Additional Mass Tag Polymers for Mass Cytometry

Abstract

A new class of mass tag polymers containing enriched metal isotopes, such as zirconium and hafnium mass tags, is provided. The chemistry of these novel mass tags differs from that of lanthanide mass tags, opening new mass channels that can be used in mass cytometry. These polymers can be used in mass cytometry, therapeutic delivery of radioisotopes, or screening for therapeutic isotopes. Embodiments include kits, methods of making, and methods of using the polymers, isotopic compositions, or both. The kits can include a polymer. The polymer can include pendant groups that chelate enriched isotopes, such as zirconium and / or hafnium. The kits can include isotopic compositions containing enriched zirconium or hafnium isotopes. The polymers can be conjugated to bioactive materials. Embodiments can also include making the kits. Embodiments include using the kits, for example, for mass cytometry.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 059,545, filed July 31, 2020, and U.S. Provisional Patent Application No. 62 / 884,548, filed August 8, 2019, the contents of both of which are incorporated herein by reference for all purposes. [Background technology]

[0002] In mass cytometry, cells are labeled with mass-tagged bioactive materials (such as antibodies or oligonucleotides), and the mass tags can be detected by mass spectrometry with single-cell resolution. These mass tags are typically lanthanide-chelating polymers carrying enriched lanthanide isotopes. The number of mass-tagged bioactive materials that can be distinguished is determined by the number of isotopes with different masses. Additional mass tags allow for the simultaneous detection of more targets in mass cytometry applications, but new chemistries may need to be developed. Summary of the Invention

[0003] Aspects of the present disclosure include kits, methods of making, and methods of using the polymers and / or isotope compositions. While specific kits and methods are described herein, any unit or combination of kit components and / or method steps is within the scope of this application.

[0004] The kit can include a polymer. The polymer can include pendant groups that chelate enriched isotopes such as zirconium and / or hafnium. The kit can further include an isotopic composition that includes enriched isotopes of zirconium or hafnium.

[0005] In certain embodiments, the polymer may include one or more pendant groups comprising a hydroxamate (e.g., hydroxamic acid), an azamacrocycle, a phenoxyamine, a salophen, a cyclam ligand, and / or a derivative thereof. The polymer may include a derivative of a hydroxamate, an azamacrocycle, a phenoxyamine, a salophen, or a cyclam that forms an eight-coordinate complex with at least one of zirconium or hafnium. For example, at least one of zirconium and hafnium may form an eight-coordinate complex with the pendant group of the polymer.

[0006] In certain embodiments, the polymer comprises a hydroxamate group, such as desferrioxamine (DFO) and / or a derivative thereof. Alternatively or additionally, the polymer may comprise an azamacrocycle, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof. The polymer may further comprise a solubility-promoting moiety, such as a PEGylated pendant group, which may aid in polymer loading. The PEGylated pendant group may be separate from the pendant group that chelates zirconium and / or hafnium. The pendant group that chelates zirconium and / or hafnium may be PEGylated (e.g., in addition to the pendant group that does not chelate zirconium and / or hafnium). For example, the pendant group may comprise a DFO derivative (e.g., comprising four hydroxamate groups) and may include a solubilizing group, such as an ether, between the hydroxamate groups. By improving the solubility of the chelating pendant group, it may be possible to incorporate more pendant groups into the polymeric mass tag without resulting in insolubility, aggregation, steric hindrance, and / or non-specific binding. For example, such a polymer may contain more than 10, more than 15, more than 20, or more than 25 chelating pendant groups (e.g., examples of DFO or its derivatives).

[0007] While PEGylation is described in the above example, any suitable solubilizing group can be used. Such solubilizing groups include charged groups such as ethers (e.g., polyethers such as polyethylene glycol), oxazolines (or polyoxazolines), and zwitterionic polymers. Oxazolines (and, e.g., derivatives thereof) are one such solubilizing group and can be used instead of or in addition to ethylene glycol groups. For example, polymeric mass tags can include polyoxazolines (e.g., poly(2-oxazolines), such as poly(2-methyl-2-oxazoline), (2-ethyl-2-oxazoline), (2-propyl-2-oxazoline)) to improve the solubility of the polymer and / or reduce aggregation, steric hindrance, and / or nonspecific binding. The solubilizing group can be charged. For example, a combination of positive and negative charges can provide a zwitterionic polymer with improved solubility and / or reduce aggregation, steric hindrance, and / or nonspecific binding.

[0008] The kits of the present application can include a polymer comprising a hydroxamate. For example, multiple pendant groups of the polymer can comprise a hydroxamate. The kits can further include an isotopic composition comprising an enriched metal isotope that can be chelated by the pendant groups.

[0009] The polymers of the present application (e.g., carrying an isotopic composition) can be conjugated to a bioactive material, such as an affinity reagent, such as an antibody. For example, the antibody can target an epitope preferentially expressed in cancer cells. The polymer can be conjugated to the antibody. The solubility of the polymer can assist in antibody binding to its epitope. In this manner, a kit can include a bioactive material conjugated to a carrying polymer described herein.

[0010] In certain embodiments, the kit can include an isotopic composition of enriched metal isotopes, such as a composition including a zirconium isotope and / or a hafnium isotope. For example, the metal isotope can be a zirconium isotope. In another example, the metal isotope is a hafnium isotope. The kit can include additional isotopic compositions including additional zirconium and / or hafnium isotopes. The isotopic compositions can be non-radioactive, e.g., for use in mass spectrometry applications (e.g., as mass tags for mass cytometry). Alternatively, the isotopic compositions can be non-radioactive, e.g., for use in radiopharmaceutical applications (e.g., biomedical imaging, such as 89-Zr PET imaging). 89 The isotopic composition may include a radioisotope such as Zr. The isotopic composition may be supported on a polymer in the kit (such that one or more pendant groups on the polymer chelate the enriched metal isotope of the isotopic composition). Alternatively, the isotopic composition may be provided separately from the polymer.

[0011] The isotopic composition may be provided in a solution (e.g., in an aprotic solvent) including an aprotic solvent (e.g., a polar aprotic solvent) such as pyridine, ethyl acetate, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and / or hexamethylphosphoramide (HMPA). Alternatively or additionally, the isotopic composition may be provided in an acidic solution. The isotopic composition may include a chloride salt form of an enriched metal isotope (e.g., an isotope of zirconium or hafnium), or may include a chloride salt form dissolved in solution. The isotopic composition may be provided in a form suitable for loading onto a polymer of the present application.

[0012] The isotopic composition can be provided separately from the polymer. For example, the isotopic composition is in solution. Alternatively, the isotopic composition can be supported on one or more pendant groups of the polymer. The polymer can be in solution. Alternatively, the polymer can be lyophilized.

[0013] The polymer may include a pendant group that aids (e.g., increases) the solubility of the polymer, such as a PEGylated pendant group. For example, the polymer may be modified to include a pendant group that aids in the solubility of the polymer before and / or after loading a metal isotope. The pendant group may include a hydrophilic group that aids in the solubility of the polymer before and after loading a metal isotope onto the pendant group. In certain embodiments, the polymer may be PEGylated.

[0014] The polymer can be functionalized to attach a bioactive material. In certain embodiments, the polymer can be functionalized with thiol-reactive chemistry, amine-reactive chemistry, or click chemistry. For example, the polymer can be functionalized for thiol reactivity (e.g., attached to a thiol group on the Fc portion of an antibody via a maleimide group).

[0015] For example, a kit may include a polymer including a plurality of pendant groups and an isotopic composition including enriched isotopes of zirconium or hafnium. The pendant groups may include polyethylene glycol (PEG) groups and / or groups including DFO or a derivative thereof. The PEG group may aid in the solubility of the polymer and / or aid in the loading of the isotopic composition onto the polymer. Each pendant group may include a DFO (or a derivative thereof) group, a PEG group, or both. The isotopic composition may be provided separately from the polymer or may be loaded onto the polymer.

[0016] The kit may further include any additional components for loading the isotopic composition onto the polymer and / or binding the loaded polymer to the bioactive material (e.g., buffers, filters, etc.) Alternatively or additionally, the kit may include additional reagents for mass cytometry, such as buffers, standards, cell barcodes, and / or reagents containing heavy atoms of different masses.

[0017] Aspects of the present application include making the kits or portions thereof discussed herein. Aspects of the present application include using the kits described herein, for example, for mass cytometry.

[0018] Aspects of the present application include a method of making a polymer for mass cytometry, the method comprising providing a polymer comprising multiple instances of pendant groups that include hydroxamates.

[0019] In certain aspects, a method of making a kit may include one or more of making a polymer, providing an isotopic composition comprising an enriched metal isotope, loading the isotopic composition (e.g., the enriched metal isotope of the isotopic composition) onto a polymer, and attaching the loaded polymer to a biologically active material.

[0020] A method of mass cytometry may include labeling cells of a biological sample with a mass-tagged bioactive material comprising an enriched isotope of zirconium or hafnium, and detecting the mass tags bound to the cells by mass spectrometry. The method may include providing a kit of the present application, for example, by obtaining the kit from a third party or by making the kit as described herein. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows the preparation of commercially available lanthanide mass tag polymers. [Figure 2] 1 shows the preparation of an exemplary mass tag polymer of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0022] Aspects of the present disclosure include kits, methods of making, and methods of using the polymers and / or isotope compositions. While specific kits and methods are described herein, any unit or combination of kit components and / or method steps is within the scope of this application.

[0023] As used herein, a sample is a biological sample, such as a cell sample or a biological fluid. A cell sample may include a cell suspension or cells on a solid support (such as a tissue). In certain embodiments, a portion of a cell may be provided. A biological sample may be obtained from any tissue, including blood or solid tissue, or from a cell culture.

[0024] As used herein, a bioactive material can be any material that binds to or modifies a portion of a biological system. For example, a bioactive material can be an antibody, amino acid, nucleoside, nucleotide, aptamer, protein, antigen, peptide, nucleic acid, oligonucleotide, enzyme, lipid, albumin, cell, carbohydrate, vitamin, hormone, nanoparticle, inorganic support, polymer, monomolecule, or drug. In certain cases, the biomolecule can be an affinity reagent that binds to a specific target based on its tertiary structure, such as an antibody (e.g., including a recombinant antibody or antibody fragment), an aptamer (e.g., a DNA aptamer or an RNA aptamer), a lectin, biotin / streptavidin, a receptor / ligand, or any other suitable biomolecule. In certain aspects, the biomolecule can be an oligonucleotide that hybridizes to a DNA or RNA target or an intermediate (e.g., an intermediate oligonucleotide in a hybridization mechanism, or an oligonucleotide attached to an antibody intermediate).

[0025] As used herein, a mass tag includes any tag that contains enriched heavy atoms, such as enriched metal isotopes. Mass tags may include polymers carrying enriched metal isotopes and, optionally, may contain conjugated bioactive materials. Mass tags may be distinguishable based on the atomic masses of their enriched metal isotopes.

[0026] As used herein, mass cytometry refers to any method for detecting mass tags in a biological sample, e.g., simultaneously detecting multiple distinguishable mass tags with single-cell resolution. Mass cytometry includes suspension mass cytometry and imaging mass cytometry (IMC). Mass cytometry may atomize and ionize mass tags in a cell sample using one or more of laser radiation, ion beam radiation, electron beam radiation, and / or inductively coupled plasma (ICP). Mass cytometry may simultaneously detect separate mass tags from a single cell, e.g., by time-of-flight (TOF) or magnetic sector mass spectrometry (MS).

[0027] Aspects of the present application include making the kits or portions thereof discussed herein. Aspects of the present application include using the kits described herein, for example, for mass cytometry or delivery of radioisotopes.

[0028] Aspects of the present application include a method of making a polymer for mass cytometry, the method comprising providing a polymer comprising multiple instances of pendant groups that include hydroxamates.

