Compounds for mass spectrometry, uses thereof and methods of making the same

Abstract

The present application relates to mass spectrometry. More specifically, the present application relates to compounds of Formula (I), (II) or (III) for mass spectrometry, their uses as tag reagents, and methods of making the same.

Description

COMPOUNDS FOR MASS SPECTROMETRY, USES THEREOF AND METHODS OF MAKING THE SAME CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of priority of co-pending U. S. Provisional Patent Application No. 63 / 683,494, which was filed August 15, 2024, the content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present application relates to mass spectrometry. More specifically, the present application relates to compounds for mass spectrometry, their uses as tag reagents, and methods of making the same.BACKGROUND

[0003] Mass cytometry is a bioanalytic tool based on atomic mass spectrometry for detecting biomarker expression on cells. The analysis of biomarker expression plays a crucial role in understanding cellular processes, vital for the prevention, diagnosis, and treatment of diseases. Biomarker expression can be analyzed by mass cytometry (MC) - a high-throughput multiparameter bioanalytical technique that uses atomic mass spectrometry with time-of-flight detection.1MC employs mass tag reagents, antibodies (Abs) labeled with heavy metal isotopes, and provides single mass isotope resolution for measuring biomarker expression, cell-by-cell, at 500 cells / sec.2The signal intensity is proportional to the number of atoms of a metal isotope attached to each Ab. Current reagents for biomarker detection are metal-chelating polymers (MCPs) with monoamides of diethylenetriamine pentaacetate (DTPA) and 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as pendant groups. These chelators are effective for binding hard metal ions such as the lanthanides, yttrium and bismuth. In current technology, MC enables the measurement of up to 50 biomarkers simultaneously.23This allows for multiplexed quantitative analysis of multiple biomarkers in cell populations, providing essential information about mechanistic interactions at the singlecell level.4The latest generation MC instrument has the capability to detect as many as 135 stable isotopes with mass-to-charge (m / z) ratios in the range of 75 (Se) to 209 (Bi).5However, the multiplexing capability of MC is currently limited to about 50 parameters. This limitation arises from the available MCPs that predominantly chelate hard metal ions.

[0004] There is a strong need to increase the number of biomarkers that can be detected on an individual cell. Metal-chelating polymers are the most commonly used mass tags for mass cytometry. These metal-chelating polymers are effective at binding heavy metal isotopes of hard metal ions. However, to increase the multiplexing capabilities of mass cytometry, one needs metal-chelating polymers able to bind soft metal ions. A new class of metal-chelating polymers based on dipicolylamine chelators was recently reported. Overcoming non-specific binding is one of the main challenges in developing new reagents for mass cytometry.

[0005] Chelators have been used to attach radioactive soft metals to Abs and other biomacromolecules. For example, dipicolylamine has been used to bind technetium (99mTc) and rhenium (188Re) isotopes6and deferoxamine has been used to bind zirconium isotopes (90Zr).7These metal complexes are small molecules which can only carry 1 metal ion per complex, and only limited numbers can be attached to a single Ab. To prepare mass tag reagents for MC, the metal complexes must be attached to polymers that in turn are attached to Abs. Only in this way can one attach sufficient copies of an isotope to an Ab to generate a useful MC signal as a mass tag. It is also essential that the binding of the metal ion to the chelator be sufficiently strong to avoid leaching, transchelation, or ion exchange during storage or application of the mass tag reagent.8

[0006] In addition to identifying potential chelators for soft metal isotopes, analogue structures must be designed with functionality that enable the chelators to be covalently attached to a polymer framework. Newly developed mass tags must then be evaluated based on several important criteria for a useful mass tag reagent for high throughput MC applications. The mass tags must be soluble in aqueous media. Ideally, they should be non-toxic, chemically stable, compatible with other mass tag reagents, and have low levels of non-specific binding (NSB).3

[0007] Several reports in the literature that focused on developing new mass tag reagents capable of binding soft or semisoft metals ions for MC applications. For instance, in 2019, Cho et al. reported the synthesis of a polymer containing pendant deferoxamine chelators. They showed that this polymer could bind natural abundance Zr(IV) ions anddemonstrated that Zr could be detected by MC was reported.8However, poor watersolubility remained a challenge, as polymers modified with too many deferoxamine groups became insoluble in water. This problem was resolved by modification of some of the polymer pendant groups with methoxy(polyethylene glycol) with 24 repeat units (mPEG24). However, the mPEG24 groups, which occupied some of the pendant groups, limited the number of deferoxamine that could be attached to the polymer and the number of Zr ions that could be bound. The low number of Zr ions per polymer limited its practical use as a polymer mass tag reagent.

[0008] Dang et al. reported the preparation of a Zr nanometal organic framework with a high degree of specificity toward the intended cell target, but NSB remained a challenge at elevated titers.9Chen et al. synthesized mesoporous porphyrinic frameworks capable of binding Sn, Pt, W, and Pd.10These frameworks showcased low NSB to Ramos cells, high sensitivity, and specificity comparable to commercial Maxpar reagents.10However, the NP synthesis remained a challenge due to the difficulties in preparing smaller (< 40 nm) particle sizes with a narrow size distribution and stable colloidal dispersions. Polymer containing dipicolylamine (DPA) chelators have been developed (WO 2023 / 279211).

[0009] NSB plays a deleterious role in many bioanalytical and biomedical applications of polymers and nanoparticles.12-15To reduce NSB, numerous strategies have been reported and adopted for a variety of different applications.16

[0010] Additional and / or improved mass tags suitable for MC applications are desirable.

[0011] Recent reports of new soft metal mass tag reagents have focused on SMC applications. The only IMC applications consist of repurposing cis-platin derivatives attached to antibodies. There are no reports of polymer mass tag reagents based upon soft metal ions that have been developed for IMC.

[0012] For example, a 2019 report described a zirconium (Zr) metal-chelating polymer (MCP) designed for SMC. However, its low water-solubility and low Zr metal content limited its applicability. The authors were able to conjugate their Zr polymer onto 3 μm or 10 μm diameter polystyrene microbeads and study the binding of the Zr polymer to these beads with IMC.14But this is a long step from a realistic application. In 2022, preparation of a tellurium-containing polymer conjugated with either a primary or a secondary Ab wasreported. The tellurium mass tags were used to stain Ramos cells (positive cell line, CD20+) and HL-60 cells (negative cell line, CD20’) and were examined by SMC. However, when the tellurium mass tag was conjugated to anti-CD20 Ab for use in a multiplexed experiment on peripheral blood mononuclear cells (PBMCs), this mass tag was unable to effectively distinguish the B cells from non-T / B cells.15These examples exemplify the difficulties with the preparation of new mass tags for improving the multiparameter capabilities of MC. In these circumstances, preparation of new mass tags suitable as probes for IMC imaging of more complex biological samples, namely tissue sections are that much more challenging.

[0013] More recently, Dang et al. reported a Zr nanoscale metal organic framework (NMOF) that carries many more Zr ions. The authors then conjugated the Zr-NMOF to anti-CD45 Abs and used the Zr NMOF-Ab conjugates in a multiparametric SMC study to stain spleen cells alongside four Maxpar reagents targeting CD19, CD3, CD4, and CD8 biomarkers. Their work showed good specificity. While there was a relatively high level of non-specific binding (NSB), they were able to observe distinct cell B and T cell populations.16

[0014] In 2022, a metal chelating polymer with dipicolylamine (DPA) pendant groups that is able to bind Re and Pt was reported. The DPA group was attached to the polymer backbone via a lysine spacer, and the remaining -COOH group was used to attach a polyethylene glycol 6-mer (PEG6) to promote water solubility. The modified polymer was further attached to an anti-CD20 Ab and incorporated into a 4-plex SMC assay, thenatRe-CD20 conjugate allowed distinct separation of B cell subsets (CD20+) from the rest of cell subsets within PBMCs at all titers. The corresponding Pt conjugate was much less effective in this 4-plex assay. Also in 2022, Chen et al. reported nanoparticles in the form of a mesoporous porphyrinic framework (MPF) that could bind Sn, Pt, W, and Pd. In their work, they prepared a Sn-loaded MPF and conjugated it to anti-CD4 Ab, and also prepared a Pt-loaded MPF conjugated to anti-CD3 Ab. The Sn-loaded MPF and Pt-loaded MPF were used together in a multiparametric assay with Maxpar reagents targeting CD8, CD19, and CD56 biomarkers.17Their work opened new mass channels for the element Sn, for MC, and showed an improvement to the Pt mass tags reported earlier in 2022. Inboth these cases, only SMC was studied and to date, the study of these materials by IMC has yet to be reported.

[0015] Since the inception of IMC back in 2014, there has only been a few reports detailing new mass tag reagents used for SMC being adopted for IMC. In 2016, the Bodenmiller group who first reported IMC as an imaging modality, used pre-existing reagents common for staining membrane structures in electron microscopy, ruthenium tetroxide (RuO4) and osmium tetroxide (OsO4) and adapted them to the SMC workflow.18Following this work, the Bodenmiller group adapted the RuO4 reagent as a suitable counterstain in IMC,19however no reports on OsO4 being adopted for IMC were reported. In 2019, Chen and co-workers prepared gold nanoparticles conjugated with DNA as probes for IMC.20

[0016] Tissue sections contain multiple cell types with different spatial arrangements and are much more complex compared with single cell suspensions.21 22Adopting reagents used for SMC to IMC has proven to be a challenging task. Mass tag reagents that work for SMC may not always work for IMC and often, these challenges go unreported.

[0017] There is a need to develop improved compounds for mass spectrometry.SUMMARY

[0018] It has been shown herein that compounds of the present application provide for metal chelators suitable as mass tag reagents.

[0019] Accordingly, the present application includes a compound of Formula IR1(I)whereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci-Csalkyl, C2-Cealkenyl, C2-Cealkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and C1-C6alkylphosphonate;n is an integer from 15 to 75, from which in 2 to 14 repetitions, each R is independently a solubility modifier selected fromwherein:y is an integer from 20 to 48,R3is a reactive functional group,and the remaining repetitions of R are each independently selected from:wherein z is an integer from 10 to 75,andR1and R2are each independently a terminal group,t is an integer from 0 to 6, andone or more A are configured to coordinate to a soft metal selected from Pt, Re, Pd and Hg.

[0020] The present application also includes A compound of Formula II:whereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci-Csalkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci-Cealkylphosphonate;n is an integer from 15 to 75, from which in 2 to 14 repetitions, each R is independently a solubility modifier selected fromR3ywherein:y is an integer from 20 to 48,R3is a reactive functional group,and the remaining repetitions of R are each independently selected from: / CLy'y’ is an integer from 20 to 48,o wherein z is an integer from 10 to 75, andR1and R2are each independently a terminal group,n is an integer from 15 to 75,t is an integer from 0 to 12,v is an integer from 1 to 3;each M is a soft metal selected from Pt, Re, Pd and Hg, and each Q is independently absent or a thiol-based solubility ligand.

[0021] The present application also includes a compound of Formula IIIwhereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci-Csalkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci-Cealkylphosphonate;n is an integer from in 2 to 14, and each R is independently a group5 R3ywherein:y is an integer from 20 to 48,R3is a reactive functional group,m is an integer from 13 to 73, and each R’ is independently selected from:wherein z is an integer from 10 to 75,andwherein n + m is an integer from 15 to 75,R1and R2are each independently a terminal group,n is an integer from 15 to 75,stat indicates that the n and m repetitions of each monomer are statistically distributed to form a polymer of Formula III;t is an integer from 0 to 12,v is an integer from 1 to 3;each M is a soft metal selected from Pt, Re, Pd and Hg, andeach Q is independently absent or a thiol-based solubility ligand.

[0022] The present application includes a kit comprisingan isotopic composition comprising multiple soft metal atoms of a single isotope of a soft metal; andan element tag comprising a compound of the present application, the compound comprising a plurality of chelating groups capable of binding at least one soft metal atom of the isotopic composition, and wherein the at least one soft metal atom is bound to a thiol-based solubility ligand;optionally wherein the kit does not comprise any radioactive soft metal selected from Pt, Re, Pd and Hg.

[0023] The present application further includes a method for preparing an element tagged reagent comprising:providing an isotopic composition comprising multiple soft metal atoms of a single isotope of a soft metal selected from Re, Pt, Pd and Hg;providing an element tag comprising a compound of the present application, comprising a plurality of chelating groups wherein each chelating group is capable of binding at least one of the soft metal atom of the isotopic composition; binding the soft metal atoms of the isotopic composition to the one or more chelating groups of the element tag;binding the soft metal atoms bound to the one or more chelating groups of the element tag, to a thiol-based solubility ligand to provide the element tagged reagent; andwherein the soft metal atoms are non-radioactive.

[0024] Also included is a method for analyzing an analyte in a biological sample, comprising:(i) incubating an element tagged affinity reagent with the analyte, the element tagged affinity reagent comprising an affinity reagent tagged with an element tag, the element tag comprising a compound of any one of claims 1 to 36 multiple soft metal atoms of a single isotope of a soft metal selected from Pt, Re, Pd and Hg, and comprising chelating groups binding at least one of the soft metal atoms, wherein the soft metal atoms are bound to a thiol-based solubility ligand, the soft metal atoms are non-radioactive, and the affinity reagent specifically binds the analyte,(ii) separating unbound element tagged affinity reagent from bound element tagged affinity reagent; and(iii) analyzing the element tag bound to the affinity reagent attached to the analyte by mass spectrometric atomic spectroscopy.

[0025] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.BRIEF DESCRIPTION OF DRAWINGS

[0026] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0027] FIG.1 is a1H NMR of PPFPA polymer 1c in CDCl3. The degree of polymerization (DP) of the polymer was determined via end-group analysis. The spectrum was normalized to 3.00 protons of the terminal CH3 of the dodecyltrithiocarbonate end group as peak a (5 0.9 ppm). Peak b is assigned to the 20H of its (CH2) alkyl chain. The integration of peak a (3H) of the terminal -CH3 of the dodecyltrithiocarbonate end group was compared to that of 22 for peak g at 5 3.1 ppm (1 H assigned to the 1 proton in the polymer backbone). In this way, the polymer was determined to have a DPn= 22

[0028] FIG.2 is a19F NMR of PPFPA polymer 1c in CDCl3.

[0029] FIG.3 is a1H NMR of P(dap-DPA) polymer 3 in D2O.

[0030] FIG. 4 is a1H NMR of Pt P(L-DPA-g-mPEGi2) polymer 6a in D2O. The mean numbers of mPEG6per repeat were determined by1H NMR by comparing peak a (δ 7.0-8.7 ppm, 8.00 H total of the two pyridyl groups) with peaks b,c,d,e (δ 2.8-4.5 ppm, 7 protons closest to the tertiary amine of the DPA pendant group and of the -NH group of the acrylamide, and the mPEG6O-CH2groups), and peaks f,g,h (δ 0.8-2.5 ppm), 3 protons on the backbone, and alkyl CH2(6H) of the lysine moiety.

[0031] FIG. 5 is a series of signal distribution histograms for the non-specific binding of A) a Maxpar control (mean, median: 1 count), B) Pt P(L-DPA-mPEG6) polymer (mean 80, median 9.8) and C) Pt P(dap DPA-mPEG6) polymer (mean 232, median 55) with PBMCs at a titer of 10 pg / mL. The value in the upper-right corner depicts the median195Pt counts as a quantitative measure of non-specific binding. All median195Pt counts less than 1 were rounded to 1.

[0032] FIG. 6 is a1H NMR of PSBMA29 (before aminolysis and maleimide capping) in D2O. The degree of polymerization (DP) of the polymer was determined via end-group analysis. The spectrum was normalized to 5.00 protons of the benzene of the dithiobenzoate end group as peak d (δ 7.4-8.3 ppm). Peak c is assigned to the O-CH2 of the pendant sulfobetaine repeat units. The integration of peak d (5H) of the benzene of the dithiobenzoate end group was compared to that of 58 for peak c at δ 4.3-4.6 ppm (2 H assigned to O-CH2). In this way, the polymer was determined to have a DPn= 29.

[0033] FIG. 7 is a1H NMR of P(L-DPA-g-mPEG12) polymer in D2O. The mean numbers of mPEG12per repeat were determined by1H NMR by comparing peak a (δ 7.0-8.7 ppm, 8.00 H total of the two pyridyl groups) with peaks b,c,d,e (δ 2.8-4.5 ppm, 7 protons closest to the tertiary amine of the DPA pendant group and of the -NH group of the acrylamide, and the mPEG12O-CH2groups), and peaks f,g,h (δ 0.8-2.5 ppm), 3 protons on the backbone, and alkyl CH2(6H) of the lysine moiety.

[0034] FIG. 8 is a1H NMR of Pt P(L-DPA-g-mPEG12) polymer in D2O.

[0035] FIG. 9 is a1H NMR of Pt P(L-DPA-g-mPEG24) polymer in D2O.

[0036] FIG. 10 is a1H NMR of PSBMA29 (after aminolysis and maleimide capping) in D2O. The degree of polymerization (DP) of the polymer was determined via end-group analysis. The spectrum was normalized to an integration of 58.43 as peak c (-O-CH2of the pendant sulfobetaine repeat units, δ 4.4-4.7 ppm) based on the integration from the1H NMR spectrum of PSBMA29 prior to aminolysis and maleimide capping shown in FIG. 6.

[0037] FIG. 11 is a1H NMR of P(L -DPA-g-PSBMA) polymer 4e in D2O. The mean numbers of SBMA29 per repeat were determined by1H NMR by comparing peak a (δ 6.8-8.5 ppm, normalized to 8.00 H total of the two pyridyl groups) with peak b (δ 4.5-4.6 ppm, 2H of the O-CH2of the methacrylate repeat unit of SBMA29).

[0038] FIG. 12 is a1H NMR of Pt P(L-DPA-g-ZW-NH2) polymer 6d in D2O.

