Method and system for super-resolution testing of therapeutics - Patents.com

Abstract

Disclosed herein is a method for generating a database of cellular characteristics from diseased and non-diseased cells using super-resolution microscopy. Also disclosed herein is a method for designing antibody-based therapeutics and identifying cancer targets for therapeutic intervention by using super-resolution microscopy. Also disclosed is a method for characterizing cell and tissue samples using super-resolution microscopy.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 275,253, filed November 3, 2021, and U.S. Provisional Patent Application No. 63 / 371,355, filed August 12, 2022, which provisional patent applications are incorporated by reference in their entireties. Summary of the Invention

[0002] Disclosed herein is a method for generating a database comprising cellular characteristics from diseased and non-diseased cells, the method comprising imaging a sample contacted with an antibody using a super-resolution microscope, generating a database comprising at least one cellular characteristic of cells in the sample, and determining whether the at least one cellular characteristic is indicative of a diseased or non-diseased cell.

[0003] In some embodiments, the at least one cellular characteristic comprises an image of an extracellular molecule. In some embodiments, the at least one cellular characteristic comprises two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises an intensity of the extracellular molecule. In some embodiments, the one or more cellular characteristics comprise all or a portion of a schematic or local representation of the extracellular molecule on a cell or tissue. In some embodiments, the at least one cellular characteristic comprises a proximity between two or more extracellular molecules. In some embodiments, the two or more extracellular molecules are of the same target or different targets. In some embodiments, the at least one cellular characteristic comprises an interaction of at least two extracellular molecules. In some embodiments, the at least one cellular characteristic comprises a distance or distances between two or more extracellular molecules. In some embodiments, the at least one cellular characteristic comprises an individual extracellular molecule or a pair of extracellular molecules. In some embodiments, the individual extracellular molecules or the pair of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual extracellular molecules or the pair of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, at least one cellular characteristic comprises individual, paired, or triad extracellular molecules. In some embodiments, the individual, paired, or triad extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, at least one cellular characteristic comprises pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules comprise the same or different extracellular molecules.In some embodiments, pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the at least one cellular characteristic comprises a geometric relationship between two or more extracellular molecules. In some embodiments, the at least one cellular characteristic comprises a translocation of at least one extracellular molecule. In some embodiments, the translocation is internalization or translocation to an extracellular surface. In some embodiments, the at least one cellular characteristic comprises an internalization of at least one extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a change in two-dimensional or three-dimensional coordinates of the extracellular molecule.

[0004] In some embodiments, the at least one cellular characteristic comprises a change in clustering of an extracellular molecule. In some embodiments, the at least one cellular characteristic comprises an oligomerization state of an extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a clustering state of an extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a clustering state of an extracellular molecule. In some embodiments, the extracellular molecule is an extracellular molecule listed in Table 1. In some embodiments, the at least one cellular characteristic is present in a cell in a tissue. In some embodiments, the at least one cellular characteristic is present in a cell in a cancer or on a normal tissue. In some embodiments, the at least one cellular characteristic is present in a normal cell, a cancer cell, or both a normal cell and a cancer cell. In some embodiments, the at least one cellular characteristic is present in a normal cell, a cancer cell, or a cell in a tumor microenvironment. In some embodiments, the at least one cellular characteristic is present in a cell in a tumor microenvironment. In some embodiments, the cell in the tumor microenvironment is selected from a B cell, a T cell, a natural killer (NK) cell, a myeloid cell, a fibroblast, and a pericyte. In some embodiments, the cells in the tumor microenvironment are selected from stromal cells, epithelial cells, or adipocytes. In some embodiments, the at least one cell characteristic is predictive of the presence of a cancer cell or a cell in the tumor microenvironment. In some embodiments, the at least one cell characteristic is predictive of the presence of a normal cell. In some embodiments, the at least one cell characteristic is present in the same cell in the tissue. In some embodiments, the at least one cell characteristic is present in at least two different cells in the tissue. In some embodiments, the at least one cell characteristic is within or between the distance of at least two different cells. In some embodiments, the at least one cell characteristic is present in a tissue structure in a normal state or in a disease state. In some embodiments, the disease state comprises a cancer disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state. In some embodiments, the tissue structure comprises an epithelium, a duct, or a blood vessel. In some embodiments, the at least one cell characteristic comprises an altered expression of an extracellular molecule.In some embodiments, the at least one cellular characteristic comprises a change in the glycosylation pattern of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a change in the enzymatic activity of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a change in intracellular or intercellular communication. In some embodiments, the at least one cellular characteristic comprises a change in intracellular or intercellular signaling or communication. In some embodiments, the at least one cellular characteristic comprises a change in cell adhesion, dynamics, or migration. In some embodiments, the at least one cellular characteristic comprises a tissue localization of the extracellular molecule. In some embodiments, the tissue localization comprises a diseased tissue or a tumor microenvironment. In some embodiments, the tissue localization comprises a cancer structure or a tumor microenvironment. In some embodiments, the extracellular molecule is internalized upon contact with the antibody. In some embodiments, different samples are evaluated for a common cellular characteristic. In some embodiments, different samples are evaluated for different cellular characteristics. In some embodiments, the method further comprises characterizing the at least one cellular characteristic based on the intensity of the imaging agent in the image. In some embodiments, the at least one cellular characteristic is evaluated after administration of the at least one ligand. In some embodiments, the at least one cellular characteristic is assessed following administration of at least one therapeutic agent or at least one therapeutic regimen.

[0005] In some embodiments, the super-resolution microscope comprises a deterministic super-resolution microscope. In some embodiments, the super-resolution microscope is a stimulated emission depletion (STED) microscope or a ground state depletion (GSD) microscope. In some embodiments, the super-resolution microscope comprises a stochastic super-resolution microscope. In some embodiments, the super-resolution microscope is a MINFLUX microscope. In some embodiments, the super-resolution microscope is a stochastic optical reconstruction microscopy (STORM) microscope. In some embodiments, the imaging step comprises tracking one or more ligands, therapeutic agents, or cell surface receptors over a period of time.

[0006] In some embodiments, the cells comprise fixed cells. In some embodiments, the cells are fixed in tissue. In some embodiments, the cells are in live tissue. In some embodiments, the cells comprise live cells. In some embodiments, the cells comprise mammalian cells. In some embodiments, the diseased cells are derived from a tumor cell line. In some embodiments, the normal cells are derived from a normal cell line. In some embodiments, the cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises frozen tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises live tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises fixed tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber is formed by a laser microdissection process.

[0007] In some embodiments, the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a single domain antibody from a camelid (VHH), a synthetically derived VHH, a single domain antibody created in a mouse, a shark antibody, an antigen binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and a scFv-Fc. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains. In some embodiments, the antibody is an immune cell engager. In some embodiments, the antibody comprises an effector domain (e.g., CD3 or CD16A). In some embodiments, the antibody based therapy is against CAR-T cells, myeloid cells, NK cells, or other cell based therapy. In some embodiments, the antibody is in a vaccine. In some embodiments, the antibody is a translated nucleic acid strand or multiple nucleic acid strands. In some embodiments, the antibody comprises a single chain polypeptide. In some embodiments, the antibody comprises a homodimer. In some embodiments, the antibody comprises a heterodimer.

[0008] In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 100 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 50 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 40 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 30 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 20 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 10 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 9 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 8 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 7 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 6 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 5 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 4 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 3 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 2 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 1 nanometer. In some embodiments, the database of imaged cell features is imaged at single molecule resolution. In some embodiments, the database of imaged cell features is imaged at single fluorophore resolution.

[0009] In some embodiments, the database of imaged cell features is imaged in a high throughput format. In some embodiments, the low throughput format includes the use of at least one 96 well plate. In some embodiments, the low throughput format includes the use of at least one 192 well plate. In some embodiments, the high throughput format includes the use of at least one 384 well plate. In some embodiments, the high throughput format includes the use of at least one 1536 well plate. In some embodiments, the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels. In some embodiments, the multiple cell features are imaged at approximately the same time. In some embodiments, the multiple cell features are imaged sequentially. In some embodiments, the multiple cell features are imaged at approximately the same time and the rest are imaged sequentially. In some embodiments, the multiple cell features are on the surface of the multiple cells. In some embodiments, the imaging of the cell features is completed in less than about 5 minutes. In some embodiments, imaging the database of imaged cell features is performed automatically.

[0010] In some embodiments, the extracellular molecule is a membrane-bound protein or a membrane-bound ligand. In some embodiments, the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0011] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. [Brief description of the drawings]

[0012] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG.").

[0013] [Figure 1] FIG. 1 illustrates a computer system that is programmed or otherwise configured to implement the methods provided herein. [Diagram 2] FIG. 1 shows an example of an application of the present disclosure in which spatially localized Kd for one or more binding molecules (e.g., VHHs) that bind to a cell surface are derived from single molecule tracking (SMT) experiments and then matched to cell characteristics in an image database. [Diagram 3] FIG. 2 shows stimulated emission depletion (STED) images of A549 cells stained with labeled anti-TROP2 VHH binder 1 (TROP2_1); the images are 34.68 × 17.04 micrometers (μm) (1734 × 852 pixels) with a resolution of 20 nanometers (nm) per pixel. [Figure 4] FIG. 1 shows STED images of A549 cells stained with labeled TROP2_1. The images are 34.68×17.04 μm (1734×852 pixels) with a resolution of 20 nm / pixel. [Figure 5A] FIG. 1 shows SKBR3 cells stained with labeled TROP2_1. The image is 34.60×43.06 μm (1730×2153 pixels) with a resolution of 20 nm / pixel. [Figure 5B] FIG. 1 shows SKBR3 cells stained with labeled anti-HER2 VHH binder 3 (HER2_3). The image is 34.60×43.06 μm (1730×2153 pixels) with a resolution of 20 nm / pixel. [Figure 6A]FIG. 1 shows MCF7 cells stained with labeled TROP2_1. The image is 63.86×58.30 μm (3193×2915 pixels) with a resolution of 20 nm / pixel. [Figure 6B] FIG. 1 shows MCF7 cells stained with labeled HER2_3. The image is 63.86×58.30 μm (3193×2915 pixels) with a resolution of 20 nm / pixel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed.

[0015] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0016] Whenever the terms "no more than," "less than," or "less than or equal to" precede the first number in a series of two or more numbers, the terms "no more than," "less than," or "less than or equal to" apply to each of the numbers in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0017] Certain inventive embodiments herein contemplate numerical ranges. When a range exists, the range includes the endpoints of the range. Furthermore, any subranges and values ​​within the range exist as if explicitly written out. The term "about" or "approximately" may mean within an acceptable error range for a particular value, which depends in part on how the value is measured or determined, for example, on the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" may mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" may be assumed to mean within an acceptable error range for the particular value.

[0018] definition As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease for which the compositions and methods described are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0019] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided polypeptides, antibody chains, and other peptides, such as linkers and connecting peptides, may contain amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptide may contain modifications to the native or naturally occurring sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as due to mutations of the host that produces the protein, or errors due to PCR amplification.

[0020] Percent sequence identity (%) to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning sequences and introducing gaps as necessary to achieve maximum sequence identity percentage, without considering any conservative substitutions as part of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Suitable parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted with user documentation to the United States Copyright Office, Washington, DC, 20559, and is registered under United States Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0021] In the situation where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A having or containing a particular % amino acid sequence identity to a given amino acid sequence B) is calculated as 100 x the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B programs, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equivalent to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equivalent to the % amino acid sequence identity of B to A. Unless otherwise specified, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0022] In some embodiments, amino acid sequence variants of the polypeptides provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may occur naturally or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the present invention, evaluating the biological activity of one or more of the polypeptides described herein, and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics, for example, antigen binding.

[0023] In some embodiments, "antigen-binding domain" or "antigen-interacting domain" refers to an immunoglobulin derivative having antigen-binding properties, i.e., an immunoglobulin polypeptide or fragment thereof that contains an antigen-binding site. Optionally, the binding domain comprises a variable domain of an antibody or fragment thereof. Each antigen-binding domain is formed by an antibody, i.e., an immunoglobulin, in which the variable heavy chain domain (VH) and the antibody variable light chain domain (VL) bind to the same epitope, but the variable heavy chain domain (VH) comprises three heavy chain complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, and the variable light chain domain (VL) comprises three light chain complementarity determining regions (CDRs), CDR1, CDR2, and CDR3. In some examples, the binding domain according to some embodiments herein lacks an immunoglobulin constant domain. In some examples, the variable light chain and heavy chain domains that form the antigen-binding site are covalently linked to each other, e.g., by a peptide linker, or in other examples, the variable light chain and heavy chain domains are non-covalently associated with each other to form the antigen-binding site. Binding domains and interaction domains also refer to antibody fragments or antibody derivatives, including, for example, Fab, Fab', F(ab')2, Fv fragments, single chain Fv, tandem single chain Fv ((scFv)2), Bispecific T-cell Engagers (BiTEs®), Dual Affinity Retargeting Antibodies (DART™), diabodies, DuoBody® IgG molecules, single domain antibodies (e.g., VHH), TriTac, etc. In further particular examples, the binding domain is multivalent, i.e., has two, three, or more binding sites for one or more antigens.

[0024] In certain embodiments, the antigen-binding or antigen-interacting domain comprises a sequence derived from the complementarity determining region of an antibody. The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).The exact amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering), Al-Lazikani et al. (1997), JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 (1996), ("Contact" numbering scheme), Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 (the "IMGT" numbering scheme), Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8;309(3):657-70 (the "Aho" numbering scheme), and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB", Protein Eng. 2000 Dec;13(12):819-24 (the "AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0025] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions provided by the insertion letter, e.g., "30a", and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0026] In certain embodiments, the antigen-binding domain or antigen-interacting domain comprises a sequence derived from the complementarity determining region of an antibody. The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) exhibit a similar overall structure, and each domain contains four conserved framework regions (FR) and three CDRs (see, for example, Kindt et al., Kuby Immunology, 6th Edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen can be isolated using the VH or VL domain from an antibody that binds the antigen to screen libraries of complementary VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0027] In certain embodiments, the antigen-binding or antigen-interacting domain is "humanized". A "humanized" polypeptide or antibody is one in which all or nearly all CDR amino acid residues are derived from non-human CDRs and all or nearly all FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has undergone humanization to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues derived from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0028] Among the polypeptides and antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or a non-human source, utilizing a human antibody repertoire, including a human antibody library, or other human antibody-encoding sequences. The term excludes humanized forms of non-human antibodies that include a non-human antigen-binding region, such as those in which all or nearly all CDRs are non-human. Although the methods and systems of the present disclosure are described herein as antibody-based therapeutics, in some embodiments, they are used to image the interaction of non-therapeutic antibodies or antibody derivatives (e.g., portions of antibodies) with cells. For example, fluorophores are attached to VHH domains and used to map surface and cellular properties of cells. In this example, surface and cellular properties are compared between diseased and normal cells.

[0029] Non-limiting examples of fluorophores or imaging agents include KK114 (also known as Abberior STAR RED), Abberior STAR ORANGE, SYBR green, SYBR blue, DAPI, propidium iodide, Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine and acridine, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and 2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, Acridine Orange, 7-AAD, Actinomycin D, LDS751, Hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Oregon Green, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes such as those containing europium and terbium, carboxytetrachlorofluorescein, 5 and / or 6-carboxyfluorescein (FAM), 5- (or 6-)iodoacetamidofluorescein, 5-{[2(and 3)-5-(acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein), Lissamine rhodamine B sulfonyl chloride, 5 and / or 6 carboxyrhodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate-4-amino-naphthalimide, phycobiliproteins, AlexaFluor Examples of suitable fluorophores include, but are not limited to, 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, quantum dots, or other fluorophores.

[0030] The polypeptides described herein may be encoded by nucleic acids. A nucleic acid is a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to introduce a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a genomic integration vector or "integration vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of inducing the expression of a gene to which it is operably linked is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In an expression vector, regulatory elements such as promoters, enhancers, polyadenylation signals, and the like, for use in controlling transcription can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may further be incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc. may be employed. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors can include sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or a restricted set of sites in the genome. Additionally, vectors can include sequences derived from transposable elements.

[0031] As used herein, the terms "homologous", "homology", or "percent homology", when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, revised Proc. Natl. Acad. Sci. USA 90:5873-5877). Such formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence homology can be determined using the most recent version of BLAST as of the filing date of this application.

[0032] Nucleic acids encoding the polypeptides described herein can be used to infect, transfect, transform, or otherwise transfect suitable cells with the nucleic acid, thus allowing production of the polypeptide for commercial or therapeutic use. Standard cell lines and methods for producing antibodies or polypeptides from large scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production," Mabs. 2010 Sep-Oct;2(5):466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing a polypeptide or antibody and is a Chinese Hamster Ovary (CHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing a polypeptide or antibody. In certain embodiments, described herein are methods of making an antibody comprising culturing cells containing nucleic acid encoding the antibody under in vitro conditions sufficient to allow production and secretion of the antibody.

[0033] Although the methods and systems described herein are described in some embodiments for use in disease state differentiation, treatment design, and therapeutic target identification, the methods and systems described herein can be used for a variety of applications. For example, the databases, methods, and systems described herein can be used for patient selection (e.g., cancer type or study group selection). In another example, the databases, methods, and systems described herein can be used for combination therapy selection (e.g., drug combination selection). In another example, the databases, methods, and systems described herein can be used for drug discovery (e.g., discovery of novel targets or therapeutic compounds). In some embodiments, the multiple extracellular receptors described herein are multiple of the same extracellular receptor. In some embodiments, the multiple extracellular receptors described herein are multiple of different extracellular receptors. In another example, the databases, methods, and systems described herein can be used in determining therapeutics that can be repurposed for different applications (e.g., determining survival rates for therapeutics approved for use in applications different from those for which the therapeutics were approved).

[0034] Although the database, method, and system of the present disclosure are described herein with respect to antibody fragments or derivatives, they can be used to test the effect of complete antibodies on a sample. For example, an antibody can be contacted with a sample, and the cellular characteristics of the sample can be recorded in the database. Examples of antibodies include, but are not limited to, IgA, IgD, IgE, IgG, IgM, IgT / Z, IgY, IgW, and the like. In some embodiments, the antibody is an antibody associated with a CAR T cell. In some embodiments, the antibody is an antibody-drug conjugate. In some embodiments, a ribonucleic acid (RNA) aptamer is used in place of the antibody. In some embodiments, a natural receptor or ligand is used in place of the antibody.

[0035] As used herein, molecular signature or molecular fingerprint is defined as all or part of the composition or state of one extracellular molecule on the cell surface that is composed of at least one cell characteristic.As used herein, cell signature or cell fingerprint is defined as all or part of the composition or state of two or more extracellular molecules on the cell surface that is composed of at least one cell characteristic.As used herein, tissue signature or tissue fingerprint is defined as the molecular signature or fingerprint or cell signature or fingerprint on the surface of two or more cells (or cell aggregates) in a tissue or sample.

[0036] Method for generating a database containing cell characteristics Cancer cells are morphologically and functionally different from normal cells, and different cell signaling pathways operate. Extracellular receptors may cluster (or appear differently) in lipid rafts during cell signaling, amplifying signals inside the cell. The specific characteristics of extracellular receptors may differ between cancer cells and normal cells. In one aspect of the present disclosure, super-resolution microscopy (SRM) at a large scale and pharmacologic relevant binders to known cancer antigens are used to build a high-content normal / cancer cell (tissue) database. This may be directly translatable to therapeutic strategies and may be used throughout testing and development. The database may be used for target selection and validation (as well as new target discovery) for monospecific, bispecific, and multispecific therapy, drug design and validation, combination therapy selection for clinical trials, cancer indication selection for clinical trials, and patient selection for clinical trials. Features are characterized as to whether the feature is present, and if so, its location, nearest neighbors, whether internalized, etc. are determined.

[0037] In some embodiments, the present disclosure provides a database of cellular interactions that can be used in a variety of different applications. In some embodiments, the database provides new information about the interactions of therapeutics (e.g., antibody-based therapeutics, small molecule therapeutics, etc.) with both diseased and healthy cells. In some embodiments, the information can then be used to improve the efficacy of the screened therapeutics by better targeting different types of cells. In some embodiments, the information in the database can be used to characterize properties in diseased and non-disease cells, and subsequently interpret patterns that provide information about diseased and non-disease cells. In some embodiments, the information in the database is used for target selection, drug design, lead and development candidate selection, patient selection for clinical trials, and / or indication selection.

[0038] In another aspect of the disclosure, the spatially localized affinity (K) of T cell engagers (TCEs) or other binding molecules (e.g., antibody-based therapeutics) to the cell surface is measured. d ) are derived from single molecule tracking (SMT) experiments and matched to the cellular properties of cells in the database to provide two-dimensional (2D) K d The SMT produces a profile (see Figure 2). The SMT is the on-rate (k on ) and off-rate (k off ) which allows for the measurement of the spatially localized affinity (K) of a binding molecule or TCE on the surface of a cell (e.g., a cancer cell or other cell). d =k off / k on When a binding molecule or TCE contains a repeating single variable domain (VHH) on the heavy chain and / or two or more different VHHs that bind to different targets on the cell surface, there is an avidity effect when those targets cluster to form multimers. Thus, whenever a target clusters to form a structure that is recognized by the binding molecule / TCE, it will have a higher affinity (lower K d ) exists. dUsing the pattern and matching it to the cell surface targeting pattern (cell characteristics) of the cells observed in the SRM images, the binding behavior of the binding molecule / TCE to the surface of a separate cell or tissue can be predicted by recording SRM images of the separate cell / tissue in the presence of the binding molecule / TCE, assuming that the target presentation / cell characteristics are approximately the same in both cells / tissues. This strategy can be beneficial, for example, to predict the binding behavior of the binding molecule / TCE to normal or cancerous cells or tissues.

[0039] Disclosed herein is a method for generating a database comprising cellular characteristics from diseased and non-diseased cells, the method comprising imaging a sample contacted with an antibody using a super-resolution microscope, generating a database comprising at least one cellular characteristic of cells in the sample, and determining whether the at least one cellular characteristic is indicative of a diseased or non-diseased cell.

[0040] Cell characteristics In some embodiments, the at least one cellular characteristic comprises an image of an extracellular molecule. In some embodiments, the at least one cellular characteristic comprises two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises an intensity of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises all or a portion of a molecular fingerprint, molecular signature, or map of the location of the extracellular molecule. In some embodiments, the one or more cellular characteristics comprise all or a portion of a schematic or local representation of the extracellular molecule on the cell or tissue. In some embodiments, the at least one cellular characteristic comprises a proximity between two or more extracellular molecules. In some embodiments, the two or more extracellular molecules are of the same target or different targets. In some embodiments, the at least one cellular characteristic comprises an interaction of at least two extracellular molecules. In some embodiments, the at least one cellular characteristic comprises a distance or distances between two or more extracellular molecules. In some embodiments, the at least one cellular characteristic comprises an individual extracellular molecule or a pair of extracellular molecules. In some embodiments, the individual or pair of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual or paired extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual or paired extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, at least one cellular characteristic comprises individual, paired, or triad extracellular molecules. In some embodiments, the individual, paired, or triad extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, at least one cellular characteristic comprises pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules. In some embodiments, pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with each other.In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules include the same or different extracellular molecules. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the at least one cellular characteristic includes a geometric relationship between two or more extracellular molecules. In some embodiments, the at least one cellular characteristic includes a translocation of at least one extracellular molecule. In some embodiments, the translocation is internalization or translocation to an extracellular surface. In some embodiments, the at least one cellular characteristic includes an internalization of at least one extracellular molecule. In some embodiments, the at least one cellular characteristic includes a change in two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the at least one cellular characteristic includes a change in clustering of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a change in clustering of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises an oligomerization state of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a clustering state of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises the name of a protein that clusters with the extracellular molecule. In some embodiments, the extracellular molecule is an extracellular molecule listed in Table 1.