[0029] In certain aspects, a method of making a kit may include one or more of making a polymer, providing an isotopic composition comprising an enriched metal isotope, loading the isotopic composition (e.g., the enriched metal isotope of the isotopic composition) onto a polymer, and attaching the loaded polymer to a biologically active material.

[0030] The kit can include a polymer. The polymer can include pendant groups that chelate enriched isotopes, such as zirconium and / or hafnium. The kit can further include an isotopic composition that includes enriched isotopes, such as isotopes of zirconium or hafnium.

[0031] The kit, kit components, and kit preparation process may include suitable storage media. For example, solvents and cosolubilizers may include, but are not limited to, water, sterile water for injection (SWFI), saline, alcohols such as ethanol and benzyl alcohol, glycols and polyalcohols such as propylene glycol and glycerin, esters of polyalcohols such as diacetin and triacetin, polyglycols and polyethers such as polyethylene glycol 400 and propylene glycol methyl ether, dioxolanes such as isopropylideneglycerin, dimethyl isosorbide, pyrrolidone derivatives such as 2-pyrrolidone, N-methyl-2-pyrrolidone, polyvinylpyrrolidone (cosolubilizer only), polyoxyethylenated fatty alcohols, esters of polyoxyethylenated fatty acids, polysorbates such as Tween™, polyoxyethylenated derivatives of polypropylene glycol such as Pluronics™. In certain aspects, the co-solubilizing agents listed above may be incorporated into the mass tag polymers described herein, for example, in place of or in addition to the PEG groups described herein. Suitable stabilizing agents include, but are not limited to, one or more monosaccharides (e.g., galactose, fructose, and fucose), disaccharides (e.g., lactose), polysaccharides (e.g., dextran), cyclic oligosaccharides (e.g., alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin), aliphatic polyols (e.g., mannitol, sorbitol, and thioglycerol), cyclic polyols (e.g., inositol), organic solvents (e.g., ethyl alcohol and glycerol), and / or aprotic solvents (e.g., pyridine, ethyl acetate, DMF, HMPA, and DMSO). The above solvents and / or stabilizers may be used in any step from polymerization to attachment of pendant groups and / or modification with chelating agents, loading the polymer with an isotopic composition, binding the polymer to a biologically active material, or storing any of the above reagents (e.g., for provision in a kit). In certain embodiments, the solution may be acidic.The acidic solution of the present application may include one or more strong acids, such as nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, perchloric acid, hydrochloric acid, and chloric acid. The acid may be present at more than 0.01% (e.g., more than 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, or 5%) and / or less than 10% (e.g., less than 5%, 2%, 1%, 0.5%, 0.2%, or 0.1%). For example, the acid may be present at 0.05% to 2%. The acidic solution may have a pH of 6 or less, 5 or less, 4.5 or less, or 4 or less. The lyophilized composition of the present application may have a moisture content (by weight) of less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0032] In any step (e.g., when the composition described herein is provided in a kit), the composition can be lyophilized. For example, the composition (e.g., a polymer, an isotope composition, a supported polymer, or a polymer conjugated to a bioactive material) can be lyophilized to a water content (e.g., by mass) of less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. Such lyophilization can allow storage in a kit before use in mass cytometry, and / or can allow flexibility in assay design if the polymer is stabilized for subsequent attachment to a bioactive material.

[0033] Chelating Agents and Chelating Pendant Groups As used herein, a chelator refers to a group of ligands that coordinate (e.g., stably coordinate) metal atoms together. The kit may include a polymer comprising one or more chelators of the present application. The chelator may be present in a pendant group of the polymer and / or may be incorporated into the polymer backbone. In certain embodiments, the chelator is included in a pendant group of the polymer.

[0034] In certain embodiments, the polymer may include one or more pendant groups comprising ligands such as hydroxamates (used interchangeably herein with hydroxamic acids), azamacrocycles, phenoxyamines, salophens, cyclams, and / or derivatives thereof. The polymer may include a chelating agent known in the art or a derivative thereof, including hydroxamates, azamacrocycles, phenoxyamines, salophens, or cyclams. In certain embodiments, the chelating agents of the present application may coordinate six or more, more than six, or eight sites of zirconium or hafnium atoms. For example, the chelating agent may form an eight-coordinate complex with at least one of zirconium and hafnium. For example, at least one of zirconium and hafnium may form an eight-coordinate complex with the pendant group of the polymer.

[0035] In certain embodiments, the polymeric chelator comprises a hydroxamate group, such as DFO and / or a derivative thereof. In certain embodiments, the chelator is a DFO derivative that has improved binding of zirconium or hafnium compared to DFO. For example, a DFO derivative may coordinate eight sites of zirconium and / or hafnium atoms and may optionally include spacing between the ligands (hydroxamate groups) to aid in binding (e.g., stable binding) of zirconium and / or hafnium.

[0036] In certain embodiments, the DFO derivative can be an octadentate derivative (i.e., chelating the metal at eight coordination sites). Alternatively or additionally, the DFO derivative can include a solubilizing group, such as an ether group, located between the hydroxamate groups. Such DFO derivatives are described for radiopharmaceutical applications in Briand et al., "A solid phase-assisted approach for the facile synthesis of a highly water-soluble zirconium-89 chelator for radiopharmaceutical development." Dalton Transactions, 46.47 (2017): 16387-16389.

[0037] Alternatively or additionally, the polymer can include an azamacrocycle, such as a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator or a derivative thereof. In certain embodiments, the chelator can include one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide (DOTAM), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene)phonic acid (DOTP), and DOTA (e.g., carrying a zirconium or hafnium isotope or provided separately from the zirconium or hafnium isotope). In certain embodiments, the chelator is a DOTA derivative that has improved binding of zirconium or hafnium (and potentially reduced binding to lanthanides) compared to DOTA. For example, the DOTA derivative can coordinate eight sites of zirconium and / or hafnium atoms and can optionally include spacing between the ligands that assists zirconium and / or hafnium in binding (e.g., stably binding). For example, the DOTA derivative can have increased binding to zirconium and / or hafnium compared to lanthanide isotopes.

[0038] a chelating agent suitable for coordinating zirconium and / or hafnium, and 89 The use of chelating agents in applications such as therapeutic delivery of Zr or detection by positron emission tomography (PET) scans has been discussed by Patra et al. (US Patent Application Publication No. 20170106206) and Wadas et al. (US Patent Application Publication No. 20190038785).

[0039] The polymer may further comprise a solubilizing group, as further described herein, such as a pegylated pendant group that may aid in polymer loading. The polymer may comprise a pegylated pendant group that is separate from the pendant group that chelates the enriched metal isotope. Alternatively or additionally, the pegylated pendant group may also comprise a chelating agent.

[0040] The chemistry of the pendant group can be optimized by the addition of various functional groups to the macrocycle. For example, the pendant arm composition, such as the combination of ligand and optional solubilizing group, the spacing of the ligand, and / or the linker composition between ligands on the same pendant arm can aid in the stable coordination of the metal isotopes described herein and can further provide other desirable properties described herein. The pendant group chelators can be specifically developed for their chemistry when attached to the polymers of the present application.

[0041] polymer A method for making a kit of the present application may include providing a polymer. Providing the polymer may include obtaining the polymer from a third party. Alternatively, providing the polymer may include polymerizing the pendant group by living polymerization. In living polymerization, chain termination and chain transfer reactions may be absent or minimal, and the chain initiation reaction rate may be faster than the chain propagation reaction rate. The resulting polymer chains may grow at a more constant rate than seen in traditional chain polymerization, and the length of the polymer may remain consistent (i.e., the length of the polymer may have a low polydispersity index, as described herein). The living polymerization used to make the polymer of the present application may include one or more of anionic polymerization, controlled radical polymerization (such as catalytic chain transfer polymerization, iniferter-mediated polymerization, stable free radical-mediated polymerization (SFRP), atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and iodine transfer polymerization), cationic polymerization, and / or ring-opening polymerization. The polymerized pendant groups may include a chelating agent, a solubilizing group, or both. Individual pendant groups of the polymer may include a chelating agent, a solubilizing group, or both. The pendant groups of the polymer may be functionalized to add a chelating agent and / or a solubilizing group after polymerization. Alternatively or additionally, at least some of the pendant groups may include a chelating agent and / or a solubilizing group before polymerization.

[0042] The polymer may have low polydispersity, e.g., to allow quantitation by mass cytometry and / or to have a consistent effect on the conjugated bioactive material. For example, the polymer may have a polydispersity index of less than 1.5, less than 1.4, less than 1.3, less than 1.2, or less than 1.1.

[0043] In certain embodiments, the polymer may comprise an organic backbone. The polymer may comprise acrylate monomers such as acrylic acid, carboxylic acid, acrylonitrile, methyl methacrylate, etc. In certain embodiments, the polymer may comprise a biopolymer such as a polysaccharide, polypeptide, or polynucleotide. The polymer may comprise an electron-rich alkene such as vinyl ether, isobutylene, styrene, and / or N-vinylcarbazole. Alternatively or additionally, the polymer may comprise ethylene, propylene, styrene, amine, hexene, aspartic acid, acrylamide, activated ester, any derivative thereof, and / or any other suitable backbone known in the art (such as any polymer suitable for living polymerization). The polymer may be a copolymer (and may support the attachment of different pendant groups). The polymers of the present application may be linear, branched, or hyperbranched. In certain embodiments, the polymer may be a linear polymer.

[0044] The polymer backbones of the present application can include any suitable number of repeat units (which can be modified, for example, to include pendant groups) in the backbone, such as greater than 2, 5, 10, 20, 30, 40, 50, 100 repeat units. For example, the polymer can include from 2 to 100 repeat units, between 5 and 80, between 10 and 50, or between 20 and 40 repeat units.

[0045] Lanthanide mass tag polymers (Maxpar® reagents) sold by Fluidigm allow for conjugation to antibodies as shown in Figure 1. Specifically, the polymer contains multiple pendant groups that can carry lanthanide isotopes. The polymer is functionalized for thiol reactivity, and reduction of the antibody (e.g., via tris(2-carboxyethyl)phosphine (TCEP)) provides thiol (-SH) groups that allow for conjugation to the polymer mass tag. A filtration-based purification step is performed separately on the reduced antibody after loading of the metal (lanthanide solution) onto the polymer.

[0046] In contrast, Figure 2 illustrates a zirconium or hafnium mass tag polymer of the present application. In addition to the metal-chelating pendant groups, the polymer may include pendant groups that aid in solubility but do not chelate the metal (e.g., metals further described herein) represented by the black pendant groups in the figure. A solution of a metal ion (M+), such as an isotope of zirconium or hafnium, can be supported on the chelating pendant group of the polymer (e.g., a pendant group comprising DFO or a derivative thereof). As discussed herein, DFO derivatives may allow for octadentate chelation (e.g., via four hydroxamate groups) and / or may include one or more solubility-aiding moieties between the hydroxamates. Notably, the M+ symbol in Figure 2 refers to any charged metal ion, such as a metal with a 4+ charge (e.g., zirconium or hafnium with a 4+ charge). Attachment of the polymer to the biomolecule (e.g., to an affinity reagent such as an antibody or fragment thereof) can be via thiol-reactive chemistry, amine-reactive chemistry, click chemistry, or any suitable conjugation mechanism known in the art. In certain aspects, the mass tag polymers of the present application can be formed by ring-opening polymerization. Certain aspects of making the subject mass tags include azide-initiated polymerization of the polymer backbone, with subsequent incorporation (e.g., by aminolysis) of pendant groups (such as a mixture of chelating and non-chelating, solubility-assisting pendant groups). Such mass tags can include azide functional groups for click chemistry-mediated conjugation. For example, amine modification or modification of amino acid motifs on biomolecules (such as antibodies) can provide dibenzocyclooctyne (DBCO) groups for reaction with azides. The design and synthesis of such mass tag polymers can have solubility that allows for metal loading and / or conjugation to antibodies, and can further reduce aggregation, steric hindrance, and / or nonspecific binding. Furthermore, such an approach may allow for simpler synthesis, increased yields, mass tags that are capable of conjugation to moieties other than thiols, and / or the incorporation of more metals.

