellular domain) and off-target (human serum albumin/HSA 1, HSA 2) antigens, as well as exemplary antibody clonotype and isotype. Antigen specificities are assigned based on clonotype and not based on the individual antibody Ag UMI information.

[0506] Graphic representations of the binding affinity of antibodies in the serum sample to spike protein, spike protein RBD, spike protein NTD and spike protein ECD domains is provided in Figure 10A-10F and Figure 11.

[0507] Figure 10 is a dimensional reduction of the antigen count, indicative of binding affinity, data provided as geometric mean UMIs, available for antibodies in the donor sample described in Example 1. Each point at a particular location in panels A-F represents an exact clonotype and is representative of data of one or more cells bearing that antibody. Coloring of the points in panels A-F indicates geometric mean antigen UMI counts for the antigen named in the panel, which is indicative of binding affinity of the antibody for the antigen. Panel A provides geometric mean UMIs for spike protein. Panel B provides geometric mean UMIs for the spike protein NTD fragment. Panel C provides geometric mean UMIs for the spike protein RBD fragment. Panel D provides geometric mean UMIs for the spike protein ECD fragment. Panel E provides geometric mean UMIs for control antigen HSA. Panel F shows geometric mean UMIs associated with antibody expression, z.e., antibody expression by a cell bearing the antibody.

[0508] FIG. 11 is a visualization display of mapped binding affinities of the spike protein antibodies from the patient sample described in Example 1, according to their affinity for one or more spike protein antigens or antigen fragments (one or more of full-length spike protein, spike protein RBD fragment, spike protein NTD fragment, spike protein ECD fragment). Each small circle, or dot, represents an antigen-specific B cell expressing a single antibody. Each small circle, or dot, is color coded according to Ig isotype or subisotype.

EXAMPLE 7

Testing - preparation and identification of antibodies in mouse models

[0509] Animals: BALB/c mice were immunized on DO with 50 pg of SARS-CoV-2- S protein (His Tag, Super stable trimer: Aero Biosystems, Cat. #: SPN-C52H9). They received a booster immunization with 25 pg of the S protein on D14, D28, D42, and a final boost (50 pg) on D51. Samples (plasma, lymph nodes, spleen, and femur and tibia) were taken from the mice on D56.

[0510] Sample preparation:

[0511] Splenocytes : briefly, samples were filtered through a 70 pm filter, washed with cold buffer (e.g., PBS + 10% serum), centrifuged (e.g., at 300 g for 5 minutes), and lysed with ACK lysis buffer and then washed prior to cell counting.

[0512] Lymphocytes : lymphocytes were obtained from femur/tibia samples as follows: samples were flushed with cold PBS + 10% serum by a 23G needle syringe. The sample was then centrifuged (e.g., at 300 g for 5 minutes), then washed with cold buffer (e.g., PBS + 10% serum, filtered through a 70 pm filter prior to cell counting.

[0513] Bone marrow briefly, bone marrow samples were filtered through a 70 pm filter, washed with cold buffer (e.g., PBS + 10% serum), centrifuged (e.g., at 300 g for 5 minutes), and lysed with ACK lysis buffer and then washed prior to cell counting.

[0514] Antigen sourcing preparation and conjugation: Biotinylated antigens were sourced from suppliers and conjugated to TotalSeqC reagents as follows:

[0515] Cell labelling: Cells were subjected to Fc block, and then stained with the above antigens and additional barcoded antibodies for lOx Single Cell immune profiling, as described in Example 4 above.

[0516] Antigen-specific enrichment via FACS: Cells were initially gated on being single, live (7AADnegative) and PE-Cy7-CD19+, then sorted based on PE status into master mix and water. Standard gene expression, V(D)J, and barcoded antigen libraries were constructed using the lOx 5Ύ2 Single Cell Immune Profiling kit per manufacturer's instructions.

[0517] Sequencing and analysis: The libraries were sequenced as described herein (see Example 6). PE positive B cells were detected as being associated with the D614G reporter barcode sequence 0995. PE positive B cells were detected as being associated with the HSA reporter barcode sequence 0955. PE positive B cells were also detected as being associated with the NTD reporter barcode sequence 0961.

[0518] Over 700 antibodies were identified that had binding affinity for Spike antigen or Spike antigen NTD. Data from exemplary antibodies, representative of exemplary clonotypes, is provided in Table 4, below.