[0041] [Table 1-1]

[0042] [Table 1-2]

[0043] [Table 1-3]

[0044]

Table 1-4

[0045]

Table 1-5

[0046]

Table 1-6

[0047]

Table 1-7

[0048]

Table 1-8

[0049]

Table 1-9

[0050]

Table 1-10

[0051]

Table 1-11

[0052]

Table 1-12

[0053] In some embodiments, the at least one cell characteristic is present in cells in a tissue. In some embodiments, the at least one cell characteristic is present in cells in a cancer or on a normal tissue. In some embodiments, the at least one cell characteristic is present in normal cells, cancer cells, or both normal and cancer cells. In some embodiments, the at least one cell characteristic is present in normal cells, cancer cells, or cells in a tumor microenvironment. In some embodiments, the at least one cell characteristic is present in cells in a tumor microenvironment. In some embodiments, the cells in the tumor microenvironment are selected from B cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and pericytes. In some embodiments, the cells in the tumor microenvironment are selected from stromal cells, epithelial cells, or adipocytes. In some embodiments, the at least one cell characteristic is predictive of the presence of a cancer cell, or a cell in a tumor microenvironment. In some embodiments, the at least one cell characteristic is predictive of the presence of a normal cell. In some embodiments, the at least one cell characteristic is present in the same cell in a tissue. In some embodiments, the at least one cell characteristic is present in at least two different cells in a tissue. In some embodiments, the at least one cellular characteristic is within or between the distance of at least two different cells. In some embodiments, the at least one cellular characteristic is present in a tissue structure in a normal state or a disease state. In some embodiments, the disease state comprises a cancer disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state. In some embodiments, the tissue structure comprises an epithelium, a duct, or a blood vessel. In some embodiments, the at least one cellular characteristic comprises a change in the expression of an extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a change in the glycosylation pattern of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a change in the enzymatic activity of the extracellular molecule. In some embodiments, the at least one cellular characteristic comprises a change in intracellular or intercellular communication. In some embodiments, the at least one cellular characteristic comprises a change in intracellular or intercellular signaling or communication. In some embodiments, the at least one cellular characteristic comprises a change in cell adhesion, mechanics, or migration.In some embodiments, the at least one cellular characteristic comprises tissue localization of the extracellular molecule. In some embodiments, the tissue localization comprises diseased tissue or tumor microenvironment. In some embodiments, the tissue localization comprises cancer structure or tumor microenvironment. In some embodiments, the extracellular molecule is internalized upon contact with the antibody. In some embodiments, different samples are evaluated for a common cellular characteristic. In some embodiments, different samples are evaluated for different cellular characteristics. In some embodiments, the method further comprises characterizing the at least one cellular characteristic based on the intensity of the contrast agent in the image. In some embodiments, the at least one cellular characteristic is evaluated after administration of at least one ligand. In some embodiments, the at least one cellular characteristic is evaluated after administration of at least one therapeutic agent or at least one treatment regimen.

[0054] Super-resolution microscope In some embodiments, the super-resolution microscope comprises a deterministic super-resolution microscope. In some embodiments, the deterministic super-resolution microscopy uses light structuring to determine spatial information below the diffraction limit of a sample. For example, the deterministic super-resolution microscope can use a depletion ring to refine the excitation spot of the microscope. In some embodiments, the super-resolution microscope is a stimulated emission depletion (STED) microscope, a ground state depletion (GSD) microscope, a saturated structured illumination microscopy (SSIM), a super-resolution orthogonal deterministic imaging (SODI), or the like. In some embodiments, the super-resolution microscope is a MINFLUX microscope. For example, the super-resolution technology can be the MINFLUX technology developed by Abberior Instruments. MINFLUX is a super-resolution fluorescence microscopy that can provide resolution in the 1-3 nanometer (nm) range by localizing individual switchable fluorophores with a probing donut-shaped excitation beam (see, e.g., Gwosch et al., "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells," Nature Methods, vol. 17, pp. 217-220, February 2020). In some embodiments, the super-resolution microscope uses stochastic super-resolution techniques (e.g., is a stochastic super-resolution microscope). In some embodiments, the stochastic super-resolution techniques use chance to improve emitter localization (e.g., by providing a flux such that only one emitter in a region is stochastically activated). In some embodiments, the super-resolution microscope is a stochastic optical reconstruction microscopy (STORM) microscope. In some embodiments, the stochastic super-resolution technique is direct stochastic optical reconstruction microscopy (dSTORM), photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), spectral precision distance microscopy (SPDM), localization microscopy (e.g., single molecule localization microscopy (SMLM)), cryogenic optical localization in three dimensions (COLD), DNA-PAINT, or the like.In some embodiments, super-resolution microscopy or multiplexing methods described in Jungmann et al. (U.S. Pat. No. 10,294,510 and European Patent No. 3027773) are used. In some embodiments, imaging involves tracking antibody-based therapeutics or cell surface receptors over a period of time.

[0055] To quantify extracellular proteins, the observed fluorescence intensity can be normalized using the intrinsic molecular brightness of a single fluorophore attached to the antibody. This can be done by labeling double strands of DNA with a single fluorophore and attaching these at low concentration to a small area of ​​the coverslip used for imaging, ensuring that single molecules are appropriately spaced. During imaging, an image is acquired in the small area using the same parameters as those used to image the cell, and the single molecule intensity from this area is used to calibrate the intensity acquired in the image of the rest of the cell.

[0056] In some embodiments, the super-resolution microscope can be configured to capture time series images of a sample. For example, a sample can be contacted with an antibody and imaged over time to obtain a time series of cellular characteristics. In this example, the time resolved data of the cellular characteristics can provide further data regarding the mechanism of action of the treatment as well as efficacy. In some embodiments, the imaging includes tracking the therapeutic agent, cellular characteristic, or cell surface receptor for a period of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000 milliseconds or more. In some embodiments, imaging includes tracking the therapeutic agent, cell feature, or cell surface receptor for a period of up to about 50,000, 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 milliseconds or less.

[0057] Cell / tissue microarrays In some embodiments, the cells comprise fixed cells. In some embodiments, the cells are fixed in tissue. In some embodiments, the cells are in live tissue. In some embodiments, the cells comprise live cells. In some embodiments, the cells comprise mammalian cells. In some embodiments, the diseased cells are derived from a tumor cell line. In some embodiments, the normal cells are derived from a normal cell line. In some embodiments, the cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises frozen tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises live tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises fixed tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber is formed by a laser microdissection process.

[0058] antibody In some embodiments, the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a single domain antibody from a camelid (VHH), a synthetically derived VHH, a single domain antibody created in a mouse, a shark antibody, an antigen binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and a scFv-Fc. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains. In some embodiments, the antibody is an immune cell engager. In some embodiments, the antibody comprises an effector domain (e.g., CD3 or CD16A). In some embodiments, the antibody based therapy is against CAR-T cells, myeloid cells, NK cells, or other cell based therapy. In some embodiments, the antibody is in a vaccine. In some embodiments, the antibody is a translated nucleic acid strand or multiple nucleic acid strands. In some embodiments, the antibody comprises a single chain polypeptide. In some embodiments, the antibody comprises a homodimer. In some embodiments, the antibody comprises a heterodimer.

[0059] resolution In some embodiments, the database of imaged cellular characteristics is imaged at or above (e.g., on the order of) the resolution of the super-resolution microscope. For example, a super-resolution microscope with a resolution limit of 5 nanometers can image a sample at a resolution of up to 5 nanometers. In some embodiments, the super-resolution microscope has a resolution of at least about 250, 225, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nanometers or less. In some embodiments, the super-resolution microscope has a resolution of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250 nanometers or more. In some embodiments, the resolution of the super-resolution microscope is in a range defined by any two of the preceding values, for example in some embodiments, the resolution of the super-resolution microscope is between 1 and 50 nanometers.

[0060] In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 100 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 50 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 40 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 30 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 20 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 10 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 9 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 8 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 7 nanometers. In some embodiments, the database of imaged cell characteristics is imaged at a resolution of at least about 6 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 5 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 4 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 3 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 2 nanometers. In some embodiments, the database of imaged cell features is imaged at a resolution of at least about 1 nanometer. In some embodiments, the database of imaged cell features is imaged at single molecule resolution. In some embodiments, the database of imaged cell features is imaged at single fluorophore resolution.

[0061] High Throughput Format In some embodiments, the database of imaged cellular features is imaged in a high throughput format. In some embodiments, the low throughput format includes the use of at least one 96 well plate. In some embodiments, the low throughput format includes the use of at least one 192 well plate. In some embodiments, the high throughput format includes the use of at least one 384 well plate. In some embodiments, the high throughput format includes the use of at least one 1536 well plate. In some embodiments, the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels. In some embodiments, the multiple cellular features are imaged at approximately the same time. In some embodiments, the multiple cellular features are imaged sequentially. In some embodiments, the multiple cellular features are imaged at approximately the same time and the rest are imaged sequentially. In some embodiments, the multiple cellular features are on the surface of multiple cells. In some embodiments, the imaging of the cellular features is completed in less than 5 minutes. In some embodiments, imaging the database of cellular features is performed automatically.

[0062] extracellular molecules In some embodiments, the extracellular molecule is a membrane-bound protein or a membrane-bound ligand. In some embodiments, the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0063] sample In some embodiments, the sample can be tested to determine whether it has cellular characteristics in common with a second sample from the same source (e.g., patient) or a different source. In some embodiments, the evaluation includes one or more of genetic testing (e.g., nucleic acid detection or sequencing), protein testing (e.g., identifying the presence or absence of a given protein), size testing (e.g., determining the size distribution of cells present in the sample), label testing (e.g., determining the presence or absence of one or more labels on the cells), and the like, or any combination thereof. In some embodiments, the sample is evaluated after administration of at least one therapeutic agent or at least one therapeutic regimen (e.g., an antibody-based therapeutic regimen, a small molecule-based therapeutic regimen, a biological-based regimen, and the like). For example, the sample can be contacted with a therapeutic regimen and subsequently evaluated to determine the effect of the regimen and whether the sample is worthy of full characterization. In some embodiments, the sample is evaluated after administration of a natural ligand or a naturally occurring agent or cofactor. In some embodiments, pre-assessment of samples can reduce full characterization of samples that do not exhibit the property of interest (e.g., the effect or lack thereof of a treatment regimen), reducing waste of time and resources.

[0064] In some embodiments, the cells include live cells. For example, the cells can be collected from a living organism and used as collected. In another example, the cells can be obtained from a maintained cell line. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells include mammalian cells. In some embodiments, the mammalian cells are from a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, yak, camel, or human. In some embodiments, the cells include non-living cells. In certain embodiments, the non-living cells are dead cells. In some embodiments, the cells include fixed cells. In some embodiments, the fixed cells are fixed as described elsewhere herein.

[0065] In some embodiments, the disease state cells are derived from tumor cell lines. Examples of tumor cell lines include, but are not limited to, 3T3 cells, A549 cells, HeLa cells, Jurkat cells, and those available from commercial cell line suppliers, such as MilliporeSigma and ThermoFischer.

[0066] In some embodiments, the cells are imaged on a tissue microarray. For example, the cells are part of the tissue microarray and are imaged as such. In some embodiments, the tissue microarray includes multiple tissue samples contained within a single object. In some embodiments, the multiple tissue samples are multiple different tissue samples. In some embodiments, the use of a tissue microarray allows for the screening of a variety of different tissue types in less time than screening each tissue type individually. In some embodiments, the tissue microarray includes one or more of frozen tissues (e.g., tissues currently or previously preserved by freezing), live tissues (e.g., currently living tissues), fixed tissues (e.g., tissues fixed in a matrix or otherwise preserved (e.g., by the use of aldehydes, alcohols, oxidizing agents, etc.), and the like, or any combination thereof. In some embodiments, the tissue microarray is formed by a laser microdissection process. In some embodiments, the tissue microarray is formed by a needle-coring process. In some embodiments, the tissue microarray is formed by a microtome process.

[0067] How to design antibody-based therapeutics Disclosed herein is a method for designing antibody-based therapeutics, comprising the steps of imaging extracellular molecules on cells for therapeutic intervention using super-resolution microscopy, characterizing cellular properties of the imaged extracellular molecules, and designing an antibody format based on the characterization of the cellular properties.

[0068] Cell characteristics In some embodiments, the cellular characteristic comprises an image of the extracellular molecule. In some embodiments, the cellular characteristic comprises two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the cellular characteristic comprises an intensity of the extracellular molecule. In some embodiments, the cellular characteristic comprises all or a portion of a molecular fingerprint, molecular signature, or map of the location of the extracellular molecule. In some embodiments, the cellular characteristic comprises all or a portion of a schematic or local representation of the extracellular molecule on the cell or tissue. In some embodiments, the cellular characteristic comprises a proximity between two or more extracellular molecules. In some embodiments, the two or more extracellular molecules are of the same target or different targets. In some embodiments, the cellular characteristic comprises an interaction of at least two extracellular molecules. In some embodiments, the cellular characteristic comprises a distance or distances between two or more extracellular molecules. In some embodiments, at least one cellular characteristic comprises an individual extracellular molecule or a pair of extracellular molecules. In some embodiments, the individual or pair of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual or pair of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, at least one cellular characteristic comprises individual, paired, or triad extracellular molecules. In some embodiments, the individual, paired, or triad extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, at least one cellular characteristic comprises pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules comprise the same or different extracellular molecules.In some embodiments, pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the cellular characteristic comprises a geometric relationship between two or more extracellular molecules. In some embodiments, the cellular characteristic comprises translocation of an extracellular molecule. In some embodiments, the translocation is internalization or translocation to an extracellular surface. In some embodiments, the cellular characteristic comprises internalization of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in two-dimensional or three-dimensional coordinates of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in clustering of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in clustering of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in clustering of an extracellular molecule. In some embodiments, the cellular characteristic comprises a translocation of at least one cellular receptor. In some embodiments, the cellular characteristic comprises an oligomerization state of an extracellular molecule. In some embodiments, the cellular characteristic comprises a clustering state of an extracellular molecule. In some embodiments, the cellular characteristic includes the name of a protein that clusters with the extracellular molecule. In some embodiments, the extracellular molecule includes an extracellular molecule listed in Table 1. In some embodiments, the cellular characteristic is present in a cell in a tissue. In some embodiments, the cellular characteristic is present in a cell in a cancer or on a normal tissue. In some embodiments, the cellular characteristic is present in a normal cell, a cancer cell, or both a normal cell and a cancer cell. In some embodiments, the cellular characteristic is present in a normal cell, a cancer cell, or a cell in a tumor microenvironment. In some embodiments, the cellular characteristic is present in a cell in a tumor microenvironment. In some embodiments, the cell in the tumor microenvironment is selected from a B cell, a T cell, a natural killer (NK) cell, a myeloid cell, a fibroblast, and a pericyte. In some embodiments, the cell in the tumor microenvironment is selected from a stromal cell, an epithelial cell, or an adipocyte. In some embodiments, the cellular characteristic is predictive of the presence of a cancer cell, or a cell in a tumor microenvironment. In some embodiments, the cellular characteristic is predictive of the presence of a normal cell. In some embodiments, the cellular characteristic is present in the same cell in a tissue. In some embodiments, the cellular characteristic is present in at least two different cells in a tissue.In some embodiments, the cellular characteristic is within or between the distance of at least two different cells. In some embodiments, the cellular characteristic is present in a tissue structure in a normal or diseased state. In some embodiments, the disease state comprises a cancer disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state. In some embodiments, the cellular characteristic comprises a change in expression of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in glycosylation pattern of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in enzymatic activity of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in intracellular or intercellular communication. In some embodiments, the cellular characteristic comprises a change in intracellular or intercellular signaling or communication. In some embodiments, the cellular characteristic comprises a change in cell adhesion, mechanics, or migration. In some embodiments, the cellular characteristic comprises tissue localization of an extracellular molecule. In some embodiments, the tissue localization comprises a diseased tissue structure or a tumor microenvironment. In some embodiments, the tissue localization comprises a cancer structure or a tumor microenvironment. In some embodiments, the extracellular molecule signals upon contact with an antibody-based therapy. In some embodiments, the different samples are evaluated for a common cellular characteristic. In some embodiments, the different samples are evaluated for different cellular characteristics. In some embodiments, the method further comprises characterizing the cellular characteristic based on the intensity of the contrast agent in the image. In some embodiments, the cellular characteristic is evaluated after administration of at least one ligand. In some embodiments, the cellular characteristic is evaluated after administration of at least one therapeutic agent or treatment regimen.

[0069] Super-resolution microscopy In some embodiments, the super-resolution microscope comprises a deterministic super-resolution microscope. In some embodiments, the deterministic super-resolution microscopy uses light structuring to determine spatial information below the diffraction limit of a sample. For example, the deterministic super-resolution microscope can use a depletion ring to refine the excitation spot of the microscope. In some embodiments, the super-resolution microscope is a stimulated emission depletion (STED) microscope, a ground state depletion (GSD) microscope, a saturated structured illumination microscopy (SSIM), a super-resolution orthogonal deterministic imaging (SODI), or the like. In some embodiments, the super-resolution microscope is a MINFLUX microscope. For example, the super-resolution technology can be the MINFLUX technology developed by Abberior Instruments. MINFLUX is a super-resolution fluorescence microscopy that can provide resolution in the 1-3 nanometer (nm) range by localizing individual switchable fluorophores with a probing donut-shaped excitation beam (see, e.g., "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells" by Gwosch et al., Nature Methods, vol. 17, pp. 217-220, February 2020). In some embodiments, the super-resolution microscope uses stochastic super-resolution techniques (e.g., is a stochastic super-resolution microscope). In some embodiments, the stochastic super-resolution technique uses chance to improve emitter localization (e.g., by providing a flux such that only one emitter in a region is stochastically activated). In some embodiments, the super-resolution microscope is a stochastic optical reconstruction microscopy (STORM) microscope. In some embodiments, the stochastic super-resolution technique is direct stochastic optical reconstruction microscopy (dSTORM), photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), spectral precision distance microscopy (SPDM), localization microscopy (e.g., single molecule localization microscopy (SMLM)), cryogenic optical localization in three dimensions (COLD), DNA-PAINT, or the like.In some embodiments, super-resolution microscopy or multiplexing methods described in Jungmann et al. (U.S. Pat. No. 10,294,510 and European Patent No. 3027773) are used. In some embodiments, imaging involves tracking antibody-based therapeutics or cell surface receptors over a period of time.

[0070] To quantify extracellular proteins, the observed fluorescence intensity can be normalized using the intrinsic molecular brightness of a single fluorophore attached to the antibody. This can be done by labeling double strands of DNA with a single fluorophore and attaching these at low concentration to a small area of ​​the coverslip used for imaging, ensuring that single molecules are appropriately spaced. During imaging, an image is acquired in the small area using the same parameters as those used to image the cell, and the single molecule intensity from this area is used to calibrate the intensity acquired in the image of the rest of the cell.

[0071] In some embodiments, the super-resolution microscope can be configured to capture time series images of a sample. For example, a sample can be contacted with an antibody-based therapy and imaged over time to obtain a time series of cellular characteristics. In this example, time resolved data on cellular characteristics can provide further data on the mechanism of action of the therapy as well as efficacy. In some embodiments, imaging includes tracking a therapeutic agent, cellular characteristic, or cell surface receptor for a period of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000 milliseconds or more. In some embodiments, imaging includes tracking the therapeutic agent, cell feature, or cell surface receptor for a period of up to about 50,000, 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 milliseconds or less.

[0072] Cell / tissue microarrays In some embodiments, the cells comprise fixed cells. In some embodiments, the cells are fixed in tissue. In some embodiments, the cells are in live tissue. In some embodiments, the cells comprise live cells. In some embodiments, the cells comprise mammalian cells. In some embodiments, the cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises frozen tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises live tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises fixed tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber is formed by a laser microdissection process.

[0073] antibody In some embodiments, the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a single domain antibody from a camelid (VHH), a synthetically derived VHH, a single domain antibody created in a mouse, a shark antibody, an antigen binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and a scFv-Fc. In some embodiments, the antibody based therapy is a bispecific antibody. In some embodiments, the antibody based therapy is a multispecific antibody. In some embodiments, the antibody based therapy comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains. In some embodiments, the antibody based therapy comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains. In some embodiments, the antibody based therapy comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains. In some embodiments, the antibody based therapy is an immune cell engager. In some embodiments, the antibody based therapy comprises an effector domain (e.g., CD3 or CD16A). In some embodiments, the antibody based therapy is for CAR-T cells, myeloid cells, NK cells, or other cell-based therapy. In some embodiments, the antibody based therapy is in a vaccine. In some embodiments, the antibody based therapy is a translated nucleic acid strand or multiple nucleic acid strands. In some embodiments, the antibody based therapy comprises a single chain polypeptide. In some embodiments, the antibody based therapy comprises a homodimer. In some embodiments, the antibody based therapy comprises a heterodimer. In some embodiments, the antibody based therapy is present in picomolar to nanomolar concentrations. In some embodiments, the antibody based therapy is present in sub-picomolar concentrations.

[0074] resolution In some embodiments, the database of imaged cellular characteristics is imaged at or above (e.g., on the order of) the resolution of the super-resolution microscope. For example, a super-resolution microscope with a resolution limit of 5 nanometers can image a sample at a resolution of up to 5 nanometers. In some embodiments, the super-resolution microscope has a resolution of at least about 250, 225, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nanometers or less. In some embodiments, the super-resolution microscope has a resolution of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250 nanometers or more. In some embodiments, the resolution of the super-resolution microscope is in a range defined by any two of the preceding values, for example in some embodiments, the resolution of the super-resolution microscope is between 1 and 50 nanometers.

[0075] In some embodiments, the cellular feature is imaged at a resolution of at least about 100 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 50 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 40 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 30 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 20 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 10 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 9 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 8 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 7 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 6 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 5 nanometers. In some embodiments, the cellular feature is imaged at a resolution of at least about 4 nanometers. In some embodiments, the cellular characteristics are imaged at a resolution of at least about 3 nanometers. In some embodiments, the cellular characteristics are imaged at a resolution of at least about 2 nanometers. In some embodiments, the cellular characteristics are imaged at a resolution of at least about 1 nanometer. In some embodiments, the cellular characteristics are imaged at single molecule resolution. In some embodiments, the cellular characteristics are imaged at single fluorophore resolution.

[0076] High Throughput Format In some embodiments, the cellular features are imaged in a high throughput format. In some embodiments, the low throughput format includes the use of at least one 96 well plate. In some embodiments, the low throughput format includes the use of at least one 192 well plate. In some embodiments, the high throughput format includes the use of at least one 384 well plate. In some embodiments, the high throughput format includes the use of at least one 1536 well plate. In some embodiments, the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels. In some embodiments, the multiple cellular features are imaged at approximately the same time. In some embodiments, the multiple cellular features are imaged sequentially. In some embodiments, the multiple cellular features are imaged at approximately the same time and the rest are imaged sequentially. In some embodiments, the multiple cellular features are on the surface of a multiple number of cells. In some embodiments, the imaging of the cellular features is completed in less than about 5 minutes. In some embodiments, the imaging of the cellular features is performed automatically.

[0077] extracellular molecules In some embodiments, the extracellular molecule is a membrane-bound protein or a membrane-bound ligand. In some embodiments, the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0078] sample In some embodiments, the sample can be tested to determine whether it has cellular characteristics in common with a second sample from the same source (e.g., patient) or a different source. In some embodiments, the evaluation includes one or more of genetic testing (e.g., nucleic acid detection or sequencing), protein testing (e.g., identifying the presence or absence of a given protein), size testing (e.g., determining the size distribution of cells present in the sample), label testing (e.g., determining the presence or absence of one or more labels on the cells), and the like, or any combination thereof. In some embodiments, the sample is evaluated after administration of at least one therapeutic agent or at least one therapeutic regimen (e.g., an antibody-based therapeutic regimen, a small molecule-based therapeutic regimen, a biological-based regimen, and the like). For example, the sample can be contacted with a therapeutic regimen and subsequently evaluated to determine the effect of the regimen and whether the sample is worthy of full characterization. In some embodiments, the sample is evaluated after administration of a natural ligand or a naturally occurring agent or cofactor. In some embodiments, pre-assessment of samples can reduce full characterization of samples that do not exhibit the property of interest (e.g., the effect or lack thereof of a treatment regimen), reducing waste of time and resources.

[0079] In some embodiments, the cells include live cells. For example, the cells can be collected from a living organism and used as collected. In another example, the cells can be obtained from a maintained cell line. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells include mammalian cells. In some embodiments, the mammalian cells are from a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, yak, camel, or human. In some embodiments, the cells include non-living cells. In certain embodiments, the non-living cells are dead cells. In some embodiments, the cells include fixed cells. In some embodiments, the fixed cells are fixed as described elsewhere herein.

[0080] In some embodiments, the disease state cells are derived from tumor cell lines. Examples of tumor cell lines include, but are not limited to, 3T3 cells, A549 cells, HeLa cells, Jurkat cells, and those available from commercial cell line suppliers, such as MilliporeSigma and ThermoFischer.