[0047] In certain embodiments, the polymer comprises a hydroxamate group, such as desferrioxamine (DFO) and / or a derivative thereof. Alternatively or additionally, the polymer may comprise an azamacrocycle, such as DOTA or a derivative thereof. The polymer may further comprise a solubility-enhancing moiety, such as a PEGylated pendant group, which may aid in polymer loading. The PEGylated pendant group may be separate from the pendant group that chelates zirconium and / or hafnium. The pendant group that chelates zirconium and / or hafnium may be PEGylated (e.g., in addition to pendant groups that do not chelate zirconium and / or hafnium). For example, the pendant group may comprise a DFO derivative (e.g., comprising four hydroxamate groups) and may include a solubility-enhancing group, such as an ether, between the hydroxamate groups. Improving the solubility of the chelating pendant group may allow more pendant groups to be incorporated into the polymeric mass tag without resulting in insolubility, aggregation, steric hindrance, and / or nonspecific binding. For example, such polymers may include more than 10, more than 15, more than 20, or more than 25 chelating pendant groups (eg, examples of DFO or its derivatives).

[0048] While PEGylation is described in the above example, any suitable solubilizing group can be used. Such solubilizing groups include charged groups such as ethers (e.g., polyethers such as polyethylene glycol), oxazolines (or polyoxazolines), and zwitterionic polymers. Oxazolines (and, e.g., derivatives thereof) are one such solubilizing group and can be used instead of or in addition to ethylene glycol groups. For example, polymeric mass tags can include polyoxazolines (e.g., poly(2-oxazolines), such as poly(2-methyl-2-oxazoline), (2-ethyl-2-oxazoline), (2-propyl-2-oxazoline)) to improve the solubility of the polymer and / or reduce aggregation, steric hindrance, and / or nonspecific binding. The solubilizing group can be charged. For example, a combination of positive and negative charges can provide a zwitterionic polymer with improved solubility and / or reduce aggregation, steric hindrance, and / or nonspecific binding.

[0049] Pendant group modification The polymer may include a pendant group, such as a PEGylated pendant group, that aids (e.g., increases) the solubility of the polymer. For example, the polymer may be modified to include a pendant group that aids in the solubility of the polymer before and / or after loading a metal isotope. Here, the pendant group includes a hydrophilic group that aids in the solubility of the polymer before and after loading a metal isotope onto the pendant group. In this manner, one or more pendant groups of the polymer may include a chain of repeating hydrophilic groups (e.g., that aid in the solubility of the polymer). For example, the coordinating pendant group may include a hydrophilic group and / or may be separate from the pendant group that includes a hydrophilic group. The chain of repeating hydrophilic groups may not affect the coordination chemistry of the coordinating pendant group of the polymer. The hydrophilic group may include a PEG group. The assisted (e.g., increased) solubility of the polymer may aid in (e.g., increase) the loading of the metal isotope in solution.

[0050] In certain aspects, pendant groups (e.g., bearing chelators and / or solubilization-promoting groups) can be incorporated during polymerization of the backbone. Alternatively or additionally, pendant groups, solubilization groups (e.g., chains), or both can be attached to functional groups provided by the polymer backbone, e.g., by any attachment chemistry known in the art. For example, a ratio of chelator to solubilization groups can be added to the polymer to obtain a ration of pendant groups bearing chelators relative to pendant groups bearing solubilization groups (and no chelators). Suitable attachment chemistries may include carboxyl-to-amine reactive chemistries (e.g., reaction with carbodiimides), amine reactive chemistries (e.g., reaction with N-hydroxysuccinimide (NHS) esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphine, etc.), sulfhydryl reactive chemistries (e.g., reaction with maleimides, haloacetyls (bromoacetyl or iodoacetyl), pyridyl disulfides, thiosulfonates, vinyl sulfones, etc.), aldehyde reactive chemistries (e.g., reaction with hydrazides, alkoxyamines, etc.), and hydroxyl reactive chemistries (e.g., reaction with isothiocyanates). Alternative attachment methods include click chemistries such as strain-promoted click chemistry (e.g., with DBCO-azide or trans-cyclooctene (TCO)-tetrazine).

[0051] The polymer may include solubilization-promoting groups, at least some of which may be organized in a chain. The solubilization-promoting groups used herein do not need to coordinate metal atoms. The polymer may be PEGylated to aid (e.g., increase) solubility. For example, the polymer may include at least 50, at least 100, at least 200, or at least 500 PEG units (e.g., PEG groups). The PEG units may be distributed across multiple pendant groups such that multiple pendant groups of the polymer may be PEGylated. For example, at least some pendant groups may include more than 5, more than 10, more than 20, more than 30, or more than 40 PEG units (e.g., organized in a chain). The number of PEG units in the polymer may aid (e.g., increase) the loading of the metal isotope into the polymer. In certain embodiments, less than 50% of all pendant groups of the polymer chelate zirconium and / or hafnium, and more than 50% of all pendant groups of the polymer include multiple PEG units. For example, less than 60% to more than 30%, such as less than 50% to more than 40% of the pendant groups of the polymer may comprise a chelating agent.

[0052] In certain embodiments, PEGylation of a polymer can include attaching a chain of PEG units to a pendant group of the polymer. The chain can include 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more PEG units. The PEGylated pendant group can include a chelator or can be separate from the pendant group that includes a chelator. The amount, distribution, and / or proportion of the chelator and solubilizing group (e.g., PEG) can aid in loading of the isotopic composition onto the polymer. For example, the amount, distribution, and / or proportion of the chelator and solubilizing group (e.g., PEG) can maximize (e.g., within 80%, 90%, or 95% of the maximum) the amount of isotopic composition (e.g., enriched isotope of the composition) that can be loaded onto the polymer. Polymer loading is discussed further herein.

[0053] While PEGylation is described in the above examples, any suitable solubilizing group can be used. Such solubilizing groups include ethers (or polyethers), oxazolines (or polyoxazolines), and charged groups, for example, in zwitterionic polymers. Oxazolines (and, for example, their derivatives) are one such solubilizing group and can be used instead of or in addition to ethylene glycol groups. For example, polymeric mass tags can include polyoxazolines (e.g., poly(2-oxazolines)) to improve the solubility of the polymer and / or reduce aggregation, steric hindrance, and / or nonspecific binding. The solubilizing group can be charged. For example, a combination of positive and negative charges can provide a zwitterionic polymer with improved solubility and / or reduce aggregation, steric hindrance, and / or nonspecific binding.

[0054] The polymer (e.g., before loading, after loading, and / or after conjugation to a bioactive material) can be non-aggregating (e.g., can be resistant to aggregation). For example, more than 90%, more than 95%, more than 98%, more than 99%, or substantially all of the polymer can be non-aggregating. As described herein, the polymer can be unloaded with an isotopic composition, loaded with an isotopic composition, and / or conjugated to a bioactive material. The polymer can be in solution as described herein. For example, more than 90%, more than 95%, more than 98%, more than 99%, or substantially all of the polymer can be non-aggregating. The polymer provided in the kit (e.g., with additional components described herein) can be stable for at least 1 month, at least 3 months, at least 6 months, or at least 1 year.

[0055] The polymers of the present application can include any suitable number of pendant groups (e.g., attached to repeat units of the polymer backbone), such as greater than 2, 5, 10, 20, 30, 40, 50, 100 pendant groups. For example, the polymer can include from 2 to 100 pendant groups, between 5 and 80, between 10 and 50, or between 20 and 40 pendant groups.

[0056] Isotopic Composition of the Present Application The kit may include an isotopic composition of enriched metal isotopes, such as a composition including a lanthanide isotope or a transition isotope. The enriched metal isotope may include an isotope from Group 3, 4, 6, 7, 9, 10, 11, 13, or 15 of the periodic table of elements. In certain embodiments, the isotopic composition may include a Group 4 isotope, such as a zirconium isotope or a hafnium isotope. For example, the metal isotope may be a zirconium isotope. In another example, the metal isotope is a hafnium isotope. The kit may include additional isotopic compositions including additional zirconium and / or hafnium isotopes. The isotopic composition may be non-radioactive. Alternatively, the isotopic composition may be 89 The isotopic composition may include a radioisotope such as Zr. The isotopic composition may be supported on a polymer in the kit (such that one or more pendant groups on the polymer chelate the enriched metal isotope of the isotopic composition). Alternatively, the isotopic composition may be provided separately from the polymer.

[0057] In certain embodiments, the kit can include an isotopic composition of enriched metal isotopes, such as a composition including zirconium and / or hafnium isotopes. For example, the metal isotope can be a zirconium isotope. The zirconium isotope can be a naturally occurring isotope, 90 Zr, 91 Zr, 92 Zr, 94 Zr, 96 The zirconium isotope may be Zr. The zirconium isotope may be non-radioactive. Alternatively, the zirconium isotope may be 89 In another example, the metal isotope is a hafnium isotope. 174 Hf, 176 Hf, 177 Hf, 178 Hf, 179 Hf, or 180 It can be Hf.

[0058] As described herein, the kits of the present application can include a polymer comprising a hydroxamate. For example, multiple pendant groups of the polymer can comprise a hydroxamate. The kits can further include an isotopic composition comprising an enriched metal isotope that can be chelated by the pendant groups.

[0059] The isotopic composition may be provided in a solution comprising (e.g., in) an aprotic solvent (e.g., a polar aprotic solvent) such as pyridine, ethyl acetate, DMF, DMSO, and / or HMPA. Alternatively or additionally, the isotopic composition may be provided in an acidic solution, e.g., a solution having a pH of 6 or less, 5 or less, 4.5 or less, e.g., between 4 and 6. The isotopic composition may include a chloride salt form of an enriched metal isotope (e.g., a crystallized chloride salt form of an isotope of zirconium or hafnium), or may include a chloride salt form dissolved in solution. For example, the chloride salt can be dissolved at a concentration of greater than 0.1 mg / ml (e.g., greater than 0.2 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, or 50 mg / ml) and / or less than 100 mg / ml (e.g., less than 50 mg / ml, 20 mg / ml, 10 mg / ml, or 5 mg / ml). For example, the chloride salt can be dissolved at 0.5 mg / ml to 20 mg / ml. The isotope composition can be provided in a form suitable for loading onto the polymer of the present application.

[0060] The polymers of the present application may be provided with or may carry non-lanthanide metal isotopes, such as zirconium or hafnium. In certain embodiments, the metal isotope may be a zirconium isotope. In certain embodiments, the metal isotope may be a hafnium isotope.

[0061] As described herein, a metal isotope can be an enriched metal isotope, where a single isotope is present at a higher abundance than in naturally occurring metals. For example, an enriched metal isotope can be present at greater than 95%, 99%, or 99.9% purity. The isotopic enrichment can be due to bombardment of precursor elements. For example, yttrium 89 ( 89Y) is converted to zirconium by proton bombardment. 89 ( 89 Alternatively, enrichment can be by atomic weight (i.e., mass-based), for example, by centrifugation or sector mass spectrometry (e.g., calutron).

[0062] A salt form of the enriched metal isotope can be provided to enable solubilization and / or loading in the polymer of the present application. The salt can be a chloride salt or an oxalate salt. In certain embodiments, the salt form can be a chloride form, such as an oxychloride form or a tetrachloride form. For example, the enriched metal isotope can be zirconium tetrachloride or hafnium tetrachloride.

[0063] Therapeutic uses include: 89 Radioisotopes such as Zr may be desirable. For example, to a polymer conjugated to an antibody. 89 Zr loading and delivery by antibodies 89 The number of Zr atoms can be increased.

[0064] In mass cytometry applications, multiple distinguishable mass tags containing different enriched isotopes and attached to different bioactive materials may be used. Furthermore, mass tags for mass cytometry may be free of radioactive isotopes, as such isotopes may be hazardous to the user. In this way, zirconium or hafnium isotopes for mass cytometry may be used. 89 Zr may be excluded and may be enriched by atomic weight. In such cases, naturally occurring metals such as zirconium or hafnium may be processed by both 1) isotopically enriching by molecular weight and 2) obtaining a form suitable for loading onto the polymers of the present application.