Table 4: Binding affinity of exemplary antibodies, representative of exemplary clonotypes. The integer values displayed in the table below represent median antigen UMI counts for each of the individual on-target (Wildtype Spike (S), Spike N-terminal binding domain (S- NTD)) and off-target (human serum albumin/HSA) antigens, as well as antibody isotype and source tissue (splenocyte, lymph node) of exemplary clonotypes.

[0519] These results indicate that the BEAM-seq workflows disclosed herein can also advantageously discover target-specific antibodies, including target domain specific antibodies to particular target domains, using a variety of mouse samples.

[0520] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herpein presented.

Claims

1. A method comprising:

(a) partitioning a reaction mixture into a plurality of partitions, wherein the reaction mixture comprises:

(i) a plurality of cells expressing antibodies or anti gen -binding fragments thereof,

(ii) a target antigen, and

(iii) a fragment of the target antigen; wherein the target antigen is coupled to a first reporter oligonucleotide and the fragment of the target antigen is coupled to a second reporter oligonucleotide, wherein the reaction mixture comprises a cell bound to the target antigen, the fragment of the target antigen, or both the target antigen and the fragment of the target antigen, wherein the partitioning provides a partition comprising:

(i) the partitioned cell bound to the target antigen, the fragment of the target antigen or both the target antigen and the fragment of the target antigen, and

(ii) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode sequence, and

(b) generating barcoded nucleic acid molecules, wherein the barcoded nucleic acid molecules comprise:

(i) a first barcoded nucleic acid molecule comprising a sequence of the first or second reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof, and

(ii) a second barcoded nucleic acid molecule comprising a nucleic acid sequence encoding the antibody, or antigen-binding fragment thereof, expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

2. The method of claim 1, wherein the method is for: identifying or characterizing an antibody, or antigen-binding fragment thereof, identifying an antibody, or antigen-binding fragment thereof, having binding affinity to a region of interest of a target antigen, or mapping binding affinity of an antibody, or an antigen-binding fragment thereof, to a region of interest of a target antigen.

3. The method of any preceding claim, wherein the barcoded nucleic molecules are generated in the partition.

4. A method comprising:

(i) a plurality of cells expressing antibodies or antigen-binding fragments thereof, and

(ii) a plurality of non-overlapping fragments of a target antigen; and wherein a first fragment of the non-overlapping fragments of the target antigen is coupled to a first reporter oligonucleotide and a second fragment of the non-overlapping fragments of the target antigen is coupled to a second reporter oligonucleotide, wherein the reaction mixture comprises a cell bound to the first fragment, wherein the partitioning provides a partition comprising:

(i) the partitioned cell bound to the first fragment, and

(i) a first barcoded nucleic acid molecule comprising a sequence of the first reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof, and

(ii) a second barcoded nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof expressed by the cell or reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

5. The method of claim 4, wherein the method is for: identifying or characterizing an antibody, or antigen-binding fragment thereof, identifying an antibody, or antigen-binding fragment thereof, having binding affinity to a region of interest of a target antigen, or mapping binding affinity of an antibody, or an antigen-binding fragment thereof, to a region of interest of a target antigen.

6. The method of claim 4 or 5, wherein the barcoded nucleic molecules are generated in the partition.

7. The method of any of claims 1-3, wherein the partitioned cell is bound to the target antigen.

8. The method of any of claims 1-3, wherein the partitioned cell is bound to the fragment of the target antigen.

9. The method of any of claims 1-3, wherein the partitioned cell is bound to the target antigen and the fragment of the target antigen.

10. The method of any of claims 4-6, wherein the partitioned cell is bound to the first and the second fragment of the target antigen.

11. The method of any preceding claim, wherein the first and the second reporter oligonucleotide comprise: (i) a first and a second reporter barcode sequence, respectively and (ii) a capture handle sequence.

12. The method of claim 11, wherein a first nucleic acid barcode molecule of the plurality of barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence and wherein a second nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to an mRNA or DNA analyte.

13. The method of claim 12, wherein the capture handle sequence is configured to couple to the capture sequence by complementary base pairing.