[0039] FIG. 13 is a1H NMR of Pt P(L-DPA-g-PSBMA) polymer 6e in D2O.

[0040] FIG. 14 is a signal distribution histograms for the non-specific binding of A) P(L-DPA-g-mPEG12) polymer , B) P(L-DPA-g-mPEG24) polymer , C) P(L-DPA-g-ZW-NH2) polymer and D) P(L-DPA-g-PSBMA) polymer with PBMCs at a titer of 10 pg / mL. The value in the upper corner depicts the median195Pt counts as a quantitative measure of non-specific binding.

[0041] FIG. 15 is a time dependent1H NMR of polymer 6a treated with a molar excess of glutathione at ca. 5 equiv. per pendant group in D2O.

[0042] FIG. 16 presents bi-exponential decay curves of free glutathione (D = 4.6×10-10m2 / s), and polymer-bound glutathione (D = 6.9×10-11m2 / s).

[0043] FIG. 17 presents signal distribution histograms for the comparison of non-specific binding of Pt-polymers before and after treatment with glutathione. The top row is for samples without GSH treatment. A) polymer 6a, B) polymer 6c, C) polymer 6d, D) polymer 6e, and E) polymer 7a. The bottom row is for the same samples after GSH treatment. F) polymer 8a, G) polymer 8c, H) polymer 8d, I) polymer 8e, and J) polymer 9a with PBMCs at a titer of 10 pg / mL. The value in the upper corner depicts the median195Pt counts as a quantitative measure of non-specific binding. Median195Pt counts less than 1 were rounded to 1.

[0044] FIG. 18A are normalized electrophoretic mobility distribution histograms of mPEG6-grafted polymers , , , and measured in ultrapure water at concentrationsca. 1 mg / mL; FIG 18B are GPC traces of polymer 4a (before metalation), polymer 6a (after Pt metalation), and polymer 8a (after GSH treatment); FIG. 18C are normalized electrophoretic mobility distribution histograms of mPEG24-grafted polymers 2, 4c, 6c, and 8c measured in ultrapure water at concentrations ca. 1 mg / mL; FIG. 18D are normalized electrophoretic mobility distribution histograms of ZW-NH2-grafted polymers 2, 4d, 6d, and 8d measured in ultrapure water at concentrations ca. 1 mg / mL.

[0045] FIG. 19 is a series of graphs showing functional tests ofnatPt polymer 8a-CD20 andnatPt polymer 8c-CD20 on PBMCs. Both polymers were treated with GSH. Biaxial scatter plots of170Er_CD3 vs196Pt-CD20 performed at a titer of 10.0 pg / mL of A) Maxpar control (here the x-axis refers to147Sm-CD20 used to detect the B-cell population, B) polymer 8a-CD20 (PEG6) and C) polymer 8c-CD20 (PEG24).

[0046] FIG. 20A, 20B, 20C, 20D and 20E are various plots for polymer 11-1. FIG. 20A is a1H NMR of Pt polymer 11-1 (treated with GSH, DP = 22) in D2O. The mean numbers of mPEG24 / N3-PEG23 grafted per repeat were determined by1H NMR by comparing peak a (δ 7.1-9.4 ppm, 8.00 H total of the two pyridyl groups) with peaks b,c,d,e (δ 3.2-4.2 ppm, 7 protons closest to the tertiary amine of the DPA pendant group and of the -NH group of the acrylamide, and the mPEG24O-CH2groups), and peaks f,g,h (δ 0.6-2.9 ppm), 3 protons on the backbone, and alkyl CH2(6H) of the lysine moiety; FIG. 20B is a1H NMR of Pt polymer 10 (DP = 55) in D2O. The mean numbers of mPEG24 / N3-PEG23 grafted per repeat were determined by1H NMR by comparing peak a (δ 7.2-9.1 ppm, 8.00 H total of the two pyridyl groups) with peaks b,c,d,e (δ 2.6-4.3 ppm, 7 protons closest to the tertiary amine of the DPA pendant group and of the -NH group of the acrylamide, and the mPEG24O-CH2groups), and peaks f,g,h (δ 0.5-2.4 ppm), 3 protons on the backbone, and alkyl CH2(6H) of the lysine moiety; FIG. 20C are normalized SEC curves of DP=22 polymer , and polymer obtained from the aqueous SEC; FIG. 20D are normalized SEC curves of DP=55 polymer , and polymer obtained from the aqueous SEC; FIG. 20E are normalized electrophoretic mobility distribution histograms of polymer 2, 1-1, 10, and 11-1, measured in ultrapure water at concentrations ca. 1 mg / mL..

[0047] FIG. 21 is a series of plots of functional tests ofnatPt-polymer22-CD20 on PBMCs. Biaxial scatter plots of170Er_CD3 vs196Pt_CD20 performed at three different titers, A) low titer (1.11 pg / mL), B) medium titer (3.33 pg / mL), and C) high titer (10.0pg / mL). The data were manually separated with lines into 4 distinct quadrants. Signals from T cells (CD45+, CD3+) appear in the upper left quadrant of each panel. Corresponding signals from B cells (CD45+, CD20+) appear in the lower right quadrant, and signals from non-B / T cells (CD45+, CD20-, CD3-) appear in the bottom left quadrant.

[0048] FIG. 22 is a series of plots of functional tests ofnatPt-polymer II-155-CD20 on PBMCs. Biaxial scatter plots of170Er_CD3 vs196Pt_CD20 performed at three different titers, A) low titer (1.11 pg / mL), B) medium titer (3.33 pg / mL), and C) high titer (10.0 pg / mL). The data were manually separated with lines into 4 distinct quadrants. Signals from T cells (CD45+, CD3+) appear in the upper left quadrant of each panel. Corresponding signals from B cells (CD45+, CD20+) appear in the lower right quadrant, and signals from non-B / T cells (CD45+, CD20-, CD3-) appear in the bottom left quadrant.

[0049] FIG. 23 is a series of plots of functional tests of isotopically enriched196Pt polymer 11-1 (with GSH treatment) (top) and polymer 10 (without GSH treatment) (bottom) on PBMCs. Biaxial scatter plots of170Er_CD3 vs196Pt_CD20 performed at three different titers, A) low titer of 0.56 pg / mL, B) medium titer of 1.67 pg / mL, and C) high titer of 5.0 pg / mL. Functional tests ofnatPt polymer 10 (without GSH treatment) on PBMCs. Biaxial scatter plots of170Er_CD3 vs196Pt_CD20 performed at three different titers, D) low titer of 0.56 pg / mL, E) medium titer of 1.67 pg / mL, and F) high titer of 5.0 pg / mL. Histograms of196Pt counts on T cells, B cells, and non-B / T cells for G) polymer 11-1 -Ab and H) polymer 10-Ab.

[0050] FIG. 24 is a series of plots of functional tests of isotopically enriched196Pt polymer 11-155-CD3 (with GSH treatment) (top) and196Pt polymer 1055-CD3 (without GSH treatment) (bottom) on PBMCs. Biaxial scatter plots of170Er_CD3 vs196Pt_CD20 performed at three different titers, A) low titer of 0.56 pg / mL, B) medium titer of 1.67 pg / mL, and C) high titer of 5.0 pg / mL. Functional tests ofnatPt polymer 1-1 (without GSH treatment) on PBMCs. Biaxial scatter plots of170Er_CD3 vs196Pt_CD20 performed at three different titers, D) low titer of 0.56 pg / mL, E) medium titer of 1.67 pg / mL, and F) high titer of 5.0 pg / mL. Histograms of 196Pt counts on T cells, B cells, and non-B / T cells for G)196Pt polymer55-CD3 and H) polymer55-CD3.

[0051] FIG. 25 is a series of images obtained from IMC for ROI 1. Top: greyscale images of A)166Er-CD45RA Maxpar control, B) GSH-capped polymer 11-1 (DP=22), C) GSH-capped polymer 11-1 (DP=55). Bottom: Images of the mass channels on IMC. For image D) light grey is assigned to166Er-CD45RA, and for E & F) light grey is assigned to196Pt-CD45RA. For all images D-F) white is assigned to170Er-CD3, and dark grey is assigned to191Ir-DNA intercalator. Grey colour refers to co-localization of light grey channels (166Er-CD45RA or196Pt-CD45RA) with dark grey channels (191Ir).

[0052] FIG 26 is a series of Images obtained from IMC for ROI 2. Top: greyscale images of A)166Er-CD45RA Maxpar control, B) polymer 11-1 (with GSH) (DP=22), C) polymer 11-1 (DP=55) on the upper right, and Maxpar control on the bottom left. Bottom: Images of the mass channels on IMC. For image D) light grey is assigned to166Er-CD45RA, and for E & F) light grey is assigned to196Pt-CD45RA. For all images D-F) white is assigned to170Er-CD3, and dark grey is assigned to191Ir-DNA intercalator.

[0053] FIG. 27 is a series of images obtained from IMC for ROI 1. Top: greyscale images of A)166Er-CD45RA Maxpar control, B) polymer 1022-CD45RA (without GSH), C) polymer 1055-CD45RA (without GSH). Bottom: Images of the mass channels on IMC. For image D) light grey is assigned to166Er-CD45RA, and for E & F) light grey is assigned to196Pt-CD45RA. For all images D-F) white is assigned to170Er-CD3, and dark grey is assigned to191Ir-DNA intercalator. Scale bar (lower right corner) represents 100 μm.

[0054] FIG. 28 is a series of images obtained from IMC for ROI 3. Top: greyscale images of A)166Er-CD45RA Maxpar control, B) polymer 10 (with GSH) (DP=22), C) polymer 10 (DP=55) on the upper right, and Maxpar control on the bottom left. Bottom: Images of the mass channels on IMC. For image D) light grey is assigned to166Er-CD45RA, and for E & F) light grey is assigned to196Pt-CD45RA. For all images D-F) white is assigned to170Er-CD3, and dark grey is assigned to191Ir-DNA intercalator

[0055] FIG. 29 is a series of images obtained from IMC for ROI 4. Top: greyscale images of A)166Er-CD45RA Maxpar control, B) polymer 11-1 (with GSH) (DP=22), C) polymer 11-1 (DP=55) on the upper right, and Maxpar control on the bottom left. Bottom: Images of the mass channels on IMC. For image D) light grey is assigned to166Er-CD45RA, and for E & F) light grey is assigned to196Pt-CD45RA. For all images D-F) white is assigned to170Er-CD3, and dark grey is assigned to191Ir-DNA intercalator.

[0056] FIG. 30 is a series of images in the152Sm mass channel obtained for the 4 ROI of human tonsil tissue from IMC stained with (left column, A, C, F, I) Maxpar Ab cocktail, (middle column, B, D, G, J) Maxpar Ab cocktail plus polymer ll-1-Ab (DP=22), and (right column, C, E, H, K) Maxpar Ab cocktail plus polymer ll-1-Ab (DP=55). Values in the upper right of each image represent the max threshold value.

[0057] FIG. 31 is a series of images in the141Pr mass channel obtained for the 4 ROI of human tonsil tissue from IMC stained with (left column, A, C, F, I) Maxpar Ab cocktail, (middle column, B, D, G, J) Maxpar Ab cocktail plus polymer ll-1-Ab (DP=22), and (right column, C, E, H, K) Maxpar Ab cocktail plus polymer ll-1-Ab (DP=55). Values in the upper right of each image represent the max threshold value.

[0058] FIG. 32 is a1H NMR of PPFPA in CDCl3. The degree of polymerization (DP) of the polymer was determined via end-group analysis. The spectrum was normalized to 3.00 protons of the terminal CH3 of the dodecyltrithiocarbonate end group as peak a (5 0.9 ppm). Peak b is assigned to the 20H of its (CH2) alkyl chain. The integration of peak a (3H) of the terminal -CH3 of the dodecyltrithiocarbonate end group was compared to that of 22 for peak g at 53.1 ppm (1 H assigned to the 1 proton in the polymer backbone). In this way, the polymer was determined to have a DPn= 22.

[0059] FIG. 33 is a1H NMR of lysine-substituted DPA polymer in D2O. The mean numbers of DPA per repeat were determined by1H NMR by comparing peak a (δ 7.0-8.7 ppm, 8.00 H total of the two pyridyl groups) with peaks c,d,e (δ 2.9-4.5 ppm, 7 protons closest to the tertiary amine of the DPA pendant group and of the -NH group of the acrylamide), and peaks f,g,h (δ 0.7-2.5 ppm), 3 protons on the backbone, and alkyl CH2(6H) of the lysine moiety.

[0060] FIG. 34 is a1H NMR of polymer 1-1 (DP = 22) in D2O. The mean numbers of mPEG24 / N3-PEG23 grafted per repeat were determined by1H NMR by comparing peak a (δ 6.6-8.7 ppm, 8.00 H total of the two pyridyl groups) with peaks b,c,d,e (δ 2.7-4.5 ppm, 7 protons closest to the tertiary amine of the DPA pendant group and of the -NH group of the acrylamide, and the mPEG24O-CH2groups), and peaks f,g,h (δ 0.9-2.4 ppm), 3 protons on the backbone, and alkyl CH2(6H) of the lysine moiety.

[0061] FIG. 35 is a1H NMR of polymer 11 in D2O.

[0062] FIG. 36 is a normalized SEC trace of polymer 1-1, polymer 11, polymer II-5, polymer II-7.

[0063] FIG. 37 is an Electrophoretic Mobility plots of polymer 1-1, polymer 11, and polymer II-5, polymer II-7.

[0064] FIG. 38 is a1H NMR of polymer in D2O.

[0065] FIG. 39 is a1H NMR of polymer II-7 in D2O.

[0066] FIG. 40 are signal distribution histograms for the non-specific binding of polymer II-5, II-7, and Maxpar Ab cocktail (Maxpar control) at a titer of 5 pg / mL. The Maxpar reagents in the control are employed at titers optimized for PBMC samples by Standard BioTools Inc.

[0067] FIG. 41 are functional tests ofnatHg polymers ll-5-CD8a and ll-7-CD8a at three different titers, low (0.56 pg / mL), medium (1.67 pg / mL), and high (5.0 pg / mL) Biaxial scatter plot of162Dy_CD8a vs145Nd_CD4 of A) Maxpar control. For the Hg polymers, the x-axis162Dy_CD8a is replaced with202Hg_CD8a. Biaxial scatter plot of145Nd_CD4 vs202Hg_CD8a of polymer ll-5-CD8a B) low titer, C) medium titer, and D) high titer, and of polymer ll-7-CD8a E) low titer, F) medium titer, and G) high titer. Histograms of202Hg counts on CD8a T cells (CD3+; CD8a+), CD4 T cells (CD3+; CD4+) and non-T cells (CD3-) for H) polymer ll-5-CD8a and I) polymer ll-7-CD8a.

[0068] FIG.42 is normalized electrophoretic mobility distribution histograms of polymer 10, polymer 11-1, and polymer II-8 measured in ultrapure water at concentrations of ca. 1 mg / mL.

[0069] FIG.43 shows MC functional tests of STS polymer ll-8-anti-CD20 Ab conjugates on PBMCs. Biaxial scatter plot of (a) Maxpar control containing the 10-plex Ab cocktail plotted as170Er-CD3 vs147Sm-CD20. For the other biaxial scatter plots (b−d),147Sm-CD20 was replaced withnatPt-CD20 and are plotted as170Er-CD3 vs196Pt-CD20: (b) low titer (1.11 pg / mL), (c) medium titer (3.33 pg / mL), and (d) high titer (10.0 pg / mL). The data were manually separated with blue lines into four distinct quadrants. Signals from T cells (CD45+, CD3+) appear in the upper left quadrant of each panel. Corresponding signals from B cells (CD45+, CD20+) appear in the lower right quadrant, and signals from non-B, T cells (CD45+, CD20-, CD3-) appear in the bottom left quadrant.

[0070] FIG.44 shows MC functional tests of STS polymers: polymer ll-8-195Pt-CD4 and polymer ll-8196Pt-CD19 Ab conjugates on PBMCs. Biaxial scatter plot of (a) Maxpar control containing the 10-plexAb cocktail plotted as170Er-CD3 vs142Nd-CD19. For the biaxial scatter plots (b-d),142Nd-CD19 was replaced with196Pt-CD19 and the data are plotted as170Er-CD3 vs196Pt-CD19: (b) low titer (1.11 pg / mL), (c) medium titer (3.33 pg / mL), and (d) high titer (10.0 pg / mL). The data were manually separated with blue lines into four distinct quadrants. Signals from T cells (CD45+, CD3+) appear in the upper left quadrant of each panel. Corresponding signals from B cells (CD45+, CD19+) appear in the lower right quadrant, and signals from non-B, T cells (CD45+, CD19-, CD3-) appear in the bottom left quadrant. Biaxial scatter plot of (e) Maxpar control containing the 10-plex Ab cocktail plotted as145Nd-CD4 vs146Nd-CD8a. For the biaxial scatter plots (f- h),145Nd-CD4 was replaced with195Pt-CD4 and the data are plotted as195Pt-CD4 vs146Nd-CD8a: (f) low titer (1.11 pg / mL), (g) medium titer (3.33 pg / mL), and (h) high titer (10.0 pg / mL). The data were manually separated with blue lines into four distinct quadrants. Signals from helper T cells (CD45+, CD3+, CD4+, CD8a-) appear in the upper left quadrant of each panel. Corresponding signals from cytotoxic T cells (CD45+, CD3+, CD8a+, CD4-) appear in the lower right quadrant.

[0071] FIG.45 are images obtained from the IMC of tissue samples from ROI1. Greyscale images of (a)166Er-CD45RA Maxpar control, (b) GSH-capped196Pt polymer (polymer ), and (c) thiosulfate-capped Pt polymer (polymer ). Pseudocolored images of the (d)166Er-CD45RA Maxpar control, (e) GSH-capped196Pt polymer (polymer ), and (f) thiosulfate-capped Pt polymer (polymer II-8). In panel (D), red represents166Er- CD45RA and green represents173Yb-CD45RO. In panels (e) and (f), red represents196Pt-CD45RA and green represents173Yb-CD45RO. Scale bar represents 200 pm (top left corner of each image). Max threshold values are displayed in the upper right corner of each image.DETAILLED DESCRIPTIONI. Definitions

[0072] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0073] As used in this application and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0074] The term “consisting” and its derivatives as used herein are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0075] The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and / or steps.