[0065] A method of making a kit of the present application can include providing an isotopic composition. In certain embodiments, the method can include providing both a polymer and an isotopic composition. Providing the isotopic composition can include obtaining the isotopic composition from a third party. Alternatively, providing the isotopic composition can include one or more of enriching an isotope (such as an isotope of zirconium or hafnium) and / or converting the isotope to a salt.

[0066] Salt forms (such as chloride salts or oxalate salts) can be dissolved in solution and supported on the polymers of the present application. Zirconium or hafnium can be provided as a salt or in solution. In certain embodiments, zirconium or hafnium can be prepared using methods such as those described in "Standardized methods for the production of high specific-activity zirconium-containing compounds" by Holland et al. (Holland, Jason P., Yiauchung Sheh, Jason S. Lewis, "Standardized methods for the production of high specific-activity zirconium-containing compounds"). 89 ", Nuclear medicine and biology 36, No. 7 (2009) pp. 729-739), Mohandas et al. ("Electrochemical deoxidation of solid zirconium dioxide in molten calcium chloride" by Mohandas, K.S., D.J. Fray, Metallurgical and materials transactions B 40.5 (2009) pp. 685-699), or Tuyen et al. ("Preparation of High Quality Zirconium Oxychloride from Zircon of Vietnam" by Tuyen, Ngo Van et al.).

[0067] Providing an isotopic composition can include purifying zirconium or hafnium from a source, e.g., purifying an oxide form of zirconium or hafnium from sand, or otherwise obtaining an oxide form of zirconium or hafnium. In certain embodiments, methods can include obtaining dry zirconium oxide (e.g., sodium zirconate) from sand by alkaline decomposition, e.g., after addition of a strong base (e.g., sodium hydroxide) and incubation at a temperature of at least 500°C, at least 600°C, at least 700°C, or between 500°C and 800°C. Providing an isotopic composition can include enriching an isotope of zirconium or hafnium, or obtaining an enriched isotope of zirconium or hafnium. For example, an oxide form of an isotope of zirconium or hafnium can be enriched by mass, e.g., by calutron (magnetic sector type). Alternative means of obtaining a specific isotope are known in the art and include centrifugation or impact. For example, 89 Zr is 89 The isotopic composition may be obtained by bombardment (e.g., proton bombardment), such as cyclotron bombardment of Y. The isotopic composition may be provided as a salt, such as a chloride salt (e.g., oxychloride or tetrachloride). In certain embodiments, oxide forms of enriched zirconium or hafnium isotopes may be converted to oxychloride salts by the addition of a strong acid, such as (hydrochloric acid). Such conversion may be carried out at elevated temperatures, such as at least 80°C, at least 90°C, or at least 95°C. Alternatively, zirconium tetrachloride may be obtained by exposure to chloride gas, such as by electrolysis to chloride gas.

[0068] Aspects of the present application may include providing an enriched metal isotope (e.g., by obtaining an enriched metal isotope as described above or by performing one or more of the steps described above). In certain aspects, the enriched metal isotope may be provided in a form suitable for loading onto a polymer (e.g., as further described herein).

[0069] Polymer Support The kit may include a metal loading buffer for loading the isotope composition onto the polymer. The metal loading buffer may be mixed with the isotope composition in solution before loading onto the polymer of the present application. The metal loading buffer may be an acidic solution (e.g., a strong acid, such as one or more of nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, perchloric acid, hydrochloric acid, and chloric acid). The isotope composition may be provided in a form suitable for loading onto the polymer of the present application. Alternatively or additionally, the loading buffer may include an acetate salt (e.g., an alkali acetate), such as ammonium acetate or sodium acetate, and / or an acetate salt paired with another alkali, such as a carbonate or bicarbonate.

[0070] The isotopic composition can be provided separately from the polymer. For example, the isotopic composition is in solution. Alternatively, the isotopic composition can be supported on one or more pendant groups of the polymer. The polymer can be in solution. Alternatively, the polymer can be lyophilized.

[0071] In certain embodiments, at least 5 atoms of the enriched metal isotope are loaded onto the polymer, for example, at least 10 atoms, 20 atoms, 30 atoms, 40 atoms, 50 atoms, or 100 atoms of the enriched metal isotope can be loaded onto the polymer, e.g., between 5 and 50 atoms, between 10 and 40 atoms, or between 20 and 30 atoms.

[0072] The isotope composition can be stably bound by the polymer (e.g., such that the metal atom of the isotope composition does not dissociate from the pendant group of the polymer under physiological and / or experimental conditions). For example, less than 10% of the isotope composition supported on the polymer can be lost to a competing free chelator (e.g., DFO, DOTA, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), derivatives thereof, etc.). The competing free (e.g., unsupported) chelator can be chemically similar or identical to the supported chelator of the polymer and can be mixed with the supported polymer under physiological conditions. Dissociation of the original isotope-chelator complex can be measured by high performance liquid chromatography (HPLC), MS, or fluorescence. Alternatively or additionally, greater than 90%, greater than 95%, greater than 98%, or greater than 99% of the isotopic composition may remain bound to the polymer under physiological conditions and / or experimental conditions (such as mass cytometry assays).

[0073] At least some of the pendant groups of the polymer can coordinate a metal, such as zirconium and / or hafnium. The pendant groups (e.g., coordinating pendant groups) can coordinate at least six coordination sites for the metal isotope, or more than six coordination sites (e.g., eight coordination sites for the metal isotope).

[0074] The pendant group (e.g., one or more pendant groups) of the polymer can comprise DFO or a derivative thereof. For example, the pendant group can comprise a derivative of DFO that coordinates more than six coordination sites for a metal isotope. Here, the pendant group can comprise a DFO derivative that includes four hydroxamate groups. The pendant group can comprise a DFO derivative that includes a first hydroxamate group (e.g., of the same pendant group) that is separated from the nearest hydroxamate group by at least eight bonds. In certain embodiments, the chelator can include more than four hydroxamate groups such that eight sites are stably coordinated even if one group dissociates from the metal atom.

[0075] An embodiment of preparing a kit can include loading an isotopic composition onto a polymer of the present application. The loading step can be in the presence of a solution containing an aprotic solvent such as pyridine, ethyl acetate, DMF, DMSO, and / or HMPA. Alternatively or additionally, loading can be carried out in the presence of an acid, for example, an acidic solution as described herein. Alternatively or additionally, loading can be in the presence of an acetate (e.g., an alkali acetate), such as ammonium acetate or sodium acetate, and / or an acetate paired with another alkali, such as a carbonate or bicarbonate.

[0076] Conjugation of mass tags to bioactive materials The kit may further include a bioactive material conjugated to a mass tag (e.g., a polymeric mass tag), e.g., via a covalent bond. For example, the bioactive material may be an affinity reagent (such as an antibody) or an oligonucleotide. The bioactive material (e.g., affinity reagent) may be in solution or lyophilized. Embodiments of making the kit may further include conjugating a polymer (e.g., carrying an isotopic composition) to the bioactive material.

[0077] For example, the bioactive material can be an antibody, amino acid, nucleoside, nucleotide, aptamer, protein, antigen, peptide, nucleic acid, oligonucleotide, enzyme, lipid, albumin, cell, carbohydrate, vitamin, hormone, nanoparticle, inorganic support, polymer, monomolecule, or drug. In certain cases, the biomolecule can be an affinity reagent that binds to a specific target based on its tertiary structure, such as an antibody (including, for example, a recombinant antibody or antibody fragment), an aptamer (e.g., a DNA aptamer or an RNA aptamer), a lectin, biotin / streptavidin, a receptor / ligand, or any other suitable biomolecule. In certain aspects, the biomolecule can be an oligonucleotide that hybridizes to a DNA or RNA target, or an intermediate (such as an intermediate oligonucleotide in a hybridization mechanism, or an oligonucleotide attached to an antibody intermediate).

[0078] The mass tag can be conjugated to a bioactive material. The bioactive material can include an oligonucleotide, an affinity reagent (e.g., an antibody, an aptamer, a lectin, or another specific binding partner such as a protein that binds to a ligand or an artificially selected peptide), or a biosensor (e.g., that deposits or binds under conditions such as hypoxia, protein synthesis, the cell cycle, and / or cell death). The bioactive material can bind to a target, such as an endogenous target or intermediate. The affinity reagent can include a tertiary structure that specifically binds to an analyte non-covalently. In examples where the bioactive material includes an antibody, the term antibody generally includes recombinant antibodies and fragments thereof (e.g., only including the Fab portion). In examples where the bioactive material comprises an oligonucleotide, the oligonucleotide may hybridize (directly or indirectly) to an endogenous target such as DNA or RNA (e.g., mRNA, miRNA, siRNA, etc.), hybridize to an oligonucleotide intermediate through a hybridization mechanism (e.g., for signal amplification), and / or hybridize (directly or indirectly) to an oligonucleotide conjugated to an antibody (or other affinity reagent) intermediate. In certain embodiments, the polymer may be separated from the bioactive material by any suitable linker, such as a PEG linker.

[0079] Alternatively, the kit may provide a polymer suitable for conjugation to a bioactive material by any chemistry described herein or known to one of skill in the art. For example, the polymers of the present application may include end-group functionalization with, for example, maleimide, biotin, azide, or any other reactive group discussed herein.

[0080] A variety of suitable conjugation means are known in the art. For example, mass tags can be conjugated to bioactive materials by, for example, covalent bonds (e.g., amine chemistry, thiol chemistry, phosphate chemistry, enzymatic reactions, redox reactions (e.g., with metal halides), and affinity intermediates (e.g., streptavidin or biotin), or forms of click chemistry such as strain-promoted click chemistry or metal-catalyzed click chemistry).

[0081] The polymer can be functionalized to attach a bioactive material. In certain embodiments, the polymer can be functionalized with thiol-reactive chemistry, amine-reactive chemistry, or click chemistry. For example, the polymer can be functionalized for thiol reactivity (e.g., attached to a thiol group on the Fc portion of an antibody via a maleimide group).

[0082] Additional Kit Components Any combination of the above components may be provided in a kit, which may include a polymer, an isotopic composition, a polymer carrying an isotopic composition, a polymer and an isotopic composition provided separately, or a polymer carrying an isotopic composition and conjugated to an antibody.

[0083] The kit may further include any additional components for loading the isotopic composition onto the polymer and / or binding the loaded polymer to the bioactive material (e.g., buffers, filters, etc.) Alternatively or additionally, the kit may include additional reagents for mass cytometry, such as buffers, standards, cell barcodes, and / or reagents comprising heavy atoms of different masses (e.g., mass tags attached to or provided for attachment to the bioactive material).

[0084] The kit may include additional isotopic compositions that are distinguishable from the above-described isotopic compositions (e.g., have enriched isotopes of different masses). The additional isotopic compositions may include zirconium, hafnium, and / or lanthanide isotopes. For example, the kit may further include an additional polymer containing multiple pendant groups that chelate (e.g., stably chelate) lanthanides but not zirconium or hafnium. In certain embodiments, the kit may include multiple antibodies (e.g., against different targets) covalently attached to polymers carrying distinct isotopic compositions. Such a collection of antibodies may be provided together in a single panel. The panel may be provided in solution or in a lyophilized mixture containing less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% water by mass.

[0085] Mass cytometry methods Mass tags containing one or more enriched isotopes of zirconium and / or hafnium can be analyzed by mass spectrometry, for example, single cells, tissues, or biological solutions can be analyzed.

[0086] In certain embodiments, one or more enriched isotopes of zirconium and / or hafnium may be used in mass cytometry workflows, such as suspension mass cytometry or imaging mass cytometry (IMC). As used herein, mass cytometry refers to the elemental analysis of mass tags in a biological sample. Mass cytometry may have cellular or better resolution. In certain embodiments, the elemental analysis may be mass spectrometry, such as time-of-flight mass spectrometry or magnetic sector mass spectrometry. Individual mass tags may contain enriched isotopes or unique combinations of isotopes that distinguish them from other mass tags.