[0081] In some embodiments, the cells are imaged on a tissue microarray. For example, the cells are part of the tissue microarray and are imaged as such. In some embodiments, the tissue microarray includes multiple tissue samples contained within a single object. In some embodiments, the multiple tissue samples are multiple different tissue samples. In some embodiments, the use of a tissue microarray allows for the screening of a variety of different tissue types in less time than screening each tissue type individually. In some embodiments, the tissue microarray includes one or more of frozen tissues (e.g., tissues currently or previously preserved by freezing), live tissues (e.g., currently living tissues), fixed tissues (e.g., tissues fixed in a matrix or otherwise preserved (e.g., by the use of aldehydes, alcohols, oxidizing agents, etc.), and the like, or any combination thereof. In some embodiments, the tissue microarray is formed by a laser microdissection process. In some embodiments, the tissue microarray is formed by a needle coring process. In some embodiments, the tissue microarray is formed by a microtome process.

[0082] Treatment Design In one aspect, the disclosure provides a method for designing an antibody-based therapeutic comprising imaging cell surface receptors on cells for therapeutic intervention, characterizing a cellular signature of the imaged cell surface receptors, and identifying an antibody format based on the characterization of the cellular signature.

[0083] In certain embodiments, antibodies useful for treating cancer or tumors are disclosed herein. In some embodiments, antibody-based therapy is useful for treating immunological, neurological, antiviral, cardiovascular, and / or autoimmune diseases. Treatment refers to a method that seeks to improve or ameliorate the disease being treated. Treatments for cancer include, but are not limited to, reducing tumor volume, reducing tumor volume growth, increasing progression-free survival, or overall life expectancy. In certain embodiments, treatment achieves remission of the cancer being treated. In certain embodiments, treatment encompasses use as a prophylactic or maintenance administration intended to prevent recurrence or progression of a previously treated cancer or tumor. Treatment in the treatment of bacterial or viral diseases includes, but is not limited to, reducing one or more symptoms associated with the viral or bacterial disease, such as reducing fever, nausea, diarrhea, vomiting, sore throat, cough, runny nose, and / or rash. Treatment of bacterial or viral diseases can reduce the overall level of viruses or bacteria in the body, shorten the period during which an individual can infect others, or shorten the overall disease or recovery time. Treatment of autoimmune or inflammatory diseases can include, but are not limited to, reducing overall or autoantibody levels, or reducing overall or autoimmune responses. Treatment can also be associated with certain symptoms of autoimmune diseases that are associated with excessive antibody or cellular immune responses. Treatment of fibrotic diseases can reduce or delay the appearance of fibrotic tissue or collagen deposition in tissue. Treatment of cardiovascular diseases can increase the signs of cardiovascular health, including lowering blood pressure, reducing atherosclerotic lesions, or increasing cardiac function, as indicated by the ability to pump blood. Those skilled in the art will understand that not all individuals respond equally or at all to treatment administered, but these individuals are considered candidates for treatment.

[0084] In certain embodiments, the single chain polypeptide is for use in the treatment of a viral infection. In certain embodiments, the single chain polypeptide is for use in the treatment of a bacterial infection. In certain embodiments, the single chain polypeptide is for use in the treatment of a solid tumor cancer. In certain embodiments, the single chain polypeptide is for use in the treatment of a hematological cancer. In certain embodiments, the single chain polypeptide is for use in the treatment of an inflammatory disease. In certain embodiments, the single chain polypeptide is for use in the treatment of an autoimmune disease. In certain embodiments, the single chain polypeptide is for use in the treatment of a cardiovascular disease. In certain embodiments, the single chain polypeptide is for use in the treatment of a fibrotic disease.

[0085] The single-chain polypeptide molecules described herein are intended for use as pharmaceutical agents. Administration can be performed by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intralesional, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The dosage regimen is determined by the attending physician and other clinical factors. The dosage for a patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time and route of administration, the type of treatment, general health, and other drugs administered simultaneously.

[0086] An "effective dose" refers to an amount of an active ingredient that is sufficient to affect the course and severity of the disease, leading to the reduction or remission of such pathology. + An "effective dose" useful for treating and / or preventing cancer may be determined using known methods. The maximum tolerated dose (MTD) and maximum responsive dose (MRD) can be determined by established animal and human experimental protocols, as well as the examples provided herein.

[0087] In some embodiments, the administration of the polypeptide herein is carried out at a dosage level determined and intended by a physician.In certain therapeutic applications, the polypeptide is administered to a patient already suffering from cancer in an amount sufficient to cure or at least partially prevent the symptoms of cancer.The amount effective for this use depends on the severity and course of cancer, treatment history, the patient's health condition, weight, and response to drugs, and the judgment of the treating physician.The therapeutically effective amount is optionally determined by methods including, but not limited to, dose escalation clinical trials such as those described in the examples.

[0088] Methods for identifying cancer targets for therapeutic intervention Disclosed herein is a method for identifying cancer targets for therapeutic intervention, comprising: generating a database of cellular characteristics by imaging cellular characteristics of a plurality of extracellular molecules contacted with an antibody using a super-resolution microscope; and selecting, as a cancer target for therapeutic intervention, an extracellular molecule that comprises at least one cancer target cellular characteristic when contacted with the antibody.

[0089] Cell characteristics In some embodiments, the cellular characteristic comprises an image of the extracellular molecule. In some embodiments, the cellular characteristic comprises two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the cellular characteristic comprises an intensity of the extracellular molecule. In some embodiments, the cellular characteristic comprises all or a portion of a molecular fingerprint, molecular signature, or map of the location of the extracellular molecule. In some embodiments, the cellular characteristic comprises all or a portion of a schematic or local representation of the extracellular molecule on the cell or tissue. In some embodiments, the cellular characteristic comprises a proximity between two or more extracellular molecules. In some embodiments, the two or more extracellular molecules are of the same target or different targets. In some embodiments, the cellular characteristic comprises an interaction of at least two extracellular molecules. In some embodiments, the cellular characteristic comprises a distance or distances between two or more extracellular molecules. In some embodiments, the cellular characteristic comprises an individual extracellular molecule or a pair of extracellular molecules. In some embodiments, the individual or pair of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual or pair of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the cellular characteristic comprises individual, paired, or triad extracellular molecules. In some embodiments, the individual, paired, or triad extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the cellular characteristic comprises pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules comprise the same or different extracellular molecules.In some embodiments, pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the cellular characteristic comprises a geometric relationship between two or more extracellular molecules. In some embodiments, the cellular characteristic comprises translocation of an extracellular molecule. In some embodiments, the translocation is internalization or translocation to an extracellular surface. In some embodiments, the cellular characteristic comprises internalization of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in two-dimensional or three-dimensional coordinates of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in clustering of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in clustering of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in clustering of an extracellular molecule. In some embodiments, the cellular characteristic comprises a translocation of at least one cellular receptor. In some embodiments, the cellular characteristic comprises an oligomerization state of an extracellular molecule. In some embodiments, the cellular characteristic comprises a clustering state of an extracellular molecule. In some embodiments, the cellular characteristic comprises the name of a protein that clusters with the extracellular molecule. In some embodiments, the extracellular receptor comprises an extracellular receptor listed in Table 1. In some embodiments, the cellular characteristic is present on a cell in a tissue. In some embodiments, the cellular characteristic is present on a cell in a cancer or on a normal tissue. In some embodiments, the cellular characteristic is present on a normal cell, a cancer cell, or both a normal cell and a cancer cell. In some embodiments, the cellular characteristic is present on a normal cell, a cancer cell, or a cell in a tumor microenvironment. In some embodiments, the cellular characteristic is present on a cell in a tumor microenvironment. In some embodiments, the cell in the tumor microenvironment is selected from a B cell, a T cell, a natural killer (NK) cell, a myeloid cell, a fibroblast, and a pericyte. In some embodiments, the cell in the tumor microenvironment is selected from a stromal cell, an epithelial cell, or an adipocyte. In some embodiments, the cellular characteristic is predictive of the presence of a cancer cell, or a cell in a tumor microenvironment. In some embodiments, the cellular characteristic is predictive of the presence of a normal cell. In some embodiments, the cellular characteristic is present on the same cell in a tissue. In some embodiments, the cellular characteristic is present on at least two different cells in a tissue.In some embodiments, the at least one cellular characteristic is within or between at least two different cellular distances. In some embodiments, the cellular characteristic is present in a tissue structure in a normal or diseased state. In some embodiments, the disease state comprises a cancer disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state. In some embodiments, the cellular characteristic comprises a change in expression of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in glycosylation pattern of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in enzymatic activity of an extracellular molecule. In some embodiments, the cellular characteristic comprises a change in intracellular or intercellular communication. In some embodiments, the cellular characteristic comprises a change in intracellular or intercellular signaling or communication. In some embodiments, the cellular characteristic comprises a change in cell adhesion, mechanics, or migration. In some embodiments, the cellular characteristic comprises tissue localization of an extracellular receptor. In some embodiments, the tissue localization comprises a cancer structure or a tumor microenvironment. In some embodiments, the cellular characteristic comprises localization of at least one extracellular receptor. In some embodiments, the extracellular receptor signals upon contact with the antibody. In some embodiments, the different samples are evaluated for a common cellular characteristic. In some embodiments, the different samples are evaluated for different cellular characteristics. In some embodiments, the method further comprises characterizing the cellular characteristic based on the intensity of the imaging agent in the image. In some embodiments, the cellular characteristic is evaluated after administration of at least one ligand. In some embodiments, the cellular characteristic is evaluated after administration of at least one therapeutic agent or at least one therapeutic regimen. In some embodiments, the cellular characteristic present in diseased cells and absent in non-diseased cells is subsequently used for the development of antibody-based therapies.

[0090] Super-resolution microscope In some embodiments, the super-resolution microscope comprises a deterministic super-resolution microscope. In some embodiments, the deterministic super-resolution microscopy uses light structuring to determine spatial information below the diffraction limit of a sample. For example, the deterministic super-resolution microscope can use a depletion ring to refine the excitation spot of the microscope. In some embodiments, the super-resolution microscope is a stimulated emission depletion (STED) microscope, a ground state depletion (GSD) microscope, a saturated structured illumination microscopy (SSIM), a super-resolution orthogonal deterministic imaging (SODI), or the like. In some embodiments, the super-resolution microscope is a MINFLUX microscope. For example, the super-resolution technology can be the MINFLUX technology developed by Abberior Instruments. MINFLUX is a super-resolution fluorescence microscopy that can provide resolution in the 1-3 nanometer (nm) range by localizing individual switchable fluorophores with a probing donut-shaped excitation beam (see, e.g., "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells" by Gwosch et al., Nature Methods, vol. 17, pp. 217-220, February 2020). In some embodiments, the super-resolution microscope uses stochastic super-resolution techniques (e.g., is a stochastic super-resolution microscope). In some embodiments, the stochastic super-resolution technique uses chance to improve emitter localization (e.g., by providing a flux such that only one emitter in a region is stochastically activated). In some embodiments, the super-resolution microscope is a stochastic optical reconstruction microscopy (STORM) microscope. In some embodiments, the stochastic super-resolution technique is direct stochastic optical reconstruction microscopy (dSTORM), photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), spectral precision distance microscopy (SPDM), localization microscopy (e.g., single molecule localization microscopy (SMLM)), cryogenic optical localization in three dimensions (COLD), DNA-PAINT, or the like.In some embodiments, super-resolution microscopy or multiplexing methods described in Jungmann et al. (U.S. Pat. No. 10,294,510 and European Patent No. 3027773) are used. In some embodiments, the imaging step includes tracking one or more ligands, therapeutic agents, or cell surface receptors over a period of time.

[0091] To quantify extracellular proteins, the observed fluorescence intensity can be normalized using the intrinsic molecular brightness of a single fluorophore attached to the antibody. This can be done by labeling double strands of DNA with a single fluorophore and attaching these at low concentration to a small area of ​​the coverslip used for imaging, ensuring that single molecules are appropriately spaced. During imaging, an image is acquired in the small area using the same parameters as those used to image the cell, and the single molecule intensity from this area is used to calibrate the intensity acquired in the image of the rest of the cell.

[0092] In some embodiments, the super-resolution microscope can be configured to capture time series images of a sample. For example, a sample can be contacted with an antibody and imaged over time to obtain a time series of cellular characteristics. In this example, the time resolved data of the cellular characteristics can provide further data regarding the mechanism of action of the treatment as well as efficacy. In some embodiments, the imaging includes tracking the therapeutic agent, cellular characteristic, or cell surface receptor for a period of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000 milliseconds or more. In some embodiments, imaging includes tracking the therapeutic agent, cell feature, or cell surface receptor for a period of up to about 50,000, 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 milliseconds or less.

[0093] Cell / tissue microarrays In some embodiments, the cells comprise fixed cells. In some embodiments, the cells are fixed in tissue. In some embodiments, the cells are in live tissue. In some embodiments, the cells comprise live cells. In some embodiments, the cells comprise mammalian cells. In some embodiments, the cells are from a tumor cell line. In some embodiments, the cells are from a normal cell line. In some embodiments, the cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises frozen tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises live tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises fixed tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber is formed by a laser microdissection process.

[0094] antibody In some embodiments, the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a single domain antibody from a camelid (VHH), a synthetically derived VHH, a single domain antibody created in a mouse, a shark antibody, an antigen binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and a scFv-Fc. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains. In some embodiments, the antibody is an immune cell engager. In some embodiments, the antibody comprises an effector domain (e.g., CD3 or CD16A). In some embodiments, the antibody based therapy is against CAR-T cells, myeloid cells, NK cells, or other cell based therapy. In some embodiments, the antibody is in a vaccine. In some embodiments, the antibody is a translated nucleic acid strand or multiple nucleic acid strands. In some embodiments, the antibody comprises a single chain polypeptide. In some embodiments, the antibody comprises a homodimer. In some embodiments, the antibody comprises a heterodimer.

[0095] resolution In some embodiments, the database of imaged cellular characteristics is imaged at or above (e.g., on the order of) the resolution of the super-resolution microscope. For example, a super-resolution microscope with a resolution limit of 5 nanometers can image a sample at a resolution of up to 5 nanometers. In some embodiments, the super-resolution microscope has a resolution of at least about 250, 225, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nanometers or less. In some embodiments, the super-resolution microscope has a resolution of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250 nanometers or more. In some embodiments, the resolution of the super-resolution microscope is in a range defined by any two of the preceding values, for example in some embodiments, the resolution of the super-resolution microscope is between 1 and 50 nanometers.

[0096] In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 100 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 50 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 40 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 30 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 20 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 10 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 9 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 8 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 7 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 6 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 5 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 4 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 3 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 2 nanometers. In some embodiments, the database of cell characteristics is imaged at a resolution of at least about 1 nanometer. In some embodiments, the database of cell characteristics is imaged at single molecule resolution. In some embodiments, the database of cell characteristics is imaged at single fluorophore resolution.

[0097] High Throughput Format In some embodiments, the database of cell features is imaged in a high throughput format. In some embodiments, the low throughput format includes the use of at least one 96 well plate. In some embodiments, the high throughput format includes the use of at least one 192 well plate. In some embodiments, the high throughput format includes the use of at least one 384 well plate. In some embodiments, the high throughput format includes the use of at least one 1536 well plate. In some embodiments, the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels. In some embodiments, the multiple cell features are imaged at approximately the same time. In some embodiments, the multiple cell features are imaged sequentially. In some embodiments, the multiple cell features are imaged at approximately the same time and the rest are imaged sequentially. In some embodiments, the multiple cell features are on the surface of the multiple cells. In some embodiments, the imaging of the cell features is performed automatically.

[0098] extracellular molecules In some embodiments, the extracellular molecule is a membrane-bound protein or a membrane-bound ligand. In some embodiments, the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein. In some embodiments, the selected extracellular molecule is present in diseased cells and is not present in non-diseased cells.

[0099] sample In some embodiments, the sample can be tested to determine whether it has cellular characteristics in common with a second sample from the same source (e.g., patient) or a different source. In some embodiments, the evaluation includes one or more of genetic testing (e.g., nucleic acid detection or sequencing), protein testing (e.g., identifying the presence or absence of a given protein), size testing (e.g., determining the size distribution of cells present in the sample), label testing (e.g., determining the presence or absence of one or more labels on the cells), and the like, or any combination thereof. In some embodiments, the sample is evaluated after administration of at least one therapeutic agent or at least one therapeutic regimen (e.g., an antibody-based therapeutic regimen, a small molecule-based therapeutic regimen, a biological-based regimen, and the like). For example, the sample can be contacted with a therapeutic regimen and subsequently evaluated to determine the effect of the regimen and whether the sample is worthy of full characterization. In some embodiments, the sample is evaluated after administration of a natural ligand or a naturally occurring agent or cofactor. In some embodiments, pre-assessment of samples can reduce full characterization of samples that do not exhibit the property of interest (e.g., the effect or lack thereof of a treatment regimen), reducing waste of time and resources.

[0100] In some embodiments, the cells include live cells. For example, the cells can be collected from a living organism and used as collected. In another example, the cells can be obtained from a maintained cell line. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells include mammalian cells. In some embodiments, the mammalian cells are from a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, yak, camel, or human. In some embodiments, the cells include non-living cells. In certain embodiments, the non-living cells are dead cells. In some embodiments, the cells include fixed cells. In some embodiments, the fixed cells are fixed as described elsewhere herein.

[0101] In some embodiments, the disease state cells are derived from tumor cell lines. Examples of tumor cell lines include, but are not limited to, 3T3 cells, A549 cells, HeLa cells, Jurkat cells, and those available from commercial cell line suppliers, such as MilliporeSigma and ThermoFischer.

[0102] In some embodiments, the cells are imaged on a tissue microarray. For example, the cells are part of the tissue microarray and are imaged as such. In some embodiments, the tissue microarray includes multiple tissue samples contained within a single object. In some embodiments, the multiple tissue samples are multiple different tissue samples. In some embodiments, the use of a tissue microarray allows for the screening of a variety of different tissue types in less time than screening each tissue type individually. In some embodiments, the tissue microarray includes one or more of frozen tissues (e.g., tissues currently or previously preserved by freezing), live tissues (e.g., currently living tissues), fixed tissues (e.g., tissues fixed in a matrix or otherwise preserved (e.g., by the use of aldehydes, alcohols, oxidizing agents, etc.), and the like, or any combination thereof. In some embodiments, the tissue microarray is formed by a laser microdissection process. In some embodiments, the tissue microarray is formed by a needle coring process. In some embodiments, the tissue microarray is formed by a microtome process.

[0103] Treatment Design In one aspect, the disclosure provides a method for designing an antibody-based therapeutic comprising imaging cell surface receptors on cells for therapeutic intervention, characterizing a cellular signature of the imaged cell surface receptors, and identifying an antibody format based on the characterization of the cellular signature.

[0104] In certain embodiments, antibodies useful for treating cancer or tumors are disclosed herein. In some embodiments, antibody-based therapy is useful for treating immunological, neurological, antiviral, cardiovascular, and / or autoimmune diseases. Treatment refers to a method that seeks to improve or ameliorate the disease being treated. Treatments for cancer include, but are not limited to, reducing tumor volume, reducing tumor volume growth, increasing progression-free survival, or overall life expectancy. In certain embodiments, treatment achieves remission of the cancer being treated. In certain embodiments, treatment encompasses use as a prophylactic or maintenance administration intended to prevent recurrence or progression of a previously treated cancer or tumor. Treatment in the treatment of bacterial or viral diseases includes, but is not limited to, reducing one or more symptoms associated with the viral or bacterial disease, such as reducing fever, nausea, diarrhea, vomiting, sore throat, cough, runny nose, and / or rash. Treatment of bacterial or viral diseases can reduce the overall level of viruses or bacteria in the body, shorten the period during which an individual can infect others, or shorten the overall disease or recovery time. Treatment of autoimmune or inflammatory diseases can include, but are not limited to, reducing overall or autoantibody levels, or reducing overall or autoimmune responses. Treatment can also be associated with certain symptoms of autoimmune diseases that are associated with excessive antibody or cellular immune responses. Treatment of fibrotic diseases can reduce or delay the appearance of fibrotic tissue or collagen deposition in tissue. Treatment of cardiovascular diseases can increase the signs of cardiovascular health, including lowering blood pressure, reducing atherosclerotic lesions, or increasing cardiac function, as indicated by the ability to pump blood. Those skilled in the art will understand that not all individuals respond equally or at all to treatment administered, but these individuals are considered candidates for treatment.

[0105] In certain embodiments, the single chain polypeptide is for use in the treatment of a viral infection. In certain embodiments, the single chain polypeptide is for use in the treatment of a bacterial infection. In certain embodiments, the single chain polypeptide is for use in the treatment of a solid tumor cancer. In certain embodiments, the single chain polypeptide is for use in the treatment of a hematological cancer. In certain embodiments, the single chain polypeptide is for use in the treatment of an inflammatory disease. In certain embodiments, the single chain polypeptide is for use in the treatment of an autoimmune disease. In certain embodiments, the single chain polypeptide is for use in the treatment of a cardiovascular disease. In certain embodiments, the single chain polypeptide is for use in the treatment of a fibrotic disease.

[0106] The single-chain polypeptide molecules described herein are intended for use as pharmaceutical agents. Administration can be performed by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intralesional, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The dosage regimen is determined by the attending physician and other clinical factors. The dosage for a patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time and route of administration, the type of treatment, general health, and other drugs administered simultaneously.

[0107] An "effective dose" refers to an amount of an active ingredient that is sufficient to affect the course and severity of the disease, leading to the reduction or remission of such pathology. + An "effective dose" useful for treating and / or preventing cancer may be determined using known methods. The maximum tolerated dose (MTD) and maximum responsive dose (MRD) can be determined by established animal and human experimental protocols, as well as the examples provided herein.

[0108] In some embodiments, the administration of the polypeptide herein is carried out at a dosage level determined and intended by a physician.In certain therapeutic applications, the polypeptide is administered to a patient already suffering from cancer in an amount sufficient to cure or at least partially prevent the symptoms of cancer.The amount effective for this use depends on the severity and course of cancer, treatment history, the patient's health condition, weight, and response to drugs, and the judgment of the treating physician.The therapeutically effective amount is optionally determined by methods including, but not limited to, dose escalation clinical trials such as those described in the examples.

[0109] Methods for generating databases of cellular characteristics of diseased and non-diseased cells - Patents.com Disclosed herein is a method for generating a database of cellular characteristics of diseased and non-disease cells, the method comprising the steps of contacting the cells with an antibody and then imaging a cell selected from the diseased and non-disease cells by super-resolution microscopy to provide an image of one or more cellular characteristics of the cell, storing the one or more cellular characteristics of the cell in the database, and repeating the imaging and storing of one or more additional cells selected from the diseased and non-disease cells to generate the database of cellular characteristics of the diseased and non-disease cells.

[0110] In some embodiments, the method further comprises determining a difference between one or more cellular characteristics of the diseased and non-disease cells stored in the database, and developing a therapeutic strategy to target the diseased cells based on the difference between one or more cellular characteristics of the diseased and non-disease cells stored in the database. In some embodiments, the diseased cells include cancer disease cells, immunological disease cells, neurological disease cells, antiviral disease cells, cardiovascular disease cells, or autoimmune disease cells. In some embodiments, the antibody binds to an antigen on the cell surface, and the antigen is associated with the disease. In some embodiments, the antibody is associated with an imaging agent. In some embodiments, the antibody is directly associated with an imaging agent. In some embodiments, the antibody is indirectly associated with an imaging agent. In some embodiments, the method further comprises determining a difference between one or more cellular characteristics of the diseased and non-disease cells stored in the database.

[0111] Localized K d In some embodiments, the method further comprises determining the local affinity (K d ) and correlating it with one or more cellular characteristics of the cells stored in the database; and d and storing a correlation between the one or more cellular properties of the cell in a database. In some embodiments, the method further comprises: d In some embodiments, the method further comprises determining a localized K for binding of the antibody to the second cell. d based on similarity of one or more cellular characteristics of the cell to one or more cellular characteristics of a second cell.