14. The method of claim 12 or 13, wherein the capture sequence configured to couple to the mRNA or DNA analyte is configured to couple to the mRNA analyte, and wherein the capture sequence configured to couple to the mRNA analyte comprises a polyT sequence.

15. The method of claim 11, wherein a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further comprises a capture sequence configured to couple to non-templated nucleotides appended to a cDNA reverse transcribed from an mRNA analyte by a reverse transcriptase comprising terminal transferase activity.

16. The method of claim 15, wherein the non-templated nucleotides comprise a cytosine.

17. The method of claim 16, wherein the capture sequence configured to couple to the cDNA comprises a guanine.

18. The method of claim 17, wherein coupling of the capture sequence to the non-templated cytosine permits reverse transcription of the cDNA to extend into the nucleic acid barcode molecule.

19. The method of any claims 1-8 or any of claims 11-18 as they depend from claims 1-8, further comprising determining sequences of the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule.

20. The method of claim 19, further comprising identifying the antibody or antigen binding fragment thereof based on the determined sequence of the second barcoded nucleic acid molecule.

21. The method of claim 19 or claim 20, further comprising assessing the affinity of the antibody or antigen binding fragment thereof based on the determined sequence of the first barcoded nucleic acid molecule.

22. The method of claim 21, further comprising: identifying or characterizing the antibody or antigen-bind fragment thereof as comprising: the characteristic of binding a region of interest of the target antigen, or as having binding affinity to the region of interest to the target antigen, or as having its binding affinity mapped to the region of interest to the target antigen based on the assessed affinity of the antibody or antigen binding fragment thereof.

23. The method of claim 9 or 10, or any of claims 11-18 as they depend from claim 9 or 10, wherein the first barcoded nucleic acid molecule generated comprises a sequence of the first reporter oligonucleotide or reverse complement thereof.

24. The method of claim 23, further comprising at (b): generating a third barcoded nucleic acid molecule, wherein the third barcoded nucleic acid molecule comprises a sequence of the second reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

25. The method of claim 24, further comprising determining sequences of the (i) second barcoded nucleic acid molecule and the (ii) first and/or third barcoded nucleic acid molecule.

26. The method of claim 25, further comprising identifying the antibody or antigen binding fragment thereof based on the determined sequence of the second barcoded nucleic acid molecule.

27. The method of any of claims 25-26, further comprising assessing the affinity of the antibody or antigen binding fragment thereof based on the determined sequence of the first and/or the third barcoded nucleic acid molecule.

28. The method of claim 27, further comprising: identifying or characterizing the antibody or antigen-bind fragment thereof as comprising: the characteristic of binding a region of interest of the target antigen, or as having binding affinity to the region of interest to the target antigen, or as having its binding affinity mapped to the region of interest to the target antigen based on the assessed affinity of the antibody or antigen binding fragment thereof..

29. The method of any preceding claim, further comprising, prior to the (a) partitioning, isolating and/or enriching the plurality of cells.

30. The method of claim 29, wherein the enriching the plurality of cells comprises sorting cells of the plurality of cells according to their binding to: (i) the target antigen and/or the fragment of target antigen as recited in claims 1-3 and claims as they depend therefrom; or (ii) the first and/or second fragments of the target antigen as recited in claims 4-6 and claims as they depend therefrom.

31. The method of any preceding claim, wherein the first and/or second reporter oligonucleotides are conjugated to labelling agents.

32. The method of claim 31, wherein the labelling agents are magnetic or fluorescent.

33. The method of claim 32, wherein the labelling agents comprise a fluorescent label.

34. The method of any preceding claim, wherein the reaction mixture further comprises a second cell bound to the (i) target antigen and/or the fragment of the target antigen as recited in claims 1-3 and claims as they depend therefrom; or (ii) the first and/or second fragments of the target antigen as recited in claims 4-6 and claims as they depend therefrom.

35. The method of claim 34, wherein the partitioning further provides the second cell in a second partition.

36. The method of any of claims 1-3, wherein the partitioned cell is bound to the target antigen, the fragment of the target antigen, and a further fragment of the target antigen.

37. The method of any of claims 4-6, wherein the partitioned cell is bound to the first, the second, and a further fragment of the target antigen.

38. The method of claim 36 or 37, wherein the further fragment of the target antigen is bound to a further reporter oligonucleotide.