[0076] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0077] As used in the present application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a compound” should be understood to present certain aspects with one compound, or two or more additional compounds.

[0078] In embodiments comprising an “additional” or “second” component, such as an additional or second compound, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0079] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present. The term “and / or” with respect to enantiomers, prodrugs, salts and / or solvates thereof means that the compounds of the application exist as individual enantiomers, prodrugs, salts and hydrates, as well as a combination of, for example, a salt of a solvate of a compound of the application.

[0080] As used herein, the term “one or more” item includes a single item selected from the list as well as mixtures of two or more items selected from the list.

[0081] The term “compound of the application” or “compound of the present application” and the like as used herein refers to a compound of Formula (I) or salts, solvates and / or prodrugs thereof.

[0082] The term “composition of the application” or “composition of the present application” and the like as used herein refers to a composition comprising one or more compounds of the application.

[0083] The term “suitable” as used herein means that the selection of the particular composition or conditions would depend on the specific steps to be performed, the identity of the components to be transformed and / or the specific use for the compositions, but the selection would be well within the skill of a person trained in the art.

[0084] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0085] The term “alkyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cni-n2”.For example, the term C1-10alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0086] The term “alkylene”, whether it is used alone or as part of another group, means straight or branched chain, saturated alkylene group, that is, a saturated carbon chain that contains substituents on two of its ends. The number of carbon atoms that are possible in the referenced alkylene group are indicated by the prefix “Cni-n2”. For example, the term C2-6alkylene means an alkylene group having 2, 3, 4, 5 or 6 carbon atoms.

[0087] The term “alkenyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkyl groups containing at least one double bond. The number of carbon atoms that are possible in the referenced alkylene group are indicated by the prefix “Cni-n2”. For example, the term C2-6alkenyl means an alkenyl group having 2, 3, 4, 5 or 6 carbon atoms and at least one double bond.

[0088] The term “alkynyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkynyl groups containing at least one triple bond. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cni-n2”. For example, the term C2-6alkynyl means an alkynyl group having 2, 3, 4, 5 or 6 carbon atoms.

[0089] The term “aryl” as used herein, whether it is used alone or as part of another group, refers to carbocyclic groups containing at least one aromatic ring and contains either 6 to 10 carbon atoms.

[0090] The term “heterocyclyl” as used herein, whether it is used alone or as part of another group, refers to cyclic groups containing at least one non-aromatic ring containing from 3 to 10 atoms in which one or more of the atoms are a heteroatom selected from O, S and N and the remaining atoms are C. Heterocyclyl groups are either saturated or unsaturated (i.e. contain one or more double bonds). When a heterocycloalkyl group contains the prefix Cni-n2 this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as selected from O, S and N and the remaining atoms are C. Heterocyclyl groups are optionally benzofused.

[0091] The term “heteroaryl” as used herein, whether it is used alone or as part of another group, refers to cyclic groups containing at least one heteroaromatic ring containing 5-10 atoms in which one or more of the atoms are a heteroatom selected from O, S and N and the remaining atoms are C. When a heteroaryl group contains the prefix Cni-n2this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as defined above. Heteroaryl groups are optionally benzofused.

[0092] All cyclic groups, including aryl, heteroaryl, and heterocyclyl groups, contain one or more than one ring (i.e. are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged or spirofused.II. Compounds and Compositions of the Application

[0093] It has been shown herein that compounds of the present application provide for metal chelators suitable as mass tag reagents.

[0094] Accordingly, the present application includes a compound of Formula Iwhereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci- Csalkyl, C2-Cealkenyl, C2-Cealkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci- Cealkylphosphonate;n is an integer from 15 to 75, from which in 2 to 14 repetitions, each R is independently a solubility modifier selected fromwherein:y is an integer from 20 to 48,R3is a reactive functional group,and the remaining repetitions of R are each independently selected from:y’ is an integer from 20 to 48;.0sowherein z is an integer from 10 to 75,andR1and R2are each independently a terminal group,t is an integer from 0 to 6, andone or more A are configured to coordinate to a soft metal selected from Pt, Re, Pd and Hg.

[0095] The present application also includes A compound of Formula II:whereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci-Csalkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci-Cealkylphosphonate;n is an integer from 15 to 75, from which in 2 to 14 repetitions, each R is independently a solubility modifier selected fromR3ywherein:y is an integer from 20 to 48,R3is a reactive functional group,and the remaining repetitions of R are each independently selected from: / CLy'y’ is an integer from 20 to 48,o wherein z is an integer from 10 to 75, andR1and R2are each independently a terminal group,n is an integer from 15 to 75,t is an integer from 0 to 12,v is an integer from 1 to 3;each M is a soft metal selected from Pt, Re, Pd and Hg, and each Q is independently absent or a thiol-based solubility ligand.

[0096] The present application also includes a compound of Formula IIIwhereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci-Csalkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci-Cealkylphosphonate;n is an integer from in 2 to 14, and each R is independently a group5 R3ywherein:y is an integer from 20 to 48,R3is a reactive functional group,m is an integer from 13 to 73, and each R’ is independently selected from:wherein z is an integer from 10 to 75,andwherein n + m is an integer from 15 to 75,R1and R2are each independently a terminal group,n is an integer from 15 to 75,stat indicates that the n and m repetitions of each monomer are statistically distributed to form a polymer of Formula III;t is an integer from 0 to 12,v is an integer from 1 to 3;each M is a soft metal selected from Pt, Re, Pd and Hg, andeach Q is independently absent or a thiol-based solubility ligand.

[0097] In some embodiments, each A is independently a pyridine, a thiazole or an imidazole, each of which being substituted or unsubstituted.

[0098] In some embodiments, each reactive functional group is independently a maleimide, a maleimide-thiol conjugate, an azide, a carboxylic acid, an amine, a fluorophenyl ester, a sulfotetrafluorophenyl electrophile, an aldehyde, an isothiocyanate derivative, a tetrazine, a trans-cyclooctyne or a combination thereof. In some embodiments, one or more of the reactive functional groups is conjugated to a biomolecule (e.g. lipid, carbohydrate, nucleic acid or protein).

[0099] In some embodiments, the biomolecule is an affinity reagent. For example, the affinity reagent can be a protein (e.g. peptide) or nucleic acid (e.g. oligonucleotide) that binds a specific target.

[0100] In some embodiments, the affinity reagent is an antibody.

[0101] The antibody is not limited and can be any kind of antibody including antibody fragments, single chain antibodies, monoclonal antibody and the like.

[0102] In some embodiments, the compound of the application is for use as a metal polymer chelator (MPC).

[0103] In some embodiments, the one or more A are configured to chelate a soft metal selected from Re and Pt. In some embodiments, the soft metal is a naturally occurring isotope or isotopically enriched. The term “isotopically enriched” refers to when the relative abundance of the isotopes of a given element are altered, thus producing a form of the element that has been enriched in one particular isotope and depleted in its other isotopic forms.

[0104] In some embodiments, the soft metal is configured to be substituted with one or more solubility ligands being a thiol-based ligand. In some embodiments, the thiol-based ligand is glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonate, or a combination thereof.

[0105] In some embodiments, the thiol-based ligand is a zwitterion or has an anionic charge, or imparts a zwitterionic or an anionic charge shift to a metal loaded compound or complex, for example as measured by electrophoretic mobility. For example, addition of thiol based ligand may impart to a metal loaded complex an electrophoretic mobility that is at least -1 pm cm / (V s) or at least -1.5 pm cm / (V s) or at least -2 pm cm / (V s) when compared to the metal loaded complex alone. Without being bound to theory, the thiol-based ligand does not have large hydrophobic groups as part of the molecule that would negatively impact aqueous solubility and would have a suitable size such that steric hindrance does not affect their chelation with the metal. In some embodiments, the thiol-based ligand has a high nucleophilic substitution coefficient for kinetic stability of the complexation. In some embodiments, the thiol-based ligand is a dipeptide comprising a cysteine and another amino acid that together have a zwitterion and / or negative charge. For example, the thiol-based ligand is cysteine-glycine.

[0106] In some embodiments, each terminal group comprises, a linker, a spacer, a capping group, a reactive group, or a combination thereof.

[0107] In some embodiments, the compound conjugated to the antibody is for use as a mass tag for targeting specific cells for detection in suspension mass cytometry (SMC) or imaging mass cytometry (IMC). In some embodiments, the mass tag is for use with further mass cytometry mass tags in a multiplex mass cytometry assay.

[0108] In the present disclosure, soft metal mass tags chelators are provided. As demonstrated herein, modifications of dipicolylamine (DPA) pendant groups produce mass tags that when conjugated to polymers and / or affinity agents, have reduced nonspecific binding. In some embodiments, the present disclosure demonstrates that PEG chains such as mPEG24- and a poly(sulfobetaine methacrylate) (PSBMA, such as DP = 29) attached to the DPA unit are very effective at reducing the Pt signal associated withNSB in PBMC samples. Replacing for example, the Pt-CI bond in the chelated complex with a Pt-S bond by reaction with thiol containing compounds such as glutathione, thiosulfate, thioglycolate or mercaptopropionic acid, is also effective at reducing nonspecific binding. These modifications provide polymer mass tags for applications to SMC and to IMC with reduced NSB.

[0109] Polymers studded with DPA chelators, either as a lysine based DPA (L-DPA) or DPA derivatives based upon 2,3-diaminopropanoic acid (dap) as pendant groups, are disclosed herein. In some embodiments, these chelators were then used to incorporate Pt2+as potential mass tags for mass cytometry applications. The pendant DPA groups of these polymers were modified to promote water solubility and to reduce non-specific binding to cells. Length of the PEG chains attached to the DPA was varied for example, mPEGe, mPEG-12, and mPEG24 were tested and two zwitterionic substituents were attached. Finally, the polymers were treated with glutathione to replace the Pt-CI bond in the Pt complex with the Pt-S- bond from GSH. The polymers were tested after metalation with Pt to observe the level of non-specific binding with CD45+ B cells based on the195Pt median counts analyzed using mass cytometry.

[0110] In some embodiments, modifications of the pendant groups with mPEG24 were shown to lead to a significant reduction in NSB of the polymer of the application. Attaching a small molecule zwitterionic group to the DPA gave high levels of NSB, but a zwitterionic polymer PSBMA29 reduced NSB comparably to the results with mPEG24. Finally, glutathione attached for example via a ligand exchange reaction, provided the greatest reduction in NSB, and this modification could be used in conjunction with the PEG and zwitterionic modifications to further reduce NSB.

[0111] In some embodiments, the polymers include conjugation to affinity reagents via a reactive functional group. A reactive functional group may include, for example, a maleimide, a maleimide-thiol conjugate, an azide, a carboxylic acid, an amine, a fluorophenyl ester, a sulfotetrafluorophenyl electrophile, an aldehyde, an isothiocyanate derivative, a tetrazine, a trans-cyclooctyne, or a combination thereof.

[0112] In some embodiments, conjugation of the polymer to an affinity reagent include trans-cyclooctyne (TCO)Ztetrazine chemistry methodologies, which include, for example, conjugation of TCO to the antibody using Mal-PEG3-TCO. For example, theTCO / tetrazine chemistry methodologies include modification of the solubility modifier pendant group with a tetrazine group or a TCO group. For example, the TCO / tetrazine chemistry methodologies include modification of the solubility modifier pendant group azido-PEG23-amine to react the azide ends with DBCO-PEGntetrazine or with to TCO-PEGntetrazine, where n is an integer from 2 to 24.

[0113] In some embodiments, conjugation of the polymer to an affinity reagent include binding the polymer to a lysine residue of an affinity reagent which include, for example, an antibody or a peptide as an affinity reagent. For example, the polymer include modification with NHS esters, sulfotetrafluorophenyl electrophile, sulfonyl halides, iminoboronates, diazonium salts, aldehydes, isothiocyanates and / or isocyanates which include coupling via the amine of one of the lysine residues of the affinity reagent which include, for example, an antibody or a peptide. For example, the polymer include modification with carboxylic acids that are activated via EDC / NHS chemistry for coupling with amines in affinity reagents which include, for example, an antibody or a peptide. For example, the polymer include modification with phenyloxadiazole sulfone derivatives, 3-arylpropiolonitriles and / or benzyl isothiocyanate for coupling with the thiol of one of the cysteine residues of the affinity reagent which include, for example, an antibody or a peptide.

[0114] In some embodiments, the metal in the soft metal mass tag chelator is conjugated to one or more thiol-based ligands which include, for example, glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid and / or 2, 3-dimercapto-1 -propanesulfonate.

[0115] In some embodiments, the thiol-based ligand comprises glutathione.

[0116] In some embodiments, the thiol-based ligand comprises thiosulfate. The thiosulfate may for example be sodium thiosulfate, ammonium thiosulfate, potassium thiosulfate or calcium thiosulfate.

[0117] Examples of compounds encompassed are provided in Table 1.Table 1. Exemplary Compounds of the ApplicationIII. Kits and Methods of the Application

[0118] The present application includes a kit comprisingan isotopic composition comprising multiple soft metal atoms of a single isotope of a soft metal; andan element tag comprising a compound of the present application, the compound comprising a plurality of chelating groups capable of binding at least one soft metal atom of the isotopic composition, and wherein the at least one soft metal atom is bound to a thiol-based solubility ligand;optionally wherein the kit does not comprise any radioactive soft metal selected from Pt, Re, Pd and Hg.

[0119] In some embodiments, the element tag is functionalized to bind a biomolecule and / or is attached to a biomolecule. In some embodiments, the biomolecule is an oligonucleotide or an antibody.

[0120] In some embodiments, the kit further comprises a second isotopic composition, wherein the second isotopic composition comprises multiple additional soft metal atoms of a second single isotope of a soft metal that is different from the single isotope of the soft metal of the isotopic composition.

[0121] The present application further includes a method for preparing an element tagged reagent comprising:providing an isotopic composition comprising multiple soft metal atoms of a single isotope of a soft metal selected from Re, Pt, Pd and Hg;providing an element tag comprising a compound of the present application, comprising a plurality of chelating groups wherein each chelating group is capable of binding at least one of the soft metal atom of the isotopic composition; binding the soft metal atoms of the isotopic composition to the one or more chelating groups of the element tag;binding the soft metal atoms bound to the one or more chelating groups of the element tag, to a thiol-based solubility ligand to provide the element tagged reagent; andwherein the soft metal atoms are non-radioactive.

[0122] In some embodiments, the isotopic composition does not comprise a natural mixture of isotopes.

[0123] In some embodiments, the method further comprises providing a second isotopic composition wherein the second isotopic composition comprises multiple second soft metal atoms of a second single isotope of a non-radioactive soft metal that is different from the single isotope of the non-radioactive soft metal of the isotopic composition.

[0124] Also included is a method for analyzing an analyte in a biological sample, comprising:(i) incubating an element tagged affinity reagent with the analyte, the element tagged affinity reagent comprising an affinity reagent tagged with an element tag, the element tag comprising a compound of any one of claims 1 to 36 multiple soft metal atoms of a single isotope of a soft metal selected from Pt, Re, Pd and Hg, and comprising chelating groups binding at least one of the soft metal atoms, wherein the soft metal atoms are bound to a thiol-based solubility ligand, the soft metal atoms are non-radioactive, and the affinity reagent specifically binds the analyte,(ii) separating unbound element tagged affinity reagent from bound element tagged affinity reagent; and(iii) analyzing the element tag bound to the affinity reagent attached to the analyte by mass spectrometric atomic spectroscopy.

[0125] In some embodiments, incubating the element tagged affinity reagent with the analyte comprises: incubating two or more differential element tagged affinity reagents with two or more analytes, wherein the element tagged affinity reagents specifically bind with the two or more analytes to produce two or more differentially tagged analytes, wherein analyzing the element tag bound to the affinity reagent comprises analyzing the differential element tags bound to the two or more analytes by mass spectrometric atomic spectroscopy. In some embodiments, the element tagged affinity reagent is configured to bind to an analyte in a biological sample, and the biological sample comprises cells.EXAMPLESExample 1Preparation of a polymer with a DPA derivative

[0126] H-dap DPA chelator A DPA derivative based upon 2,3-diaminopropanoic acid (dap) which contains 3 fewer CH2groups than lysine was synthesized.o oScheme 1. Synthesis of H-dap DPA chelator, i. 2-picolyl chloride hydrochloride, K2CO3, room temp., 2 h. i(b). KI, reflux, overnight, ii. 6 M HCl, ultrapure water, reflux, 2 h.

[0127] H-dap(Boc)-OMe HCI was reacted with 2-picolyl chloride in the presence of potassium iodide under reflux via a Finkelstein SN2 reaction (Scheme 1). The product H-dap-OMe DPA (1a) was purified via flash column chromatography. The resulting product was further hydrolyzed with 6 M HCI to remove both the -OMe ester and Boc, followed by neutralization with NaOH after hydrolysis. The crude product was washed with ultrapure water to yield the final H-dap DPA product (1b). The level of NSB for a lysine-based chelator (L-DPA) and the DPA derivative based upon 2,3-diaminopropanoic acid (dap) which contains 3 fewer CH2groups than lysine was assessed. The lysine-based chelator (L-DPA) was prepared as indicated in Example 2.