[0112] Cell characteristics In some embodiments, the one or more cellular characteristics include an image of an extracellular molecule. In some embodiments, the one or more cellular characteristics include two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the one or more cellular characteristics include an intensity of the extracellular molecule. In some embodiments, the one or more cellular characteristics include all or a portion of a molecular fingerprint, molecular signature, or map of the location of the extracellular molecule. In some embodiments, the one or more cellular characteristics include all or a portion of a schematic or local representation of the extracellular molecule on a cell or tissue. In some embodiments, the one or more cellular characteristics include a proximity between two or more extracellular molecules. In some embodiments, the two or more extracellular molecules are of the same target or different targets. In some embodiments, the one or more cellular characteristics include an interaction of at least two extracellular molecules. In some embodiments, the one or more cellular characteristics include a distance or distances between two or more extracellular molecules. In some embodiments, the one or more cellular characteristics include individual or paired extracellular molecules. In some embodiments, the individual or paired extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual or paired extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual or paired extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the one or more cellular characteristics include individual, paired, or triad extracellular molecules. In some embodiments, the individual, paired, or triad extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the one or more cellular characteristics include pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules. In some embodiments, pairs, triplets, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with each other.In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules comprise the same or different extracellular molecules. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher clusters of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the one or more cellular characteristics comprise a geometric relationship between two or more extracellular molecules. In some embodiments, the one or more cellular characteristics comprise a translocation of at least one extracellular molecule. In some embodiments, the translocation is internalization or translocation to an extracellular surface. In some embodiments, the one or more cellular characteristics comprise an internalization of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics comprise a change in two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the one or more cellular characteristics comprise a change in clustering of the extracellular molecule. In some embodiments, the one or more cellular characteristics comprise a change in clustering of the extracellular molecule. In some embodiments, the one or more cellular characteristics include an oligomerization state of the extracellular molecule. In some embodiments, the one or more cellular characteristics include a clustering state of the extracellular molecule. In some embodiments, the one or more cellular characteristics include the name of a protein that clusters with the extracellular molecule. In some embodiments, the extracellular molecule is an extracellular molecule listed in Table 1. In some embodiments, the one or more cellular characteristics are present on a cell in a tissue. In some embodiments, the one or more cellular characteristics are present on a cell in a cancer or on a normal tissue. In some embodiments, the one or more cellular characteristics include an oligomerization state of the extracellular molecule. In some embodiments, the one or more cellular characteristics include an oligomerization state of an antigen on the cell surface. In some embodiments, the oligomerization state is selected from dimers, trimers, tetramers, pentamers, and higher number states of oligomers. In some embodiments, the one or more cellular characteristics include a clustering state of the extracellular molecule. In some embodiments, the one or more cellular characteristics include a clustering state of an antigen on the cell surface.In some embodiments, the one or more cellular characteristics include an interaction between two or more extracellular molecules. In some embodiments, the one or more cellular characteristics include tissue localization of the extracellular molecule. In some embodiments, the extracellular molecule is selected from the extracellular molecules listed in Table 1. In some embodiments, the one or more cellular characteristics include translocation of at least one cellular receptor. In some embodiments, the method further includes characterizing the one or more cellular characteristics based on the intensity of the imaging agent in the image.

[0113] antibody In some embodiments, the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a single domain antibody from a camelid (VHH), a synthetically derived VHH, a single domain antibody created in a mouse, a shark antibody, an antigen binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and a scFv-Fc. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains. In some embodiments, the antibody is an immune cell engager. In some embodiments, the antibody comprises an effector domain (e.g., CD3 or CD16A). In some embodiments, the antibody based therapy is against CAR-T cells, myeloid cells, NK cells, or other cell based therapy. In some embodiments, the antibody is in a vaccine. In some embodiments, the antibody is a translated nucleic acid strand or multiple nucleic acid strands. In some embodiments, the antibody comprises a single chain polypeptide. In some embodiments, the antibody comprises a homodimer. In some embodiments, the antibody comprises a heterodimer.

[0114] Super-resolution microscope In some embodiments, the super-resolution microscopy includes stimulated emission depletion (STED) microscopy. In some embodiments, the super-resolution microscopy includes ground state depletion (GSD) microscopy, saturated structured illumination microscopy (SSIM), super-resolution orthogonal deterministic imaging (SODI), and the like. In some embodiments, the super-resolution microscopy includes MINFLUX microscopy. For example, the deterministic super-resolution technology can be MINFLUX technology developed by Abberior Instruments. MINFLUX is a super-resolution fluorescence microscopy that can provide resolution in the range of 1 to 3 nanometers (nm) by localizing individual switchable fluorophores with a probing donut-shaped excitation beam (see, for example, Gwosch et al., "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells," Nature Methods, vol. 17, pp. 217-220, February 2020). In some embodiments, the super-resolution microscope uses a stochastic super-resolution technology (e.g., stochastic super-resolution microscope). In some embodiments, stochastic super-resolution techniques use chance to improve emitter localization (e.g., by providing a flux such that only one emitter in a region is stochastically activated). In some embodiments, the super-resolution microscopy includes stochastic optical reconstruction microscopy (STORM) microscopy. In some embodiments, the stochastic super-resolution technique is direct stochastic optical reconstruction microscopy (dSTORM), photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), spectral precision distance microscopy (SPDM), localization microscopy (e.g., single molecule localization microscopy (SMLM)), cryogenic optical localization in three dimensions (COLD), DNA-PAINT, etc. In some embodiments, the super-resolution microscopy or multiplexing methods described in Jungmann et al. (U.S. Pat. No. 10,294,510 and European Patent No. 3027773) are used. In some embodiments, the imaging includes tracking extracellular molecules of cells over a period of time.

[0115] To quantify extracellular proteins, the observed fluorescence intensity can be normalized using the intrinsic molecular brightness of a single fluorophore attached to the antibody. This can be done by labeling double strands of DNA with a single fluorophore and attaching these at low concentration to a small area of ​​the coverslip used for imaging, ensuring that single molecules are appropriately spaced. During imaging, an image is acquired in the small area using the same parameters as those used to image the cell, and the single molecule intensity from this area is used to calibrate the intensity acquired in the image of the rest of the cell.

[0116] In some embodiments, the super-resolution microscope can be configured to capture time series images of a sample. For example, a sample can be contacted with an antibody-based therapy and imaged over time to obtain a time series of cellular characteristics. In this example, time resolved data on cellular characteristics can provide further data on the mechanism of action of the therapy as well as efficacy. In some embodiments, imaging includes tracking a therapeutic agent, cellular characteristic, or cell surface receptor for a period of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000 milliseconds or more. In some embodiments, imaging includes tracking the therapeutic agent, cell feature, or cell surface receptor for a period of up to about 50,000, 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 milliseconds or less.

[0117] Cell / tissue microarrays In some embodiments, the cells comprise fixed cells. In some embodiments, the cells are fixed in tissue. In some embodiments, the cells are in live tissue. In some embodiments, the cells comprise live cells. In some embodiments, the cells comprise mammalian cells. In some embodiments, the diseased cells are derived from a tumor cell line. In some embodiments, the normal cells are derived from a normal cell line. In some embodiments, the cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises frozen tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises live tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises fixed tissue.

[0118] resolution In some embodiments, imaging is performed at or above (e.g., on the order of) the resolution of the super-resolution microscope. For example, a super-resolution microscope with a resolution limit of 5 nanometers can image a sample at a resolution of up to 5 nanometers. In some embodiments, the super-resolution microscope has a resolution of at least about 250, 225, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nanometers or less. In some embodiments, the super-resolution microscope has a resolution of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250 nanometers or more. In some embodiments, the resolution of the super-resolution microscope is in a range defined by any two of the preceding values, for example in some embodiments, the resolution of the super-resolution microscope is between 1 and 50 nanometers.

[0119] In some embodiments, imaging is performed at a resolution of at least about 100 nanometers. In some embodiments, imaging is performed at a resolution of at least about 50 nanometers. In some embodiments, imaging is performed at a resolution of at least about 40 nanometers. In some embodiments, imaging is performed at a resolution of at least about 30 nanometers. In some embodiments, imaging is performed at a resolution of at least about 20 nanometers. In some embodiments, imaging is performed at a resolution of at least about 10 nanometers. In some embodiments, imaging is performed at a resolution of at least about 5 nanometers. In some embodiments, imaging is performed at a resolution of at least about 2 nanometers. In some embodiments, imaging is performed at a resolution of at least about 1 nanometer. In some embodiments, imaging is performed at single molecule resolution. In some embodiments, imaging is performed at single fluorophore resolution.

[0120] High Throughput Format In some embodiments, imaging is performed in a high-throughput format. In some embodiments, the high-throughput format includes the use of at least one 96-well plate. In some embodiments, the high-throughput format includes the use of at least one 192-well plate. In some embodiments, the high-throughput format includes the use of at least one 384-well plate. In some embodiments, the high-throughput format includes the use of at least one 1536-well plate. In some embodiments, the high-throughput format includes the use of slides, coverslips, or flow cells containing multiple channels.

[0121] extracellular molecules In some embodiments, the extracellular molecule is a membrane-bound protein or a membrane-bound ligand. In some embodiments, the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0122] sample In some embodiments, the sample can be tested to determine whether it has cellular characteristics in common with a second sample from the same source (e.g., patient) or a different source. In some embodiments, the evaluation includes one or more of genetic testing (e.g., nucleic acid detection or sequencing), protein testing (e.g., identifying the presence or absence of a given protein), size testing (e.g., determining the size distribution of cells present in the sample), label testing (e.g., determining the presence or absence of one or more labels on the cells), and the like, or any combination thereof. In some embodiments, the sample is evaluated after administration of at least one therapeutic agent or at least one therapeutic regimen (e.g., an antibody-based therapeutic regimen, a small molecule-based therapeutic regimen, a biological-based regimen, and the like). For example, the sample can be contacted with a therapeutic regimen and subsequently evaluated to determine the effect of the regimen and whether the sample is worthy of full characterization. In some embodiments, the sample is evaluated after administration of a natural ligand or a naturally occurring agent or cofactor. In some embodiments, pre-assessment of samples can reduce full characterization of samples that do not exhibit the property of interest (e.g., the effect or lack thereof of a treatment regimen), reducing waste of time and resources.

[0123] In some embodiments, the cells include live cells. For example, the cells can be collected from a living organism and used as collected. In another example, the cells can be obtained from a maintained cell line. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells include mammalian cells. In some embodiments, the mammalian cells are from a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, yak, camel, or human. In some embodiments, the cells include non-living cells. In certain embodiments, the non-living cells are dead cells. In some embodiments, the cells include fixed cells. In some embodiments, the fixed cells are fixed as described elsewhere herein.

[0124] In some embodiments, the disease state cells are derived from tumor cell lines. Examples of tumor cell lines include, but are not limited to, 3T3 cells, A549 cells, HeLa cells, Jurkat cells, and those available from commercial cell line suppliers, such as MilliporeSigma and ThermoFischer.

[0125] In some embodiments, the cells are imaged on a tissue microarray. For example, the cells are part of the tissue microarray and are imaged as such. In some embodiments, the tissue microarray includes multiple tissue samples contained within a single object. In some embodiments, the multiple tissue samples are multiple different tissue samples. In some embodiments, the use of a tissue microarray allows for the screening of a variety of different tissue types in less time than screening each tissue type individually. In some embodiments, the tissue microarray includes one or more of frozen tissues (e.g., tissues currently or previously preserved by freezing), live tissues (e.g., currently living tissues), fixed tissues (e.g., tissues fixed in a matrix or otherwise preserved (e.g., by the use of aldehydes, alcohols, oxidizing agents, etc.), and the like, or any combination thereof. In some embodiments, the tissue microarray is formed by a laser microdissection process. In some embodiments, the tissue microarray is formed by a needle coring process. In some embodiments, the tissue microarray is formed by a microtome process.

[0126] Method for characterizing a cell or tissue sample Disclosed herein is a method of characterizing a cell or tissue sample, the method comprising: contacting the cell or tissue sample with an antibody that binds to at least one extracellular molecule on the cell or tissue sample and that is associated with an imaging agent detectable by super-resolution microscopy; imaging the cell or tissue sample by super-resolution microscopy after contacting the cell or tissue sample with the antibody to provide an image of the at least one extracellular molecule on a cell surface of the cell or tissue sample; and characterizing one or more cellular properties of the cell or tissue sample based on the image of the at least one extracellular molecule on the cell or tissue sample.

[0127] In some embodiments, the method further comprises providing a multiplexed image of the cell or tissue sample by i) contacting the cell or tissue sample with a plurality of antibodies, each associated with an imaging agent, and ii) imaging the cell or tissue sample by super-resolution microscopy after contacting the cell or tissue sample with each of the plurality of antibodies, and further comprises characterizing one or more cellular properties of the cell or tissue sample based on the multiplexed image.

[0128] In some embodiments, the imaging agent is a fluorophore. In some embodiments, the imaging agent is directly or indirectly bound to a fluorophore. In some embodiments, the method further comprises characterizing one or more cellular properties based on the two-dimensional or three-dimensional coordinates of at least one extracellular molecule or the intensity of the imaging agent in the image. In some embodiments, the method further comprises characterizing one or more cellular properties based on the intensity of the imaging agent in the image. In some embodiments, the at least one cellular property is evaluated after administration of at least one ligand. In some embodiments, the at least one cellular property is evaluated after administration of at least one therapeutic agent or treatment regimen.

[0129] In some embodiments, the method further comprises determining which of the one or more cellular characteristics are matched on the cancer cell line for one or more indications. In some embodiments, the method further comprises determining which of the one or more cellular characteristics are unique to the cancer cell line for one or more indications. In some embodiments, the method further comprises determining which of the one or more cellular characteristics are matched on non-disease state cells. In some embodiments, the method further comprises determining which of the one or more cellular characteristics are different on the cancer cell and the non-disease state cells. In some embodiments, the method further comprises determining which of the one or more cellular characteristics are the same on the cancer cell and the non-disease state cells. In some embodiments, the method further comprises determining which of the one or more cellular characteristics are matched on the cancer cell. In some embodiments, the method further comprises determining which of the one or more cellular characteristics are matched on the cancer cell between patients in one or more indications. In some embodiments, the method further comprises determining which of the one or more cellular characteristics predict clinical toxicity. In some embodiments, the method further comprises detecting the binding form of the antibody based on the image.

[0130] Cell characteristics In some embodiments, the one or more cellular characteristics include clustering of at least one extracellular molecule. In some embodiments, the clustered extracellular molecules are functional and involved in signal transduction, internalization, motility, and / or enzymatic activity. In some embodiments, the clustered extracellular molecules are non-functional and aggregated or unable to be internalized or shed. In some embodiments, the one or more cellular characteristics are associated with a single extracellular molecule on a cell or tissue sample. In some embodiments, the one or more cellular characteristics are associated with an extracellular molecule pair on a cell or tissue sample. In some embodiments, the one or more cellular characteristics are associated with a triplet, tetrad, quintuplet, or other higher order cluster of extracellular molecules on a cell or tissue sample. In some embodiments, the single extracellular molecule, extracellular molecule pair, triplet, tetrad, quintuplet, or other higher order cluster of extracellular molecules includes one or more other proteins. In some embodiments, the extracellular molecule pair, triplet, tetrad, quintuplet, or other higher order extracellular molecule cluster is composed of a single extracellular molecule or multiple extracellular molecules, respectively. In some embodiments, the one or more cellular characteristics include one or more cellular characteristics of a multiplicity of extracellular molecules. In some embodiments, the one or more cellular characteristics include the presence of at least one extracellular molecule on the cell or tissue sample. In some embodiments, the one or more cellular characteristics include the absence of at least one extracellular molecule on the cell or tissue sample. In some embodiments, the one or more cellular characteristics include the abundance of at least one extracellular molecule on the cell or tissue sample. In some embodiments, the one or more cellular characteristics include the distribution of at least one extracellular molecule on the cell or tissue sample. In some embodiments, the one or more cellular characteristics include the density of at least one extracellular molecule on the cell or tissue sample.

[0131] In some embodiments, the one or more cellular characteristics include an oligomeric state of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics include a heteromeric state of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics include one or more nearest neighbors of at least one extracellular molecule. In some embodiments, the one or more nearest neighbors are selected from proteins, extracellular receptors, antigens, carbohydrates, and lipids. In some embodiments, the one or more cellular characteristics include a distance between separate molecules of a single extracellular molecule. In some embodiments, the one or more cellular characteristics include a pairwise distance between extracellular molecules of a pair of extracellular molecules. In some embodiments, the one or more cellular characteristics include a pairwise distance between extracellular molecules of an extracellular molecule triad, an extracellular molecule tetrad, an extracellular molecule quintuplet, or other higher order extracellular molecule cluster. In some embodiments, the one or more cellular characteristics include a lateral, anterior, or anterior movement of at least one extracellular molecule.

[0132] In some embodiments, the one or more cellular characteristics include one or more cellular characteristics on the same cell and / or between different cells. In some embodiments, the one or more cellular characteristics include an intercellular interaction between at least one extracellular molecule and one or more extracellular proteins on separate cells. In some embodiments, the one or more cellular characteristics include an absence of an intercellular interaction between at least one extracellular molecule and one or more extracellular proteins on separate cells. In some embodiments, the intercellular interaction includes an oligomer or cluster of two or more extracellular proteins on separate cells. In some embodiments, the intracellular interaction is selected from a tumor cell-tumor cell interaction, a tumor cell-tumor microenvironment cell interaction, and a tumor microenvironment cell-tumor microenvironment cell interaction. In some embodiments, the cells in the tumor microenvironment are selected from B cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and pericytes. In some embodiments, the cells in the tumor microenvironment are selected from stromal cells, epithelial cells, or adipocytes.

[0133] In some embodiments, the one or more cellular characteristics predict the presence of a cancer cell, or a cell in a tumor microenvironment. In some embodiments, the one or more cellular characteristics predict the presence of a normal cell. In some embodiments, the one or more cellular characteristics are indicative of a signaling event. In some embodiments, the one or more cellular characteristics define a molecular signature, a cellular signature, or a tissue sample signature. In some embodiments, the one or more cellular characteristics include an image of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics include two-dimensional or three-dimensional coordinates of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics include an intensity of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics include all or a portion of a molecular fingerprint, molecular signature, or map of the location of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics include all or a portion of a schematic or local representation of the extracellular molecule on a cell or tissue. In some embodiments, the one or more cellular characteristics include a proximity between two or more extracellular molecules. In some embodiments, the two or more extracellular molecules are of the same extracellular molecule or different extracellular molecules. In some embodiments, the one or more cellular characteristics include an interaction of at least two extracellular molecules. In some embodiments, the one or more cellular characteristics include a distance or distances between two or more extracellular molecules. In some embodiments, the one or more cellular characteristics include individual extracellular molecules or pairs of extracellular molecules. In some embodiments, the individual or pairs of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the individual or pairs of extracellular molecules are within a distance of about 1 nanometer of each other. In some embodiments, the individual or pairs of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers of each other. In some embodiments, the one or more cellular characteristics include individual, pairs, or triplet extracellular molecules. In some embodiments, the individual, pairs, or triplet extracellular molecules are adjacent to or in contact with each other.In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the one or more cellular characteristics include pairs, triplets, tetrads, quintuplets, or higher-order clusters of extracellular molecules. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher-order clusters of extracellular molecules are adjacent to or in contact with each other. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher-order clusters of extracellular molecules include the same or different extracellular molecules. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher-order clusters of extracellular molecules are within a distance of about 1 nanometer from each other. In some embodiments, the pairs, triplets, tetrads, quintuplets, or higher-order clusters of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other. In some embodiments, the one or more cellular characteristics include a geometric relationship between two or more extracellular molecules.

[0134] In some embodiments, the one or more cellular characteristics include the translocation of at least one extracellular molecule. In some embodiments, the translocation is internalization or translocation to the extracellular surface. In some embodiments, the at least one cellular characteristic includes the internalization of at least one extracellular molecule. In some embodiments, the one or more cellular characteristics include a change in the two-dimensional or three-dimensional coordinates of the extracellular molecule. In some embodiments, the one or more cellular characteristics include a change in the clustering of the extracellular molecule. In some embodiments, the one or more cellular characteristics include a change in the clustering of the extracellular molecule. In some embodiments, the one or more cellular characteristics include an oligomerization state of the extracellular molecule. In some embodiments, the one or more cellular characteristics include a clustering state of the extracellular molecule. In some embodiments, the one or more cellular characteristics include the name of a protein that clusters with the extracellular molecule. In some embodiments, the extracellular molecule is an extracellular molecule listed in Table 1.

[0135] In some embodiments, the method further comprises repeating the method on one or more additional cell or tissue samples comprising diseased or non-disease cells, and storing the one or more cellular characteristics of each of the cell or tissue samples in a database to provide a database of cellular characteristics of diseased and non-disease cells.

[0136] Localized K d In some embodiments, the method further comprises determining the local affinity (K d ) to one or more cellular characteristics of the cell or tissue sample stored in a database; d and storing a correlation between the one or more cellular characteristics of the cell or tissue sample in a database. In some embodiments, the method further comprises: d In some embodiments, the method further comprises determining a localized K for binding of the antibody to the second cell or tissue sample. d based on similarity of one or more cellular characteristics of the cell or tissue sample to one or more cellular characteristics of a second cell or tissue sample.

[0137] antibody In some embodiments, the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a single domain antibody from a camelid (VHH), a synthetically derived VHH, a single domain antibody created in a mouse, a shark antibody, an antigen binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and a scFv-Fc. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains. In some embodiments, the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains. In some embodiments, the antibody is an immune cell engager. In some embodiments, the antibody comprises an effector domain (e.g., CD3 or CD16A). In some embodiments, the antibody based therapy is against CAR-T cells, myeloid cells, NK cells, or other cell based therapy. In some embodiments, the antibody is in a vaccine. In some embodiments, the antibody is a translated nucleic acid strand or multiple nucleic acid strands. In some embodiments, the antibody comprises a single chain polypeptide. In some embodiments, the antibody comprises a homodimer. In some embodiments, the antibody comprises a heterodimer.

[0138] Super-resolution microscope In some embodiments, the super-resolution microscope is a deterministic super-resolution microscope. In some embodiments, the deterministic super-resolution microscopy uses light structuring to determine spatial information below the diffraction limit of the sample. For example, the deterministic super-resolution microscope can use a depletion ring to refine the excitation spot of the microscope. In some embodiments, the super-resolution microscopy includes stimulated emission depletion (STED) microscopy. In some embodiments, the super-resolution microscopy includes ground state depletion (GSD) microscopy. In some embodiments, the deterministic super-resolution technique is saturated structured illumination microscopy (SSIM), super-resolution orthogonal deterministic imaging (SODI), etc. In some embodiments, the super-resolution microscopy includes MINFLUX microscopy. For example, the deterministic super-resolution technique can be the MINFLUX technique developed by Abberior Instruments. MINFLUX is a super-resolution fluorescence microscopy that can provide resolution in the 1-3 nanometer (nm) range by localizing individual switchable fluorophores with a probing donut-shaped excitation beam (see, e.g., "MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells" by Gwosch et al., Nature Methods, vol. 17, pp. 217-220, February 2020). In some embodiments, the super-resolution microscope uses stochastic super-resolution techniques (e.g., is a stochastic super-resolution microscope). In some embodiments, the stochastic super-resolution technique uses chance to improve emitter localization (e.g., by providing a flux such that only one emitter in a region is stochastically activated). In some embodiments, the super-resolution microscopy includes stochastic optical reconstruction microscopy (STORM) microscopy. In some embodiments, the stochastic super-resolution technique is direct stochastic optical reconstruction microscopy (dSTORM), photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), spectral precision distance microscopy (SPDM), localization microscopy (e.g., single molecule localization microscopy (SMLM)), cryogenic optical localization in three dimensions (COLD), DNA-PAINT, or the like.In some embodiments, the super-resolution microscopy or multiplexing methods described in Jungmann et al. (US Pat. No. 10,294,510 and EP 3027773) are used.

[0139] To quantify extracellular proteins, the observed fluorescence intensity can be normalized using the intrinsic molecular brightness of a single fluorophore attached to the antibody. This can be done by labeling double strands of DNA with a single fluorophore and attaching these at low concentration to a small area of ​​the coverslip used for imaging, ensuring that single molecules are appropriately spaced. During imaging, an image is acquired in the small area using the same parameters as those used to image the cell, and the single molecule intensity from this area is used to calibrate the intensity acquired in the image of the rest of the cell.

[0140] In some embodiments, the super-resolution microscope can be configured to capture time series images of a sample. For example, a sample can be contacted with an antibody-based therapy and imaged over time to obtain a time series of cellular characteristics. In this example, time resolved data on cellular characteristics can provide further data on the mechanism of action of the therapy as well as efficacy. In some embodiments, imaging includes tracking a therapeutic agent, cellular characteristic, or cell surface receptor for a period of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000, 50,000 milliseconds or more. In some embodiments, imaging includes tracking the therapeutic agent, cell feature, or cell surface receptor for a period of up to about 50,000, 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 milliseconds or less.