39. The method of claim 38, wherein the first, the second reporter and the further oligonucleotide comprise: (i) a first, a second, and a further reporter sequence, respectively, and (ii) a capture handle sequence.

40. The method of claim 39, wherein a first nucleic acid barcode molecule of the plurality of barcode molecules comprises a capture sequence configured to couple to the capture handle sequence and wherein a second nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to an mRNA or a DNA analyte.

41. The method of claim 40, wherein the capture handle sequence is configured to couple to the capture sequence by complementary base pairing.

42. The method of claim 40 or 41, wherein the capture sequence configured to couple to the mRNA or DNA analyte is configured to couple to the mRNA analyte, and wherein the capture sequence configured to couple to the mRNA analyte comprises a polyT sequence.

43. The method of claim 39, wherein a first nucleic acid barcode molecule of the plurality of barcode molecules comprises a capture sequence configured to couple to the capture handle sequence and wherein a second nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to non- templated nucleotides appended to a cDNA reverse transcribed from an mRNA analyte by a reverse transcriptase comprising terminal transferase activity.

44. The method of claim 43, wherein the non-templated nucleotides comprise a cytosine.

45. The method of claim 44, wherein the capture sequence configured to couple to the cDNA comprise a guanine.

46. The method of claim 45, wherein coupling of the capture sequence to the non-templated cytosine permits reverse transcription of the cDNA to extend into the nucleic acid barcode molecule.

47. The method of any of claims 36-46, wherein the first barcoded nucleic acid molecule generated in the partition comprises a sequence of the first reporter oligonucleotide or reverse complement thereof.

48. The method of claim 47, further comprising at (b): generating a third barcoded nucleic acid molecule, wherein the third barcoded nucleic acid molecule comprises a sequence of the second reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

49. The method of claim 47 or 48, further comprising at (b): generating a fourth barcoded nucleic acid molecule, wherein the fourth barcoded nucleic acid molecule comprises a sequence of the further reporter oligonucleotide or a reverse complement thereof and the partition-specific barcode sequence or reverse complement thereof.

50. The method of claim 49, further comprising determining sequence of the first, second, third and/or fourth barcoded nucleic acid molecule.

51. The method of claim 50, further comprising identifying the antibody or antigen binding fragment thereof based on the determined sequence of the second barcoded nucleic acid molecule.

52. The method of claim 50 or 51, further comprising assessing the affinity of the antibody or antigen binding fragment thereof based on the determined sequence of the first, third, and/or fourth barcoded nucleic acid molecule.

53. The method of claim 52, further comprising: identifying or characterizing the antibody or antigen-bind fragment thereof as comprising: the characteristic of binding a region of interest of the target antigen, or as having binding affinity to the region of interest to the target antigen, or as having its binding affinity mapped to the region of interest to the target antigen, based on the assessed affinity of the antibody or antigen-binding fragment thereof.

54. The method of any of claims 36-53, further comprising, prior to the (a) partitioning, isolating and/or enriching the plurality of cells.

55. The method of claim 54, wherein the enriching the plurality of cells comprises sorting cells of the plurality of cells according to their binding to the (i) target antigen, the fragment of target antigen and/or the further fragment of the target antigen as recited in claim 36 and claims as they depend therefrom; or (ii) the first and/or second fragments of the target antigen as recited in claim 37 and claims as they depend therefrom.

56. The method of any of claims 38-55, wherein the first, second and/or further reporter oligonucleotides are conjugated to labelling agents.

57. The method of claim 56, wherein the labelling agents are magnetic or are fluorescent.

58. The method of claim 57, wherein the labelling agents comprise a fluorescent label.

59. The method of any of claims 36-58, wherein the reaction mixture further comprises a second cell bound to (i) the target antigen, the fragment of target antigen and/or the further fragment of the target antigen as recited in claim 36 and claims as they depend therefrom; or (ii) the first and/or second fragments of the target antigen as recited in claim 37 and claims as they depend therefrom.

60. The method of claim 59, wherein the partitioning provides the second cell in a second partition.

61. The method of any of the preceding claims further comprising determining a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof.

62. The method of claim 61, wherein the nucleic acid sequence encoding the antibody or fragment thereof encodes one or more of a complementarity determining region (CDR), a framework (FWR), a variable heavy chain domain (VH), or a variable light chain domain (VL) of the antibody or fragment thereof.