[0128] Preparation of dipicolylamine-pendant polymers. In order to compare the properties of metal chelating polymers with dap DPA pendant groups with those with L-DPA pendant groups, both polymers containing both pendant groups were prepared in parallel. Poly(pentafluorophenyl acrylate) (PPFPA, polymer 1c) by RAFT polymerization was synthesized.11’33Polymer 1c was characterized bynGPC= 8000 g mol-1and a relatively narrow dispersity (D = 1.3) based on GPC analysis in THF with PMMA standards. The degree of polymerization (DP) was determined via end-group analysis by1H NMR analysis (FIG. 1 ), and fluorine peaks were confirmed by19F NMR (FIG. 2).o o oScheme 2. Synthetic Scheme for the synthesis of L-DPA polymers (2, 4a, 6a) with lysine-based chelators and dap DPA polymers (3, 5a, 7a), iii. L-DPA or dap DPA in DMF. iv. mPEG6-amine, DMTMM, ultrapure water, room temp., 1 day; v. K2PtCl4in DMSO, 45 °C, 24 h.

[0129] The DPA metal-chelating polymers were synthesized as depicted in Scheme 2. Polymer 2 was prepared by reacting polymer 1c with L-DPA in DMF at room temperature overnight as described in Example 2. Polymer 3 was prepared using a similar protocol by reaction with H-dap DPA. The19F NMR spectra following both reactions showed the disappearance of the three fluorine peaks at -153.1 ppm, -156.7 ppm, and -162.2 ppm, characteristic of the PFPA pendant groups in polymer 3 confirming complete aminolysis reactions. The mean numbers of DPA per repeat were determined by1H NMR (FIG. 3) by comparing peak f (δ 6.0-8.8 ppm, 8.00 H of the two pyridyl groups) with peaks d,e (δ 3.0-4.5 ppm, 5 protons closest to the tertiary amine of the DPA pendant group), and peaks a,b,c (δ 0.5-2.8 ppm, 3 protons on the backbone, and -CH2neighboring the NH2).1H NMR for19F NMR spectra for polymer 2 (L-DPA pendants) were obtained. Both analyses showed near-quantitative conjugation of dap DPA and L-DPA to PPFPA.

[0130] Covalent attachment of mPEGe to the DPA pendant groups. In previous experiments, the polymer with L-DPA pendant groups (2) became insoluble when loaded with Re2+or Pt2+. To overcome this problem, mPEG6was attached as an amide to the -COOH group of the chelator. The polymer 4a in Scheme 2 maintained solubility aftermetal loading. Here, in parallel with the synthesis of 4a, mPEG6was attached to the dapDPA polymer 3. This polymer is denoted 5a in Scheme 2. These two polymers serve asreferences for comparing the effect of other polymer pendant groups on NSB after metalloading.

[0131] 1H NMR spectra were used to quantify the extent of PEGylation. Based on the 8 Hof the aromatic pyridyl groups (δ 6.9 - 8.5 ppm) as a reference, the relative integration ofthe grafted mPEG6polymer (δ 2.5 - 4.3 ppm) shows an integration of 35 for polymer 4aand 29 for polymer 5a. To give the best estimate of the number of mPEG6grafted to thepolymer, the1H NMR was compared before and after PEGylation to find the difference inintegration at δ 2.5 - 4.3 ppm. This difference provides the integration of only the newlygrafted mPEG6groups. The number of PEGs per pendant group was then estimatedusing equation 1. The PEGylation was quantitative for polymer 4a and ca. 0.88 mPEG6per DPA repeat unit for 5a. / Integration of PEG \ # of PEG per DPA = (1 )protons per DPA J

[0132] Loading the polymers with Pt2+. Pt2+was incorporated into polymers 4a and 5a by treating their solutions in DMSO with an excess of K2PtCl4 at 45 °C for 24 h as depicted in Scheme 2. The excess Pt salts were removed by washing with a combination of ultrapure water and 50 mM NaCI solution several times via spin filtration with Amicon Ultra-15 10 kDa MWCO (2700 x g, 15 min). The Pt metal content was determined by ICP-OES giving values of 18 Pt / polymer for the L-DPA polymer 6a and 19 Pt / polymer for the dap DPA polymer 7a. The1H NMR for polymer 6a can be found in FIG. 4.Example 2Materials

[0133] Sodium triacetoxyborohydride (STAB), hydrogen chloride solution 4 M in dioxane, pentafluorophenol, sodium hydroxide pellets, magnesium sulfate (MgSO4), sodium chloride (NaCI) 2-(chloromethyl)pyridine hydrochloride, potassium iodide (KI), aluminum oxide (activated, Brockmann I), concentrated hydrochloric acid (HCI), 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1 -propanol ester, 2,2'-Azobis(2-methylpropionitrile) (AIBN), trifluoroacetic acid (TFA), 1,3-propanesultone, Di-tert-butyldicarbonate, 3-(dimethylamino)-1 -propylamine, A / -(3-dimethylaminopropyl)- / \ / -ethylcarbodiimide hydrochloride (EDC), / V-hydroxysuccinimide (NHS), ethanolamine and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA monomer), 4,4'-azobis(4-cyanopentanoic acid), potassium tetrachloroplatinate(ll) (K2PtCl4), L-glutathione reduced (GSH), were purchased from Sigma Aldrich. 4-(4,6-Dimethoxy-s-triazin-2-yl)-4-methyl-morpholinium Chloride (DMTMM) was purchased from Toronto Research Chemicals. N-boc ethylenediamine was purchased from Alfa Aesar. mPEG6-NH2and nitric acid (67-70 %) optima grade were purchased from Fisher Scientific. mPEG24-amine were purchased from Broadpharm. 2-pyridinecarboxaldehyde was purchased from TCI. Ns-Boc-L-lysine and H-dap(Boc)-OMe HCI were purchased from Bachem. All chemicals listed were used as received without further purification. Ultrapure water (18.2 MO cm) was obtained using a Thermo Barnstead GenPure xCAD Ultrapure Water system.1d 1e Scheme 3. Synthesis of lysine DPA chelator

[0134] In a round-bottom flask (150 mL), Ns-Boc-L-lysine (2 g, 8.12 mmol, 1 equiv.) and STAB (5.2 g, 24.5 mmol, 3 equiv.) were added and suspended in dichloroethane (50 mL). The mixture was stirred and degassed with N2 gas. After 15 min, 2-pyridinecarboxaldehyde (1.85 g, 17.3 mmol, 2.1 equiv.) in dichloroethane (5 mL) was added and the reaction was stirred at room temperature for 6 h until a yellow solution was formed. Next, the reaction mixture was diluted with 100 mL dichloroethane and washed with water (75 mL x 4) in a separatory funnel (500 mL). The organic layer was recovered and dried with MgSO4. The sample was dried by rotary evaporation to afford 769 mg (22 %) of the product 1d as a yellow solid.1H NMR (500 MHz, CDCl3) 58.52 (m, 2 H), 5 7.63 (td, 2 H), 57.37-7.09 (m, 4 H), 54.71 (t, 1 H), 54.08 (d, 4 H), 53.46 (dd, 1 H), 53.11 (d, 2 H), 5 1.98 (dd, 2 H), 5 1.56-1.34 (m, 13 H).

[0135] In a vial (4 mL), Boc-L-DPA 1d (500 mg 1.17 mmol) was dissolved in dichloromethane (4.5 mL) and cooled to 0 °C. Next, 4 M HCI in dioxane (2 mL) was added to the Boc-L-DPA solution and immediately a beige precipitate was observed. The reaction mixture was continually stirred for 6 h at room temperature. After 6 h, the solution was decanted from the precipitates. The precipitate was re-dissolved in MeOH (1 mL), re-precipitated over diethyl ether (10 mL) and centrifuged at 2700 x g for 15 min to form a pellet. The supernatant was discarded, and the pellet was re-dissolved in MeOH (1 mL) and precipitated again in diethyl ether (10 mL). This process was repeated a total of 3 times. The pellet was then dried under high vacuum to afford Boc-L-DPA as a light-yellow solid. Prior to use, this product was neutralized and basified using the protocol below.

[0136] L-DPA was dissolved in ultrapure (1 mL) and the solution was made basic by dropwise addition of a 5 M NaOH solution until pH 13 was reached (measured by pH paper). The product was lyophilized overnight. After drying, the product was re-dissolved in dichloromethane (10 mL) and the solution was centrifuged at 2700 x g for 15 min topellet out the salts from the neutralization reaction. The pelleted salts were removed by decanting out the dichloromethane layer containing the product. The dichloromethane was dried by rotary evaporation to afford 362 mg (72 %) of the free-base L-DPA product 1e as a yellow solid.1H NMR (500 MHz, D2O) 58.71 (ddd, 2 H), 58.52 (td, 2 H), 58.08 (d, 2 H), 57.95 (ddd, 2 H), 54.45 (m, 4 H), 53.58 (dd, 1 H), 53.01 (t, 2 H), 5 1.93 (m, 2 H), 5 1.70 (tt, 2 H), 5 1.53 (m, 2 H).

[0137] The synthesis was adapted from a procedure in the literature.1In a round-bottom flask (100 mL), H-Dap(Boc)-OMe hydrochloride (500 mg, 1.86 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (30 mL) and degassed with N2(g) for 30 minutes while stirring with a magnetic stir bar. Next, 2-(chloromethyl)pyridine hydrochloride (671 mg, 4.1 mmol, 2.2 equiv.), K2CO3 (820 mg, 5.9 mmol, 3.2 equiv.) were successively added. After stirring for 2 h at room temperature, KI (679 mg, 4.1 mmol, 2.2 equiv.) was added and the reaction was refluxed at 85 °C overnight until a dark orange-red solution was formed. The sample was dried by rotary evaporation to remove acetonitrile and the dried solids were re-dissolved in dichloromethane. The dichloromethane was washed with water (3 x 100 mL) in a separatory funnel. The organic layer was collected and dried via rotary evaporation. The crude product was further purified by flash column chromatography with aluminum oxide (activated, Brockmann I) using a gradient elution starting from 100 % dichloromethane to 95:5 dichloromethane / ethyl acetate. The collected fractions were monitored by TLC in 95:5 dichloromethane / ethyl acetate and similar fractions were combined and dried in vacuo to afford 269 mg (36 %) of product 1a as a brown solid.1H NMR (500 MHz, CD2Cl2) 58.51 (d, 2 H), 57.56 (tt, 2 H), 57.28 (d, 2 H), 57.12 (dd, 2 H), 5 4.01 (d, 3 H), 53.81-3.15 (m, 7 H), 52.11-1.18 (m, 11 H).

[0138] In a round-bottom flask (100 mL), Boc-Dap-DPA ester 1a (187 mg, 0.46 mmol) was dissolved in 6 M solution of HCI (40 mL). The solution was refluxed overnight at 110 °C. After 24 h, the crude product was neutralized by dropwise addition of 5 M NaOH (pH monitored with pH paper) and the basified product was dried via rotary evaporation. The crude product was re-dissolved over dichloromethane and centrifuged at 2700 x g for 15 min to pellet out the salts from the neutralization. The dichloromethane layer containing the desired product was decanted out from the salt pellet and the dichloromethane was dried in vacuo to afford 131 mg (100 %) of Dap-DPA product 1b as a dark brown solid.1HNMR (500 MHz, D2O) 58.12 (d, 2 H), 57.44 (td, 2 H), 57.11 (d, 2 H), 57.01 (m, 2 H), 5 3.91-3.70 (m, 4 H), 53.27 (ddd, 1 H), 52.94-2.78 (m, 2 H).1,4-Dioxane Anhydrous DMF 0 °C, 2h R. L, 3 days R.t., 18 h- / H - \ / °3S^^N^^N-Boc - O3SX^XXNX / XXNH2+DCM1g 4 M HCI in Dioxane Scheme 4. Preparation of zwitterionic molecule 3-((3-aminopropyl)-dimethyl- ammonio)propane-1 -sulfonate (ZW-NH2)

[0139] ZW-NH2 was prepared following a previously reported synthetic procedure.2In a round-bottom flask (100 mL), di-tert-butyldicarbonate (15.6 g, 71.5 mmol) was added to a solution of 3-(dimethylamino)-1 -propylamine (4.9 g, 6 mL, 47.7 mmol) in 1,4-dioxane (50 mL). The solution was stirred at 0 °C for 2 h and further stirred overnight at room temperature for 18 h. After 18 h, the solvent was removed by rotary evaporation and Ultrapure™ (50 mL) was added to the crude product. The product was extracted with ethyl acetate (30 mL) three times. The ethyl acetate was dried to afford 6 g (62 %) of a light-yellow oil as Boc-protected 3-(dimethylamino)-1 -propylamine 1f.

[0140] In a round-bottom flask (50 mL), Boc-protected 3-(dimethylamino)-1 -propylamine 1f (2.5 g, 0.01235 mol, 1 equiv.) was dissolved in anhydrous DMF (15 mL). Next, 1,3-propanesultone (2.113 g, 0.0173 mol, 1.4 equiv.) was added to the Boc-protected 3-(dimethylamino)-l -propylamine solution and stirred for 3 days at room temperature. The crude product was dried under high vacuum to remove DMF until a slightly yellow viscous oil remained. The viscous oil was washed with diethyl ether (30 mL), followed by another wash of ethyl acetate (30 mL) to remove any unreacted 1,3-propanesultone. The oil was freeze-dried to afford 5 g (120 %) of a highly hygroscopic sticky white solid 1g.

[0141] In a round-bottom flask (100 mL), Boc-protected ZW-NH2 1g (4 g) was dissolved in dichloromethane (50 mL), and cooled to 0 °C, resulting in a slightly turbid slightly white-colored solution. After cooling to 0 °C, 4 M HCI in 1,4-dioxane (5 mL) was added to the solution and left to stir for 1 h. After 1 h, the solution became clear, and a solid white chunk precipitate had formed. The solvent was removed by rotary evaporation to dryness and purified by successive (3') precipitation from dichloromethane with isopropanol / methanol (10:5:1 v / v). The precipitated gooey white chunks were lyophilized to afford 2.47 g (90 %) of the product 1h.1H NMR (500 MHz, D2O) 53.62-3.43 (m, 4 H), 5 3.23-3.10 (m, 8 H), 53.07-2.96 (t, 2 H), 52.33-2.17 (dddd, 4 H).Scheme 5. Synthesis of Poly(SBMA)-NH2 via RAFT polymerization

[0142] In an vial (8 mL), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (270 mg, 0.97 mmol, 1 equiv.), EDC (285 mg, 1.83 mmol, 2 equiv.), and NHS (211 mg, 1.83 mmol, 2 equiv.) were mixed together, dissolved in anhydrous acetonitrile (4 mL) and vortexed for15 min. Next, N-Boc-ethylenediamine (300 mg, 1.87 mmol, 2 equiv.) was added to the 8 mL vial containing the RAFT agent mixture and the reaction mixture was stirred overnight at room temperature. The reaction was monitored using thin-layer chromatography (solvent 1:1 dichloromethane / ethyl acetate). After 24 h, the reaction was dried by rotary evaporation and purified via silica gel column chromatography using a gradient elution starting from 100% dichloromethane to 1:1 dichloromethane / ethyl acetate. The fractions were monitored by thin-layer chromatography and similar fractions were combined and concentrated by rotary evaporation affording 80 mg (20 %) of a red-pink gooey solid as the product 1i.1H NMR (500 MHz, CDCl3) 57.90 (dd, 2 H), 57.56 (m, 1 H), 57.39 (m, 2 H), 56.46 (br s, 1 H), 54.94 (br s, 1 H), 53.41- 3.22 (m, 4 H), 52.52 (m, 4 H), 5 1.44 (s, 9 H).

[0143] In a round-bottom flask (5 mL), N-Boc-ethylenediamine modified 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid RAFT agent 1i (45 mg, 0.11 mmol, 1 equiv.), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide monomer (629 mg, 2.25 mmol, 21 equiv.), 4,4'-azobis(4-cyanopentanoic acid) initiator (3.04 mg, 0.011 mmol, 0.1 equiv.) were dissolved in 2,2,2-trifluoroethanol (4 mL). The round-bottom flask was fitted with a rubber septum and the solution was degassed vigorously with N2(g) gas for 30 minutes. The reaction was then stirred at 70 °C for 7 h. Next, the reaction was exposed to air and TFA (1.7 mL) was added to the polymer mixture and stirred overnight at room temperature overnight for 18 h. Next, the polymer mixture was precipitated over 7:3 methanol / diethyl ether (10 mL) and centrifuged at 2700 x g for 15 min to afford a pink pellet. The pellet was re-dissolved once more in 2,2,2-trifluoroethanol (1 mL), vortexed until fully dissolved and re-precipitated over 7:3 MeOH / diethyl ether (10 mL). The pellet was collected and dried under high vacuum to obtain 416 mg of the polymer as a pink powder.

[0144] The degree of polymerization (DP) of the polymer was determined via end-group analysis using1H NMR (FIG. 6). The spectrum was normalized to 5.00 protons of the benzene of the dithiobenzoate end group as peak d (δ 7.4-8.3 ppm). Peak c is assigned to the O-CH2 of the pendant sulfobetaine repeat units. The integration of peak d (5H) of the benzene of the dithiobenzoate end group was compared to that of 58 for peak c at 54.3-4.6 ppm (2 H assigned to O-CH2). In this way, the polymer was determined to have a DPn = 29.

[0145] The Boc was removed by hydrolysis with 7:3 trifluoroethanol / trifluoroacetic acid overnight at room temperature for 18 h. The PSBMA polymer was purified by successive (3') precipitation from 7:3 trifluorothanol / trifluoroacetic acid with 1:1 methanol / diethyl ether. The collected pellet was dried by high vacuum.