[0141] Cell / tissue microarrays In some embodiments, the cell or tissue comprises a fixed cell. In some embodiments, the cell or tissue comprises a live cell. In some embodiments, the cell or tissue comprises a mammalian cell. In some embodiments, imaging the cell or tissue comprises imaging the cell on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises frozen tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises live tissue. In some embodiments, the tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber comprises fixed tissue.

[0142] resolution In some embodiments, the database of imaged cellular characteristics is imaged at or above (e.g., on the order of) the resolution of the super-resolution microscope. For example, a super-resolution microscope with a resolution limit of 5 nanometers can image a sample at a resolution of up to 5 nanometers. In some embodiments, the super-resolution microscope has a resolution of at least about 250, 225, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nanometers or less. In some embodiments, the super-resolution microscope has a resolution of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250 nanometers or more. In some embodiments, the resolution of the super-resolution microscope is in a range defined by any two of the preceding values, for example in some embodiments, the resolution of the super-resolution microscope is between 1 and 50 nanometers.

[0143] In some embodiments, imaging is performed at a resolution of at least about 100 nanometers. In some embodiments, imaging is performed at a resolution of at least about 50 nanometers. In some embodiments, imaging is performed at a resolution of at least about 40 nanometers. In some embodiments, imaging is performed at a resolution of at least about 30 nanometers. In some embodiments, imaging is performed at a resolution of at least about 20 nanometers. In some embodiments, imaging is performed at a resolution of at least about 10 nanometers. In some embodiments, imaging is performed at a resolution of at least about 5 nanometers. In some embodiments, imaging is performed at a resolution of at least about 2 nanometers. In some embodiments, imaging is performed at a resolution of at least about 1 nanometer. In some embodiments, imaging is performed at single molecule resolution. In some embodiments, imaging is performed at single fluorophore resolution.

[0144] High Throughput Format In some embodiments, imaging is performed in a high-throughput format. In some embodiments, the high-throughput format includes the use of at least one 96-well plate. In some embodiments, the high-throughput format includes the use of at least one 192-well plate. In some embodiments, the high-throughput format includes the use of at least one 384-well plate. In some embodiments, the high-throughput format includes the use of at least one 1536-well plate. In some embodiments, the high-throughput format includes the use of slides, coverslips, or flow cells containing multiple channels.

[0145] extracellular molecules In some embodiments, the at least one extracellular molecule is a membrane-bound protein or a membrane-bound ligand. In some embodiments, the at least one extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, and a structural protein.

[0146] sample In some embodiments, the sample can be tested to determine whether it has cellular characteristics in common with a second sample from the same source (e.g., patient) or a different source. In some embodiments, the evaluation includes one or more of genetic testing (e.g., nucleic acid detection or sequencing), protein testing (e.g., identifying the presence or absence of a given protein), size testing (e.g., determining the size distribution of cells present in the sample), label testing (e.g., determining the presence or absence of one or more labels on the cells), and the like, or any combination thereof. In some embodiments, the sample is evaluated after administration of at least one therapeutic agent or at least one therapeutic regimen (e.g., an antibody-based therapeutic regimen, a small molecule-based therapeutic regimen, a biological-based regimen, and the like). For example, the sample can be contacted with a therapeutic regimen and subsequently evaluated to determine the effect of the regimen and whether the sample is worthy of full characterization. In some embodiments, the sample is evaluated after administration of a natural ligand or a naturally occurring agent or cofactor. In some embodiments, pre-assessment of samples can reduce full characterization of samples that do not exhibit the property of interest (e.g., the effect or lack thereof of a treatment regimen), reducing waste of time and resources.

[0147] In some embodiments, the cells include live cells. For example, the cells can be collected from a living organism and used as collected. In another example, the cells can be obtained from a maintained cell line. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells include mammalian cells. In some embodiments, the mammalian cells are from a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, yak, camel, or human. In some embodiments, the cells include non-living cells. In certain embodiments, the non-living cells are dead cells. In some embodiments, the cells include fixed cells. In some embodiments, the fixed cells are fixed as described elsewhere herein.

[0148] In some embodiments, the disease state cells are derived from tumor cell lines. Examples of tumor cell lines include, but are not limited to, 3T3 cells, A549 cells, HeLa cells, Jurkat cells, and those available from commercial cell line suppliers, such as MilliporeSigma and ThermoFischer.

[0149] In some embodiments, the cells are imaged on a tissue microarray. For example, the cells are part of the tissue microarray and are imaged as such. In some embodiments, the tissue microarray includes multiple tissue samples contained within a single object. In some embodiments, the multiple tissue samples are multiple different tissue samples. In some embodiments, the use of a tissue microarray allows for the screening of a variety of different tissue types in less time than screening each tissue type individually. In some embodiments, the tissue microarray includes one or more of frozen tissues (e.g., tissues currently or previously preserved by freezing), live tissues (e.g., currently living tissues), fixed tissues (e.g., tissues fixed in a matrix or otherwise preserved (e.g., by the use of aldehydes, alcohols, oxidizing agents, etc.), and the like, or any combination thereof. In some embodiments, the tissue microarray is formed by a laser microdissection process. In some embodiments, the tissue microarray is formed by a needle coring process. In some embodiments, the tissue microarray is formed by a microtome process.

[0150] Computer Systems The present disclosure provides a computer system programmed to implement the methods of the present disclosure. In some embodiments, the computer system of the present disclosure may implement a script in Imagej or other similar image processing program. FIG. 1 shows a computer system (101) programmed or otherwise configured to instruct a super-resolution microscope. The computer system (101) may regulate various aspects of the present disclosure, such as, for example, the operation of the microscope and the analysis of data acquired by the microscope. The computer system (101) may be a user's electronic device or a computer system located remotely relative to the electronic device. The electronic device may be a mobile electronic device.

[0151] The computer system (101) comprises a central processing unit (CPU, also referred to herein as "processor" and "computer processor") (105), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system (101) also comprises memory or memory locations (110) (e.g., random access memory, read-only memory, flash memory), electronic storage (115) (e.g., hard disk), communication interfaces (120) (e.g., network adapters) for communicating with one or more other systems, and peripheral devices (125), such as cache, other memory, data storage, and / or electronic display adapters. The memory (110), storage (115), interface (120), and peripheral devices (125) communicate with the CPU (105) via a communication bus (solid line), such as a motherboard. The storage (115) may be a data storage device (i.e., a data repository) for storing data. The computer system (101) may be operatively coupled to a computer network ("network") (130) with the aid of a communication interface (120). The network (130) may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network (130) may possibly be a telecommunications and / or data network. The network (130) may comprise one or more computer servers, which may enable distributed computing, such as cloud computing. The network (130) may possibly implement a peer-to-peer network with the aid of the computer system (101), thereby enabling devices to be coupled to the computer system (101) to behave as clients or servers.

[0152] The CPU (105) may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory (110). The instructions may be directed to the CPU (105), which may then be programmed or otherwise configured to perform the methods of the present disclosure. Examples of operations performed by the CPU (105) may include fetch, decode, execute, and writeback.

[0153] The CPU (105) may be part of a circuit, such as an integrated circuit. One or more other components of the system (101) may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0154] The storage device (115) can store files such as drivers, libraries, and saved programs. The storage device (115) can store user data, such as user preferences and user programs. The computer system (101) can optionally include one or more additional data storage devices external to the computer system (101), such as located on a remote server that communicates with the computer system (101) via an intranet or the Internet.

[0155] The computer system (101) can communicate with one or more remote computer systems via the network (130). For example, the computer system (101) can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad®, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone®, Android-enabled device, Blackberry®), or a personal digital assistant. A user can access the computer system (101) via the network (130). For example, the computer instructs a cloud (e.g., remote) computing resource to execute an image processing program. In this example, the cloud computing resource includes one or more GPUs to improve the processing speed of the image.

[0156] The methods described herein can be implemented by a machine (e.g., a computer processor) stored in an electronic storage location of the computer system (101), such as, for example, memory (110) or electronic storage (115). Machine executable or machine readable code can be provided in the form of software. In use, the code can be executed by the processor (105). In some cases, the code can be retrieved from storage (115) and stored in memory (110) for easy access by the processor (105). In some situations, the electronic storage (115) can be eliminated and the machine executable instructions are stored in memory (110).

[0157] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled at run time. The code may be provided in a programming language that can be selected to enable execution of the code in a pre-compiled or as-compiled manner. The code may include pre-compiled libraries. For example, the code may include a scripting language configured to utilize compiled libraries.

[0158] Aspects of the systems and methods provided herein, such as the computer system (101), may be embodied in programming. Various aspects of the technology may be considered as "products" or "articles of manufacture" in the form of machine (or processor) executable code and / or associated data, typically executed or embodied in some type of machine-readable medium. The machine executable code may be stored in electronic storage such as memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media may include any or all of the tangible memory of a computer or processor, or associated modules such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory storage at any time in software programming. All or part of the software may be constantly communicated via the Internet or various other telecommunications networks. Such communication may, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, another type of media that may carry software elements includes light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, via 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, etc., may also be considered as media that carry software. As used herein, unless limited 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.

[0159] Thus, a machine-readable medium such as a computer executable code may take many forms, including but not limited to a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices in any computer that may be used to implement, for example, the databases shown in the drawings. Volatile storage media include dynamic memories, such as the main memory of such a computer platform. Specific transmission media include coaxial cables, copper wire, and optical fibers with 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. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards paper tape, any other physical storage media having a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves carrying data or instructions, cables or links carrying such carrier waves, 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.

[0160] The computer system (101) may include or communicate with an electronic display (135) that includes a user interface (UI) (140), for example, to preview the field of view of the super-resolution microscope. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0161] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented by software when executed by the central processing unit (105). The algorithms can, for example, identify cellular characteristics in an image.

[0162] Embodiment Embodiment 1 includes a method of generating a database comprising cellular characteristics from diseased and non-diseased cells, the method comprising imaging a sample contacted with an antibody using a super-resolution microscope, generating a database comprising at least one cellular characteristic of cells in the sample, and determining whether the at least one cellular characteristic is indicative of a diseased or non-diseased cell.

[0163] Embodiment 2 includes the method of embodiment 1, wherein the at least one cellular characteristic includes an image of an extracellular molecule.

[0164] Embodiment 3 includes the method of embodiment 1 or 2, wherein the at least one cellular characteristic includes two- or three-dimensional coordinates of an extracellular molecule.

[0165] Embodiment 4 includes the method of any one of embodiments 1 to 3, wherein the at least one cellular characteristic includes the intensity of an extracellular molecule.

[0166] Embodiment 5 includes the method of any one of embodiments 1 to 4, wherein the at least one cellular characteristic comprises all or a portion of a molecular fingerprint, molecular signature, or map of the location of extracellular molecules.

[0167] Embodiment 6 includes the method of any one of embodiments 1 to 5, wherein the at least one cellular characteristic comprises all or a portion of a molecular fingerprint, molecular signature, or map of the location of extracellular molecules.

[0168] Embodiment 7 includes the method of any one of embodiments 1 to 6, wherein the at least one cellular characteristic includes proximity between two or more extracellular molecules.

[0169] Embodiment 8 includes the method of embodiment 7, wherein the two or more extracellular molecules are for the same target or different targets.

[0170] Embodiment 9 includes the method of any one of embodiments 1 to 8, wherein the at least one cellular characteristic includes an interaction of at least two extracellular molecules.

[0171] Embodiment 10 includes the method of any one of embodiments 1 to 9, wherein the at least one cellular characteristic includes a distance or distances between two or more extracellular molecules.

[0172] Embodiment 11 includes the method of any one of embodiments 1 to 10, wherein the at least one cellular characteristic includes an individual or pair of extracellular molecules.

[0173] Embodiment 12 includes the method of embodiment 11, wherein the individual or paired extracellular molecules are adjacent to or in contact with each other.

[0174]

[0031] Embodiment 13 includes the method of embodiment 11 or 12, wherein the individual or paired extracellular molecules are within a distance of about 1 nanometer from each other.

[0175]

[0031] Embodiment 14 includes the method of embodiment 11 or 12, wherein the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other.

[0176] Embodiment 15 includes the method of any one of embodiments 1 to 14, wherein the at least one cellular characteristic includes an individual extracellular molecule, or a pair or triplet of extracellular molecules.

[0177] Embodiment 16 includes the method of embodiment 15, wherein the individual extracellular molecules, pairs of extracellular molecules, or triplet of extracellular molecules are adjacent to or in contact with each other.

[0178] Embodiment 17 includes the method of embodiment 15, wherein the individual extracellular molecules, pairs of extracellular molecules, or triplet of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other.

[0179] Embodiment 18 includes the method of any one of embodiments 1 to 17, wherein at least one cellular characteristic includes a pair, triplet, tetrad, quintuplet, or higher order cluster of extracellular molecules.

[0180] Embodiment 19 includes the method of embodiment 18, wherein the pairs, triads, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with one another.

[0181] Embodiment 20 includes the method of embodiment 18, wherein the pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules comprise the same or different molecules.

[0182] Embodiment 21 includes the method of any one of embodiments 1 to 20, wherein the at least one cellular characteristic includes a geometric relationship between two or more extracellular molecules.

[0183] Embodiment 22 includes the method of any one of embodiments 1 to 21, wherein the at least one cellular characteristic includes the translocation of at least two extracellular molecules.

[0184] Embodiment 23 includes the method of embodiment 22, wherein the transfer is internalization or transfer to the extracellular surface.

[0185] Embodiment 24 includes the method of any one of embodiments 1 to 23, wherein the at least one cellular characteristic includes internalization of at least two extracellular molecules.

[0186] Embodiment 25 includes the method of any one of embodiments 1 to 24, wherein the at least one cellular characteristic includes a change in two-dimensional or three-dimensional coordinates between extracellular molecules.

[0187] Embodiment 26 includes the method of any one of embodiments 1 to 25, wherein the at least one cellular characteristic includes a change in two-dimensional or three-dimensional coordinates between extracellular molecules.

[0188] Embodiment 27 includes the method of any one of embodiments 1 to 26, wherein the at least one cellular characteristic comprises a change in clustering between extracellular molecules.

[0189] Embodiment 28 includes the method of any one of embodiments 1 to 27, wherein the at least one cellular characteristic includes an oligomerization state of an extracellular molecule.

[0190] Embodiment 29 includes the method of any one of embodiments 1 to 28, wherein the at least one cellular characteristic includes a clustering state of extracellular molecules.

[0191] Embodiment 30 includes the method of any one of embodiments 1 to 29, wherein the at least one cellular characteristic includes the name of a protein that clusters with the extracellular molecule.

[0192] Embodiment 31 includes the method of any one of embodiments 2 to 30, wherein the extracellular molecule is an extracellular molecule listed in Table 1.

[0193] Embodiment 32 includes the method of any one of embodiments 1 to 31, wherein at least one cellular characteristic is present in cells in a tissue.

[0194] Embodiment 33 includes the method of any one of embodiments 1 to 31, wherein at least one cellular characteristic is present in cells in a cancer or on normal tissue.

[0195] Embodiment 34 includes the method of any one of embodiments 1 to 31, wherein at least one cellular characteristic is present in normal cells, cancer cells, or both normal cells and cancer cells.

[0196] Embodiment 35 includes the method of any one of embodiments 1 to 31, wherein the at least one cellular characteristic is present in a normal cell, a cancer cell, or a cell in the tumor microenvironment.

[0197] Embodiment 36 includes the method of any one of embodiments 1 to 31, wherein the at least one cellular characteristic is present in cells in the tumor microenvironment.

[0198] Embodiment 37 includes the method of embodiment 35 or 36, wherein the cells in the tumor microenvironment are selected from B cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and pericytes.

[0199] Embodiment 38 includes the method of embodiment 35 or 36, wherein the cells in the tumor microenvironment are selected from stromal cells, epithelial cells, or adipocytes.

[0200] Embodiment 39 includes the method of any one of embodiments 1 to 38, wherein at least one cellular characteristic is predictive of the presence of a cancer cell or a cell in the tumor microenvironment.

[0201] Embodiment 40 includes the method of any one of embodiments 1 to 38, wherein at least one cellular characteristic is predictive of the presence of a normal cell.

[0202] Embodiment 41 includes the method of any one of embodiments 1 to 40, wherein at least one cellular characteristic is present in the same cells in a tissue.

[0203] Embodiment 42 includes the method of any one of embodiments 1 to 40, wherein at least one cellular characteristic is present in at least two different cells in the tissue.

[0204] Embodiment 43 includes the method of embodiment 42, wherein at least one cellular characteristic is within or between the distances of at least two different cells.

[0205] Embodiment 44 includes the method of any one of embodiments 1 to 43, wherein the at least one cellular characteristic is present in a tissue structure in a normal or diseased state.

[0206] Embodiment 45 includes the method of embodiment 44, wherein the disease state includes a cancer disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state.

[0207] Embodiment 46 includes the method of embodiment 44 or 45, wherein the tissue structure comprises epithelium, duct, or blood vessel.

[0208] Embodiment 47 includes the method of any one of embodiments 1 to 46, wherein the at least one cellular characteristic includes a change in expression of an extracellular molecule.

[0209] Embodiment 48 includes the method of any one of embodiments 1 to 47, wherein the at least one cellular characteristic comprises an alteration in the glycosylation pattern of an extracellular molecule.

[0210] Embodiment 49 includes the method of any one of embodiments 1 to 48, wherein the at least one cellular characteristic includes a change in an enzymatic activity of an extracellular molecule.

[0211] Embodiment 50 includes the method of any one of embodiments 1 to 49, wherein the at least one cellular characteristic includes a change in intracellular or intercellular communication.

[0212] Embodiment 51 includes the method of any one of embodiments 1 to 49, wherein the at least one cellular characteristic includes a change in signaling or communication within or between cells.

[0213] Embodiment 52 includes the method of any one of embodiments 1 to 51, wherein the at least one cellular characteristic includes a change in cell adhesion, mechanics, or migration.

[0214] Embodiment 53 includes the method of any one of embodiments 1 to 52, wherein the at least one cellular characteristic includes tissue localization of an extracellular molecule.

[0215] Embodiment 54 includes the method of embodiment 53, wherein the tissue localization includes diseased tissue or the tumor microenvironment.

[0216] Embodiment 55 includes the method of embodiment 53, wherein the tissue localization includes a cancer structure or a tumor microenvironment.

[0217] Embodiment 56 includes the method of any one of embodiments 2-31, 47-49, and 53, wherein the extracellular molecule is internalized upon contact with the antibody.

[0218] Embodiment 57 includes the method of any one of embodiments 1 to 56, wherein different samples are assessed for a common cellular characteristic.

[0219] Embodiment 58 includes the method of any one of embodiments 1 to 57, wherein different samples are evaluated for different cellular characteristics.

[0220] Embodiment 59 includes the method of any one of embodiments 1 to 58, further comprising characterizing at least one cellular property based on the intensity of the contrast agent in the image.

[0221] Embodiment 60 includes the method of any one of embodiments 1 to 59, wherein at least one cellular characteristic is assessed after administration of at least one ligand.

[0222] Embodiment 61 includes the method of any one of embodiments 1 to 60, wherein at least one cellular characteristic is assessed following administration of at least one therapeutic agent or at least one therapeutic regimen.

[0223] Embodiment 62 includes a method according to any one of embodiments 1 to 61, wherein the super-resolution microscope includes a deterministic super-resolution microscope.

[0224] Embodiment 63 includes the method of any one of embodiments 1 to 61, wherein the super-resolution microscope is a stimulated emission depletion (STED) microscope or a ground state depletion (GSD) microscope.

[0225] Embodiment 64 includes the method of any one of embodiments 1 to 61, wherein the super-resolution microscope includes a stochastic super-resolution microscope.

[0226] Embodiment 65 includes the method of any one of embodiments 1 to 61, wherein the super-resolution microscope is a MINFLUX microscope.

[0227] Embodiment 66 includes the method of any one of embodiments 1 to 61, wherein the super-resolution microscope is a stochastic optical reconstruction microscopy (STORM) microscope.

[0228] Embodiment 67 is a method according to any one of embodiments 1 to 66, wherein the imaging step comprises tracking one or more ligands, therapeutic agents, or cell surface receptors over a period of time.

[0229] Embodiment 68 includes the method of any one of embodiments 1 to 67, wherein the cells include fixed cells.

[0230] Embodiment 69 includes the method of any one of embodiments 1 to 68, wherein the cells are fixed in a tissue.

[0231] Embodiment 70 includes the method of any one of embodiments 1 to 69, wherein the cells are in a living tissue.

[0232] Embodiment 71 includes the method of any one of embodiments 1 to 70, wherein the cells include live cells.

[0233] Embodiment 72 includes the method of any one of embodiments 1 to 71, wherein the cell comprises a mammalian cell.

[0234] Embodiment 73 includes the method of any one of embodiments 1 to 72, wherein the disease state cells are derived from a tumor cell line.

[0235] Embodiment 74 includes the method of embodiment 44, wherein the normal state cells are derived from a normal cell line.

[0236] Embodiment 75 includes the method of any one of embodiments 1 to 74, wherein the cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber.

[0237] Embodiment 76 includes the method of embodiment 75, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber comprises frozen tissue.

[0238] Embodiment 77 includes the method of embodiment 75, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber comprises live tissue.

[0239] Embodiment 78 includes the method of embodiment 75, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber comprises fixed tissue.

[0240] Embodiment 79 includes the method of embodiment 75, wherein the tissue microarray, cover slip, flow cell, chip, or microfluidic chamber is formed by a laser microdissection process.

[0241]

[0036] Embodiment 80 includes the method of any one of embodiments 1 to 79, wherein the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a camelid-derived single domain antibody (VHH), a synthetically derived VHH, a murine engineered single domain antibody, a shark antibody, an antigen-binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and an scFv-Fc.

[0242] Embodiment 81 includes the method of any one of embodiments 1 to 80, wherein the antibody comprises a bispecific antibody.

[0243] Embodiment 82 includes the method of any one of embodiments 1 to 80, wherein the antibody is a multispecific antibody.

[0244] Embodiment 83 includes the method of any one of embodiments 1 to 82, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains.

[0245] Embodiment 84 includes the method of any one of embodiments 1 to 82, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains.

[0246] Embodiment 85 includes the method of any one of embodiments 1 to 82, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains.

[0247] Embodiment 86 includes the method of any one of embodiments 1 to 85, wherein the antibody is an immune cell engager.

[0248] Embodiment 87 includes the method of any one of embodiments 1 to 85, wherein the antibody comprises an effector domain (e.g., CD3 or CD16A).

[0249] Embodiment 88 includes the method of any one of embodiments 1 to 85, wherein the antibody-based therapy is for CAR-T cells, myeloid cells, NK cells, or other cell-based therapy.

[0250] Embodiment 89 includes the method of any one of embodiments 1 to 88, wherein the antibody is in a vaccine.

[0251] Embodiment 90 includes the method of any one of embodiments 1 to 89, wherein the antibody is a translated nucleic acid strand or multiple nucleic acid strands.

[0252] Embodiment 91 includes the method of any one of embodiments 1 to 89, wherein the antibody comprises a single chain polypeptide.

[0253] Embodiment 92 includes the method of any one of embodiments 1 to 91, wherein the antibody comprises a homodimer.

[0254] Embodiment 93 includes the method of any one of embodiments 1 to 91, wherein the antibody comprises a heterodimer.

[0255] Embodiment 94 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 50 nanometers.

[0256] Embodiment 95 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 40 nanometers.

[0257]

[0041] Embodiment 96 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 30 nanometers.

[0258] Embodiment 97 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 20 nanometers.

[0259] Embodiment 98 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 10 nanometers.

[0260] Embodiment 99 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 9 nanometers.

[0261] Embodiment 100 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 8 nanometers.

[0262] Embodiment 101 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 7 nanometers.

[0263] Embodiment 102 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 6 nanometers.

[0264] Embodiment 103 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 5 nanometers.

[0265] Embodiment 104 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 4 nanometers.

[0266] Embodiment 105 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 3 nanometers.

[0267] Embodiment 106 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 2 nanometers.

[0268] Embodiment 107 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged with a resolution of at least about 1 nanometer.

[0269] Embodiment 108 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged at single molecule resolution.

[0270] Embodiment 109 includes the method of any one of embodiments 1 to 93, wherein the database of imaged cell characteristics is imaged at single fluorophore resolution.

[0271] Embodiment 110 includes the method of any one of embodiments 1 to 109, wherein the database of imaged cell characteristics is imaged in a high-throughput format.