[0087] Mass tags may include heavy atoms, such as atoms with masses greater than 80 amu. Mass tags may include transition elements, lanthanides, noble metals, and / or metalloids. At least some mass tags may include organic polymers containing multiple pendant groups to which enriched metal isotopes are attached. Such polymers may improve the signal in the metal isotope channel compared to mass tags containing a single isotope. However, mass tags may cause steric hindrance and / or poor solubility may reduce the binding or specificity of affinity reagents to which they are attached. Mass tags used in mass cytometry may be free of radioactive isotopes, as radioactive isotopes may pose a risk to users and may be unnecessary for mass spectrometric detection.

[0088] Mass tags can be conjugated to bioactive materials, for example, by covalent bonds (e.g., amine chemistry, thiol chemistry, phosphate chemistry, enzymatic reactions, or forms of click chemistry such as strain-promoted click chemistry or metal-catalyzed click chemistry). The bioactive material can be an affinity reagent (e.g., an antibody or fragment thereof, an aptamer, a lectin, etc.), or an oligonucleotide probe that hybridizes to an endogenous target (e.g., DNA or RNA) or intermediate (e.g., an antibody-oligonucleotide intermediate and / or oligonucleotide hybridization mechanism). As described herein, suitable attachment chemistries can include carboxyl-amine reactive chemistries (e.g., reaction with carbodiimides), amine reactive chemistries (e.g., reaction with NHS esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphine, etc.), sulfhydryl reactive chemistries (e.g., reaction with maleimides, haloacetyls (bromoacetyl or iodoacetyl), pyridyl disulfides, thiosulfonates, vinyl sulfones, etc.), aldehyde reactive chemistries (e.g., reaction with hydrazides, alkoxyamines, etc.), hydroxyl reactive chemistries (e.g., reaction with isothiocyanates, etc.). Alternative attachment methods include click chemistries, such as strain-promoted click chemistry (e.g., with DBCO-azide or TCO-tetrazine).

[0089] Additional reagents for mass cytometry include metal-containing biosensors (e.g., that deposit or bind under conditions such as hypoxia, protein synthesis, cell cycle, and / or cell death) and / or metal-containing histochemical compounds that bind to structures (e.g., DNA, cell membranes, layers) based on their chemical properties. Such mass tags may contain only a single chelator (e.g., one of the DFOs described herein or its derivatives). In addition, mass tags (e.g., of this application or other mass tags) may be combined to provide a unique barcode for labeling a specific sample or experimental condition prior to pooling with other samples or experimental conditions. In embodiments in which zirconium and / or hafnium mass tags are used for barcoding, the mass tag may be a polymeric mass tag (e.g., attached to an antibody) or a small molecule mass tag (e.g., a single chelator functionalized for attachment, such as covalent attachment to an intracellular moiety). For example, the barcode mass tag may be a derivative of DFO containing a functional group for attachment to a cell. Attachment may be, for example, via thiol-reactive or amine-reactive chemistry. For example, the functional group can be a maleimide, which reacts with thiols (e.g., thiols presented by cysteines on intracellular proteins). In another example, the functional group can be an isothiocyanate, which reacts with amines. A DFO derivative can contain three hydroxamate groups or four hydroxamate groups (i.e., retaining a zirconium or hafnium atom in eight coordination sites). As described herein, mass tags can be modified with one or more solubility-promoting moieties. The solubility-promoting moieties can be hydrophilic or charged. Oppositely charged groups can combine to provide a zwitterionic mass tag. The hydrophilic solubility-promoting moiety can be, for example, ethylene glycol (e.g., a chain of ethylene glycol units in PEG) or oxazoline (e.g., in polyoxazoline). In certain embodiments, solubility-promoting moieties, such as individual ethylene glycol units, can be positioned between the hydroxamate groups of a DFO derivative. Barcoding reagents can be prepared as known in the art.For example, a barcoding kit can include separate mixtures, each containing a unique combination of mass tag barcodes (e.g., a unique combination of zirconium and / or hafnium isotopes). The mixture of mass tag barcodes can be applied to cells of a particular sample, after which the cells can be pooled across samples. The pooled samples can be stained with mass-tagged antibodies and analyzed together (e.g., in the same cell suspension). The combination of barcode mass tags detected by mass cytometry in a given cellular event can be used to identify which sample the cell came from (e.g., to classify the cellular event into a particular sample dataset). In certain aspects, barcodes can be used to barcode live cells (e.g., prior to fixation and / or permeabilization of the cells in a staining protocol).

[0090] Mass tags may be sampled, micronized, and ionized prior to elemental analysis. For example, mass tags in a biological sample may be sampled, micronized, and / or ionized by radiation, such as a laser beam, ion beam, or electron beam. Alternatively or additionally, mass tags may be micronized and ionized by a plasma, such as an inductively coupled plasma (ICP). In suspension mass cytometry, whole cells containing mass tags may be flowed into an ICP-MS, such as an ICP-TOF-MS. In imaging mass cytometry, a form of radiation may remove (and optionally ionize and micronize) portions (e.g., pixels, regions of interest) of a solid biological sample, such as a tissue sample, containing mass tags. Examples of IMC include laser ablation (LA)-ICP-MS and secondary ion mass spectrometry (SIMS)-MS of mass-tagged samples. In certain embodiments, the ion optics may deplete ions other than the isotopes of the mass tags. For example, ion optics can filter out lighter ions (e.g., C, N, O), organic molecular ions. In ICP applications, ion optics can filter out gases such as Ar and / or Xe, for example, via a high-pass quadrupole filter. In certain embodiments, IMC can provide images of mass tags (e.g., targets associated with mass tags) with cellular or subcellular resolution.

[0091] The one or more mass tags detected by mass cytometry may comprise an enriched isotope of zirconium or hafnium, as described herein. In certain cases, an antibody containing zirconium or hafnium (e.g., an enriched isotope of zirconium or hafnium) may be administered to an animal subject, and the distribution of zirconium in the subject's tissues may be assessed by IMC. For example, a pulse-chase experiment in which the same antibody tagged with different zirconium isotopes is administered at different times may allow imaging of zirconium metabolism and / or distribution over time. Such an assay may: 89 This may be done to screen one or more antibodies for delivery of Zr (e.g., without off-target effects).

[0092] A method of mass cytometry may include labeling cells of a biological sample with a mass-tagged bioactive material comprising an enriched isotope of zirconium or hafnium, and detecting the mass tags bound to the cells by mass spectrometry. The method may include providing a kit of the present application, for example, by obtaining the kit from a third party or by making the kit as described herein.

[0093] In certain aspects, a method of mass cytometry can include providing a first mass-tagged affinity reagent, the affinity reagent conjugated to a polymer, the polymer comprising multiple instances of pendant groups comprising hydroxamates, and the polymer carrying an isotopic composition comprising an enriched metal isotope. The method can further include labeling cells of a biological sample with multiple mass-tagged affinity reagents, including the first mass-tagged affinity reagent. The method can further include detecting the mass tags bound to the cells by mass spectrometry. Cells can be detected with single-cell resolution, for example, by suspension mass cytometry or by sampling individual cells (or portions of cells) from a solid support.

[0094] In certain aspects, a method of mass cytometry comprises detecting a plurality of mass tags bound to a cell, wherein at least one mass tag of the plurality of mass tags comprises an enriched isotope of zirconium or hafnium.

[0095] Additional Uses The polymers of the present application may be used in combination with other polymers for uses other than mass cytometry. 89 Radioactive isotopes such as Zr may be carried. For example, 89 Zr-loaded polymers target specific tissues or cell types (e.g., cancer cells). 89 It can be attached to a bioactive material such as an antibody that targets Zr. 89 Zr may be detected and / or imaged by means other than mass cytometry, for example by PET scanning. 89Zr-loaded polymers can be used for therapeutic applications such as radiation therapy in human subjects or to investigate potential therapies in animal models. 67 Ga, 68 Ga, 90 Y, 177 Lu, or 225 Ac etc. 89 Radioisotopes other than Zr may be used.

[0096] In this way, the kits and methods of the present application provide a concentrated 89 In certain embodiments, the isotopic composition may include the Zr isotope. 89 Zr can be conjugated to a polymer. The polymer can be for attachment to a bioactive material or can be provided conjugated to a bioactive material such as an antibody. For example, the antibody can target an epitope preferentially expressed in cancer cells.

[0097] The method of use involves the use of a polymer of the invention (e.g., 89 The method may further comprise administering to the animal subject a radioisotope (loaded with Zr and bound to a therapeutic bioactive material). Such a method may further comprise detecting the radioisotope by another means, such as, for example, mass cytometry or PET scanning.

[0098] Examples of applications include anti-tumor agents such as HERCEPTIN™ (trastuzumab), RITUXAN™ (rituximab), ZEVALIN™ (ibritumomab tiuxetan), LYMPHOCIDE™ (epratuzumab), GLEEVAC™, and BEXXAR™ (iodine-131 tositumomab), Neulasta, Provenge, nivolumab, and blinatumomab.

[0099] Other anti-neoplastic agents / compounds that can be used in combination with the compounds of the invention include anti-angiogenic compounds such as ERBITUX™ (IMC-C225), kinase domain receptor (KDR) inhibitors (e.g., antibodies and antigen-binding regions that specifically bind to kinase domain receptors), anti-VEGF agents (e.g., antibodies or antigen-binding regions that specifically bind to VEGF, or soluble VEGF receptors or ligand-binding regions thereof) such as AVASTIN™ or VEGF-TRAP™, and anti-vascular endothelial growth factor (anti-VEGF) receptor agents (e.g., antibodies or antigen-binding regions that specifically bind to VEGF), epidermal growth factor receptor (EGF) inhibitors such as ABX-EGF (panitumumab), IRESSA™ (gefitinib), TARCEVA™ (erlotinib), anti-Ang1 agents, and anti-Ang2 agents (e.g., antibodies or antigen-binding regions that specifically bind to EGFR or its receptors, e.g., Tie2 / Tek). These include anti-EGFR (eg, epidermal growth factor receptor) inhibitors (e.g., antibodies or antigen-binding regions that specifically bind to EGFR), and anti-Tie2 kinase inhibitors (e.g., antibodies or antigen-binding regions that specifically bind to Tie2 kinase).

[0100] Other anti-angiogenic compounds / agents that can be used in combination with the compounds of the invention include Campath, IL-8, B-FGF, Tek antagonists, anti-TWEAK agents (e.g., antibodies or antigen-binding regions that specifically bind, or soluble TWEAK receptor antagonists, ADAM disintegrin domains that antagonize the binding of integrins to their ligands, anti-eph receptor and / or anti-ephrin antibodies or antigen-binding regions that specifically bind, and anti-platelet-derived growth factor (PDGF)-BB antagonists (e.g., antibodies or antigen-binding regions that specifically bind), as well as antibodies or antigen-binding regions that specifically bind to PDGF-BB ligands, and platelet-derived growth factor receptor (PDGFR) kinase inhibitors (e.g., antibodies or antigen-binding regions that specifically bind).