[0146] The polymer was re-dissolved in ultrapure water (2 mL) and ethanolamine (2 mL) was added and stirred for 3 hours and to aminolyze the RAFT end group. The resulting polymer 1j was purified by spin filtration with an Amicon™ Ultra-15, 10 kDa MWCO spin filter (7 times, 2700 x g for 15 min) until the filtrate was clear. The retentate (~2 mL water) was subsequently reacted with TCEP solution (0.5 M, 1.4 mL, ~14 equiv. relative to the polymer) was added and stirred at room temperature for 10 min. Maleimide (150 mg, 1.54 mmol, 30 equiv. per polymer) was added to the polymer solution and the reaction was stirred at room temperature for 2 h. The reaction was washed using an Amicon Ultra-4, 10 kDa MWCO spin filter (5 times, 2700 x g for 15 min) with ultrapure water. The retentate was lyophilized to obtain 324 mg of the polymer 1k as a pink powder. We refer to this polymer as SBMA29-NH2 or PSBMA29. MnSEC= 5000 Da, D = 1.1.Scheme 6. Synthesis of DPA polymers

[0147] PPFPA was prepared using a previously reported synthetic procedure.3In a roundbottom flask (5 mL), 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester RAFT agent (32.1 mg, 0.072 mmol, 1 equiv.), AIBN (1.10 mg, 0.0067 mmol, 0.1 equiv.), and PFPA monomer (500 mg, 2.1 mmol, 29 equiv.) were dissolved in 1,4-dioxane (1.8 mL) and vigorously degassed with N2(g) for 30 min. After 30 min, the reaction was stirred at 70 °C. After 7 h, the reaction was stopped by exposing it to air and precipitating the polymerization solution in cold hexane. The polymer was pelleted out via centrifugation at 2700 x g for 5 min. The polymer was re-dissolved in chloroform and reprecipitated over cold hexane followed by pellet via centrifugation. This process was repeated a total of 3 times. The polymer was re-dissolved in chloroform and dried underhigh vacuum to afford 297 mg of a yellow solid as PPFPA polymer 1c.nGPC= 8000 g mol-1, D = 1.3.

[0148] The degree of polymerization (DP) of the polymer was determined via end-group analysis using1H NMR (FIG. 1). The spectrum was normalized to 3.00 protons of the terminal CH3 of the dodecyltrithiocarbonate end group as peak a (50.9 ppm). Peak b is assigned to the 20H of the structure’s (CH2)w alkyl chain. -The integration of peak a (3H) of the terminal -CH3 of the dodecyltrithiocarbonate end group was compared to that of 22 for peak g at 5 3.1 ppm (1 H assigned to the 1 proton in the polymer backbone). In this way, the polymer was determined to have a DPn= 22.

[0149] In a vial (4 mL) PPFPA(DP=22, M=5200 Da, 78.7 mg, 0.015 mmol) and L-DPA 1e (212 mg, 0.61 mmol, 2.1 equiv. per PFP pendant group) were dissolved in DMF (2 mL) and stirred overnight at room temperature. After 18 h, ethanolamine (200 pL) was added and stirred for 3 h. The reaction mixture was precipitated over diethyl ether (10 mL) and then centrifuged to a brown pellet at 2700 x g for 15 min. The pellet was then air dried to remove diethyl ether and then re-dissolved in ultrapure and washed using an Am icon Ultra-4, 10 kDa MWCO spin filter (5 times, 2700 x g for 15 min) with ultrapure water until the filtrate was clear. The retentate was lyophilized overnight to afford 62 mg of a light yellow-beige solid.

[0150] The polymer was re-dissolved in 1 mL ultrapure water and TCEP solution (0.5 M, 200 pL, ~14 equiv. relative to the polymer) was added and stirred at room temperature for 10 min. Maleimide (22.6 mg, 0.23 mmol, 30 equiv. per polymer) was added to the polymer solution and the reaction was stirred at room temperature for 2 h. The reaction was washed using an Amicon Ultra-4, 3 kDa MWCO spin filter (5 times, 2700 x g for 30 min) with ultrapure water. The retentate was lyophilized overnight to afford 62 mg of a light yellow-beige solid as P(L-DPA) polymer 2. MnSEC= 4000 g mol’1, D = 1.1.

[0151] PPFPA (DP=22, M=5200 Da, 12 mg, 0.002 mmol) and Dap-DPA 1 b (26 mg, 0.087 mmol, 2 equiv. per PFP pendant group) were dissolved in DMF (0.75 mL) in a 4 mL vial and was stirred overnight at room temperature. After 18 h, ethanolamine (200 pL) was added and stirred for 3 h. The reaction mixture was precipitated over diethyl ether (10 mL) and then centrifuged to a brown pellet at 2700 x g for 15 min. The pellet was then air dried to remove diethyl ether and then re-dissolved in water and washed using AmiconUltra-4, 10k MWCO spin filters (5 times, 2700 x g for 15 min) over ultrapure until the filtrate was clear. The retentate was lyophilized overnight to afford 11 mg of a dark yellow solid

[0152] The polymer was re-dissolved in 1 mL ultrapure water and TCEP solution (0.5 M, 200 pL, ~14 equiv. relative to the polymer) was added and stirred at room temperature for 10 min. Maleimide (22.6 mg, 0.23 mmol, 30 equiv. per polymer) was added to the polymer solution and the reaction was stirred at room temperature for 2 h. The reaction was washed using an Amicon Ultra-4, 3 kDa MWCO spin filter (5 times, 2700 x g for 30 min) with ultrapure water. The retentate was lyophilized overnight to afford 10 mg of a yellow solid as P(dap-DPA) polymer 3. MnSEC= 5000 g mol’1, D = 1.25.6Scheme 7. Grafting of methoxy (polyethylene glycol)s (mPEGs) and zwitterions to P(DPA)

[0153] In a vial (4 mL), P(DPA) 2 (DP = 22, 50 mg) was dissolved in ultrapure (400 pL). Amide coupling agent, DMTMM (182 mg, 0.66 mmol, 5 equiv. per pendant group) in ultrapure (600 pL) was added to the P(DPA) solution and pre-reacted for 10 min. After 10 min of stirring at room temperature, water solubilizing agent (0.118 mmol, 3 equiv. per pendant group) was added to the vial containing the polymer solution. After 1 day, the reaction was washed via spin filtration over Amicon Ultra-15, 10 kDa MWCO spin filters (6 times, 2700 x g for 15 min) using ultrapure. The retentate was lyophilized overnight to afford the polymers 4a to 4c and 5a. MnSEC= 4500 g mol’1, D = 1.15. for 4a, MnSEC=6000 g mol-1, D = 1.2. for 4b. MnSEC= 15000 g mol’1, D = 1.3. for 4c. MnSEC= 6500 g mol-1, D = 1.5 for 5a.

[0154] In a vial (4 mL), P(DPA) 2 (DP = 22, 5 mg) was dissolved in ultrapure (400 pL). Amide coupling agent, DMTMM (17.65 mg, 0.064 mmol, 5 equiv. per pendant group) in ultrapure (200 pL) was added to the P(DPA) 2 solution and pre-reacted for 10 min. After 10 min of stirring at room temperature, ZW-NH2 solution 1g (14.3 mg, 0.064 mmol, 5 equiv. per pendant group, in 250 pL ultrapure water) was added to the vial containing the polymer solution. The polymer mixture was adjusted to pH 9 with 0.5 M NaOH. After 1 day, the reaction was washed via spin filtration overAmicon Ultra-15, 10 kDa MWCO spin filters (6 times, 2700 x g for 15 min) using ultrapure. The retentate was lyophilized overnight to afford the polymers 4d. MnSEC= 4500 g mol’1, D = 1.27. for 4d.

[0155] In a vial (4 mL), P(DPA) 2 (DP = 22, 3.2 mg) was dissolved in ultrapure (200 pL). Amide coupling agent, DMTMM (11.56 mg, 0.042 mmol, 5 equiv. per pendant group) in ultrapure (200 pL) was added to the P(DPA) 2 solution and pre-reacted for 10 min. After 10 min of stirring at room temperature, PSBMA29-NH21k (100 mg, 0.017 mmol, 2 equiv. per pendant group, in 250 pL ultrapure water) was added to the vial containing the polymer solution. The polymer mixture was adjusted to pH 9 with 0.5 M NaOH. After 1 day, the reaction was washed via spin filtration overAmicon Ultra-15, 10 kDa MWCO spin filters (6 times, 2700 x g for 15 min) using ultrapure. The retentate was lyophilized overnight to afford the polymers 4e. MnSEC= 7000 g mol’1, D = 1.14.Scheme 8. General Procedure for the Pt loading of polymer with K2PtCl4

[0156] For polymers 4a-c, 5a-c. In a vial (8 mL), polymers 4a to 4c or 5a to 5c (10 mg) were dissolved in DMSO (200 pL). A solution of K2PtCl4 (400 pL, 50 mM in DMSO) was added to the polymer solution and the mixture was heated at 45 °C for 24 h. After 24 h, the reaction mixture (200 pL aliquot) was added to a Amicon Ultra-15, 10 kDa MWCO spin filter pre-filled with 15 mL of ultrapure water to avoid damaging the regenerated cellulose ester membrane of the filter and then washed by centrifugation (2700 x g, 15 min). The process was repeated 3 times, until all 600 pL of the crude reaction mixture was transferred to the spin filter. The crude polymer solution was further washed with 50 mM NaCI solution (3 times, 2700 x g, 15 min), and finally another 3 times with ultrapure (3 times, 2700 x g, 15 min). The retentate was lyophilized to afford polymers 6a-c and 7a.

[0157] For polymer 4d and 5d. A similar protocol to the method for polymers 6a to 6c was used, however a mixed solvent system using TFE / DMSO was required due to the solubility of the zwitterionic modified polymers in DMSO. In a vial (8 mL) polymer 4d and 5d were dissolved in DMSO (0.2 mL) and TFE (0.6 mL). A solution of K2PtCl4 (400 pL, 50 mM in DMSO) was added to the polymer solution and the mixture was heated at 45 °C for 24 h. After 24 h, the reaction mixture (200 pL aliquot) was added to a Amicon Ultra-15, 10 kDa MWCO spin filter pre-filled with 15 mL of ultrapure water to avoid damaging theregenerated cellulose ester membrane of the filter and then washed by centrifugation (2700 x g, 15 min). The process was repeated 3 times, until all 600 pL of the crude reaction mixture was transferred to the spin filter. The crude polymer solution was further washed with 50 mM NaCI solution (3 times, 2700 x g, 15 min), and finally another 3 times with Ultrapure (3 times, 2700 x g, 15 min). The retentate was lyophilized to afford polymer 6d and 7d.

[0158] For polymer 4e and 5e. A similar protocol to the method for polymer 4d and 5d were used, however a greater ratio of TFE / DMSO was required due to the solubility of the zwitterionic modified polymers in DMSO. In a vial (8 mL) polymer 4e or 5e were dissolved in DMSO (0.2 mL) and TFE (1.2 mL). A solution of K2PtCl4 (400 pL, 50 mM in DMSO) was added to the polymer solution and the mixture was heated at 45 °C for 24 h. After 24 h, the reaction mixture (200 pL aliquot) was added to an Amicon Ultra-15, 10 kDa MWCO spin filter pre-filled with 15 mL of ultrapure water to avoid damaging the regenerated cellulose ester membrane of the filter and then washed by centrifugation (2700 x g, 15 min). The process was repeated 3 times, until all 600 pL of the crude reaction mixture was transferred to the spin filter. The crude polymer solution was further washed with 50 mM NaCI solution (3 times, 2700 x g, 15 min), and finally another 3 times with ultrapure (3 times, 2700 x g, 15 min). The retentate was lyophilized to afford polymers 6e and 7e.Ligand exchange with glutathione (GSH)

[0159] In a vial (4 mL), polymers 6a-e and 7a (5 mg) were dissolved in ultrapure water (300 pL). GSH (1 or 5 equiv. based upon the DPA content of the polymer) was added to polymer solution and the mixture was stirred at room temperature for 2 h. After 2 h, the reaction mixture was purified by washing with ultrapure water using an Amicon Ultra-410 kDa MWCO spin filter (5 times, 2700 x g, 15 min). The retentate was lyophilized to afford polymers 8a-e and 9a, respectively.Example 3Non-specific binding assays

[0160] To compare the level of NSB of the dap polymer 7a with that of the -L-DPA polymer 6a, suspensions of human peripheral blood mononuclear cells (PBMCs) were treated witha cocktail containing the Pt-polymers at a titer of 10 pg / mL plus Maxpar reagents. The Maxpar reagents consisted of 10 labeled antibodies (see Table 2) designed for a 10-plex assay to identify the B-cells, T-cells, and non-B, T cells in the sample. The data were processed via a gating strategy to look at the entire ensemble of PBMC (CD66-; CD45+) cells.Materials

[0161] Pt-polymers prepared as described in Example 1. Maxpar reagents were provided by Standard BioTools Canada. For measurements of non-specific binding, a cocktail containing ten different Ab conjugates labelled with different metal isotopes at optimum concentrations were mixed with the various Pt polymers at a polymer concentration of 10 pg / mL. The Abs, clones and metal labels are listed in Table 2. An experiment run with the Ab cocktail without Pt polymer was run as a “Maxpar control.”Table 2. Maxpar Antibodies and their metal isotopes that made up the 10-plex assay Antibody Clone MetalCD3 UCHT1170ErCD14 RMO52160GdCD33 WM53158GdCD4 RPA-T4145NdCD66b 80H3152SmCD16 3G8148NdCD56 NCAM16.2163DyCD45 HI3089YCD20 2H7147Sm162CD8a RPA-T8Dy

[0162] These conjugates include CD3 labeled with170Er, CD14 labeled with160Gd, CD33 labeled with158Gd, CD4 labeled with145Nd, CD66b labeled with152Sm, CD16 labeled with148Nd, CD56 labeled with163Dy, CD45 labeled with89Y, CD20 labeled with147Sm, and CD8a labeled with162Dy.Cell staining experiments with PBMCs

[0163] PBMCs and CTL anti-aggregate wash solution were thawed in a 37 °C water bath. A buffer solution containing CTL media (500 pL) was added to Gibco RPMI 1640 cell culture media (9.5 mL). PBMCs (ca.10 million cells / mL, each sample vial is calculated to achieve roughly 3 million samples per individual staining experiment) were added to thebuffer solution, mixed well, and centrifuged (8 min, 453 x g). The supernatant was carefully removed with a micropipette and the pellet containing the PBMCs was resuspended in 10 mL RPMI containing FBS (10 %). An aliquot (10 pL) was mixed with 10 pL trypan blue and analyzed with a hemocytometer to count the cells in the solution.20 pL Rh-intercalator (500 pM) was added to the PBMC solution (10 mL in RPMI with FBS) and was incubated at room temperature for 15 minutes. The PBMC solution was centrifuged (5 min, 400 x g) and the supernatant was removed. The PBMC pellet was washed with 10 mL Maxpar PBS and centrifuged again (5 min, 400 x g). The supernatant was removed and the cell pellet was resuspended in desired volume of CSB to achieve ca. 3 million cells per and the mixture was incubated at room temperature for 10 min.

[0164] PBMC solution (50 pL) was added to a Maxpar cocktail (50 pL) in a centrifuge tube (2 mL) and vortexed gently for 30 minutes at room temperature. The cells were washed with CSB media (1 mL) and centrifuged (5 min, 400 x g). The supernatant was removed and the cell pellet was racked three times to loosen and disperse the cell pellets CSB media was added to the dispersed cell pellet and the washing procedure was repeated.

[0165] PBMCs were fixed by addition of formaldehyde in PBS (1.6 %, 1 mL) into each tube and incubated for 10 minutes at room temperature. The cells were centrifuged (5 min, 800 x g) and the supernatant was removed. Ir DNA intercalator (500 pM, 2.5 pL) was added to 10 mL fix & perm cell fixation and permeabilization buffer to afford a final concentration of 125 nM. The Ir DNA intercalator buffer solution (1 mL) was added to each tube containing PBMCs and was vortexed for 2 seconds and incubated overnight at 4 °C in the fridge. CSB media (1 mL) was added to each sample tube and centrifuged (5 min, 800 x g). The supernatant was removed and the pellet was washed again by repeating the procedure with CSB media (2 mL). The samples were then further washed with Maxpar cell acquisition solution (2 mL), gently vortexed and centrifuged down (5 min, 800 x g). The supernatant was discarded, and the cell pellet was washed once again with Maxpar (2 mL). EQ4 beads (1X, 1.5 mL CAS + beads solution) were added to each sample tube. The cell concentration was adjusted to 1 x 106cells / mL and then run on the Helios instrument for data acquisition.Non-specific binding (NSB) studies

[0166] These results are presented in FIG. 5. It can be seen in FIG. 5C that the dap DPA polymer 7a exhibits a higher mean (and median)195Pt intensity value (232, 55 counts) than the L-DPA polymer 4a (80, 9.8 counts). In these measurements, the mean signal is always higher than the median signal. Both polymers show high levels of NSB compared to the Maxpar control. The control represents the signal (1 count) monitored in the195Pt channel, gated for all (CD66-; CD45+) cells, for the 10 antibodies labeled with metals other than Pt. This tiny signal is representative of background noise and represents the level of signal one should obtain if there were no NSB with the Pt-labeled polymers.

[0167] It can be noted that the degree of PEGylation was slightly lower in polymer 7a (0.88 mPEGe per DPA repeat unit), but this difference cannot explain the large discrepancy in the NSB. The dap DPA polymer is significantly harder to purify than the L-DPA polymer, as can be seen with the new impurities observed in the1H NMR spectrum after hydrolysis of the Boc-protecting group.PEG length and other pendant group modifications

[0168] The consequences of replacing the mPEGe pendant group in polymer 4a with longer mPEG chains were tested. These reactions are presented in Scheme 9.