[0272] Embodiment 111 includes the method of embodiment 110, wherein the high throughput format includes the use of at least one 96-well plate.

[0273] Embodiment 112 includes the method of embodiment 110, wherein the high throughput format includes the use of at least one 192-well plate.

[0274] Embodiment 113 includes the method of embodiment 110, wherein the high throughput format includes the use of at least one 384-well plate.

[0275] Embodiment 114 includes the method of embodiment 110, wherein the high throughput format includes the use of at least one 1536-well plate.

[0276] Embodiment 115 includes the method of embodiment 110, wherein the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels.

[0277] Embodiment 116 includes the method of any one of embodiments 1 to 115, wherein multiple cellular characteristics are imaged substantially simultaneously.

[0278] Embodiment 117 includes the method of any one of embodiments 1 to 115, wherein a plurality of cellular characteristics are imaged sequentially.

[0279]

[0042] Embodiment 118 includes the method of any one of embodiments 1 to 115, wherein a plurality of cellular characteristics are imaged substantially simultaneously and the remainder are imaged sequentially.

[0280] Embodiment 119 includes the method of any one of embodiments 116-118, wherein the plurality of cell features are on the surface of a plurality of cells.

[0281] Embodiment 120 includes a method according to any one of embodiments 1 to 119, wherein imaging of cellular characteristics is completed in less than about 5 minutes.

[0282] Embodiment 121 includes the method of any one of embodiments 1 to 120, wherein imaging the database of imaged cell characteristics is performed automatically.

[0283] Embodiment 122 includes the method of any one of embodiments 2 to 121, wherein the extracellular molecule is a membrane-associated protein or a membrane-associated ligand.

[0284] Embodiment 123 includes the method of any one of embodiments 2 to 121, wherein the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0285] Embodiment 124 includes a method of designing an antibody-based therapy, comprising the steps of imaging extracellular molecules on cells for therapeutic intervention using super-resolution microscopy, characterizing cellular properties of the imaged extracellular molecules, and designing an antibody format based on the characterization of the cellular properties.

[0286] Embodiment 125 includes the method of embodiment 124, wherein the cellular characteristics include images of extracellular molecules.

[0287] Embodiment 126 includes the method of embodiment 124 or 125, wherein the cellular characteristic includes two-dimensional or three-dimensional coordinates of an extracellular molecule.

[0288] Embodiment 127 includes the method of any one of embodiments 124 to 126, wherein the cellular characteristic includes the intensity of an extracellular molecule.

[0289] Embodiment 128 includes the method of any one of embodiments 124 to 127, wherein the cellular characteristic includes all or part of a molecular fingerprint, a molecular signature, or a map of the location of extracellular molecules.

[0290] Embodiment 129 includes the method of any one of embodiments 124 to 128, wherein the cellular characteristics include all or part of a general or local representation of an extracellular molecule on a cell or tissue.

[0291] Embodiment 130 includes the method of any one of embodiments 124 to 129, wherein the cellular characteristic includes proximity between two or more extracellular molecules.

[0292] Embodiment 131 includes the method of embodiment 130, wherein the two or more extracellular molecules are for the same target or different targets.

[0293] Embodiment 132 includes the method of any one of embodiments 124 to 131, wherein the cellular characteristic includes the interaction of at least two extracellular molecules.

[0294] Embodiment 133 includes the method of any one of embodiments 124 to 132, wherein the cellular characteristic includes a distance or distances between two or more extracellular molecules.

[0295] Embodiment 134 includes the method of any one of embodiments 124 to 133, wherein the cellular characteristics include individual or paired extracellular molecules.

[0296] Embodiment 135 includes the method of embodiment 134, wherein the individual or paired extracellular molecules are adjacent to or in contact with each other.

[0297] Embodiment 136 includes the method of embodiment 134 or 135, wherein the individual or paired extracellular molecules are within a distance of about 1 nanometer from each other.

[0298] Embodiment 137 includes the method of embodiment 134 or 135, wherein the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other.

[0299] Embodiment 138 includes the method of any one of embodiments 124 to 133, wherein the cellular characteristic includes an individual extracellular molecule, a pair of extracellular molecules, or a triplet of extracellular molecules.

[0300] Embodiment 139 includes the method of embodiment 138, wherein individual extracellular molecules, pairs of extracellular molecules, or triplet of extracellular molecules are adjacent to or in contact with each other.

[0301] Embodiment 140 includes the method of embodiment 138, wherein the individual extracellular molecules, pairs of extracellular molecules, or triplet of extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other.

[0302] Embodiment 141 includes the method of any one of embodiments 124 to 133, wherein the cellular signature comprises a pair, triplet, tetrad, quintuplet, or higher order cluster of extracellular molecules.

[0303] Embodiment 142 includes the method of embodiment 141, wherein the pairs, triads, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with one another.

[0304] Embodiment 143 includes the method of embodiment 141, wherein the pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules include the same or different extracellular molecules.

[0305] Embodiment 144 includes the method of any one of embodiments 124 to 143, wherein the cellular characteristic includes a geometric relationship between two or more extracellular molecules.

[0306] Embodiment 145 includes the method of any one of embodiments 124 to 144, wherein the cellular characteristic includes the translocation of an extracellular molecule.

[0307] Embodiment 146 includes the method of embodiment 145, wherein the transfer is internalization or transfer to the extracellular surface.

[0308] Embodiment 147 includes the method of any one of embodiments 124 to 146, wherein the cellular characteristic includes internalization of an extracellular molecule.

[0309] Embodiment 148 includes the method of any one of embodiments 124 to 147, wherein the cellular characteristic includes a change in two-dimensional or three-dimensional coordinates of an extracellular molecule.

[0310] Embodiment 149 includes the method of any one of embodiments 124 to 148, wherein the cellular characteristic comprises a change in clustering of extracellular molecules.

[0311] Embodiment 150 includes the method of any one of embodiments 124 to 149, wherein the cellular characteristic includes a change in clustering of extracellular molecules.

[0312] Embodiment 151 includes the method of any one of embodiments 124 to 150, wherein the cellular characteristic includes translocation of at least one cellular receptor.

[0313] Embodiment 152 includes the method of any one of embodiments 124 to 151, wherein the cellular characteristic includes an oligomerization state of an extracellular molecule.

[0314] Embodiment 153 includes the method of any one of embodiments 124 to 152, wherein the cellular characteristic includes a clustering state of extracellular molecules.

[0315] Embodiment 154 includes the method of any one of embodiments 124 to 153, wherein the cellular signature includes the name of a protein that clusters with the extracellular molecule.

[0316] Embodiment 155 includes the method of any one of embodiments 124 to 154, wherein the extracellular molecule includes an extracellular molecule listed in Table 1.

[0317] Embodiment 156 includes the method of any one of embodiments 124 to 155, wherein the cellular characteristic is present in a cell in a tissue.

[0318] Embodiment 157 includes the method of any one of embodiments 124 to 156, wherein the cellular characteristic is present in a cell in a cancer or on a normal tissue.

[0319] Embodiment 158 ​​includes the method of any one of embodiments 124 to 156, wherein the cellular characteristic is present in normal cells, cancer cells, or both normal cells and cancer cells.

[0320] Embodiment 159 includes the method of any one of embodiments 124 to 156, wherein the cellular characteristic is present in a normal cell, a cancer cell, or a cell in the tumor microenvironment.

[0321] Embodiment 160 includes the method of any one of embodiments 124 to 156, wherein the cellular characteristic is present in a cell in the tumor microenvironment.

[0322] Embodiment 161 includes the method of embodiment 159 or 160, wherein the cells in the tumor microenvironment are selected from B cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and pericytes.

[0323] Embodiment 162 includes the method of embodiment 159 or 160, wherein the cells in the tumor microenvironment are selected from stromal cells, epithelial cells, or adipocytes.

[0324] Embodiment 163 includes the method of any one of embodiments 124 to 162, wherein the cell characteristic predicts the presence of a cancer cell or a cell in the tumor microenvironment.

[0325] Embodiment 164 includes the method of any one of embodiments 124 to 162, wherein the cell characteristic is predictive of the presence of a normal cell.

[0326] Embodiment 165 includes the method of any one of embodiments 124 to 164, wherein the cellular characteristic is present in a cell in a tissue.

[0327] Embodiment 166 includes the method of any one of embodiments 124 to 164, wherein the cellular characteristic is present in at least two different cells in the tissue.

[0328] Embodiment 167 includes the method of embodiment 166, wherein the cell characteristic is within or between the distances of at least two different cells.

[0329] Embodiment 168 includes the method of any one of embodiments 124 to 167, wherein the cellular characteristic is present in a tissue structure in a normal or diseased state.

[0330] Embodiment 169 includes the method of embodiment 168, wherein the disease state includes a cancer disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state.

[0331] Embodiment 170 includes the method of any one of embodiments 124 to 169, wherein the cell characteristic comprises a change in expression of an extracellular molecule.

[0332] Embodiment 171 includes the method of any one of embodiments 124 to 170, wherein the cellular characteristic comprises an alteration in the glycosylation pattern of an extracellular molecule.

[0333] Embodiment 172 includes the method of any one of embodiments 124 to 171, wherein the cellular characteristic comprises a change in an enzymatic activity of an extracellular molecule.

[0334] Embodiment 173 includes the method of any one of embodiments 124 to 172, wherein the cellular characteristic includes a change in intracellular or intercellular communication.

[0335] Embodiment 174 includes the method of any one of embodiments 124 to 173, wherein the cellular characteristic includes a change in signaling or communication within or between cells.

[0336] Embodiment 175 includes the method of any one of embodiments 124 to 174, wherein the cell characteristic includes a change in cell adhesion, mechanics, or migration.

[0337] Embodiment 176 includes the method of any one of embodiments 124 to 175, wherein the cellular characteristic includes tissue localization of an extracellular molecule.

[0338] Embodiment 177 includes the method of embodiment 176, wherein the tissue localization includes a diseased tissue structure or microenvironment.

[0339] Embodiment 178 includes the method of embodiment 176, wherein the tissue localization includes a cancer structure or a tumor microenvironment.

[0340] Embodiment 179 includes the method of any one of embodiments 124 to 178, wherein the extracellular molecule signals upon contact with the antibody-based therapy.

[0341] Embodiment 180 includes the method of any one of embodiments 124 to 179, wherein different samples are evaluated for a common cellular characteristic.

[0342] Embodiment 181 includes the method of any one of embodiments 124 to 180, wherein different samples are evaluated for different cellular characteristics.

[0343] Embodiment 182 includes the method of any one of embodiments 124 to 181, further comprising a step of characterizing cellular properties based on the intensity of the contrast agent in the image.

[0344] Embodiment 183 includes the method of any one of embodiments 124 to 182, wherein the cell characteristic is evaluated after administration of at least one ligand.

[0345] Embodiment 184 includes the method of any one of embodiments 124 to 183, wherein the cellular characteristic is assessed after administration of at least one therapeutic agent or at least one therapeutic regimen.

[0346] Embodiment 185 includes a method according to any one of embodiments 124 to 184, wherein the super-resolution microscope includes a deterministic super-resolution microscope.

[0347] Embodiment 186 includes a method according to any one of embodiments 124 to 184, wherein the super-resolution microscope is a stimulated emission depletion (STED) microscope or a ground state depletion (GSD) microscope.

[0348] Embodiment 187 includes a method according to any one of embodiments 124 to 184, wherein the super-resolution microscope includes a stochastic super-resolution microscope.

[0349] Embodiment 188 includes a method described in any one of embodiments 124 to 184, wherein the super-resolution microscope is a MINFLUX microscope.

[0350] Embodiment 189 includes a method described in any one of embodiments 124 to 184, wherein the super-resolution microscope is a stochastic optical reconstruction microscopy (STORM) microscope.

[0351] Embodiment 190 includes the method of any one of embodiments 124 to 189, wherein the imaging step includes tracking an antibody-based therapy or a cell surface receptor over a period of time.

[0352] Embodiment 191 includes the method of any one of embodiments 124 to 190, wherein the cells include fixed cells.

[0353] Embodiment 192 includes the method of any one of embodiments 124 to 191, wherein the cells are fixed in a tissue.

[0354] Embodiment 193 includes the method of any one of embodiments 124 to 190, wherein the cells are in a living tissue.

[0355] Embodiment 194 includes the method of any one of embodiments 124 to 190, wherein the cells include live cells.

[0356] Embodiment 195 includes the method of any one of embodiments 124 to 194, wherein the cell comprises a mammalian cell.

[0357] Embodiment 196 includes a method according to any one of embodiments 124 to 195, wherein cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber.

[0358] Embodiment 197 includes the method of embodiment 196, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber comprises frozen tissue.

[0359] Embodiment 198 includes the method of embodiment 196, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber comprises live tissue.

[0360] Embodiment 199 includes the method of embodiment 196, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber contains fixed tissue.

[0361] Embodiment 200 includes the method of embodiment 196, in which the tissue microarray, cover slip, flow cell, chip, or microfluidic chamber is formed by a laser microdissection process.

[0362]

[0043] Embodiment 201 includes the method of any one of embodiments 124 to 200, wherein the antibody-based therapy comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a camelid-derived single domain antibody (VHH), a synthetically derived VHH, a murine engineered single domain antibody, a shark antibody, an antigen-binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and an scFv-Fc.

[0363] Embodiment 202 includes the method of any one of embodiments 124 to 201, wherein the antibody-based therapy is a bispecific antibody.

[0364] Embodiment 203 includes the method of any one of embodiments 124 to 201, wherein the antibody-based therapy is a multispecific antibody.

[0365] Embodiment 204 includes the method of any one of embodiments 124 to 203, wherein the antibody-based therapy comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains.

[0366] Embodiment 205 includes the method of any one of embodiments 124 to 203, wherein the antibody-based therapy comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains.

[0367] Embodiment 206 includes the method of any one of embodiments 124 to 203, wherein the antibody-based therapy comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains.

[0368] Embodiment 207 includes the method of any one of embodiments 124 to 206, wherein the antibody-based therapy is an immune cell engager.

[0369] Embodiment 208 includes the method of any one of embodiments 124 to 206, wherein the antibody-based therapy includes an effector domain (eg, CD3 or CD16A).

[0370] Embodiment 209 includes the method of any one of embodiments 124 to 206, wherein the antibody-based therapy is for CAR-T cells, myeloid cells, NK cells, or other cell-based therapy.

[0371] Embodiment 210 includes the method of any one of embodiments 124 to 209, wherein the antibody-based therapy is in a vaccine.

[0372] Embodiment 211 includes the method of any one of embodiments 124 to 210, wherein the antibody-based therapy is a translated nucleic acid strand or multiple nucleic acid strands.

[0373] Embodiment 212 includes the method of any one of embodiments 124 to 210, wherein the antibody-based therapy comprises a single-chain polypeptide.

[0374] Embodiment 213 includes the method of any one of embodiments 124 to 212, wherein the antibody-based therapy comprises a homodimer.

[0375] Embodiment 214 includes the method of any one of embodiments 124 to 212, wherein the antibody-based therapy comprises a heterodimer.

[0376] Embodiment 215 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 50 nanometers.

[0377] Embodiment 216 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 40 nanometers.

[0378] Embodiment 217 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 30 nanometers.

[0379] Embodiment 218 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 20 nanometers.

[0380] Embodiment 219 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 10 nanometers.

[0381] Embodiment 220 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 9 nanometers.

[0382] Embodiment 221 includes a method according to any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 8 nanometers.

[0383] Embodiment 222 includes a method according to any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 7 nanometers.

[0384] Embodiment 223 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 6 nanometers.

[0385] Embodiment 224 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 5 nanometers.

[0386] Embodiment 225 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 4 nanometers.

[0387] Embodiment 226 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 3 nanometers.

[0388] Embodiment 227 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 2 nanometers.

[0389] Embodiment 228 includes a method according to any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with a resolution of at least about 1 nanometer.

[0390] Embodiment 229 includes the method of any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with single molecule resolution.

[0391] Embodiment 230 includes a method according to any one of embodiments 124 to 214, wherein the cellular characteristics are imaged with single fluorophore resolution.

[0392] Embodiment 231 includes the method of any one of embodiments 124 to 230, wherein the cellular characteristics are imaged in a high-throughput format.

[0393] Embodiment 232 includes the method of embodiment 231, wherein the high throughput format includes the use of at least one 96-well plate.

[0394] Embodiment 233 includes the method of embodiment 231, wherein the high throughput format includes the use of at least one 192-well plate.

[0395] Embodiment 234 includes the method of embodiment 231, wherein the high throughput format includes the use of at least one 384-well plate.

[0396] Embodiment 235 includes the method of embodiment 231, wherein the high throughput format includes the use of at least one 1536-well plate.

[0397] Embodiment 236 includes the method of embodiment 231, wherein the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels.

[0398] Embodiment 237 includes a method according to any one of embodiments 124 to 236, in which multiple cellular characteristics are imaged substantially simultaneously.

[0399] Embodiment 238 includes a method according to any one of embodiments 124 to 236, wherein multiple cellular characteristics are imaged sequentially.

[0400] Embodiment 239 includes the method of any one of embodiments 124 to 236, wherein a plurality of cellular characteristics are imaged substantially simultaneously and the remainder are imaged sequentially.

[0401] Embodiment 240 includes the method of any one of embodiments 237-239, wherein the plurality of cell features are on the surface of a plurality of cells.

[0402] Embodiment 241 includes a method according to any one of embodiments 124 to 240, wherein imaging of cellular characteristics is completed in less than about 5 minutes.

[0403] Embodiment 242 includes the method of any one of embodiments 124 to 241, wherein imaging of cell characteristics is performed automatically.

[0404] Embodiment 243 includes the method of any one of embodiments 124 to 242, wherein the extracellular molecule is a membrane-associated protein or a membrane-associated ligand.

[0405] Embodiment 244 includes the method of any one of embodiments 124 to 242, wherein the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0406]

[00131] Embodiment 245 includes a method of identifying cancer targets for therapeutic intervention, comprising: generating a database of cellular characteristics by imaging cellular characteristics of a plurality of extracellular molecules contacted with an antibody using a super-resolution microscope; and selecting an extracellular molecule that comprises at least one cancer target cellular characteristic when contacted with the antibody as a cancer target for therapeutic intervention.

[0407] Embodiment 246 includes the method of embodiment 245, wherein the cellular characteristics include images of extracellular molecules.

[0408] Embodiment 247 includes the method of embodiment 245 or 246, wherein the cellular characteristic includes two-dimensional or three-dimensional coordinates of an extracellular molecule.

[0409] Embodiment 248 includes the method of any one of embodiments 245 to 247, wherein the cellular characteristic includes the intensity of an extracellular molecule.

[0410] Embodiment 249 includes the method of any one of embodiments 245 to 248, wherein the cellular characteristic includes all or part of a molecular fingerprint, a molecular signature, or a map of the location of extracellular molecules.

[0411] Embodiment 250 includes a method according to any one of embodiments 245 to 249, wherein the cellular characteristics include all or part of a general or local representation of an extracellular molecule on a cell or tissue.

[0412] Embodiment 251 includes the method of any one of embodiments 245 to 250, wherein the cellular characteristic includes proximity between two or more extracellular molecules.

[0413] Embodiment 252 includes the method of embodiment 251, wherein the two or more extracellular molecules are for the same target or different targets.

[0414] Embodiment 253 includes the method of any one of embodiments 245 to 252, wherein the cellular characteristic includes the interaction of at least two extracellular molecules.

[0415] Embodiment 254 includes the method of any one of embodiments 245 to 253, wherein the cellular characteristic includes a distance or distances between two or more extracellular molecules.

[0416] Embodiment 255 includes the method of any one of embodiments 245 to 254, wherein the cellular characteristics include individual or paired extracellular molecules.

[0417] Embodiment 256 includes the method of embodiment 255, wherein the individual or paired extracellular molecules are adjacent to or in contact with each other.

[0418] Embodiment 257 includes the method of embodiment 255 or 256, wherein the individual or paired extracellular molecules are within a distance of about 1 nanometer of each other.

[0419] Embodiment 258 includes the method of embodiment 255 or 256, wherein the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other.

[0420] Embodiment 259 includes the method of any one of embodiments 245 to 258, wherein the cellular characteristic includes an individual extracellular molecule, a pair of extracellular molecules, or a triplet of extracellular molecules.

[0421] Embodiment 260 includes the method of embodiment 259, wherein the individual, paired, or triad extracellular molecules are adjacent to or in contact with each other.

[0422] Embodiment 261 includes the method of embodiment 259, wherein the individual, paired, or triad extracellular molecules are within a distance of about 1 nanometer to about 100 nanometers from each other.

[0423] Embodiment 262 includes the method of any one of embodiments 245 to 261, wherein the cellular signature comprises a pair, triplet, tetrad, quintuplet, or higher order cluster of extracellular molecules.

[0424] Embodiment 263 includes the method of embodiment 262, wherein the pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules are adjacent to or in contact with one another.

[0425] Embodiment 264 includes the method of embodiment 262, wherein the pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules include the same or different molecules.

[0426] Embodiment 265 includes the method of any one of embodiments 245 to 264, wherein the cellular characteristic includes a geometric relationship between two or more extracellular molecules.

[0427] Embodiment 266 includes the method of any one of embodiments 245 to 265, wherein the cellular characteristic includes the translocation of an extracellular molecule.

[0428] Embodiment 267 includes the method of embodiment 266, wherein the transfer is internalization or transfer to the extracellular surface.

[0429] Embodiment 268 includes the method of any one of embodiments 245 to 267, wherein the cellular characteristic includes internalization of an extracellular molecule.

[0430] Embodiment 269 includes the method of any one of embodiments 245 to 268, wherein the cellular characteristic includes a change in two- or three-dimensional coordinates of an extracellular molecule.

[0431] Embodiment 270 includes the method of any one of embodiments 245 to 269, wherein the cellular characteristic includes a change in clustering of extracellular molecules.

[0432] Embodiment 271 includes the method of any one of embodiments 245 to 270, wherein the cellular characteristic includes a change in clustering of extracellular molecules.

[0433] Embodiment 272 includes the method of any one of embodiments 245 to 271, wherein the cellular characteristic includes translocation of at least one cellular receptor.

[0434] Embodiment 273 includes the method of any one of embodiments 245 to 272, wherein the cellular characteristic includes an oligomerization state of an extracellular molecule.

[0435] Embodiment 274 includes the method of any one of embodiments 245 to 273, wherein the cellular characteristic includes a clustering state of extracellular molecules.

[0436] Embodiment 275 includes the method of any one of embodiments 245 to 274, wherein the cellular signature includes names of proteins that cluster with the extracellular molecule.

[0437] Embodiment 276 includes the method of any one of embodiments 245 to 275, wherein the extracellular receptor comprises an extracellular receptor listed in Table 1.

[0438] Embodiment 277 includes the method of any one of embodiments 245 to 276, wherein the cellular characteristic is present in a cell in a tissue.

[0439] Embodiment 278 includes the method of any one of embodiments 245 to 277, wherein the cellular characteristic is present in a cell in a cancer or on a normal tissue.

[0440] Embodiment 279 includes the method of any one of embodiments 245 to 278, wherein the cellular characteristic is present in normal cells, cancer cells, or both normal cells and cancer cells.

[0441] Embodiment 280 includes the method of any one of embodiments 245 to 279, wherein the cellular characteristic is present in a normal cell, a cancer cell, or a cell in the tumor microenvironment.

[0442] Embodiment 281 includes the method of any one of embodiments 245 to 280, wherein the cellular characteristic is present in a normal cell, a cancer cell, or a cell in the tumor microenvironment.

[0443] Embodiment 282 includes the method of embodiment 280 or 281, wherein the cells in the tumor microenvironment are selected from B cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and pericytes.

[0444] Embodiment 283 includes the method of embodiment 280 or 281, wherein the cells in the tumor microenvironment are selected from stromal cells, epithelial cells, or adipocytes.

[0445] Embodiment 284 includes the method of any one of embodiments 245 to 283, wherein the cell characteristic predicts the presence of a cancer cell or a cell in the tumor microenvironment.

[0446] Embodiment 285 includes the method of any one of embodiments 245 to 283, wherein the cell characteristic is predictive of the presence of a normal cell.

[0447] Embodiment 286 includes the method of any one of embodiments 245 to 285, wherein the cellular characteristics are present in the same cells in a tissue.

[0448] Embodiment 287 includes the method of any one of embodiments 245 to 285, wherein the cellular characteristic is present in at least two different cells in the tissue.