63. The method of any of claims 1-60, further comprising determining an amino acid sequence of the antibody or antigen-binding fragment thereof.

64. The method of claim 63, wherein the amino acid sequence of the antibody or fragment thereof comprises a sequence of one or more of a CDR, FWR, VH or VL of the antibody or fragment thereof.

65. The method of any preceding claim, wherein a barcoded nucleic acid molecule further comprises a unique molecular identifier (UMI) sequence.

66. The method of any preceding claim, wherein the reaction mixture further comprises a non-target antigen coupled to a reporter oligonucleotide.

67. The method of any of the preceding claims, wherein the plurality of cells is obtained from a subject who has been exposed, or who is suspected of being exposed, to the target antigen.

68. The method of claim 67, wherein the subject is a mammal, optionally wherein the mammal is a human.

69. The method of claim 68, wherein the plurality of cells comprise B cells, optionally wherein the B cells comprise memory B cells.

70. The method of any preceding claim, wherein the target antigen is a GPCR, a viral glycoprotein, an influenza hemagglutin, a glycan, an glycan conjugate, a soluble cytokine, a cell-based co-stimulatory molecule, a cell-based co-inhibitory molecule, an ion channel, or a growth factor.

71. The method of any of claims 1-69, wherein the target antigen is a coronavirus protein.

72. The method of claim 71, wherein the coronavirus protein is a spike (S) protein, optionally wherein the coronavirus S protein is a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) S protein or a fragment thereof, optionally wherein the coronavirus S protein is a fragment of the SARS-CoV-2 S protein, optionally wherein the fragment of the SARS-CoV-2 S protein comprises a receptor binding domain, a N-terminal domain, and/or an extracellular domain of the SARS-CoV-2 S protein.

73. The method of any preceding claim, wherein the partition is a microwell or droplet.

74. The method of any preceding claim, wherein the fragment or non-overlapping fragments of the target antigen are not complexed with a MHC molecule.

75. A partition comprising: a cell expressing an antigen-binding molecule (ABM), wherein the ABM is bound to an antigen and a fragment of the antigen; and wherein the antigen is coupled to a first reporter oligonucleotide and the fragment of the antigen is coupled to a second reporter oligonucleotide.

76. The partition of claim 75, wherein the first and the second reporter oligonucleotide comprise: (i) a first and second reporter sequence, respectively and (ii) a capture handle sequence, wherein the first reporter sequence is specific to the antigen and the second reporter sequence is specific to the fragment of the antigen.

77. A partition comprising: a cell expressing an ABM, wherein the ABM is bound to a first and a second fragment of an antigen; and wherein the first and the second fragment of the antigen are coupled to a first and a second reporter oligonucleotide, respectively.

78. The partition of claim 77, wherein the first and the second fragment are non-overlapping fragments of the antigen.

79. The partition of claim 77 or 78, wherein the first and the second reporter oligonucleotide comprise: (i) a first and second reporter sequence, respectively and (ii) a capture handle sequence, wherein the first reporter sequence is specific to the first fragment of the antigen and the second reporter sequence is specific to the second fragment of the antigen.

80. The partition of claim 76 or 79, further comprising a plurality of nucleic acid barcode molecules comprising a partition-specific sequence, optionally wherein a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further comprises a capture sequence configured to couple to an mRNA or a DNA analyte, optionally wherein the capture sequence configured to couple to the mRNA or DNA analyte is configured to couple to the mRNA analyte, and wherein the capture sequence configured to couple to the mRNA analyte comprises a polyT sequence.

81. The partition of claim 76 or 79, further comprising a primer, optionally wherein the primer is configured to couple to an mRNA or a DNA analyte, optionally wherein the partition further comprises a reverse transcriptase comprising terminal transferase activity, optionally wherein a first nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules further comprises a capture sequence configured to couple to the capture handle sequence and wherein a second nucleic acid barcode molecule of the plurality nucleic acid barcode molecules further comprises a capture sequence configured to couple to a cDNA reverse transcribed from an mRNA analyte by the reverse transcriptase comprising terminal transferase activity.

82. The partition of claim 80 or 81, wherein the capture handle sequence is configured to couple to the capture handle sequence by complementary base pairing.