[0169] Polymers 4b and 4c were prepared by modifying polymer 2 with mPEG-12- and mPEG24- amine, respectively, using the same protocol used to attach mPEGe. It was found by1H NMR that the extent of PEGylation for mPEG-12 was quantitative to obtain polymer 4b (FIG. 7). The grafting of the longer mPEG24 posed a greater challenge due to the increased steric effects, a common problem for ‘graft-to’ approaches to modify polymers.3435PEGylation efficiencies of 80-96 % were achieved. The Pt polymers 6b and 6c (1H NMR, FIG. 8, FIG. 9, respectively) were then prepared by metalation of polymers 4b and 4c with K2PtCl4 in DMSO.Scheme 9. Synthetic scheme for the metalation of polymers with Pt and the treatment of the Pt-polymers with glutathione, xi. l<2PtCl4, DMSO, 45 °C, 24 h. xii. Glutathione, ultrapure water, room temp., 2 h.

[0170] Zwitterionic groups have been investigated as a means of reducing NSB and these results were compared to the PEG systems.36-38To test the effect of zwitterionic pendant groups on NSB, a zwitterion small molecule ZW-NH2 ((d) was synthesized as shown in Scheme 9) using the protocol from Wang et al.39It was then coupled to the -COOH groups of the DPA chelators in polymer 2 employing a DMTMM-activated amidation reaction to obtain polymer 4d. The characterization of polymer 4d by1H NMR is described in Example 2.

[0171] In parallel, RAFT polymerization was used to prepare the zwitterionic polymer, poly(sulfobetaine methacrylate) (PSBMA, DP=29, (e) in Scheme 9,1H NMR, FIG. 10) with an NH2-group on the initiating end, prepared with a Boc-protected NH2-dithiobenzoate RAFT agent (synthesis and polymer characterization details are in Example 2. It was then coupled to polymer 2 to form polymer 4e (1H NMR, FIG. 11). Pt-polymers 6d and 6e (1H NMR, FIG. 12, FIG. 13, respectively) were prepared by metalating the zwitterion-grafted polymers 4d and 4e. Since the zwitterion-grafted polymers were insoluble in DMSO, a 1:1 (v / v) mixture of trifluoroethanol and DMSO was employed for the metalation reaction. The number of Pt ions determined by ICP-OES perpolymer was ca. 19 for both polymers. The Pt content of all the metalated polymers examined here are collected in Table 3.Table 3. Pt metal content of polymers determined by ICP-OES expressed in mg Pt per mg of polymer and number of Pt per polymer.Sample Metal per polymer Metal content by(mg metal / mg ICP-OESpolymer) (# of Pt / polymer of DP=22)Polymer 6a 0.173 18 / 22Polymer 6b 0.143 19 / 22Polymer 6c 0.099 22 / 22Polymer 6d 0.201 19 / 22Polymer 6e 0.072 19 / 22Polymer 7a 0.192 18 / 22Polymer 8a 0.117 14 / 22Polymer 8c 0.096 21 / 22Polymer 8d 0.139 16 / 22Polymer 9a 0.141 16 / 22

[0172] For completeness, similar synthesis transformations were also carried out on the dap polymer 5b to 5e to obtain polymer 7b to 7e.

[0173] Pt polymers 6b to 6e were then examined for NSB, using the same staining and gating strategy as described above. It can be seen in FIG. 14A, B that the median signal intensity was 2.9 counts for L-DPA-mPEGi2 and 2.2 counts for L-DPA-mPEG24, respectively. The corresponding signal for L-DPA-mPEGe in this sample batch was 5.7 counts. From these results, a general trend can be seen in which the longer grafted mPEG chains were more effective at lowering the NSB.

[0174] It can be seen in FIG. 14C that the ZW-NH2 groups increased the extent of NSB, with a median195Pt signal of 1900 counts. In striking contrast, polymer 6e showed195Pt signal of 1.8 counts, almost completely suppressing NSB. It can be concluded that PSBMA29 is as effective as mPEG24 in reducing NSB.Ligand exchange with a water solubilizing thiol-containing group

[0175] Pt2+incorporation into the polymer introduces two other aspects that can contribute to NSB. It renders the repeat unit (+) charged, and this can promote binding to negatively charged cell surfaces. In addition, the Pt-CI bond is labile, opening the possibility forinteraction with thiol groups associated with proteins in solution or on the cell surface. Pt polymers were treated with glutathione (GSH) to assess its effects on NSB.

[0176] The Pt-polymer 6a was treated with glutathione and the time-evolution of the reaction was followed by1H NMR (FIG. 15). There are two notable changes observed in the1H NMR spectrum. There is a chemical shift change in the aromatic region from 57.3-8.9 ppm to 5 7.4-9.3 ppm. This change occurred within the first 15 minutes of reacting and no observable changes were observed even after 24 hours, indicating that the reaction between the Pt and glutathione is very rapid. The second change is the new peaks observed at 5 1.7-2.5 ppm which correspond to glutathione bound to the polymer which are quite broad. These peaks closely resemble the glutathione peaks from 5 2-3 ppm. The broadness of these peaks is another indication that the glutathione is bound to the polymer. Similar experiments with glycine, cysteine and histidine have shown that the -SH group of cysteine and glutathione binds to the Pt metal, whereas glycine and histidine having no thiols show no reaction by1H NMR.

[0177] To further confirm the binding of glutathione (GSH) to the polymer, mPEG24-grafted polymer 6c was reacted with GSH and DOSY NMR was employed to analyze the diffusion of the polymers. The self-diffusion coefficient measured by DOSY NMR is sensitive to the size of the diffusing molecules.41’42To better discern between polymer-bound GSH and the unbound small molecule GSH, mPEG24-modified polymer 6c was used due to its larger size which would allow a greater difference to be seen in the diffusion coefficients.DOSY NMR and mass cytometry experiments

[0178] DOSY experiments were first ran on GSH and the diffusion coefficient was determined to be D = 4.6x1 O’10m2 / s for the peak at 5 2.95 ppm. The DOSY NMR measurement was then ran on the Pt-polymer 6c after reacting it with GSH and purifying by spin filtration. The diffusion coefficient was determined to be D = 6.9x1 O’11m2 / s for the broad peak from 52.3 to 2.5 ppm, which corresponds to GSH bound to the polymer. The decay curve profile for both free-GSH and polymer bound-GSH plotted from the DOSY data is shown in FIG. 16. Comparison of the diffusion coefficients between the two samples demonstrates an order of magnitude difference between the free-GSH and bound-GSH. This result further confirms the displacement of Pt-CI with the thiol of GSH.

[0179] ICP-OES was then ran to check the metal content of the polymer after the reaction with GSH to determine if exposure to excess GSH led to loss of any Pt metal due to the transchelation effect from a competing ligand. The metal content decreased slightly after treatment with 5 equivalents of GSH for 2 hours from 22 Pt per polymer to about 19 Pt per polymer.

[0180] A suspension of PBMC cells were treated at a titer of 10 pg / mL and results are presented for CD45+ B cells. Prior to treatment with GSH the polymer samples show low to high NSB levels (FIG. 18A-E), depending on the pendant stealth group attached to the DPA.

[0181] A batch of polymer 8a was prepared by incubating polymer 6a with glutathione for 2 hours at room temperature in ultrapure water. GSH treated polymer 8a (mPEGe) shows a195Pt median count value of 0.6 (FIG. 17F), compared with polymer 6a without GSH treatment, that showed a195Pt median count of 9.8 (FIG. 17A). To further validate the effect of GSH on reducing NSB, polymers 6c, 6d, 6e and 7a were further treated, all of which previously showed high levels of NSB, with GSH to afford polymer 8c, 8d, 8e and 9a, respectively. Polymer 6c (mPEG24) showed a195Pt median count of 2.3 (FIG. 17B), and after GSH treatment, polymer 8c shows a value of 1.7 (FIG. 17G), indicating a slight decrease in the observed NSB.Polymer 6d (ZW-NH2) originally showed the highest NSB with a195Pt median count of 1900 (FIG. 17C), but after treatment with GSH, polymer 8d showed a195Pt median count of 0.5 (rounded to 1 in FIG. 17H). Polymer 6e (PSBMA29) showed a low195Pt median count of 1.8 (FIG. 17D), which was reduced even further after GSH treatment (polymer 8e, 0.5 counts, FIG. 171). The GSH treatment was so effective that its effect was also examined on the dap polymer 7a (mPEGe) that showed a195Pt median count of 55 (FIG.17E). Treatment with GSH formed polymer 9a, in which the195Pt median count decreased significantly to 0.7 (rounded to 1 in FIG. 17J). A comprehensive Table containing the mean195Pt counts on all PBMCs, B cells, and T cells and median195Pt counts for all PBMCs can be found in Table 4.Table 4. Tabulated values of the mean195Pt counts of Pt-polymers for all PBMCs (CD45+), B cells (CD20+), All T cells (CD3+), CD8a T cells (CD3+; CD8+), CD4a T cells (CD3+; CD4+), and median195Pt counts for all PBMCs (CD45+).Sample Name Mean195Pt Signal Median195pf SignalAll B Cells All T CD8a T CD4 T AllPBMCs Cells Cells Cells PBMCs Polymer 6aa12.6 6.56 15.1 16.9 13.6 5.70 Polymer 6bb3.42 3.14 3.37 3.34 3.38 2.87 Polymer 6cc2.56 2.38 2.49 2.42 2.51 2.17 Polymer 8aa d1.92 1.86 1.89 1.81 1.93 1.57 Polymer 8ccd2.72 2.66 2.00 1.94 2.03 1.68 Maxpar Control 2.22 2.22 2.18 2.11 2.21 1.83a. Pendant mPEGe; b. mPEGn; c. mPEG24; d. GSH

[0182] Without wishing to be bound by theory, the reaction of the Pt-labeled polymer with GSH may make three contributions toward reducing NSB. First, GSH may increase the water-solubility of the polymer. For example, while polymer 2 is water soluble, the Pt complex of polymer 2 is insoluble in water. This polymer regains water solubility after reacting the Pt-DPA groups with GSH. Second, it may prevent the Pt-CI from interacting with thiols from cysteine residues on the cell surface. Third, and possibly most important, it may reduce the cationic nature of the Pt-DPA metal complex.

[0183] One hint that GSH reduces the cationic charge of the polymer comes from aqueous size exclusion chromatography (SEC) measurements. The columns on the instrument are effective at analyzing neutral polymers and anionic polyelectrolytes, but positively charged water-soluble polymers do not elute due to adsorption onto the stationary phase.43Prior to GSH treatment, Pt polymers such as 6a do not elute from the SEC columns (no Rl signal is observed). After treatment with GSH, polymer 8a can be run through the SEC with an observable Rl trace.

[0184] More direct evidence was obtained by electrophoretic mobility measurements. These results are presented in FIG. 18. Polymer 2 (metal free) shows an electrophoretic mobility of ca. -1 pm.cmA / .s, which is close to neutral. After PEGylation with mPEGe,polymer 4a shows a value of ca. -4 pm.cm / V.s, indicating that this polymer is negatively charged. Upon metalation of this polymer to form polymer 6a, the electrophoretic mobility increases substantially to +3 pm.cm / V.s, indicating that the Pt-polymer is positively charged. After treatment with GSH, polymer 8a, shows an electrophoretic mobility of ca.0.5 indicating the polymer has become neutral. Electrophoretic mobility plots for mPEG24-grafted polymers, and ZW-NH2-grafted polymers are shown in FIG. 18C and FIG. 18D respectively. This demonstrates the shielding effect of GSH to block the charges of the Pt-DPA complex that can contribute to NSB via electrostatic interactions between the polymers and cells.Example 4A Platinum mass tag suitable for suspension mass cytometry and imaging mass cytometry

[0185] Pt MCP were prepared with reduced NSB. Differences in the polymer structure help improve conjugation to Abs by the addition of azide groups to the end of the pendant groups (Schemes 10 & 11). The Pt-polymer was conjugated to two types of Abs (anti-CD20 and anti-CD3) and both conjugates were used in an 11 -plex assay with Maxpar reagents. Both Pt-polymer Ab conjugates were effective at distinguishing B cells and T cells, respectively in PBMC suspensions by SMC. The Pt-polymers were conjugated with CD45RA and were used to stain human tonsil tissue in a 5-plex assay studied by IMC. The Pt mass tags were effective at visualizing human tonsil tissue with image quality comparable to the Maxpar reagents. The Pt MCP is a reagent for Pt mass channels, without interfering with existing Maxpar reagents and can be incorporated for multiplexed assays for both SMC and IMC.RESULTS AND DISCUSSIONPreparation of a P(DPA)-Pt polymer with end-group azide functionality for Ab conjugation

[0186] A P(DPA)-Pt polymer (c.f. 1 in Scheme 10) containing an azide end group derived from the RAFT agent during the synthesis of the PPFPA polymer scaffold was prepared. The synthesis of the Pt polymer was modified to reduce NSB with cells studied using mass cytometry. Methoxy (polyethylene glycol) (mPEGn) chain (PEG24) attached to theDPA chelator was more effective in reducing NSB than shorter grafted PEGs (mPEGe or mPEG-12). Treatment of the Pt polymers with glutathione (GSH) to replace the Pt-CI bond also greatly reduced NSB levels in experiments with peripheral blood mononuclear cells (PBMCs). GSH substitution reduced NSB such that that polymer 8a (with mPEGe pendants) and polymer 8c (with mPEG24 pendants) showed similar NSB levels in experiments with PBMCs. In this synthesis design, the terminal azide group was envisioned to be used as a handle to conjugate an Ab for MC applications.o o2 8a 8cScheme 10. Structures of azido end-group Pt polymers 8a and 8c, and their Ab conjugates with an anti-CD20 Ab. Pt-CI-Polymer 2 is the precursor for the synthesis of Pt polymers 8a and 8c. It was PEGylated prior to complexation with Pt2+.

[0187] Ab conjugation was performed at Standard BioTools Inc. using the strain-promoted azide alkyne click (SPAAC) reaction between the azide group on the polymers and a dibenzocyclooctyne (DBCO)-modified Ab. In these experiments, lysine amino groups on anti-CD20 were modified to contain ca. 10 DBCO groups per Ab. After Ab conjugation,both polymer-Ab conjugates were purified as described in Example 5 and used for cell staining experiments with PBMCs and analyzed by suspension MC (SMC).

[0188] Cell staining and mass cytometry experiments with PBMCs. In two separate experiments, PBMCs were stained with a cocktail of Maxpar reagents along with either polymer 8a-CD20 or 8c-CD20, to evaluate their performance as mass tag reagents. Anti-CD20 targets the antigen CD20 expressed on B cells, whereas T cells have very low expression of CD20 and can be regarded as CD20- in our experiments. For these polymer-Ab conjugates to be effective SMC reagents, 1) the mass tag should have low NSB with cells other than its intended target, 2) it should have high specificity to its intended target, 3) the metal signal should be sufficiently high to achieve good separation of the cell populations, 4) the mass tag must be compatible with existing mass tags (namely Maxpar reagents).

[0189] A common test of new reagents for SMC is to see if they can distinguish B cells from T cells, with appreciable signal in the desired mass channel. FIG. 19A shows an example of separation of cell populations in PBMCs achieved with current Maxpar reagents. The T cells in the upper left quadrant are detected with170Er-CD3. The B cells in the lower left quadrant are detected with147Sm-CD20. The ca. 13% of signal in the lower left quadrant is due to non-T non-B cells. When this experiment is repeated with Pt-polymer 8a-CD20 replacing the Maxpar147Sm-CD20 reagent, a specific binding is detected (FIG. 19B). The signal intensity is much weaker than that of147Sm-CD20. Experiments with Pt-polymer 8c-CD20 show no signal for detection of CD20 on B cells (FIG. 19C). The fact that weak signals were seen for the polymer with PEGe pendants and no signal from the polymer with PEG24 pendants suggests there is likely a significant steric effect caused by the longer pendant PEG24 chains that interferes with the Ab reacting with the azide on the short end group of the polymer.Preparation of a Pt polymer with multiple pendant azido groups

[0190] To overcome the problem with the short chain azide and steric effects, we reevaluated the design strategy for conjugating Pt polymers with Abs. The previous design contained only a single azide group with a short 3-carbon spacer. In this new design, a larger number of azide groups per polymer were incorporated and also a longer spacer between the azide and the polymer backbone, allowing it to be further extended out fromthe polymer. Azido-PEG23-amine (N3-PEG23-NH2) has a much longer spacer for the azide group and can be grafted to the DPA polymer similarly to the grafting of mPEG6-NH2and mPEG24-NH2 to the DPA pendants of Polymer 2 (Scheme 10).Polymer synthesis and characterization

[0191] To proceed, polymer 2 (Scheme 10) was treated with a 7:3 mole ratio of mPEG24-NH2 / N3-PEG23-NH2 to introduce a random mixture of pendant groups. This assumes equal reactivity of the amino-PEGs. To explore the influence of polymer length, polymer 2 samples with two different degrees of polymerization, DP 22 and 55, were used. These structures are presented as polymer 3 in Scheme 11. While the choice of a 7:3 mole ratio of azido to methoxy end groups was arbitrary, the goal was to ensure Ab conjugation and at the same time avoid extensive cross-linking during the conjugation reaction. For example, mole ratio of 9:1, 8:2, 6:4 and 5:5 are possible embodiments. Polymers were metalated by treatment with K2PtCl4 as the platinum(ll) source as described in Example 5.Scheme 11. Synthetic route for the preparation of Pt polymers containing pendant azide functionality. Polymer 2 was used to prepare the pendant azide polymer I-1, which was further modified to prepare polymer 10 by metalation with tePtCk Polymer 10 was treated with glutathione to replace the Pt-CI bond with Pt-GSH to obtain polymer 11-1. Thestructure of the Ab conjugated Pt polymers 10 and 11-1 is shown as 10-Ab and 11-1 -Ab. DP = n + m.