[0449] Embodiment 288 includes the method of any one of embodiments 245 to 287, wherein the cell characteristic is within or between the distances of at least two different cells.

[0450] Embodiment 289 includes the method of any one of embodiments 245 to 288, wherein the cellular characteristic is present in a tissue structure in a normal or diseased state.

[0451] Embodiment 290 includes the method of embodiment 289, wherein the disease state includes a cancer disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state.

[0452] Embodiment 291 includes the method of any one of embodiments 245 to 290, wherein the cell characteristic comprises a change in expression of an extracellular molecule.

[0453] Embodiment 292 includes the method of any one of embodiments 245 to 291, wherein the cellular characteristic comprises an alteration in the glycosylation pattern of an extracellular molecule.

[0454] Embodiment 293 includes the method of any one of embodiments 245 to 292, wherein the cellular characteristic comprises a change in an enzymatic activity of an extracellular molecule.

[0455] Embodiment 294 includes the method of any one of embodiments 245 to 293, wherein the cellular characteristic includes a change in intracellular or intercellular communication.

[0456] Embodiment 295 includes the method of any one of embodiments 245 to 293, wherein the cellular characteristic includes a change in signaling or communication within or between cells.

[0457] Embodiment 296 includes the method of any one of embodiments 245 to 295, wherein the cell characteristic includes a change in cell adhesion, mechanics, or migration.

[0458] Embodiment 297 includes the method of any one of embodiments 245 to 296, wherein the cellular characteristic includes tissue localization of an extracellular receptor.

[0459] Embodiment 298 includes the method of embodiment 297, wherein the tissue localization includes a cancer structure or a tumor microenvironment.

[0460] Embodiment 299 includes the method of any one of embodiments 245 to 298, wherein the cellular characteristic includes internalization of at least one extracellular receptor.

[0461] Embodiment 300 includes the method of any one of embodiments 245-299, wherein the extracellular receptor transduces a signal upon contact with the antibody.

[0462] Embodiment 301 includes a method according to any one of embodiments 245 to 300, in which different samples are evaluated for a common cellular characteristic.

[0463] Embodiment 302 includes the method of any one of embodiments 245 to 301, wherein different samples are evaluated for different cellular characteristics.

[0464] Embodiment 303 includes the method of any one of embodiments 245 to 302, further comprising a step of characterizing cellular properties based on the intensity of the contrast agent in the image.

[0465] Embodiment 304 includes the method of any one of embodiments 245 to 303, wherein the cell characteristic is evaluated after administration of at least one ligand.

[0466] Embodiment 305 includes the method of any one of embodiments 245 to 304, wherein the cellular characteristics are evaluated after administration of at least one therapeutic agent or at least one therapeutic regimen.

[0467] Embodiment 306 includes a method according to any one of embodiments 245 to 305, wherein the super-resolution microscope includes a deterministic super-resolution microscope.

[0468] Embodiment 307 includes a method according to any one of embodiments 245 to 305, wherein the super-resolution microscope is a stimulated emission depletion (STED) microscope or a ground state depletion (GSD) microscope.

[0469] Embodiment 308 includes the method of embodiment 307, in which the STED microscope is a MINFLUX microscope.

[0470] Embodiment 309 includes a method described in any one of embodiments 245 to 305, wherein the super-resolution microscope includes a stochastic super-resolution microscope.

[0471] Embodiment 310 includes a method according to any one of embodiments 245 to 305, wherein the super-resolution microscope is a stochastic optical reconstruction microscopy (STORM) microscope.

[0472] Embodiment 311 is a method according to any one of embodiments 245 to 310, wherein the imaging step comprises tracking one or more ligands, therapeutic agents, or cell surface receptors over a period of time.

[0473] Embodiment 312 includes the method of any one of embodiments 245 to 311, wherein the cells include fixed cells.

[0474] Embodiment 313 includes the method of any one of embodiments 245 to 311, wherein the cells are fixed in a tissue.

[0475] Embodiment 314 includes the method of any one of embodiments 245 to 311, wherein the cells are in a living tissue.

[0476] Embodiment 315 includes the method of any one of embodiments 245 to 311, wherein the cells include live cells.

[0477] Embodiment 316 includes the method of any one of embodiments 245 to 315, wherein the cell comprises a mammalian cell.

[0478] Embodiment 317 includes the method of any one of embodiments 245 to 316, wherein the cells are derived from a tumor cell line.

[0479] Embodiment 318 includes the method of any one of embodiments 245 to 316, wherein the cells are derived from a normal cell line.

[0480] Embodiment 319 includes a method according to any one of embodiments 245 to 318, wherein cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber.

[0481] Embodiment 320 includes the method of embodiment 319, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber contains frozen tissue.

[0482] Embodiment 321 includes the method of embodiment 319, in which the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber contains live tissue.

[0483] Embodiment 322 includes the method of embodiment 319, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber contains fixed tissue.

[0484] Embodiment 323 includes the method of embodiment 319, in which the tissue microarray, cover slip, flow cell, chip, or microfluidic chamber is formed by a laser microdissection process.

[0485]

[0043] Embodiment 324 includes the method of any one of embodiments 245 to 323, wherein the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a camelid-derived single domain antibody (VHH), a synthetically derived VHH, a murine engineered single domain antibody, a shark antibody, an antigen-binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and an scFv-Fc.

[0486] Embodiment 325 includes the method of any one of embodiments 245 to 324, wherein the antibody is a bispecific antibody.

[0487] Embodiment 326 includes the method of any one of embodiments 245 to 324, wherein the antibody is a multispecific antibody.

[0488] Embodiment 327 includes the method of any one of embodiments 245 to 326, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains.

[0489] Embodiment 328 includes the method of any one of embodiments 245 to 326, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains.

[0490] Embodiment 329 includes the method of any one of embodiments 245 to 326, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains.

[0491] Embodiment 330 includes the method of any one of embodiments 245 to 329, wherein the antibody is an immune cell engager.

[0492] Embodiment 331 includes the method of any one of embodiments 245 to 329, wherein the antibody comprises an effector domain (e.g., CD3 or CD16A).

[0493] Embodiment 332 includes the method of any one of embodiments 245 to 329, wherein the antibody is directed against CAR-T cells, myeloid cells, NK cells, or other cell-based therapy.

[0494] Embodiment 333 includes the method of any one of embodiments 245 to 329, wherein the antibody is in a vaccine.

[0495] Embodiment 334 includes the method of any one of embodiments 245 to 333, wherein the antibody is a translated nucleic acid strand or multiple nucleic acid strands.

[0496] Embodiment 335 includes the method of any one of embodiments 245 to 334, wherein the antibody comprises a single chain polypeptide.

[0497] Embodiment 336 includes the method of any one of embodiments 245 to 335, wherein the antibody comprises a homodimer.

[0498] Embodiment 337 includes the method of any one of embodiments 245 to 335, wherein the antibody comprises a heterodimer.

[0499] Embodiment 338 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 50 nanometers.

[0500] Embodiment 339 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 40 nanometers.

[0501] Embodiment 340 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 30 nanometers.

[0502] Embodiment 341 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 20 nanometers.

[0503] Embodiment 342 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 10 nanometers.

[0504] Embodiment 343 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 9 nanometers.

[0505] Embodiment 344 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 8 nanometers.

[0506] Embodiment 345 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 7 nanometers.

[0507] Embodiment 346 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 6 nanometers.

[0508] Embodiment 347 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 5 nanometers.

[0509] Embodiment 348 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 4 nanometers.

[0510] Embodiment 349 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 3 nanometers.

[0511] Embodiment 350 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 2 nanometers.

[0512] Embodiment 351 includes a method according to any one of embodiments 245 to 337, wherein the database of cell characteristics is imaged with a resolution of at least about 1 nanometer.

[0513] Embodiment 352 includes the method of any one of embodiments 245 to 337, wherein the database of cellular characteristics is imaged at single molecule resolution.

[0514] Embodiment 353 includes a method according to any one of embodiments 245 to 337, wherein the database of cellular characteristics is imaged at single fluorophore resolution.

[0515] Embodiment 354 includes the method of any one of embodiments 245 to 353, wherein the database of cell characteristics is imaged in a high-throughput format.

[0516] Embodiment 355 includes the method of embodiment 354, wherein the high throughput format includes the use of at least one 96-well plate.

[0517] Embodiment 356 includes the method of embodiment 354, wherein the high throughput format includes the use of at least one 192-well plate.

[0518] Embodiment 357 includes the method of embodiment 354, wherein the high throughput format includes the use of at least one 384-well plate.

[0519] Embodiment 358 includes the method of embodiment 354, wherein the high throughput format includes the use of at least one 1536-well plate.

[0520] Embodiment 359 includes the method of embodiment 354, wherein the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels.

[0521] Embodiment 360 includes a method according to any one of embodiments 245 to 359, in which multiple cellular characteristics are imaged substantially simultaneously.

[0522] Embodiment 361 includes a method according to any one of embodiments 245 to 359, in which multiple cellular characteristics are imaged sequentially.

[0523] Embodiment 362 includes a method according to any one of embodiments 245 to 359, in which a plurality of cellular characteristics are imaged substantially simultaneously and the remainder are imaged sequentially.

[0524] Embodiment 363 includes the method of any one of embodiments 360 to 362, wherein the plurality of cell features are on the surface of a plurality of cells.

[0525] Embodiment 364 includes a method according to any one of embodiments 245 to 363, in which imaging of cell characteristics is performed automatically.

[0526] Embodiment 365 includes the method of any one of embodiments 245 to 364, wherein the extracellular molecule is a membrane-associated protein or a membrane-associated ligand.

[0527] Embodiment 366 includes the method of any one of embodiments 245 to 364, wherein the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0528] Embodiment 367 includes the method of any one of embodiments 245 to 366, wherein the selected extracellular molecule is present in cells in a diseased state and is absent in cells in a non-disease state.

[0529] Embodiment 368 includes the method of any one of embodiments 245 to 366, wherein the cellular characteristics present in diseased cells but not in non-diseased cells are subsequently used to develop antibody-based therapeutics.

[0530] Embodiment 369 includes a method of generating a database of cellular characteristics of diseased and non-disease cells, comprising contacting the cells with an antibody and then imaging a cell selected from the diseased and non-disease cells by super-resolution microscopy to provide an image of one or more cellular characteristics of the cell, storing the one or more cellular characteristics of the cell in the database, and repeating the imaging and storing of one or more additional cells selected from the diseased and non-disease cells to generate the database of cellular characteristics of the diseased and non-disease cells.

[0531] Embodiment 370 includes the method of embodiment 369, wherein the disease state cell comprises a cancer disease state cell, an immunological disease state cell, a neurological disease state cell, an antiviral disease state cell, a cardiovascular disease state cell, or an autoimmune disease state cell.

[0532] Embodiment 371 includes the method of embodiment 369 or 370, wherein the antibody binds to an antigen on the surface of a cell, and the antigen is associated with a disease.

[0533] Embodiment 372 includes the method of any one of embodiments 369 to 371, wherein the antibody is associated with an imaging agent.

[0534] Embodiment 373 includes the method of any one of embodiments 369 to 372, wherein the antibody is directly associated with the imaging agent.

[0535] Embodiment 374 includes the method of any one of embodiments 369 to 372, wherein the antibody is indirectly associated with the imaging agent.

[0536] Embodiment 375 includes a method according to any one of embodiments 369 to 374, further comprising determining differences between one or more cellular characteristics of diseased cells and non-disease cells stored in the database, and developing a therapeutic strategy targeting diseased cells based on differences between one or more cellular characteristics of diseased cells and non-disease cells stored in the database.

[0537] Embodiment 376 relates to a method for determining the local affinity (K d ) and correlating it with one or more cellular characteristics of the cells stored in the database; d , and K d and storing a correlation between the one or more cellular characteristics of the cell in the database.

[0538] Embodiment 377 is directed to a method for treating a pulmonary artery disease comprising administering a localized K d 377. The method of embodiment 376, further comprising determining by single molecule tracking.

[0539] Embodiment 378 relates to a method for determining localized K binding of an antibody to a second cell. d 378. The method of embodiment 376 or 377, further comprising predicting based on similarity of one or more cellular characteristics of the cell to one or more cellular characteristics of a second cell.

[0540] Embodiment 379 includes the method of any one of embodiments 369 to 378, wherein the one or more cellular characteristics include an image of an extracellular molecule.

[0541] Embodiment 380 includes the method of any one of embodiments 369 to 379, wherein the one or more cellular characteristics include two-dimensional or three-dimensional coordinates of an extracellular molecule.

[0542] Embodiment 381 includes the method of any one of embodiments 369 to 380, wherein the one or more cellular characteristics include the intensity of an extracellular molecule.

[0543] Embodiment 382 includes a method according to any one of embodiments 369 to 381, wherein the one or more cellular characteristics include all or a portion of a molecular fingerprint, molecular signature, or map of the location of extracellular molecules.

[0544] Embodiment 383 includes a method according to any one of embodiments 369 to 382, ​​wherein the one or more cellular characteristics include all or a portion of a schematic or local representation of an extracellular molecule on a cell or tissue.

[0545] Embodiment 384 includes the method of any one of embodiments 369 to 382, ​​wherein the one or more cellular characteristics include proximity between two or more extracellular molecules.

[0546] Embodiment 385 includes the method of embodiment 384, wherein the two or more extracellular molecules are for the same target or different targets.

[0547] Embodiment 386 includes the method of any one of embodiments 369 to 385, wherein the one or more cellular characteristics include an interaction of at least two extracellular molecules.

[0548] Embodiment 387 includes the method of any one of embodiments 369 to 386, wherein the one or more cellular characteristics include a distance or distances between two or more extracellular molecules.

[0549] Embodiment 388 includes the method of any one of embodiments 369 to 387, wherein the one or more cellular characteristics include individual or paired extracellular molecules.

[0550] Embodiment 389 includes the method of embodiment 388, wherein the individual or paired extracellular molecules are adjacent to or in contact with each other.

[0551] Embodiment 390 includes the method of embodiment 388 or 389, wherein the individual or paired extracellular molecules are within a distance of about 1 nanometer from each other.

[0552] Embodiment 391 includes the method of embodiment 388 or 389, wherein the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other.

[0553] Embodiment 392 includes the method of any one of embodiments 369 to 391, wherein the one or more cellular characteristics include individual extracellular molecules, or pairs or triplet of extracellular molecules.

[0554] Embodiment 393 includes the method of embodiment 392, wherein individual extracellular molecules, or pairs or triplet extracellular molecules, are adjacent to or in contact with each other.

[0555] Embodiment 394 includes the method of embodiment 392, wherein the individual extracellular molecules, or pairs or triads of extracellular molecules, are within a distance of about 1 nanometer to about 100 nanometers from each other.

[0556] Embodiment 395 includes the method of any one of embodiments 369 to 394, wherein the one or more cellular characteristics include pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules.

[0557] Embodiment 396 includes the method of embodiment 395, wherein the pairs, triads, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with one another.

[0558] Embodiment 397 includes the method of embodiment 395, wherein the pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules include the same or different extracellular molecules.

[0559] Embodiment 398 includes the method of any one of embodiments 369 to 397, wherein the one or more cellular characteristics include a geometric relationship between two or more extracellular molecules.

[0560] Embodiment 399 includes the method of any one of embodiments 369 to 398, wherein the one or more cellular characteristics include translocation of at least one extracellular molecule.

[0561] Embodiment 400 includes the method of embodiment 399, wherein the transfer is internalization or transfer to the extracellular surface.

[0562] Embodiment 401 includes a method according to any one of embodiments 369 to 400, wherein the one or more cellular characteristics include internalization of at least one extracellular molecule.

[0563] Embodiment 402 includes the method of any one of embodiments 369 to 401, wherein the one or more cellular characteristics include a change in two-dimensional or three-dimensional coordinates of an extracellular molecule.

[0564] Embodiment 403 includes a method according to any one of embodiments 369 to 402, wherein the one or more cellular characteristics include a change in clustering of extracellular molecules.

[0565] Embodiment 404 includes a method according to any one of embodiments 369 to 403, wherein the one or more cellular characteristics include changes in clustering of extracellular molecules.

[0566] Embodiment 405 includes the method of any one of embodiments 369 to 404, wherein the one or more cellular characteristics include an oligomerization state of an extracellular molecule.

[0567] Embodiment 406 includes a method according to any one of embodiments 369 to 405, wherein the one or more cellular characteristics include a clustering state of extracellular molecules.

[0568] Embodiment 407 includes the method of any one of embodiments 369 to 406, wherein the one or more cellular characteristics include names of proteins that cluster with the extracellular molecule.

[0569] Embodiment 408 includes the method of any one of embodiments 379 to 407, wherein the extracellular molecule is an extracellular molecule listed in Table 1.

[0570] Embodiment 409 includes the method of any one of embodiments 369 to 408, wherein the one or more cellular characteristics are present in cells in the tissue.

[0571] Embodiment 410 includes the method of any one of embodiments 369 to 409, wherein the one or more cellular characteristics are present in cells in a cancer or on normal tissue.

[0572] Embodiment 411 includes the method of any one of embodiments 369 to 410, wherein the one or more cellular characteristics include an oligomerization state of an extracellular molecule.

[0573] Embodiment 412 includes the method of any one of embodiments 371 to 411, wherein the one or more cellular characteristics include an oligomerization state of an antigen on the surface of the cell.

[0574] Embodiment 413 includes the method of embodiment 411 or 412, wherein the oligomerization state is selected from dimers, trimers, tetramers, pentamers, and higher state oligomers.

[0575] Embodiment 414 includes the method of any one of embodiments 369 to 413, wherein the one or more cellular characteristics include an interaction between two or more extracellular molecules.

[0576] Embodiment 415 includes the method of any one of embodiments 369 to 414, wherein the one or more cellular characteristics include tissue localization of an extracellular molecule.

[0577] Embodiment 416 includes the method of embodiment 415, wherein the tissue localization includes diseased tissue or the tumor microenvironment.

[0578] Embodiment 417 includes the method of embodiment 415, wherein the tissue localization includes a cancer structure or a tumor microenvironment.

[0579] Embodiment 418 includes the method of any one of embodiments 369 to 417, wherein the one or more cellular characteristics include translocation of at least one cellular receptor.

[0580] Embodiment 419 includes a method according to any one of embodiments 372 to 418, further comprising a step of characterizing one or more cellular properties based on the intensity of the contrast agent in the image.

[0581]

[0081] Embodiment 420 includes the method of any one of embodiments 369 to 419, wherein the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a camelid-derived single domain antibody (VHH), a synthetically derived VHH, a murine engineered single domain antibody, a shark antibody, an antigen-binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and an scFv-Fc.

[0582] Embodiment 421 includes the method of any one of embodiments 369 to 420, wherein the antibody is a bispecific antibody.

[0583] Embodiment 422 includes the method of any one of embodiments 369 to 420, wherein the antibody is a multispecific antibody.

[0584] Embodiment 423 includes the method of any one of embodiments 369 to 422, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains.

[0585] Embodiment 424 includes the method of any one of embodiments 369 to 422, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains.

[0586] Embodiment 425 includes the method of any one of embodiments 369 to 422, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains.

[0587] Embodiment 426 includes the method of any one of embodiments 369 to 425, wherein the antibody is an immune cell engager.

[0588] Embodiment 427 includes the method of any one of embodiments 369 to 425, wherein the antibody comprises an effector domain (e.g., CD3 or CD16A).

[0589] Embodiment 428 includes the method of any one of embodiments 369 to 425, wherein the antibody is directed against CAR-T cells, myeloid cells, NK cells, or other cell-based therapy.

[0590] Embodiment 429 includes the method of any one of embodiments 369 to 428, wherein the antibody is in a vaccine.

[0591] Embodiment 430 includes the method of any one of embodiments 369 to 429, wherein the antibody is a translated nucleic acid strand or multiple nucleic acid strands.

[0592] Embodiment 431 includes the method of any one of embodiments 369 to 430, wherein the antibody comprises a single chain polypeptide.

[0593] Embodiment 432 includes the method of any one of embodiments 369 to 431, wherein the antibody comprises a homodimer.

[0594] Embodiment 433 includes the method of any one of embodiments 369 to 432, wherein the antibody comprises a heterodimer.

[0595] Embodiment 434 includes a method according to any one of embodiments 369 to 433, in which the super-resolution microscopy is stimulated emission depletion (STED) microscopy.

[0596] Embodiment 435 includes a method according to any one of embodiments 369 to 433, wherein the super-resolution microscopy is ground-state suppression (GSD) microscopy.

[0597] Embodiment 436 includes a method according to any one of embodiments 369 to 433, wherein the super-resolution microscopy is stochastic optical reconstruction microscopy (STORM) microscopy.

[0598] Embodiment 437 includes a method according to any one of embodiments 369 to 433, wherein the super-resolution microscopy method includes MINFLUX microscopy.

[0599] Embodiment 438 includes a method according to any one of embodiments 369 to 437, wherein imaging includes tracking extracellular molecules of the cells over a period of time.

[0600] Embodiment 439 includes the method of any one of embodiments 369 to 438, wherein the cells include fixed cells.

[0601] Embodiment 440 includes the method of any one of embodiments 369 to 438, wherein the cells are fixed in the tissue.

[0602] Embodiment 441 includes the method of any one of embodiments 369 to 438, wherein the cells are in a living tissue.

[0603] Embodiment 442 includes the method of any one of embodiments 369 to 438, wherein the cells include live cells.

[0604] Embodiment 443 includes the method of any one of embodiments 369 to 442, wherein the cell comprises a mammalian cell.

[0605] Embodiment 444 includes the method of any one of embodiments 369 to 443, wherein the disease state cells are derived from a tumor cell line.

[0606] Embodiment 445 includes the method of any one of embodiments 369 to 444, wherein the disease state cells are derived from a normal cell line.

[0607] Embodiment 446 includes a method according to any one of embodiments 369 to 445, wherein cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber.

[0608] Embodiment 447 includes the method of embodiment 446, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber contains frozen tissue.

[0609] Embodiment 448 includes the method of embodiment 446, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber contains live tissue.

[0610] Embodiment 449 includes the method of embodiment 446, wherein the tissue microarray, coverslip, flow cell, chip, or microfluidic chamber contains fixed tissue.

[0611] Embodiment 450 includes the method of any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 50 nanometers.

[0612] Embodiment 451 includes a method according to any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 40 nanometers.

[0613] Embodiment 452 includes a method according to any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 30 nanometers.

[0614] Embodiment 453 includes a method according to any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 20 nanometers.

[0615] Embodiment 454 includes a method according to any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 10 nanometers.

[0616] Embodiment 455 includes a method according to any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 5 nanometers.

[0617] Embodiment 456 includes a method according to any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 2 nanometers.

[0618] Embodiment 457 includes a method according to any one of embodiments 369 to 449, wherein imaging is performed with a resolution of at least about 1 nanometer.

[0619] Embodiment 458 includes the method of any one of embodiments 369 to 449, wherein imaging is performed at single molecule resolution.

[0620] Embodiment 459 includes the method of any one of embodiments 369 to 449, wherein imaging is performed at single fluorophore resolution.

[0621] Embodiment 460 includes the method of any one of embodiments 369 to 459, wherein imaging is performed in a high-throughput format.

[0622] Embodiment 461 includes the method of embodiment 460, wherein the high throughput format includes the use of at least one 96-well plate.

[0623] Embodiment 462 includes the method of embodiment 460, wherein the high throughput format includes the use of at least one 192-well plate.

[0624] Embodiment 463 includes the method of embodiment 460, wherein the high throughput format includes the use of at least one 384-well plate.

[0625] Embodiment 464 includes the method of embodiment 460, wherein the high throughput format includes the use of at least one 1536-well plate.

[0626] Embodiment 465 includes the method of embodiment 460, wherein the high throughput format includes the use of slides, coverslips, or flow cells containing multiple channels.

[0627] Embodiment 466 includes the method of any one of embodiments 379 to 465, wherein the extracellular molecule is a membrane-associated protein or a membrane-associated ligand.

[0628] Embodiment 467 includes the method of any one of embodiments 379 to 465, wherein the extracellular molecule is selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

[0629] Embodiment 468 includes a method of characterizing a cell or tissue sample, comprising contacting the cell or tissue sample with an antibody that binds to at least one extracellular molecule on the cell or tissue sample and that is associated with an imaging agent detectable by super-resolution microscopy, imaging the cell or tissue sample by super-resolution microscopy after contacting the cell or tissue sample with the antibody to provide an image of the at least one extracellular molecule on a cell surface of the cell or tissue sample, and characterizing one or more cellular properties of the cell or tissue sample based on the image of the at least one extracellular molecule on the cell or tissue sample.