[0101] Other anti-angiogenic / anti-tumor agents that can be used in conjunction with the compounds of the present invention include: These include SD-7784 (Pfizer, USA); cilengitide (Merck KGaA, Germany, EPO 770622); pegaptanib octasodium (Gilead Sciences, USA); alphastatin (BioActa, UK); M-PGA (Celgene, USA); ilomastat (Arriva, USA); emaxanib (Pfizer, USA); vatalanib (Novartis, Switzerland); 2-methoxyestradiol (EntreMed, USA); TLC ELL-12 (Elan, Ireland); anecortave acetate (Alcon, USA); alpha-D 148 Mab (Amgen, USA); CEP-7055 (Cephalon, USA); anti-Vn Mab (Crucell, Netherlands); DAC: antiangiogenic agent (ConjuChem, Canada); angiocidin (InKine Pharmaceutical, USA; KM-2550, (Kyowa Hakko, Japan); SU-0879, (Pfizer, USA); CGP-79787, (Novartis, Switzerland); the ARGENT technology of Ariad, USA; YIGSR-Stealth, (Johnson & Johnson, USA); fibrinogen-E fragment, (BioActa, UK); Trigen angiogenesis inhibitors, UK; TBC-1635, (Encysive Pharmaceuticals, USA); SC-236, (Pfizer, USA); ABT-567, (Abbott, USA); metastatin, (EntreMed, USA); angiogenesis inhibitors, (Tripep, Sweden); maspin, (Sosei, Japan); 2-methoxyestradiol, (Oncology Sciences Corporation, USA); ER-68203-00, (WVAX, USA); Benefin (Lane Labs, USA); Tz-93, (Tsumura, Japan); TAN-1120, (Takeda, Japan); FR-111142, (Fujisawa, Japan); Platelet Factor 4 (RepliGen, USA); Vascular Endothelial Growth Factor Antagonist, (Borean, Denmark); Bevacizumab (pINN), (Genentech, USA);XL 784, (Exelixis, USA); XL 647, (Exelixis, USA); MAb, alpha5beta3 integrin, second generation, (Applied Molecular Evolution, USA and MediImmune, USA); gene therapy, retinopathy (Oxford BioMedica, UK); enzastaurin hydrochloride (USAN), (Lilly, USA); CEP 7055, (Cephalon, USA and Sanofi-Synthelabo, France); BC 1, (Genoa Institute of Cancer Research, Italy); angiogenesis inhibitors (Alchemia, Australia); VEGF antagonists, (Regeneron, USA); rBPI 21 and BPI-derived antiangiogenic agents, (XOMA, USA); PI 88, (Progen, Australia); cilengitide (pINN), (Merck KGaA, Germany; Munich Technical University, Germany, Scripps Clinic and Research Foundation, USA); cetuximab (INN), (Aventis, France); AVE 8062, (Ajinomoto, Japan); AS 1404, (Cancer Research Laboratory, New Zealand); SG 292, (Telios, USA); endostatin, (Boston Children's Hospital, USA); ATN 161, (Attenuon, USA); angiostatin, (Boston Children's Hospital, USA); 2-methoxyestradiol, (Boston Children's Hospital, USA); ZD 6474, (AstraZenec, UK); ZD 6126, (Angiogene Pharmaceuticals, UK); PPI 2458, (Praecis, USA); AZD 9935, (AstraZeneca, UK); AZD 2171, (AstraZeneca, UK); vatalanib (pINN), (Novartis, Switzerland and Schering AG, Germany); tissue factor pathway inhibitor, (EntreMed, USA); pegaptanib (Pinn), (Gilead Sciences, USA);Xanthorrhizol (Yonsei University, Korea); Vaccine, Gene-Based, VEGF-2 (Scripps Clinic and Research Foundation, USA); SPV5.2 (Supratek, Canada); SDX 103 (University of California at San Diego, USA); PX 478 (ProIX, USA); Metastatin (EntreMed, USA); Troponin I (Harvard University, USA); SU 6668 (SUGEN, USA); OXI 4503 (OXiGENE, USA); o-Guanidine (Dimensional Pharmaceuticals, USA); Motuporamine C (University of British Columbia, Canada); CDP 791 (Celltech Group, UK); Atiprimod (plNN) (GlaxoSmithKline, UK); E 7820 (Eisai, Japan); CYC 381 (Harvard University, USA); AE 941, (Aeterna, Canada); Vaccine, Angiogenesis, (EntreMed, USA); Urokinase-type Plasminogen Activator Inhibitor, (Dendreon, USA); Oglufanide (pINN), (Melmotte, USA); HIF-1 alpha inhibitor, (Xenova, UK); CEP 5214, (Cephalon, USA); BAY RES 2622, (Bayer, Germany); Angiocidin, (InKine, USA); A6, (Angstrom, USA); KR 31372, (Korea Research Institute of Chemical Technology, Korea); GW 2286, (GlaxoSmithKline, UK); EHT 0101, (ExonHit, France); CP 868596, (Pfizer, USA); CP 564959, (OSI, USA); CP 547632, (Pfizer, USA); 786034, (GlaxoSmithKline, USA); KRN 633, (Kirin Brewery, Japan); Drug delivery system, intraocular, 2-methoxyestradiol (EntreMed, USA);Anginex (Maastricht University, The Netherlands and University of Minnesota, USA); ABT 510 (Abbott, USA); AAL 993 (Novartis, Switzerland); VEGI (ProteomTech, USA); Tumor necrosis factor alpha inhibitor (National Institute on Aging, USA); SU 11248 (Pfizer, USA and SUGEN, USA); ABT 518 (Abbott, USA); YH16 (Yantai Rongchang, China); S-3APG (Boston Children's Hospital, USA and EntreMed, USA); MAb, KDR (ImClone Systems, USA); MAb, alpha5beta1 (Protein Design, USA); KDR kinase inhibitor (Celltech Group, UK and Johnson & Johnson, USA); GFB 116 (University of South Florida, USA and Yale University, USA); CS 706 (Sankyo, Japan); Combretastatin A4 prodrug (Arizona State University, USA); Chondroitinase AC (IBEX, Canada); BAY RES 2690 (Bayer, Germany); AGM 1470 (Harvard University, USA, Takeda Pharmaceutical, Japan, and TAP, USA); AG 13925 (Agouron, USA); Tetrathiomolybdate (University of Michigan, USA); GCS 100 (Wayne State University, USA); CV 247 (Ivy Medical, UK); CKD 732 (Chong Kun Dang, Korea); MAb, vascular endothelial growth factor (Xenova, UK); Irsogladine (INN), (Nippon Shinyaku, Japan); RG 13577 (Aventis, France); WX 360 (Wilex, Germany); Squalamine (pIN), (Genaera, USA); RPI 4610, (Sima, USA); heparanase inhibitor, (InSight, Israel); KL 3106, (Kolon, Korea); honokiol, (Emory University, USA);ZK CDK, (Schering AG, Germany); ZK Angio, (Schering AG, Germany); ZK 229561, (Novartis, Switzerland and Schering AG, Germany); XMP 300, (XOMA, USA); VGA 1102, (Taisho Pharmaceutical, Japan); VEGF receptor modulator, (Pharmacopeia, USA); VE-cadherin-2 antagonist, (ImClone Systems, USA); vasostatin, (National Institutes of Health, USA); vaccine, Flk-1, (ImClone Systems, USA); TZ 93, (Tsumura, Japan); tumstatin (Beth Israel Hospital, USA); truncated soluble FLT 1 (vascular endothelial growth factor receptor 1), (Merck & Co, USA); Tie-2 ligand, (Regeneron, USA); 28H1 (Roche Pharmaceuticals, Switzerland) and thrombospondin 1 inhibitor (Allegheny Health, Education and Research These include anti-FAP agents such as fluticasone (Family Aid Foundation, USA);

[0102] Utilities In mass cytometry, cells are labeled with mass-tagged bioactive materials (such as antibodies or oligonucleotides) and the mass tags are detected by mass spectrometry, often with single-cell resolution. These mass tags are typically lanthanide-chelating polymers carrying enriched lanthanide isotopes. The number of mass-tagged bioactive materials that can be distinguished is the number of lanthanide isotopes with different masses. The present invention presents a new class of mass tags for mass cytometry analysis, specifically zirconium and hafnium mass tags. The coordination chemistry of these new mass tags differs from that of lanthanide mass tags.

[0103] In addition to developing a catalog of distinguishable mass tags available for mass cytometry, the mass tags described herein may be used to assess the therapeutic potential of radioactive forms that may or may not be used in mass cytometry. For example,89 Zr is a radioactive isotope of zirconium conjugated to an antibody for therapeutic use. The zirconium polymers described herein are useful when the antibody is 89 This may increase the ability to deliver Zr to target tissues and / or cell types. An important aspect of drug development is understanding the mechanism and distribution of drug delivery, for example, to screen for delivery to target tissues and / or cell types with minimal off-target distribution. Naturally occurring lanthanide isotopes traditionally used in mass cytometry are not radioactive. Thus, the mass tags provided herein enable the unique ability to detect the distribution of mass tags bearing chemically identical radioactive zirconium analogs. For example, pulse chases with different isotopes of zirconium-tagged antibodies can be performed (e.g., administered to an animal subject), and tissues can be analyzed by imaging mass cytometry. In this manner, the methods of the present application may include determining the distribution of zirconium isotopes at cellular or subcellular resolution in the context of other targets and tissue morphologies by IMC, and optionally, further by light or fluorescence microscopy. In such experiments, radioactive 89 Even if Zr is not used, the distribution of non-radioactive zirconium isotopes can be estimated to 89 In this way, the methods and kits described herein can be used to generate radioisotopes of zirconium for therapeutic use, even though the methods or kits may only use non-radioactive isotopes of zirconium. 89 Screening for Zr-tagged antibodies may be provided. In certain embodiments, 89 Zr distribution can be assayed by radioassay (eg, PET) and further examined by IMC.

[0104] Development of some kits and methods of the present invention may require understanding the value of mass cytometry with more mass channels, specialized therapeutic reagents, inorganic chemistry (e.g., for metal purification, isotope enrichment, and / or conversion to salt forms), and organic chemistry (e.g., to generate low polydispersity polymers stably loaded with multiple zirconium or hafnium isotopes). To the inventors' knowledge, polymers containing multiple zirconium or hafnium isotopes have not been reported. 89 Although isotopes such as Zr have the potential to increase therapeutic delivery, they have not been published. Such polymers may disrupt the stability of the chelated zirconium or hafnium and risk reducing antibody specificity through steric hindrance, aggregation, and / or poor solubility, all of which may risk off-target effects in therapeutic use. In certain embodiments, adjacent pendant ligands may further stabilize the association of the zirconium or hafnium isotope with the polymer.

[0105] By loading a polymer with a zirconium or hafnium isotope, the number of atoms delivered via an antibody intermediate can be increased compared to direct attachment of chelated zirconium or hafnium to an antibody. However, the development and use of the polymer can be hindered by one or more of the following: aggregation of the loaded or unloaded polymer, insufficient loading of the isotope on the polymer, isotopes in a form incompatible with loading on the polymer, disruption of coordination stability with neighboring polymer structures such as adjacent pendant ligands, steric hindrance of the polymer on antibody affinity, and variability in the size of the polymer, any of which can hinder detection and / or target-specific delivery.

[0106] Exemplary Embodiments Exemplary embodiments of the subject kits and methods are listed below.

[0107] 1. A kit comprising: a polymer comprising pendant groups that chelate zirconium and / or hafnium; and Isotopic compositions containing enriched zirconium or hafnium isotopes Includes a kit.

[0108] 2. The kit of aspect 1, wherein the enriched zirconium or hafnium isotope is supported on the pendant group.

[0109] 3. The kit of any one of aspects 1 to 2, wherein the polymer comprises a hydroxamate, azamacrocycle, phenoxyamine, salophen, and / or cyclam ligand or a derivative thereof.

[0110] 4. The kit of any one of aspects 1 to 3, wherein the polymer comprises a derivative of hydroxamate, azamacrocycle, phenoxyamine, salophen, and / or cyclam that forms an eight-coordinate complex with at least one of zirconium or hafnium.

[0111] 5. The kit of any one of aspects 1 to 4, wherein at least one of zirconium and hafnium forms an eight-coordinate complex with a pendant group of the polymer.

[0112] 6. The kit of any one of aspects 1 to 5, wherein the polymer comprises a hydroxamate.

[0113] 7. The kit of aspect 6, wherein the polymer comprises DFO or a derivative thereof.

[0114] 8. The kit of any one of aspects 1 to 7, wherein the polymer comprises an azamacrocycle.