[0192] To test the effectiveness of the design strategy for incorporating reactive azides to P(DPA), the azide content of DP 22 polymer 8c (without pendant azides) and polymer 10 (with pendant azides) was compared. Samples of the polymers were reacted with an excess of DBCO-Cy5 dye via the SPAAC reaction. Unreacted DBCO-Cy5 dye was removed, and the polymer was purified by washing the polymer via spin filtration with an Amicon Ultra-15 mL 10 kDa MWCO spin filter (2700 x g, 15 min). Washing with ultrapure water was continued until the filtrate was colourless. As a control, a P(DPA) polymer was prepared that did not contain any azide functional groups and reacted with DBCO-Cy5. Any observed Cy5 dye signal in the UV-vis spectrum of this polymer would be due to DBCO-Cy5 that that could not be removed during the purification spin filtration washes.

[0193] The number of reactive azides per polymer was then determined for all 3 polymers by UV-vis spectroscopy to measure the absorption of the Cy5 dye at A649nm. Corresponding spectra were obtained, and the mean number of azides per polymer was calculated using equation in Example 5. For the control polymer containing no azides, a value of 0.04 azides per polymer was calculated. For polymer 8a containing only a short chain terminal azide end-group, a value of only 0.4 azides per polymer was calculated. For Polymer 8a (DP 22) there was on average ca. 3 azides per polymer.

[0194] The samples of polymer 10 were treated with glutathione to replace the Pt-CI bonds. The polymers were treated with 5 equivalents of GSH for 2 hours, followed by removal of excess GSH by spin filtration. In this way, samples of polymer 11-1 were obtained. Since glutathione can act as a reducing agent, which can in principle reduce azides to amines, it was necessary to ensure that the reactive azides were not consumed upon treatment with GSH. DBCO-Cy5 was used to measure the azide content after glutathione treatment. The results are shown in Table 5.Table 5. azide content of Polymers 1-1, 10, 11-1 determined from the absorbance at A649nm measured by UV-vis on DBCO-Cy5 and calculated with eq S1.Calculated # of azides per Samplepolymer Polymer 1-1 (before Pt loading) 3.1 azides per polymerPolymer 10 (after Pt loading) 2.8 azides per polymerPolymer 11-1 (after GSH treatment) 2.9 azides per polymer

[0195] For polymer 5 (DP = 22), ca. 3 azide groups per polymer were measured. Polymers 10 and 11-1 samples were further characterized by1H NMR (FIG. 20A for DP 22 and 20B for DP 55), GPC (FIG. 20C for DP 22, FIG. 20D for DP 55), as well as electrophoretic mobility (FIG. 20E), and thenatPt metal content was determined by ICP-OES (Table 6). These measurements are described in Example 5.Table 6. Metal content ofnatPt polymers determined by ICP-OESSample Metal per polymer (mg Metal content by ICP-OES metal / mg polymer)Polymer 10 (DP=22) 22 / 22natPt per polymer0.11(before GSH)Polymer 11-1 (DP=22) 18 / 22natPt per polymer0.10(after GSH)Polymer 4 (DP=55) 56 / 55natPt per polymer0.16(before GSH)Polymer 11-1 (DP=55) 40 / 55natPt per polymer0.10(after GSH)

[0196] Then the polymer 11-1 samples, fully characterized, were reacted with DBCO-modified anti-CD20 Ab using the same protocol used to attach this Ab to the end of polymer 8a. These samples are referred to as Pt-polymer II-122-CD20 and Pt-polymer II-155-CD20, where the subscripts distinguish the two polymer chain lengths.

[0197] Cell staining experiments with Pt-polymer II-1-CD20. Cell staining experiments were carried out as described above for Pt polymer 8a-CD20 and Pt polymer 8c-CD20. As in those experiments, the Pt polymer conjugates were combined with a cocktail of Maxpar reagents and tested for their effectiveness at detecting and distinguishing B cells in PBMC samples. For the full antibody panel in the Maxpar reagents, see Table 14 in Example 5.

[0198] Cells (ca. 1,000,000) were stained with three different titers of Pt-polymer II-I22-CD20, and the results are presented in FIG. 21. FIG. 21 A shows the results for the lowest titer (1.11 pg / mL), and results for titers of 3.33 pg / mL and 10.0 pg / mL are presented respectively in FIG. 21B, C. All three panels show good resolution of B cells (lower right quadrant), T cells (upper left quadrant) and non B, T cells (lower left quadrant), and thesignal associated with CD20+ B cells increases with titer. Note that good signal intensity was obtained, even when using natural abundance Pt. A table containing the196Pt counts as well as the calculated signal-to-noise ratios for each titer are reported in Table 7. Table 7. Metal signal counts obtained from PBMCs stained with Maxpar 10-plex antibody cocktail plusnatPt-polymer-anti-CD20 Abs. The Maxpar control is used to stain PBMCs in a similar manner, however thenatPt-polymer-anti-CD20 Ab was replaced with147Sm-anti-CD20 Ab. The concentrations for cell staining are low titer: 1.11 pg / mL, med titer: 3.33 pg / mL, and high titer: 10.0 pg / mL.Mean195Pt Signal Signal-to- Signal-to- Sample Non- noise (B noise (BTiter T BName B / T cells / T cells / non- Cells Cellscells cells) B / T cells)Low 2.6 81 2.5 31 33Polymer 11-1Med 2.8 178 2.6 64 68(DP=22)aHigh 2.7 260 2.5 95 104Low 2.8 78 2.7 28 29Polymer 11-1Med 2.9 180 2.8 62 64(DP=55)aHigh 3.2 288 3.0 92 98MaxparControl ** 2.1 474 2.1 222 223 (147Sm-anti- CD20)bMaxparControl 3.52.6 217 2.4 83 89(196CisPt- pg / mLCD20)aa.196Pt channelb.147Sm channel

[0199] A more subtle test of the suitability of a new MCP reagent is whether the relative populations of T cells, B cells and non B, T cells determined with the new metal properlyreflects that determined with current Maxpar reagents. As shown in Table 8, the essentially identical cell populations found at each titer in FIG. 21 correspond to those determined with the Maxpar control. The medium titer (3.33 pg / mL) provides data with the highest signal-to-noise and represents the optimum titer in these experiments. Similar results were achieved for Pt-polymer II-155-CD20, and these results are shown in Tables 8, 9 and FIG. 22. Corresponding polymers labeled with isotopically enriched196Pt were also prepared.Table 8. Population percentages of different cell types, T cells, B cells, and non-B / T cells of PBMCs stained with the Maxpar 10-plexAb cocktail plusnatPt-anti-CD20 analyzed by SMC. The values are obtained from the 4 separated quadrants in the biaxial scatter plots of170Er_CD3 vs196Pt (FIG. 21 and FIG. 22). For the Maxpar control B cells were detected with147Sm-anti-CD20 Ab. The concentrations for cell staining are low titer: 1.11 pg / mL, med titer: 3.33 pg / mL, and high titer: 10.0 pg / mLDouble T cells B Cells Non-B / Tpositive signal Sample Titer (Upper-left (Lower-right cells (Lower- (Upper-right quadrant) quadrant) left quadrant)quadrant) Low 55.3 19.0 25.6 0.1 Polymer 11-122- Med 55.2 19.3 25.5 0.1 CD20aHigh 54.5 18.6 26.8 0.1Low 54.5 18.9 26.5 0.1 Polymer 11-155- Med 54.5 18.9 26.6 0.1 CD20aHigh 55.6 18.9 24.9 0.1 Maxpar Control**(147Sm-anti- 55.1 19.4 25.4 0.1CD20)a.196Pt channelb.147Sm channel** Maxpar titer according to the protocol.Preparation of an isotopically enriched196Pt pendant azido mass tag

[0200] Samples of polymer 10 and polymer 11-1 were prepared with isotopically enriched196Pt using the same polymer batch used for the experiments reported above. Here only 1.2 eqv K2196PtCl4 per DPA pendant groups was used compared to the 3.6 eqv used for the experiments described above. The lower equivalents led to a slightly lower metal content (17 Pt per polymer for polymer 10) as determined by ICP-OES. Here, treatment with GSH preserved the Pt content of the polymer with DP = 22. A table with the metal content for196Pt polymers 10 and 11-1 can be found in Table 9 of the SI. In the experiments described above fornatPt polymers, treatment with GSH reduced the Pt content from 22 Pt per polymer to 18 for polymer 11-1. Then the polymers labeled with196Pt were conjugated to anti-CD3 and used to stain PBMCs. The expression of CD3 on T cells is lower than the expression of CD20 on B cells and provides a more sensitive test of the reagent as a mass tag.Table 9. Metal content of196Pt polymers determined by ICP-OESSample Metal per polymer (mg Metal content by ICP-OES metal / mg polymer) (# of Pt / polymer ofDP=22)Polymer 10 (DP=22) 0.08 17 / 22196Pt per polymer(before GSH)Polymer 11-1 (DP=22) 0.08 17 / 22196Pt per polymer(after GSH)Polymer 10 (DP=55) 0.09 39 / 55196Pt per polymer(before GSH)Polymer 11-1 (DP=55) 0.08 38 / 55196Pt per polymer(after GSH)Cell staining experiments with196Pt-polymer 10-CD3 and196Pt-polymer II-1-CD3.

[0201] Cell staining experiments employed the same lot of PBMCs as used in the experiment described above. Because of the isotopic enrichment, lower titers were chosen for these experiments [0.56 pg / mL (low), 1.67 pg / mL (med), and 5.0 pg / mL (high)]. Results for 196Pt-polymer II-122-CD3 are shown in FIG. 23A, B, C, where we see good separation of T cells, B cells, and non B, T cells, and the CD3 signal increases with mas tag titer as expected. Corresponding results for196Pt-polymer IO22-CD3 (not substituted with GSH) are shown in FIG. 23C, D, E. The results are very similar to those obtained with polymer 11-1. Further information is provided in FIG. 23G, H, which displayshistograms of the three cell populations. For both polymer 10 and polymer 11-1, one sees that the CD3 signal intensity increases with the applied titer, but the NSB signal for196Pt associated with the B cell and non-B, T cell populations is small except at the highest titer. Similar results were obtained for the corresponding196Pt-polymer 1055-CD3 and196Pt-polymer II-155-CD3 with DP = 55 (Table 10 and FIG.24 A-H). While the cell populations from all three titers with these 4196Pt-labeled mass tags agree, they yield slightly higher T cell populations and slightly smaller B cell populations than are found with the Maxpar control (Table 11). One remarkable feature of these experiments is that the mPEG24 pendants reduce NSB to levels where no additional protection is provided by GSH.Table 10. Metal signal counts obtained from PBMCs stained with Maxpar 10-plex antibody cocktail plus196Pt-polymer-anti-CD3 Abs. The Maxpar control is used to stain PBMCs in a similar manner, however the196Pt-polymer-anti-CD3 Ab are replaced with170Er-anti-CD3 Ab. The concentrations for cell staining are low titer: 0.56 pg / mL, med titer: 1.67 pg / mL, and high titer: 5.0 pg / mLMean195Pt Signal Signal-to- Signal-to- Sample Non- noise (T noise (TTiter T BName B / T cells / B cells / non- Cells Cellscells cells) BT cells)Low 131 2.9 3.7 45 36Polymer 10Med 260 3.2 4.7 80 56 (DP=22)aHigh 396 4.0 6.8 99 58Low 204 3.3 4.8 63 42Polymer 10Med 381 3.8 6.6 100 58 (DP=55)aHigh 528 5.1 10.4 104 51Low 138 2.7 3.6 51 38Polymer 11-1Med 239 2.9 4.6 83 52 (DP=22)aHigh 337 3.3 6.8 103 50Polymer 11-1 Low 146 2.8 3.7 53 39 (DP=55)aMed 282 3.1 4.6 92 56High 393 3.4 6.8 117 49 MaxparControl** 2.4 2.4 2.6 N / a N / a(170Er-anti- CD3)aMaxparControl** 168 1.4 1.9 122.0 88.0(170Er-anti- CD3)ba.196Pt channelb.170Er channel** Maxpar titer according to the protocol.Table 11. Population percentages of different cell types, T cells, B cells, and non-B / T cells of PBMCs stained with the Maxpar 10-plex Ab cocktail plus196Pt-polymer-anti-CD3 Ab analyzed by SMC and obtained via a gating strategy. The values are obtained from the 4 separated quadrants in the biaxial scatter plots of196Pt_CD3 vs147Sm_CD20 (FIG. 23 and FIG. 24). The Maxpar control is used to stain PBMCs in a similar manner, however the196Pt-polymer-anti-CD3 Ab was replaced with170Er-anti-CD3 Ab. The concentrations for cell staining are low titer: 0.56 pg / mL, med titer: 1.67 pg / mL, and high titer: 5.0 pg / mL DoubleB Cells Non-B / T positiveT cells(Lower- cells signalSample Titer (Upper-leftright (Lower-left (Upperquadrant)quadrant) quadrant) right quadrant)Low 51.9 19.4 28.5 0.3Polymer 10Med 51.3 19.8 28.7 0.2(DP=22)aHigh 49.1 20.2 30.6 0.1Low 50.9 19.6 29.2 0.2Polymer 10Med 50.3 20.1 29.4 0.2(DP=55)aHigh 50.7 20.2 28.9 0.2Low 52.1 19.1 28.5 0.2Polymer 11-1Med 51.0 19.5 29.4 0.2(DP=22)aHigh 50.6 19.4 29.8 0.2 Low 52.6 19.3 27.9 0.2Polymer 11-1Med 50.9 19.5 29.5 0.2(DP=55)aHigh 49.8 19.9 30.1 0.2 MaxparControl** 52.5 17.8 29.5 0.2(170Er-anti- CD3)ba.196Pt channelb.170Er channel** Maxpar titer according to the protocol.Tissue staining experiments monitored by Imaging Mass Cytometry (IMC)

[0202] Tissue samples are more complex than cells and some reagents that work with SMC do not work for IMC. Therefore a challenging test for196Pt-polymer 10 and196Pt-polymer 11-1 is to incorporate them into IMC experiments. In these experiments, both polymers were conjugated to anti-CD45RA, which targets naive (unactivated) T cells.

[0203] Sections of paraffin-embedded human normal tonsil tissue were independently deparaffinated and subjected to antigen retrieval. These 3 tissue sections were sliced consecutively from a larger portion of tissue, and thus it is expected that each section will be similar, but not identical to one another. All 3 tissue sections were stained with a Maxpar reagent Ab cocktail in a 5-plex assay (Table 12) that contained170Er-anti-CD3 Ab to stain T cells as well as an lr DNA intercalator (detected as191lr and193lr) to stain cell nuclei. The staining of the three consecutive tissue sections differed only by the anti-CD45RA mass tag reagent. One of the three tissue sections was stained with166Er-anti-CD45RA Maxpar reagent which serves as the control. For the second and third tissue sections,196Pt-polymer II-122-CD45RA mass tag (DP=22) and196Pt-polymer II-I55-CD45RA (DP=55) were used, respectively. Details of the staining protocol are described in Example 5.Table 12. Maxpar Antibodies and their metal isotopes that made up the 5-plex assay for SMC.Antibody Clone MetalSMA 1A4141PrCD45 D9M8I152SmCD3 Polyclonal170ErCD45RO UCHL1173Yb

[0204] On each tissue section, four regions of interest (ROIs) were selected to include specific regions of the human tonsil tissue which are comprised of different types of cells. These include a region (ROI 1 ) near the edge of the tissue which contains epithelial cells, (ROI 2) containing a germinal center, a region containing both tissue and glass, and a region containing blood vessels. In this way, different cell types were examined to obtain a more comprehensive analysis regarding the specificity of the 196Pt polymer-Ab conjugates to their intended targets.

[0205] Each of the ROIs were examined by IMC via laser ablation in 1 mm2pixels. Representative images from ROI 1 containing epithelial cells is shown in FIG. 25. The images were initially analyzed by grayscale image thresholding. This technique assigns a value of 0 as black on the image. The max threshold value for an image (shown on the upper right corner of each image) is determined from the pixel of the image that contains the highest metal count and is calculated to be the 95th percentile of the metal count per pixel. Any pixel with a metal count above the max threshold is assigned the color white. This value provides a relative quantitative metric on the number of metal counts detected by the instrument after ablation of the tissue sample (1 mm2) and can be used as a measure of signal intensity (or brightness) of the image. Generally, a larger max threshold provides better image contrast as it extends the grayscale to a larger range of detected metal counts by the IMC instrument. However, different metals have different ionization potentials and different transmission coefficients, and these values affect how many ions are detected by the instrument after ablation and ionization of the samples. For example, the lanthanides have lower ionization potentials and a higher ionization efficiency (>90 %) than Pt, which has a much greater ionization potential and n ionization efficiency of only 62 %.1516For similar metal atom content in a given pixel, fewer Pt ions will be detected by the instrument than corresponding lanthanide ions.

[0206] FIG. 25A presents the grayscale image for the166Er-CD45RA Maxpar control with a max threshold value of 56.3. FIG. 25B presents the corresponding image for196Pt-polymer II-122-CD45RA (DP=22). Here the max threshold value is 18.2, while the max threshold for196Pt-polymer II-155-CD45RA (DP=55) is 22.5 (FIG. 40). Note that the longer polymer with more Pt atoms shows a higher signal intensity. While the max threshold is a useful relative quantitative metric for evaluating the performance of new mass tag reagents, it does not provide information on the quality of the images. Upon looking at the images themselves in FIG. 25. The images of polymer 11-1 -Ab (DP =22 and DP=55, FIG.4B and 4C, respectively) show comparable image quality to the Maxpar control (FIG. 25A) even with a significantly lower max threshold value. The image also shows that the polymer does not bind to glass (black regions of the tissue section). These images highlight the applicability of the prepared196Pt polymer-Ab conjugates for IMC applications.

[0207] To better study the co-localization of the 196Pt polymer-Ab conjugates compared to a counterstain of170Er-CD3, colors can be assigned to the different mass channels of the grayscale images (FIG. 26E and 26F), where light grey is assigned to196Pt-CD45RA, white is assigned to170Er-CD3, and dark grey is assigned to the191lr signal of the-DNA intercalator. One anticipates that the counterstain containing the anti-CD3 Ab should not bind in regions where196Pt-polymer anti-CD45RA Ab conjugates bind. In FIG. 26E, F, the light grey and white colors for the most part do not overlap, indicating that the 196Pt-polymer-Ab conjugates are binding specifically to their target antigens. This provides further evidence to support the specificity of our optimized196Pt-polymer-Ab conjugates for IMC.