[0630] Embodiment 469 includes the method of embodiment 468, further comprising the steps of: i) providing a multiplexed image of the cell or tissue sample by contacting the cell or tissue sample with a plurality of antibodies, each of which is associated with an imaging agent, and ii) imaging the cell or tissue sample by super-resolution microscopy after contacting the cell or tissue sample with each of the plurality of antibodies, and further comprising the step of characterizing one or more cellular characteristics of the cell or tissue sample based on the multiplexed image.

[0631] Embodiment 470 includes the method of embodiment 468 or 469, wherein the imaging agent is a fluorophore.

[0632] Embodiment 471 includes the method of any one of embodiments 468 to 470, wherein the imaging agent is directly or indirectly bound to the fluorophore.

[0633] Embodiment 472 includes a method according to any one of embodiments 372 to 418, further comprising a step of characterizing one or more cellular properties based on the two-dimensional or three-dimensional coordinates of at least one extracellular molecule or the intensity of a contrast agent in the image.

[0634] Embodiment 473 includes a method according to any one of embodiments 468 to 472, further comprising a step of characterizing one or more cellular properties based on the intensity of the contrast agent in the image.

[0635] Embodiment 474 includes a method according to any one of embodiments 468 to 473, wherein one or more cellular characteristics are evaluated after administration of at least one ligand.

[0636] Embodiment 475 includes the method of any one of embodiments 468 to 474, wherein at least one cellular characteristic is assessed following administration of at least one therapeutic agent or treatment regimen.

[0637] Embodiment 476 includes a method according to any one of embodiments 468 to 475, wherein the one or more cellular characteristics include clustering of at least one extracellular molecule.

[0638] Embodiment 477 includes the method of embodiment 476, wherein the clustered extracellular molecules are functional and are involved in signal transduction, internalization, motility, and / or enzymatic activity.

[0639] Embodiment 478 includes the method of embodiment 476, wherein the clustered extracellular molecules are non-functional and are aggregated or incapable of being internalized or shed.

[0640] Embodiment 479 includes a method according to any one of embodiments 468 to 478, wherein the one or more cellular characteristics are associated with a single extracellular molecule on the cell or tissue sample.

[0641] Embodiment 480 includes a method according to any one of embodiments 468 to 478, wherein the one or more cellular characteristics are associated with a pair of extracellular molecules on a cell or tissue sample.

[0642] Embodiment 481 includes a method according to any one of embodiments 468 to 478, wherein the one or more cellular characteristics are associated with an extracellular molecular triad, an extracellular molecular tetrad, an extracellular molecular quintuplet, or other higher order extracellular molecular cluster on a cell or tissue sample.

[0643] Embodiment 482 includes a method according to any one of embodiments 479 to 481, wherein the single extracellular molecule, pair of extracellular molecules, triad of extracellular molecules, quartet of extracellular molecules, quintet of extracellular molecules, or other higher order extracellular molecular cluster includes one or more other proteins.

[0644] Embodiment 483 includes a method according to any one of embodiments 480 to 482, wherein the extracellular molecule pair, extracellular molecule triad, extracellular molecule quartet, extracellular molecule quintet, or other higher order extracellular molecule cluster is composed of a single extracellular molecule or multiple extracellular molecules, respectively.

[0645] Embodiment 484 includes a method according to any one of embodiments 468 to 483, wherein the one or more cellular characteristics include one or more cellular characteristics of a plurality of extracellular molecules.

[0646] Embodiment 485 includes a method according to any one of embodiments 468 to 484, wherein the one or more cellular characteristics include the presence of at least one extracellular molecule on the cell or tissue sample.

[0647] Embodiment 486 includes a method according to any one of embodiments 468 to 484, wherein the one or more cellular characteristics include the absence of at least one extracellular molecule on the cell or tissue sample.

[0648] Embodiment 487 includes a method according to any one of embodiments 468 to 486, wherein the one or more cellular characteristics include the abundance of at least one extracellular molecule on the cell or tissue sample.

[0649] Embodiment 488 includes a method according to any one of embodiments 468 to 487, wherein the one or more cellular characteristics include the distribution of at least one extracellular molecule on the cell or tissue sample.

[0650] Embodiment 489 includes a method according to any one of embodiments 468 to 488, wherein the one or more cellular characteristics include the density of at least one extracellular molecule on the cell or tissue sample.

[0651] Embodiment 490 includes the method of any one of embodiments 468 to 489, wherein the one or more cellular characteristics includes the oligomeric state of at least one extracellular molecule.

[0652] Embodiment 491 includes the method of any one of embodiments 468 to 490, wherein the one or more cellular characteristics includes the heteromeric state of at least one extracellular molecule.

[0653] Embodiment 492 includes the method of any one of embodiments 468 to 491, wherein the one or more cellular characteristics include one or more nearest neighbors of at least one extracellular molecule.

[0654] Embodiment 493 includes the method of embodiment 492, wherein the one or more nearest neighbors are selected from proteins, extracellular receptors, antigens, carbohydrates, and lipids.

[0655] Embodiment 494 includes the method of any one of embodiments 479 to 493, wherein the one or more cellular characteristics include a distance between distinct molecules of a single extracellular molecule.

[0656] Embodiment 495 includes the method of any one of embodiments 480 to 494, wherein the one or more cellular characteristics include a pairwise distance between extracellular molecules of a pair of extracellular molecules.

[0657] Embodiment 496 includes a method according to any one of embodiments 481 to 495, wherein the one or more cellular characteristics include pairwise distances between extracellular molecules of an extracellular molecular triplet, an extracellular molecular tetrad, an extracellular molecular quintuplet, or other larger cluster of extracellular molecules.

[0658] Embodiment 497 includes a method according to any one of embodiments 468 to 496, wherein the one or more cellular properties include lateral, anterior, or anterior movement of at least one extracellular molecule.

[0659] Embodiment 498 includes a method according to any one of embodiments 468 to 497, wherein the one or more cellular characteristics include one or more cellular characteristics on the same cell and / or between different cells.

[0660] Embodiment 499 includes a method according to any one of embodiments 468 to 498, wherein the one or more cellular characteristics include a cellular interaction between at least one extracellular molecule and one or more extracellular proteins on separate cells.

[0661] Embodiment 500 includes a method according to any one of embodiments 468 to 498, wherein the one or more cellular characteristics include the absence of intercellular interaction between at least one extracellular molecule and one or more extracellular proteins on separate cells.

[0662] Embodiment 501 includes the method of embodiment 499 or 500, wherein the cell-cell interaction includes oligomers or clusters of two or more extracellular proteins on separate cells.

[0663] Embodiment 502 includes a method according to any one of embodiments 499 to 501, wherein the intracellular interaction is selected from tumor cell-tumor cell interaction, tumor cell-tumor microenvironment cell interaction, and tumor microenvironment cell-tumor microenvironment cell interaction.

[0664] Embodiment 503 includes the method of embodiment 502, wherein the cells in the tumor microenvironment are selected from B cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and pericytes.

[0665] Embodiment 504 includes the method of embodiment 503, wherein the cells in the tumor microenvironment are selected from B cells, T cells, natural killer (NK) cells, myeloid cells, fibroblasts, and pericytes.

[0666] Embodiment 505 includes the method of any one of embodiments 468 to 504, wherein one or more cellular characteristics predict the presence of a cancer cell or a cell in the tumor microenvironment.

[0667] Embodiment 506 includes the method of any one of embodiments 468 to 504, wherein one or more cellular characteristics predict the presence of a normal cell.

[0668] Embodiment 507 includes the method of any one of embodiments 468 to 506, wherein one or more cellular characteristics are indicative of a signaling event.

[0669] Embodiment 508 includes a method according to any one of embodiments 468 to 507, wherein the one or more cellular characteristics define a molecular signature, a cellular signature, or a tissue sample signature.

[0670] Embodiment 509 includes a method according to any one of embodiments 468 to 508, wherein the one or more cellular characteristics include an image of at least one extracellular molecule.

[0671] Embodiment 510 includes the method of any one of embodiments 468 to 509, wherein the one or more cellular characteristics include two-dimensional or three-dimensional coordinates of at least one extracellular molecule.

[0672] Embodiment 511 includes the method of any one of embodiments 468 to 510, wherein the one or more cellular characteristics include the intensity of at least one extracellular molecule.

[0673] Embodiment 512 includes a method according to any one of embodiments 468 to 511, wherein the one or more cellular characteristics include all or a portion of a molecular fingerprint, molecular signature, or map of the location of at least one extracellular molecule.

[0674] Embodiment 513 includes a method according to any one of embodiments 468 to 512, wherein the one or more cellular characteristics include all or a portion of a general or local representation of an extracellular molecule on a cell or tissue.

[0675] Embodiment 514 includes a method according to any one of embodiments 468 to 513, wherein the one or more cellular characteristics include proximity between two or more extracellular molecules.

[0676] Embodiment 515 includes the method of embodiment 514, wherein the two or more extracellular molecules are the same extracellular molecule or different extracellular molecules.

[0677] Embodiment 516 includes a method according to any one of embodiments 468 to 515, wherein the one or more cellular characteristics include an interaction of at least two extracellular molecules.

[0678] Embodiment 517 includes a method according to any one of embodiments 468 to 516, wherein the one or more cellular characteristics include a distance or distances between two or more extracellular molecules.

[0679] Embodiment 518 includes a method according to any one of embodiments 468 to 517, wherein the one or more cellular characteristics include individual or paired extracellular molecules.

[0680] Embodiment 519 includes the method of embodiment 518, wherein the individual or paired extracellular molecules are adjacent to or in contact with each other.

[0681] Embodiment 520 includes the method of embodiment 518, wherein the individual or paired extracellular molecules are within a distance of about 1 nanometer from each other.

[0682] Embodiment 521 includes the method of embodiment 518, wherein the individual or paired extracellular molecules are at a distance of about 1 nanometer to about 100 nanometers from each other.

[0683] Embodiment 522 includes the method of any one of embodiments 468 to 521, wherein the one or more cellular characteristics include individual extracellular molecules, or pairs or triplet of extracellular molecules.

[0684] Embodiment 523 includes the method of embodiment 522, wherein individual extracellular molecules, or pairs or triplet extracellular molecules, are adjacent to or in contact with each other.

[0685] Embodiment 524 includes the method of embodiment 522, wherein the individual extracellular molecules, or pairs or triads of extracellular molecules, are within a distance of about 1 nanometer to about 100 nanometers from each other.

[0686] Embodiment 525 includes the method of any one of embodiments 468 to 524, wherein the one or more cellular characteristics include pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules.

[0687] Embodiment 526 includes the method of embodiment 525, wherein the pairs, triads, tetrads, quintuplets, or higher order clusters of extracellular molecules are adjacent to or in contact with one another.

[0688] Embodiment 527 includes the method of embodiment 525, wherein the pairs, triplet, tetrad, quintuplet, or higher order clusters of extracellular molecules include the same or different molecules.

[0689] Embodiment 528 includes a method according to any one of embodiments 468 to 527, wherein the one or more cellular characteristics include a geometric relationship between two or more extracellular molecules.

[0690] Embodiment 529 includes the method of any one of embodiments 468 to 528, wherein the one or more cellular characteristics include translocation of at least one extracellular molecule.

[0691] Embodiment 530 includes the method of embodiment 529, wherein the transfer is internalization or transfer to the extracellular surface.

[0692] Embodiment 531 includes the method of any one of embodiments 468 to 530, wherein the at least one cellular characteristic includes internalization of at least two extracellular molecules.

[0693] Embodiment 532 includes a method according to any one of embodiments 468 to 531, wherein the one or more cellular characteristics include a change in two-dimensional or three-dimensional coordinates of an extracellular molecule.

[0694] Embodiment 533 includes a method according to any one of embodiments 468 to 532, wherein the one or more cellular characteristics include a change in clustering of extracellular molecules.

[0695] Embodiment 534 includes a method according to any one of embodiments 468 to 533, wherein the one or more cellular characteristics include changes in clustering of extracellular molecules.

[0696] Embodiment 535 includes the method of any one of embodiments 468 to 534, wherein the one or more cellular characteristics include an oligomerization state of an extracellular molecule.

[0697] Embodiment 536 includes a method according to any one of embodiments 468 to 535, wherein the one or more cellular characteristics include a clustering state of extracellular molecules.

[0698] Embodiment 537 includes the method of any one of embodiments 468 to 536, wherein the one or more cellular characteristics include names of proteins that cluster with the extracellular molecule.

[0699] Embodiment 538 includes the method of any one of embodiments 468 to 537, wherein at least one extracellular molecule is an extracellular molecule listed in Table 1.

[0700] Embodiment 539 includes a method according to any one of embodiments 468 to 538, further comprising repeating the method for one or more additional cell or tissue samples comprising diseased or non-disease state cells, and storing one or more cellular characteristics of each of the cell or tissue samples in a database to provide a database of cellular characteristics of diseased and non-disease state cells.

[0701] Embodiment 540 relates to a method for determining the local affinity (K d ) to one or more cellular characteristics of the cell or tissue sample stored in a database; d and storing the correlation with one or more cellular characteristics of the cell or tissue sample in a database.

[0702] Embodiment 541 is directed to a method for treating a pulmonary artery disease comprising administering localized K d 541. The method of embodiment 540, further comprising determining by single molecule tracking.

[0703] Embodiment 542 relates to a method for determining localized K for binding of an antibody to a second cell or tissue sample. d The method of any one of embodiments 540 or 541 further comprises predicting based on similarity of one or more cellular characteristics of the cell or tissue sample to one or more cellular characteristics of a second cell or tissue sample.

[0704]

[0046] Embodiment 543 includes the method of any one of embodiments 468 to 542, wherein the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a camelid-derived single domain antibody (VHH), a synthetically derived VHH, a murine engineered single domain antibody, a shark antibody, an antigen-binding fragment (Fab), a monoclonal antibody, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody, a diabody, and an scFv-Fc.

[0705] Embodiment 544 includes the method of any one of embodiments 468 to 543, wherein the antibody is a bispecific antibody.

[0706] Embodiment 545 includes the method of any one of embodiments 468 to 543, wherein the antibody is a multispecific antibody.

[0707] Embodiment 546 includes a method according to any one of embodiments 468 to 545, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VHH domains.

[0708] Embodiment 547 includes the method of any one of embodiments 468 to 546, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VL domains.

[0709] Embodiment 548 includes the method of any one of embodiments 468 to 547, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six or more VH domains.

[0710] Embodiment 549 includes the method of any one of embodiments 468 to 548, wherein the antibody is an immune cell engager.

[0711] Embodiment 550 includes the method of any one of embodiments 468 to 548, wherein the antibody comprises an effector domain (e.g., CD3 or CD16A).

[0712] Embodiment 551 includes the method of any one of embodiments 468 to 548, wherein the antibody-based therapy is for CAR-T cells, myeloid cells, NK cells, or other cell-based therapy.

[0713] Embodiment 552 includes the method of any one of embodiments 468 to 551, wherein the antibody is in a vaccine.

[0714] Embodiment 553 includes the method of any one of embodiments 468 to 552, wherein the antibody is a translated nucleic acid strand or multiple nucleic acid strands.

[0715] Embodiment 554 includes the method of any one of embodiments 468 to 553, wherein the antibody comprises a single chain polypeptide.

[0716] Embodiment 555 includes the method of any one of embodiments 468 to 554, wherein the antibody comprises a homodimer.

[0717] Embodiment 556 includes the method of any one of embodiments 468 to 555, wherein the antibody comprises a heterodimer.

[0718] Embodiment 557 includes a method according to any one of embodiments 468 to 556, wherein the super-resolution microscopy is stimulated emission depletion (STED) microscopy.

[0719] Embodiment 558 includes a method according to any one of embodiments 468 to 556, wherein the super-resolution microscopy is ground-state suppression (GSD) microscopy.

[0720] Embodiment 559 includes a method described in any one of embodiments 468 to 556, wherein the super-resolution microscopy is stochastic optical reconstruction microscopy (STORM) microscopy.

[0721] Embodiment 560 includes a method described in any one of embodiments 468 to 556, wherein the super-resolution microscopy method includes MINFLUX microscopy.

[0722] Embodiment 561 includes the method of any one of embodiments 468 to 560, wherein the cell or tissue sample comprises fixed cells.

[0723] Embodiment 562 includes the method of any one of embodiments 468 to 560, wherein the cell or tissue sample comprises live cells.

[0724] Embodiment 563 includes the method of any one of embodiments 468 to 562, wherein the cell or tissue sample comprises mammalian cells.

[0725] Embodiment 564 includes a method according to any one of embodiments 468 to 563, wherein the step of imaging a cell or tissue sample includes imaging cells on a tissue, a microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber.

[0726] Embodiment 565 includes the me...

Claims

1. 1. A method for generating a database containing cellular characteristics from diseased and non-diseased cells, the method comprising: imaging the sample contacted with the antibody using a super-resolution microscope; generating a database comprising at least one cellular characteristic of cells in said sample; and determining whether said at least one cellular characteristic is indicative of cells in said diseased state or cells in said non-disease state; A method comprising:

2. said at least one cellular characteristic (i) images of extracellular molecules; (ii) the two- or three-dimensional coordinates of an extracellular molecule; (iii) the strength of extracellular molecules; (iv) all or part of a molecular fingerprint, molecular signature, or map of the location of extracellular molecules; (v) all or part of a schematic or local representation of an extracellular molecule on a cell or tissue; (vi) proximity between two or more extracellular molecules; (vii) the interaction of at least two extracellular molecules; (viii) a distance or distances between two or more extracellular molecules; (ix) individual or paired extracellular molecules; (x) an individual extracellular molecule, a pair of extracellular molecules, or a triplet of extracellular molecules; (xi) pairs, triads, tetrads, quintuplets, or higher order clusters of extracellular molecules; (xii) a geometric relationship between two or more extracellular molecules; (xiii) translocation of at least one extracellular molecule; (xiv) internalization of at least one extracellular molecule; (xv) a change in the two-dimensional or three-dimensional coordinates of an extracellular molecule; (xvi) changes in clustering of extracellular molecules; (xvii) changes in clustering of extracellular molecules; (xviii) the oligomerization state of the extracellular molecule; (xix) clustering state of extracellular molecules; (xx) the name of the protein that clusters with the extracellular molecule; (xxi) changes in the expression of extracellular molecules; (xxii) alterations in the glycosylation patterns of extracellular molecules; (xxiii) changes in the enzymatic activity of extracellular molecules; (xxiv) changes in intracellular or intercellular communication; (xxv) alterations in intracellular or intercellular signaling or communication; (xxvi) alterations in cell adhesion, mechanics, or migration; and (xxvii) Tissue localization of extracellular molecules 2. The method of claim 1, wherein the compound is selected from the group consisting of:

3. The method described in claim 2, wherein the two or more extracellular molecules are for the same target or different targets.

4. The method described in claim 2, wherein the individual extracellular molecules or the paired extracellular molecules are (a) adjacent to or in contact with each other, (b) within a distance of about 1 nanometer from each other, or (c) at a distance of about 1 nanometer to about 100 nanometers from each other.

5. The method described in claim 2, wherein the individual extracellular molecules, the paired extracellular molecules, or the triplet of extracellular molecules are (a) adjacent to or in contact with each other, or (b) within a distance of about 1 nanometer to about 100 nanometers from each other.

6. The method described in claim 2, wherein the pairs, triplets, quartets, quintuplets, or higher-order clusters are adjacent to or in contact with each other.

7. The method described in claim 2, wherein the movement is internalization or movement to the extracellular surface.

8. The method described in claim 2, wherein the extracellular molecule is an extracellular molecule listed in Table 1. Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12

9. The method described in claim 1, wherein the at least one cellular characteristic is present in normal cells, cancer cells, both normal cells and cancer cells, or cells in the tumor microenvironment.

10. The method described in claim 1, wherein the at least one cellular characteristic predicts (a) the presence of cancer cells or cells in a tumor microenvironment, or (b) the presence of normal cells.

11. The method of claim 1, wherein the at least one cellular characteristic is (a) present in the same cell in a tissue, (b) present in at least two different cells in a tissue, (c) within or between the distances of the at least two different cells, or (d) present in a tissue structure in a normal state or a diseased state, wherein the diseased state comprises a cancerous disease state, an immunological disease state, a neurological disease state, an antiviral disease state, a cardiovascular disease state, or an autoimmune disease state, or wherein the tissue structure comprises an epithelium, a duct, or a blood vessel.

12. The method described in claim 2, wherein the tissue localization includes diseased tissue, cancer structure, or tumor microenvironment.

13. The method of claim 2, wherein the extracellular molecule is internalized upon contact with the antibody.

14. The method of claim 1, wherein different samples are evaluated for common or different cellular characteristics.

15. The method of claim 1, further comprising the step of characterizing the at least one cellular characteristic based on the intensity of the contrast agent in the image.

16. The method of claim 1, wherein the at least one cellular characteristic is evaluated (a) after administration of at least one ligand, or (b) after administration of at least one therapeutic agent or at least one therapeutic regimen.

17. The method of claim 16, wherein the imaging step includes tracking the at least one ligand, the at least one therapeutic agent, or the cell surface receptor over a period of time.

18. The method of claim 1, wherein the super-resolution microscope comprises a deterministic super-resolution microscope, a stimulated emission depletion (STED) microscope, a ground state depletion (GSD) microscope, a stochastic super-resolution microscope, a MINFLUX microscope, or a stochastic optical reconstruction microscopy (STORM) microscope.

19. The method of claim 1, wherein the diseased cells are derived from a tumor cell line or the non-diseased cells are derived from a normal cell line.

20. The method of claim 1, wherein the cells are imaged on a tissue microarray, a coverslip, a flow cell, a chip, or a microfluidic chamber.

21. The method of claim 1, wherein the antibody comprises an antibody selected from a single chain variable fragment (scFv), a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of a camelid-derived single domain antibody (VHH), a synthetic VHH, a mouse-generated single domain antibody, a shark antibody, an antigen-binding fragment (Fab), a monoclonal antibody, an F(ab') fragment, an F(ab')2 fragment, a single chain antibody, a diabody, an scFv-Fc, a bispecific antibody, and a multispecific antibody.

22. The method of claim 21, wherein the antibody comprises (a) at least one, at least two, at least three, at least four, at least five, or at least six VHH domains; (b) at least one, at least two, at least three, at least four, at least five, or at least six VL domains; or (c) at least one, at least two, at least three, at least four, at least five, or at least six VH domains.

23. The method of claim 22, wherein the antibody is an immune cell engager.

24. The method of claim 1, wherein the database of imaged cell characteristics is imaged at a resolution of at least about 50, about 40, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 nanometer.

25. The method described in claim 1, wherein the database of imaged cell characteristics is imaged at (a) single molecule resolution or (b) single fluorophore resolution.

26. The method of claim 1, wherein the database of imaged cell characteristics is imaged in a high-throughput format.

27. ​​The method described in claim 1, wherein multiple cellular characteristics are imaged (a) approximately simultaneously, (b) sequentially, or (c) some approximately simultaneously and the rest sequentially.

28. The method described in claim 27, wherein the multiple cell characteristics are on the surface of multiple cells.

29. The method of claim 2, wherein the extracellular molecule is (a) a membrane-bound protein, a membrane-bound ligand, or a protein or ligand bound to a membrane protein, or (b) selected from a protein ligand, a sugar, a lipid, a ligand, an extracellular receptor, a membrane-bound protein, a soluble protein, a structural protein, and a protein or ligand bound to a membrane protein.

30. A method described in any one of claims 1 to 29, further comprising a step of determining the local affinity (Kd) for binding of the antibody and correlating it with at least one cellular characteristic of the cells stored in the database.

31. A computer system for generating a database containing cellular characteristics from diseased and non-diseased cells, comprising: one or more processors; One or more memories having computer-executable instructions stored thereon, the computer-executable instructions, when executed, causing the computer system to receiving one or more images of the sample contacted with the antibody using a super-resolution microscope; generating a database comprising at least one cellular characteristic of cells in said sample; one or more memories for determining whether the at least one cellular characteristic is indicative of a cell in the diseased state or a cell in the non-disease state; A computer system comprising:

32. A non-transitory computer-readable medium having stored thereon computer-executable instructions that, when executed by one or more processors, cause a computer to receiving one or more images of the sample contacted with the antibody using a super-resolution microscope; generating a database comprising at least one cellular characteristic of cells in said sample; determining whether said at least one cellular characteristic is indicative of said diseased state cell or said non-disease state cell; Non-transitory computer-readable medium.

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