83. The partition of any of claims 75-82, wherein the ABM is further bound to a further fragment of the antigen, and wherein the further fragment of the antigen is coupled to a further reporter oligonucleotide, optionally wherein the further reporter oligonucleotide comprises: (i) a further reporter sequence specific to the further fragment of the antigen and (ii) a capture handle sequence.

84. The partition of any of claims 80-83, wherein a nucleic acid barcode molecule and/or reporter oligonucleotides comprises a UMI.

85. The partition of any of claims 75-84, wherein the cell is conjugated to a labelling agent, and/or wherein the partition is a microwell or droplet, and/or wherein the cell is a B cell, optionally wherein the B cell is a memory B cell or a labeled B cell, and/or wherein the fragment is not complexed with an MHC molecule, and/or wherein the ABM is selected from the group consisting of an antibody or functional fragment thereof, an immune receptor, and an immunoglobulin, optionally wherein the ABM is an immunoglobulin (Ig), optionally wherein the Ig is selected from the group consisting of IgA, IgD, IgE, IgG, and IgM.

86. A kit comprising instructions for use thereof and: a target antigen and a fragment of the target antigen, wherein the target antigen and the fragment of the target antigen are coupled to reporter oligonucleotides; and wherein the kit is for: (i) identification of an antibody, or antigen-binding fragment thereof, that has binding affinity for a region of interest of the target antigen, or (ii) mapping binding affinity for at least one region of interest of the target antigen by the antibody or antigen-binding fragment thereof or (iii) characterizing the antibody or antigen-binding fragment thereof.

87. The kit of claim 86, wherein the target antigen is coupled to a first reporter oligonucleotide and the fragment of the target antigen is coupled to a second reporter oligonucleotide.

88. The kit of claim 87, wherein the first and the second reporter oligonucleotides comprise: (i) a first and second reporter sequence, respectively and (ii) a capture handle sequence, wherein the first reporter sequence is specific to the target antigen to which it is coupled and the second reporter sequence is specific to the fragment of the target antigen to which it is coupled.

89. A kit comprising instructions for use thereof and: a plurality of fragments of a target antigen, wherein each of the plurality of fragments is coupled to reporter oligonucleotides, and wherein the kit is for: (i) identification of an antibody, or antigen-binding fragment thereof, that has binding affinity for a region of interest of the target antigen, or (ii) mapping binding affinity for at least one region of interest of the target antigen by the antibody or antigen-binding fragment thereof or (iii) characterizing the antibody or antigen-binding fragment thereof.

90. The kit of claim 89, wherein the plurality of fragments of the target antigen do not overlap in amino acid sequence.

91. The kit of claim 90, wherein a first of the plurality of fragments is coupled to a first reporter oligonucleotide and a second of the plurality of fragments is coupled to a second reporter oligonucleotide, optionally wherein the first and the second reporter oligonucleotide comprise: (i) a first and second reporter sequence, respectively, and (ii) a capture handle sequence, wherein the first reporter sequence is specific to the first of the plurality of fragments to which it is coupled and the second reporter sequence is specific to the second of the plurality of fragment to which it is coupled.

92. The kit of any of claims 86-91, further comprising a plurality of nucleic acid barcode molecules, optionally wherein a nucleic acid barcode of the plurality further comprises a capture sequence, optionally wherein the capture sequence comprises a sequence complementary to a capture handle sequence.

93. The kit of any of claims 86-92, further comprising a non-target antigen or a fragment of a non-target antigen, wherein the non-target antigen or fragment of the non-target antigen is coupled to a non-target reporter oligonucleotide, optionally wherein the non-target reporter oligonucleotide comprises a non-target reporter sequence and a capture handle sequence, optionally wherein the non-target reporter sequence is specific to the non-target antigen or fragment to which it is coupled.

94. The kit of any of claims 86-93, further comprising a further fragment of the target antigen coupled to a further reporter oligonucleotide, optionally wherein the further reporter oligonucleotide identifies the further fragment to which it is coupled.

95. A system comprising the kit of claim 86 or 89 and reagents for generating barcoded nucleic acid molecules formed by complementary base pairing of (i) the capture sequence of a nucleic acid barcode of the plurality of nucleic acid barcode molecules and (ii) the capture handle sequence of the first and/or second reporter oligonucleotide.