[0115] 9. The kit of aspect 8, wherein the polymer comprises DOTA or a derivative thereof.

[0116] 10. The kit of any one of aspects 1 to 3, wherein the polymer further comprises one or more chains of hydrophilic groups to aid in solubility.

[0117] 11. The kit of any one of aspects 1 to 10, wherein the polymer further comprises a pegylated pendant group.

[0118] 12. The kit of aspect 11, wherein the pegylated pendant group aids in loading of the polymer.

[0119] 13. The kit of aspect 12, wherein the PEGylated pendant groups are distinct from the pendant groups that chelate zirconium and / or hafnium.

[0120] 14. A method of making a kit according to any one of aspects 1 to 13.

[0121] 15. A method of mass cytometry using a kit according to any one of aspects 1 to 14.

[0122] 16. A kit comprising: a polymer comprising a plurality of pendant groups comprising hydroxamates; and an isotopic composition comprising an enriched metal isotope capable of being chelated by said pendant group; Includes a kit.

[0123] 17. The kit of aspect 16, wherein the metal isotope is a zirconium or hafnium isotope.

[0124] 18. The kit of aspect 17, wherein the metal isotope is a zirconium isotope.

[0125] 19. The kit of aspect 17, wherein the metal isotope is a hafnium isotope.

[0126] 20. The kit of any one of aspects 16-19, further comprising an additional isotopic composition comprising additional enriched zirconium and / or hafnium isotopes.

[0127] 21. The kit of any one of aspects 16 to 20, wherein the isotopic composition is non-radioactive.

[0128] 22. The kit of any one of aspects 16-21, wherein the isotopic composition is provided separately from the polymer.

[0129] 23. The kit of any one of aspects 16 to 22, wherein the isotopic composition is in solution.

[0130] 24. The kit of any one of aspects 16 to 23, wherein the isotopic composition is provided in a solution comprising an aprotic solvent.

[0131] 25. The kit of aspect 24, wherein the aprotic solvent is DMSO.

[0132] 26. The kit of any one of aspects 16 to 25, wherein the isotopic composition is provided in an acidic solution.

[0133] 27. The kit of any one of aspects 16 to 26, further comprising a metal loading buffer for loading the isotopic composition onto the polymer.

[0134] 28. The kit of aspect 27, wherein the metal-loaded buffer comprises an alkali.

[0135] 29. The kit of aspect 28, wherein the metal-loaded buffer is at an acidic pH.

[0136] 30. The kit of any one of aspects 16 to 29, wherein the isotopic composition is supported on one or more pendant groups of the polymer.

[0137] 31. The kit of any one of aspects 16 to 30, wherein the polymer is in solution.

[0138] 32. The kit of any one of aspects 16 to 30, wherein the polymer is lyophilized.

[0139] 33. The kit of any one of aspects 16 to 32, wherein at least five atoms of the enriched metal isotope are supported on the polymer.

[0140] 34. The kit of aspect 33, wherein at least 20 atoms of the enriched metal isotope are supported on the polymer.

[0141] 35. The kit of aspect 33 or 34, wherein fewer than 40 atoms of the enriched metal isotope are supported on the polymer.

[0142] 36. The kit of any one of aspects 16 to 35, wherein the isotopic composition is stably bound to the polymer.

[0143] 37. The kit of any one of aspects 16 to 36, wherein less than 10% of the isotope composition loaded on the polymer can be lost to competing free chelating agent.

[0144] 38. The kit of any one of aspects 16-37, wherein under physiological conditions, greater than 95% of the isotopic composition remains bound to the polymer.

[0145] 39. The kit of any one of aspects 16-38, wherein, when used in a mass cytometry assay, greater than 95% of the isotopic composition remains bound to the polymer.

[0146] 40. The kit of any one of aspects 16 to 39, wherein the polymer comprises a pegylated pendant group.

[0147] 41. The kit of any one of aspects 16 to 40, wherein the polymer is modified to include pendant groups that aid in the solubility of the polymer both before and after loading the metal isotope.

[0148] 42. The kit of any one of aspects 16 to 41, wherein the polymer further comprises a pendant group comprising a hydrophilic group that aids in solubility of the polymer before and after loading of the metal isotope onto the pendant group.

[0149] 43. The kit of any one of aspects 16-42, wherein the pendant group comprising a hydrophilic group that aids solubility is separate from the pendant group that comprises a hydroxamic acid.

[0150] 44. The kit of any one of aspects 16 to 43, wherein one or more pendant groups of the polymer comprise a chain of repeating hydrophilic groups.

[0151] 45. The kit of any one of aspects 16 to 44, wherein the hydrophilic group does not affect the coordination chemistry of the pendant group.

[0152] 46. ​​The kit of any one of aspects 16 to 45, wherein the increased solubility of the polymer aids in retaining the metal isotope in solution.

[0153] 47. The kit of any one of aspects 42 to 46, wherein when the polymer is conjugated to an antibody, the resulting solubility improves antibody binding to the epitope of the antibody.

[0154] 48. The kit of any one of aspects 42 to 47, wherein increasing the solubility of the polymer improves target binding of an antibody bound to the polymer.

[0155] 49. The kit of any one of aspects 42 to 48, wherein the hydrophilic group comprises at least one PEG group.

[0156] 50. The kit of any one of aspects 16 to 49, wherein the polymer is PEGylated.

[0157] 51. The kit of any one of aspects 16 to 50, wherein the polymer comprises at least 100 PEG groups.

[0158] 52. The kit of any one of aspects 16 to 51, wherein the PEG groups are distributed over multiple pendant groups.

[0159] 53. The kit of any one of aspects 16-52, wherein at least some of the pendant groups comprise more than 20 PEG groups.

[0160] 54. The kit of any one of aspects 16 to 53, wherein less than 50% of all pendant groups of the polymer chelate zirconium and / or hafnium, and more than 50% of all pendant groups of the polymer comprise multiple PEG groups.

[0161] 55. The kit of any one of aspects 16 to 54, wherein the number of PEG groups on the polymer aids in loading the metal isotope onto the polymer in solution.

[0162] 56. The kit of any one of aspects 16-55, further comprising a bioactive material conjugated to the polymer.

[0163] 57. The kit of aspect 56, wherein the bioactive material comprises an affinity reagent.

[0164] 58. The kit of aspect 57, wherein the affinity reagent is an antibody.

[0165] 59. The kit of any one of aspects 56-58, wherein the affinity reagent is in solution.

[0166] 60. The kit of any one of aspects 56-58, wherein the affinity reagent is lyophilized.

[0167] 61. The kit of any one of aspects 16 to 60, wherein one or more pendant groups of the polymer coordinate at least six coordination sites of the metal isotope.

[0168] 62. The kit of any one of aspects 16 to 60, wherein one or more pendant groups of the polymer coordinate more than six sites for the metal isotope.

[0169] 63. The kit of any one of aspects 16 to 60, wherein one or more pendant groups of the polymer coordinate eight coordination sites of the metal isotope.

[0170] 64. The kit of any one of aspects 16-61, wherein one or more pendant groups of the polymer comprise DFO or a derivative thereof.

[0171] 65. The kit of any one of aspects 16-61, wherein the one or more pendant groups of the polymer comprise a DFO derivative comprising four hydroxamate groups.

[0172] 66. The kit of any one of aspects 16-61, wherein the one or more pendant groups of the polymer comprise a DFO derivative comprising a first hydroxamate group spaced at least 8 bonds from the nearest hydroxamic group.

[0173] 67. The kit of any one of aspects 16 to 66, comprising another additional isotopic composition that is distinguishable from the isotopic composition.

[0174] 68. The kit of aspect 67, wherein one or more of the additional isotopic compositions comprises zirconium or hafnium.

[0175] 69. The kit of embodiment 67 or 68, wherein one or more of the additional isotopic compositions comprises a lanthanide.

[0176] 70. The kit of any one of aspects 16 to 69, further comprising a polymer comprising multiple pendant groups that chelate a lanthanide but not zirconium or hafnium.

[0177] 71. The kit of any one of aspects 16-70, comprising a plurality of antibodies each covalently attached to a polymer carrying a distinct isotopic composition.

[0178] 72. The kit of any one of aspects 16 to 71, wherein the polymer is formed by living polymerization.

[0179] 73. The kit of any one of aspects 16 to 72, wherein the polymer is formed by at least one of anionic polymerization, controlled radical polymerization, cationic polymerization, and ring-opening polymerization.

[0180] 74. The kit of any one of aspects 16 to 73, wherein the polymer has a polydispersity index of less than 1.5.

[0181] 75. The kit of any one of aspects 16 to 74, wherein the polymer has a polydispersity index of less than 1.2.

[0182] 76. The kit of any one of aspects 16 to 75, wherein the polymer comprises 2 to 100 pendant groups.

[0183] 77. The kit of any one of aspects 16 to 76, wherein the polymer comprises 5 to 30 instances of the pendant group.

[0184] 78. The kit of any one of aspects 16 to 77, wherein the polymer comprises at least 5 instances of the pendant group.

[0185] 79. The kit of any one of aspects 16 to 78, wherein the polymer comprises at least 10 atoms of enriched zirconium isotopes.

[0186] 80. The kit of embodiment 79, wherein the polymer comprises between 20 and 30 atoms of enriched zirconium isotopes.

[0187] 81. The kit of any one of aspects 16 to 80, wherein the polymer is functionalized at one end.

[0188] 82. The kit of aspect 81, wherein the polymer is functionalized to bind to a bioactive material.

[0189] 83. The kit of aspect 82, wherein the bioactive material comprises an affinity reagent.

[0190] 84. The kit of aspect 83, wherein the affinity reagent is an antibody.

[0191] 85. The kit of aspect 84, wherein the polymer is functionalized to bind to the bioactive material via thiol-reactive chemistry, amine-reactive chemistry, or click chemistry.

[0192] 86. A kit comprising: a polymer comprising a plurality of pendant groups; and Isotopic compositions containing enriched zirconium or hafnium isotopes Including, the pendant groups comprise DFO or a derivative thereof; the pendant groups comprise PEG groups, which aid in the solubility of the polymer and / or aid in the loading of the isotopic composition onto the polymer; kit.

[0193] 87. A method of making the kit of any one of aspects 1 to 86.

[0194] 88. A method for making a polymer for mass cytometry, said method comprising: providing a polymer comprising a plurality of instances of pendant groups comprising hydroxamates; method.

[0195] 89. The method of embodiment 88, further comprising providing an isotopic composition comprising an enriched metal isotope.

[0196] 90. The method of embodiment 89, wherein the enriched metal isotope is a chloride salt of zirconium or hafnium.

[0197] 91. The method of aspect 90, wherein the enriched metal isotope is non-radioactive.

[0198] 92. The method of any one of aspects 88 to 92, further comprising loading the isotopic composition onto the polymer.

[0199] 93. The method of any one of aspects 87-92, comprising polymerizing the pendant groups by living polymerization.

[0200] 94. The method of aspect 93, wherein the living polymerization comprises at least one of anionic polymerization, controlled radical polymerization, cationic polymerization, and ring-opening polymerization.

[0201] 95. The method of any one of aspects 87 to 93, wherein at least some of the pendant groups of the polymer comprise hydroxamates.

[0202] 96. The method of any one of aspects 87 to 94, wherein at least some of the pendant groups of the polymer are PEGylated.

[0203] 97. The method of embodiment 92, wherein the step of loading is in the presence of an aprotic solvent.

[0204] 98. The method of embodiment 96, wherein the aprotic solvent is DMSO.

[0205] 99. The method of any one of aspects 97 to 98, wherein the step of loading is in the presence of an acid.

[0206] 100. The method of aspect 89, wherein the isotopic composition is an enriched isotope of zirconium or hafnium.

[0207] 101. The method of embodiment 100, wherein the isotopic composition is a salt.

[0208] 102. The method of embodiment 101, wherein the salt is a chloride salt.

[0209] 103. The method of embodiment 102, wherein the salt is a tetrachloride salt or an oxychloride salt.

[0210] 104. The method of embodiment 105, wherein dry zirconium oxide is obtained from the sand after addition of a strong base and / or incubation at a temperature of at least 500°C.

[0211] 105. The method of any one of aspects 87-104, further comprising isotopically enriching the zirconium oxide.

[0212] 106. The method of embodiment 105, further comprising converting the enriched isotope into a salt by the addition of a strong acid.

[0213] 107. The method of embodiment 106, wherein the salt is a chloride salt and the strong acid comprises HCl.

[0214] 108. The method of aspect 106 or 107, wherein the salt is a tetrachloride or oxychloride salt.

[0215] 109. The method of any one of aspects 105 to 109, further comprising converting the enriched isotope into a salt at a temperature of at least 80°C.

[0216] 110. The method of any of aspects 86-109, further comprising functionalizing the polymer for attachment to a bioactive material.

[0217] 111. The method of any of aspects 86-110, further comprising conjugating the polymer to a bioactive material.

[0218] 112. The method of aspect 111, wherein the bioactive material comprises an affinity reagent.

[0219] 113. The method of embodiment 112, wherein the affinity reagent is an antibody.

[0220] 114. The method of any one of aspects 86 to 113, further comprising attaching pendant groups to the polymer, wherein at least some of the pendant groups comprise hydroxamate, azamacrocycle, phenoxyamine, salophen, and / or cyclam ligands or derivatives thereof.

[0221] 115. The method of embodiment 114, wherein the polymer comprises a derivative of a hydroxamate, an azamacrocycle, a phenoxyamine, a salophen, and / or a cyclam that forms an eight-coordinate complex with at least one of zirconium or hafnium.

[0222] 116. The method of any one of aspects 86 to 115, wherein at least one of zirconium and hafnium forms an eight-coordinate complex with a pendant group of the polymer.

[0223] 117. The method of any one of aspects 86 to 116, wherein the polymer comprises a hydroxamate.

[0224] 118. The method of any one of aspects 86 to 117, wherein the polymer comprises DFO or a derivative thereof.

[0225] 119. The method of any one of aspects 86 to 118, wherein the polymer further comprises one or more chains of hydrophilic groups to aid in solubility.

[0226] 120. The method of any one of aspects 86 to 119, further comprising PEGylating one or more pendant groups of the polymer.

[0227] 121. The method of embodiment 120, wherein PEGylation of the pendant group aids in loading of the polymer.

[0228] 122. The method of aspect 121, wherein the PEGylated pendant groups are distinct from the pendant groups that chelate zirconium and / or hafnium.

[0229] 123. A method of mass cytometry using a kit according to any one of embodiments 1 to 86.

[0230] 124. A method of mass cytometry comprising: labeling cells of a biological sample with a mass-tagged bioactive material comprising an enriched isotope of zirconium or hafnium; and detecting the mass tags bound to said cells by mass spectrometry; A method comprising:

[0231] 125. The method of aspect 124, wherein the mass-tagged bioactive material is provided by the kit of any one of aspects 1 to 86.

[0232] 126. The method of embodiment 124, further comprising providing the mass-tagged bioactive material by using a kit according to any one of embodiments 1 to 86.

[0233] 127. A method of mass cytometry, comprising: providing a first mass-tagged affinity reagent, the affinity reagent is conjugated to a polymer; the polymer comprises multiple instances of pendant groups comprising hydroxamates; the polymer carries an isotopic composition comprising an enriched metal isotope; providing a first mass-tagged affinity reagent; labeling cells of the biological sample with a plurality of mass-tagged affinity reagents including the first mass-tagged affinity reagent; detecting the mass tags bound to said cells by mass spectrometry; A method comprising:

[0234] 128. An isotopic composition comprising an enriched isotope of zirconium or hafnium, wherein said enriched isotope is a chloride salt.

[0235] 129. A polymer containing multiple pendant groups, including hydroxamates.

[0236] 130. A method of mass cytometry comprising detecting by mass spectrometry a plurality of mass tags bound to a cell, wherein at least one mass tag of said plurality of mass tags comprises an enriched isotope of zirconium or hafnium.

[0237] 131. The kit of aspect 7, wherein the pendant group that chelates zirconium and / or hafnium comprises a solubility-enhancing moiety.

[0238] 132. The kit of aspect 131, wherein the solubility-aiding moiety is disposed between the hydroxamates.

[0239] 133. The kit of aspect 131 or 132, wherein the solubilizing moiety is an ether.

[0240] 134. A barcoding kit for mass cytometry, comprising: mass tags comprising enriched isotopes of zirconium and / or hafnium combined in separate mixtures, each containing a unique combination of enriched isotopes; Each mixture provides a unique barcode for labeling a particular sample or experimental condition before pooling with other samples or experimental conditions. Barcoding kit.

[0241] 135. The kit of embodiment 134, wherein the mass tag is functionalized for covalent attachment.

[0242] 136. The kit of embodiment 135, wherein the mass tag is functionalized with a maleimide.

[0243] 137. The kit of aspect 134, wherein the mass tag is conjugated to an antibody.

[0244] 138. A method for making a mass tag polymer, comprising: a first step of azide-initiated polymerization to form a polymer backbone; a second step of incorporating a mixture of pendant groups, said mixture of pendant groups comprising: a chelating pendant group comprising DFO or a derivative thereof; solubility-assisting pendant groups, a second step comprising: a third step of providing an isotopic composition comprising enriched zirconium or hafnium isotopes, said isotopic composition being metal in solution; A method comprising:

[0245] 139. The method of embodiment 138, wherein the second step is by aminolysis.

[0246] 140. The method of any one of embodiments 138 and 139, further comprising a third step of loading the isotopic composition onto the polymer produced in the second step.

[0247] 141. A mass tag kit comprising: an isotopic composition comprising enriched isotopes of zirconium or hafnium, and A polymer comprising DFO or a derivative thereof and a plurality of pendant groups comprising a solubility-promoting moiety Including, the polymer is attached to an antibody or antibody fragment thereof or functionalized for attachment to the antibody or antibody fragment thereof; Mass tag kit.

[0248] 142. The kit according to aspect 141, further comprising a plurality of solubility-aiding pendant groups that do not comprise DFO or a derivative thereof.

[0249] 143. The kit of aspect 141 or 142, wherein the polymer is functionalized for attachment via thiol-reactive chemistry, amine-reactive chemistry, or click chemistry.

[0250] 144. The kit of embodiment 143, wherein the polymer is functionalized with maleimide.

[0251] 145. The kit according to aspect 143, wherein the polymer is functionalized with an NHS ester.

[0252] 146. The kit of embodiment 143, wherein the polymer is functionalized with an azide.

[0253] 147. The kit of embodiment 146, wherein the polymer was polymerized by azide initiation prior to incorporation of the pendant group.

[0254] 148. The kit according to aspect 141, wherein the isotope is non-radioactive.

[0255] 149. The kit according to aspect 141 or 142, wherein the plurality of pendant groups comprises a DFO derivative comprising four hydroxamate groups.

[0256] 150. The kit of aspect 149, wherein the DFO derivative comprises multiple solubility-promoting moieties interspersed among hydroxamates.

[0257] 151. The kit according to aspect 150, wherein the polymer provides at least 20 examples of the DFO derivative.

[0258] 152. The kit of embodiment 141, wherein at least some of the solubility-aiding moieties are hydrophilic.

[0259] 153. The kit of embodiment 152, wherein at least some of the solubility-aiding moieties comprise a polyether.

[0260] 154. The kit of aspect 153, wherein the polyether is polyethylene glycol.

[0261] 155. The kit of embodiment 152, wherein at least some of the solubility-promoting moieties comprise an ether disposed between the hydroxamates.

[0262] 156. The kit of embodiment 152, wherein at least some of the solubility-aiding moieties comprise an oxazoline.

[0263] 157. The kit of embodiment 156, wherein at least some of the solubility-aiding moieties comprise a polyoxazoline.

[0264] 158. The kit of embodiment 141, wherein the polymer is zwitterionic.

[0265] 159. The kit of aspect 141, wherein the isotopic composition is a metal in solution.

[0266] 160. The kit of embodiment 141, wherein the polymer carries the isotopic composition.

[0267] Any of the above listed examples may further include additional aspects described elsewhere in this application.

Claims

1. A kit comprising: a polymer comprising pendant groups that chelate zirconium and / or hafnium, wherein at least one of the pendant groups comprises a chelating agent and a solubilizing group; and Isotopic compositions containing enriched isotopes of zirconium or hafnium Includes a kit.

2. 10. The kit of claim 1, wherein the enriched isotope of zirconium or hafnium is supported on a pendant group.

3. The kit of claim 1 , wherein the polymer comprises a hydroxamate, azamacrocycle, phenoxyamine, salophen, and / or cyclam ligand or a derivative thereof.

4. The kit of claim 3 , wherein the polymer comprises desferrioxamine (DFO) or a derivative thereof.

5. The kit of claim 1 , wherein the polymer comprises an azamacrocycle.

6. The kit of claim 5, wherein the polymer comprises 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or a derivative thereof.

7. The kit of claim 1 , wherein the polymer further comprises one or more chains of hydrophilic groups to aid in solubility.

8. The kit of claim 1 , wherein the polymer further comprises a pegylated pendant group.

9. 9. The kit of claim 8, wherein the pegylated pendant groups are separate from the pendant groups that chelate zirconium and / or hafnium.

10. The kit of claim 1 , wherein the polymer is lyophilized.

11. 10. The kit of claim 1, wherein the polymer is modified to include pendant groups that aid in the solubility of the polymer both before and after loading the enriched isotope of zirconium or hafnium.

12. The kit of claim 1 , wherein the polymer further comprises a pendant group comprising a hydrophilic group.

13. 13. The kit of claim 12, wherein the pendant group comprising a hydrophilic group is separate from the pendant group comprising a hydroxamic acid.

14. The kit of claim 1 , wherein one or more pendant groups of the polymer comprise a chain of repeating hydrophilic groups.

15. 13. The kit of claim 12, wherein when the polymer is conjugated to an antibody, the resulting solubility improves antibody binding to the epitope of the antibody.

16. The kit of claim 12 , wherein the hydrophilic group comprises at least one polyethylene glycol (PEG) group.

17. The kit of claim 12 , wherein the polymer comprises at least 100 PEG groups.

18. 18. The kit of claim 17, wherein the PEG groups are distributed over multiple pendant groups.

19. 20. The kit of claim 18, wherein at least some of the pendant groups comprise more than 20 PEG groups.

20. 2. The kit of claim 1, wherein less than 50% of all pendant groups of the polymer chelate zirconium and / or hafnium and more than 50% of all pendant groups of the polymer comprise multiple PEG groups.

21. The kit of claim 1 , further comprising a bioactive material conjugated to the polymer.

22. 22. The kit of claim 21, wherein the bioactive material comprises an affinity reagent.

23. 23. The kit of claim 22, wherein the affinity reagent is an antibody.

24. 10. The kit of claim 1, wherein one or more pendant groups of the polymer coordinate at least six coordination sites of the enriched isotope of zirconium or hafnium.

25. The kit of claim 1 , wherein the polymer has a polydispersity index of less than 1.

5.

26. 26. The kit of claim 25, wherein the polymer has a polydispersity index of less than 1.

2.

27. The kit of claim 1, wherein the polymer comprises 2 to 100 pendant groups.

28. 28. The kit of claim 27, wherein the polymer comprises 5 to 30 instances of the pendant group.

29. A kit comprising: a polymer comprising a plurality of pendant groups, and Isotopic compositions containing enriched isotopes of zirconium or hafnium Including, the plurality of pendant groups are capable of chelating enriched isotopes of the zirconium and / or the hafnium; the plurality of pendant groups comprise DFO or a derivative thereof; the pendant groups include PEG groups, which aid in the solubility of the polymer and aid in the loading of the isotopic composition onto the polymer; kit.

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