[0208] Similar experiments were carried out for human tonsil tissue sections stained with polymers196Pt-polymer 1022-CD45RA (DP=22) and196Pt-polymer 1055-CD45RA (DP=55) (no GSH treatment). These images are shown in FIG. 27. These polymers show higher max threshold values (24.5 and 37.4, respectively) compared to the polymers treated with GSH, indicating that both lengths of polymer 10 have higher detected196Pt metal counts. However, even though max threshold values are higher for polymer 10 without GSH treatment, the observed images for DP=22 and DP =55 (FIG. 27B and 27C, respectively) show images that are blurred compared to those obtained with the Maxpar control (FIG.27 A). These higher max threshold values appear to be caused by higher NSB on regions where no CD45RA binding is expected, and this can be observed in the images in FIG.27B and 27C. Using the pseudo-colored images shown in FIG. 27 and 27F, dimmer170Er-CD3 white dots are visible, which show the presence of polymer 3 in those regions, resulting in the loss of the white coloration in the image. Images for the other 3 ROIs on human tonsil tissues stained with polymer 1-1 were also obtained.

[0209] Previous results with SMC (FIG. 23) on196Pt mass tags showed no differences between polymers treated with GSH (polymer 11-1) and polymers without GSH treatment (polymer 10). In the results for IMC, both DP=22 and DP=55196Pt polymer 10-Ab conjugates without GSH treatment (FIG. 24B and 24C) show images with much lower resolution and have much higher levels of background as can be seen by the worse image quality (blurrier) compared with FIG. 25B and 25C. These differences are likely attributed to the more complex nature of tissues compared with cells.

[0210] ROI 2 (FIG. 26) focuses on a location of the human tonsil tissue containing germinal centers. These centers are rich in B cells (CD45RA+) where we would expect substantial binding for anti-CD45RA. The region just outside of the germinal center, known as lymphoid follicles, displays T cells (CD3+), making it a useful region to image with the170Er-anti-CD3 counterstain.17 Comparing the images obtained from the staining of this tissue section with196Pt-polymer II-122-CD45RA (DP=22, FIG. 26B), and196Pt-polymer5s5-CD45RA (DP=55, FIG. 26C) shows the longer DP=55 polymer is brighter and provides better contrast in the image. Moreover, comparison of the max threshold values shows a greater difference between the longer DP=55 polymer and the DP=22 polymer. Analysis of the pseudo-colored images of ROI 2 for196Pt-polymer II-122-CD45RA (FIG.26E) and196Pt-polymer II-155-CD45RA (FIG. 26F) shows that the light grey and white colors do not overlap similar to what was seen for ROI 1, providing further support of the high specificity of the196Pt-polymer 10 conjugates to their target antigens.

[0211] For the third ROI (FIG. 28), a region containing other germinal centers were imaged and the quality of the images on the germinal centers comparable to those obtained in the second ROI. The image quality for both DP=22 and DP=55196Pt polymer-anti-CD45RA conjugates (FIG. 25B and FIG. 25C, respectively). Comparing the max threshold of the two196Pt-polymer II-1-CD45RA conjugates,196Pt-polymer II-122-CD45RA(FIG. 28B) shows a value of 20.8, while the longer196Pt-polymer II-155-CD45RA (FIG.28C) shows a higher value of 23.6. In both pseudo-colored images for the DP=22 polymer (FIG. 28E) and DP=55 polymer (FIG. 28F), minimal overlap between the light grey colour (196Pt-polymer II-1-CD45RA) and the white color (170Er-CD3) of the counterstain is seen.

[0212] For the fourth ROI (FIG. 29), a region near the edge of the tissue containing epithelial tissue and glass were observed. Again, no binding of the196Pt mass tags to glass was observed as indicated by the black spaces on the edge of all the images in FIG. 29A-F. For these images, the max threshold value of196Pt-polymer II-122-CD45RA (FIG. 29B) is 13.3 and for196Pt-polymer II-155-CD45RA is slightly higher at 14.5, consistent with all the other ROIs (1-3, FIG. 25, 26 and 28, respectively) showing the longer DP=55 polymer has a higher value and shows brighter images compared with the shorter DP=22 polymer.

[0213] Based on the IMC results on the four ROIs, the196Pt polymers are promising as IMC probes, with the longer DP=55196Pt-polymer II-155-CD45RA performing slightly better based on a higher max threshold value and brighter image quality than the DP=22196Pt-polymer II-122-CD45RA conjugate. It was found that the GSH treatment to cap the polymers generated a good image with lower background. This is contrary to the results with SMC, where not treating the196Pt polymer with GSH did not reduce the performance. The improvement in image quality with196Pt polymers treated with GSH may be due to the complexity of tissue sections which contain multiple different cell types compared to PBMCs. These more complex tissue sections may amplify the importance of the GSH to reduce the NSB further to reduce background on the images obtained from IMC.Evaluating the compatibility of196Pt-polymer-anti-CD45RA-Ab conjugate with Maxpar reagents

[0214] For a new mass reagent to be suitable for a multiplex assay for IMC, it should be compatible with the Maxpar reagents. The new mass tag must also not interfere (i.e. due to steric effects) with the binding of Maxpar mass tags to their target biomarkers. This information can be obtained by comparing IMC results for the Ab cocktail containing196Pt-polymer II-122-CD45RA or196Pt-polymer II-155-CD45RA with the Maxpar control that targets CD45RA with166Er-CD45RA. All three Ab cocktails also contain152Sm-CD45. Anti-CD45 and anti-CD45RA Abs should bind to similar domains in the tissue. Thus,152Sm should co-localize with166Er in the tissue sample stained with the Maxpar control, and it should co-localize with196Pt in the tissue sections stained with the Pt polymers.

[0215] The Maxpar Ab cocktail also contains an Ab that targets smooth muscle actin (SMA) labelled with141Pr (141Pr-SMA), which targets different antigens than196Pt-polymer II-1-CD45RA. One would expect no co-localization between these two Ab conjugates. In each of the three 5-plex assays, the max threshold values in the152Sm channel for the samples stained with Ab cocktails containing166Er-CD45RA (control sample) was compared with those containing196Pt-polymer II-122-CD45RA or196Pt-polymer II-I55-CD45RA (FIG. 30B, D, G, J and FIG. 30C, E, H, K, respectively). Some small differences are found for ROI-1. Here, the max threshold value for the152Sm channel was 46.0 for the tissue stained with the Maxpar control. The max threshold in the152Sm channel for the image obtained from tissues stained with the Maxpar Ab cocktail with196Pt-polymer 11-122-CD45RA was slightly reduced (42.0), and for the196Pt-polymer II-155-CD45RA, the max threshold value was even lower (39.0). However, for the other ROIs, the differences are much smaller.

[0216] To test whether the196Pt-Ab conjugates might interfere with non-colocalized targets, we examine their influence on max threshold values of the141Pr mass channel associated with SMA (FIG. 31). These effects are also very small. For example, in ROI-1, a max threshold of 24.9 was obtained for the141Pr mass channel in the Maxpar control. In samples containing the196Pt-Ab conjugate (DP=22 and DP=55), the141Pr mass channel for both showed a value of 28.0. The max threshold values for all of these experiments are tabulated in Table 13. From these various experiments, it is seen that the the196Pt-Ab conjugates have a minimal influence on the binding of other Maxpar Ab conjugates to these tonsil tissue samples.Table 13. Max threshold values of human tonsil tissue stained in a 5-plex assay obtained from IMC for 4 ROIs in the152Sm channel and141Pr channel. The values correspond to those in the images shown in FIG. 30 and 31.Max threshold valuesTarget Metal RO I196Pt-polymer IO22-196Pt-polymer MaxparCD45RA 1055-CD45RA1 46.0 42.0 39.02 57.7 52.8 58.5CD45152Sm3 57.0 56.8 57.64 27.8 22.9 22.81 24.9 28.0 28.02 12.0 12.0 13.0SMA141Pr3 12.0 10.0 12.04 14.0 9.0 13.0Example 5EXPERIMENTAL DETAILSInstrumentation

[0217] Gel Permeation Chromatography (GPC). GPC measurements were performed with a Waters 515 HPLC GPC system equipped with a Viscotek VE 3580 Rl detector and a Viscotek VE3210 UV / Vis detector, and Waters Styragel HR 4E THF column with a Waters Styragel Guard Column kept at room temperature (23 °C). The eluent consisted of THF containing 2.5 g / L tetra-n-butylammonium bromide (TBAB) with a flow rate maintained at 0.4 mL / min. The system was calibrated with poly(methyl methacrylate) (PMMA) standards (Mn: 645-127000 Da)

[0218] Size Exclusion Chromatography (SEC). SEC measurements were performed with a Viscotek size exclusion chromatograph equipped with a Viscotek UV2500 Detector, VE3580 refractive index detector, and Viscotek ViscoGEL G4000PWXL and G2500PWXL columns kept at 32 °C. The eluent consisted of 200 mM KNO3, 25 mM pH 8.5 phosphate buffer, 200 ppm NaNs with a flow rate maintained at 1.0 mL / min using a Viscotek VE1122 Solvent Delivery System and VE7510 SEC Degasser. The system was calibrated with poly(ethylene oxide) standards (Mn: 540-26720 Da).

[0219] Nuclear magnetic resonance spectrometry (NMR). NMR spectra were obtained using an Agilent DD2500 MHz spectrometer fitted with a XSens C13 Cold Probe.

[0220] Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Metal content was measured using a Thermo Scientific iCAP Pro ICP OES. Samples (stock solution of 10 mg / mL) were digested in reverse aqua regia by pipetting 5 pL of stock solution into a solution of (60 pL HNO3 and 10 pL HCI) and heating the mxiture at 85 °C for 2 hours in an aluminium heating block. The digested samples were then diluted to 10 mL by serial dilution with 2 % nitric acid.

[0221] Electrophoretic Mobility Distribution. Electrophoretic Mobility Distribution histograms were obtained using a Malvern Zetasizer Nano ZS instrument. Polymer samples were dissolved in ultrapure water with a concentration of 1 mg / mL. Electrophoretic mobility measurements were performed in a disposable folded capillary cell and all measurements were equilibrated at room temperature for 120 s and run in triplicate at 25 °C.Materials

[0222] Sodium triacetoxyborohydride (STAB), hydrogen chloride solution 4 M in dioxane, pentafluorophenol, sodium hydroxide pellets, concentrated hydrochloric acid (HCI), 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1 -propanol ester, 2,2'-Azobis(2-methylpropionitrile) (AIBN), ethanolamine, L-glutathione reduced (GSH), and potassium tetrachloroplatinate(ll) (K2PtCk) were purchased from Sigma Aldrich. 4-(4,6-dimethoxy-s-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) was purchased from Toronto Research Chemicals. MethoxyPEGe-amine (mPEG6-NH2) and optima grade nitric acid was purchased from Fisher Scientific. MethoxyP EG24-amine (mPEG24-NH2) and azido-PEG23-amine (N3-PEG23-NH2) were purchased from Broadpharm. 2-pyridinecarboxaldehyde was purchased from TCI. Ns-Boc-L-lysine was purchased from Bachem. All chemicals listed were used as received without further purification. Ultrapure water (18.2 MO cm) was obtained using a Thermo Barnstead GenPure xCAD Ultrapure Water system.

[0223] Maxpar reagents (control) are a cocktail containing ten different Ab conjugates labelled with a different metal isotope. These conjugates include CD3 labeled with170Er, CD14 labeled with160Gd, CD33 labeled with158Gd, CD4 labeled with145Nd, CD66blabeled with152Sm, CD16 labeled with148Nd, CD56 labeled with163Dy, CD45 labeled with89Y, CD20 labeled with147Sm, and CD8a labeled with162Dy.

[0224] The preparation of P(DPA) (Polymer 2) was synthesized as reported previously11and in Examples 1-3.Polymer synthesis

[0225] PEGylation of P(DPA) polymer 2 with PEG6-NH2 or PEG24-NH2. Polymers 8a and 8c were prepared as previously reported and as described in Examples 1-3.11In a vial (4 mL), P(DPA) polymer 2 (50 mg) was dissolved in water (400 pL). Amide coupling agent, DMTMM (182 mg, 0.66 mmol, 5 equiv. per pendant group) in water (600 pL) was added to the P(DPA) solution and pre-reacted for 10 min. After 10 min stirring at room temperature, mPEG6-NH2or mPEG24-NH2 (0.118 mmol, 3 equiv. per pendant group) was added to the vial containing the polymer solution. After 1 day, the reaction was washed via spin filtration over Amicon Ultra-15, 10 kDa MWCO spin filters (6 times, 2700 x g for 15 min) using ultrapure water. The retentate was lyophilized overnight to afford two PEGylated polymers, one grafted with mPEG6, and the second grafted with mPEG24. Pt metal loading of polymer with K2PtCl4

[0226] Pt metal loading with K2PtCl4 was performed as previously reported11and as described in Examples 1-3. In a vial (8 mL), PEGylated polymer (20 mg) was dissolved in DMSO (200 pL). A solution of K2PtCl4 (50 mM in DMSO, 3.6 eqv. per DPA pendant group) was added to the polymer solution and the mixture was heated at 45 °C for 24 h. After 24 h, an aliquot of the reaction mixture (200 pL aliquot) was added to an Amicon Ultra-15, 10 kDa MWCO spin filter pre-filled with 15 mL of ultrapure water to avoid damaging the regenerated cellulose ester membrane of the filter and then washed by centrifugation (2700 x g, 15 min). This process with was repeated 3 times so that all 600 pL of the crude reaction mixture was transferred to the spin filter. The crude polymer solution was further washed with 50 mM NaCI solution (3 times, 2700 x g, 15 min), and finally another 3 times with water (3 times, 2700 x g, 15 min). The retentate was lyophilized to afford two light yellow solids as Pt polymers, one grafted with mPEG6, and the second grafted with mPEG24(24 mg for mPEGe, and 23 mg for mPEG-12).

[0227] Glutathione capping of Pt polymers. In a vial (4 mL), Pt polymer (5 mg) was dissolved in ultrapure water (300 pL). GSH (5 equiv. based upon the DPA content of the polymer) was added to the polymer solution and the mixture was stirred at room temperature for 2 h. After 2 h, the reaction mixture was purified by washing with ultrapure water using an Amicon Ultra-4, 10 kDa MWCO spin filter (5 times, 2700 x g, 15 min). The retentate was lyophilized to afford polymers 8a and 8c (6.7 mg and 6.3 mg, respectively).

[0228] PEGylation of P(DPA) with a 7:3 ratio of mPEG24-NH2 and N3-PEG23-NH2 to obtain polymer 1-1. In a vial (4 mL), P(DPA) polymer 2 (50 mg) was dissolved in ultrapure water (400 pL). Amide coupling agent, DMTMM (182 mg, 0.66 mmol, 5 equiv. per pendant group) in water (400 pL) was added to the P(DPA) solution and pre-reacted for 10 min. After 10 min of stirring at room temperature, a 7:3 PEG mixture consisting of mPEG24-NH2 and N3-PEG23-NH2 (0.118 mmol, 3 equiv. per pendant group) dissolved in water (200 pL), was added to the vial containing the polymer solution. After 1 day, the reaction was washed via spin filtration over Amicon Ultra-15, 10 kDa MWCO spin filters (6 times, 2700 x g for 15 min) using ultrapure water. The retentate was lyophilized overnight to afford a light brown solid as polymer 3 (138 mg). MnSEC = 11000, D = 1.4.

[0229] Pt metal loading with K2PtCl4 was performed in the same manner as described above but with polymer 1-1. In short, PEGylated polymer (20 mg) was dissolved in DMSO (200 pL). A solution of K2PtCl4 (1 mL, 50 mM in DMSO, 3.6 eqv. per DPA pendant group, 0.05 mmol) was added to the polymer solution and the mixture was heated at 45 °C for 24 h and purified via spin filtration and dried as detailed above for polymers 8a and 8c. A yellow-brown solid was obtained as polymer 10 (23 mg).

[0230] GSH capping was performed in the same manner as above but with Pt polymer 10. Polymer 11-1 was obtained as a light yellow-beige solid (5.8 mg).Pt polymer-Antibody conjugationAntibody preparation

[0231] Abs were concentrated with an Amicon Ultra-0.5 mL, 50 kDa MWCO spin filter and centrifuged (11000 x g, 5 min, room temperature) down to a pellet. The supernatant is discarded between each spin cycle, and the number of spin cycles was determined based on the total volume of Ab required. After the last spin, the supernatant was discarded and400 pL of R-buffer was added to the spin filter, and centrifuged again (11000 x g, 10, min, room temperature) and the supernatant was discarded. This procedure was repeated 3 times. R-buffer was added until the volume of Ab reached 200 pL, and the Ab concentration was determined with a Nanodrop instrument. The Ab was again concentrated with the spin filter (11000 x g, 10 min, room temperature) and the supernatant was discarded. Next, TCEP (4 mM in R-buffer, 100 pL) was added to the Ab in the spin filter and gently pipetted to mix well. The Ab mixture was heated at 25 °C with a Thermomixer for 30 minutes.

[0232] After 30 minutes, C-buffer (300 pL) was added to the spin filter containing the partially reduced Ab to reach a total volume of 420 pL. The Ab was washed by centrifugation (11000 x g, 10 min, room temperature) and the supernatant was discarded. C-buffer (400 pL) was added and centrifuged again, and the supernatant was discarded. Next, 80 pL of C-buffer was added and pipetted gently to mix well.Preparation of a DBCO-modified Ab.

[0233] Mal-PEG4-DBCO (c = 8.10 mg / mL, 2.5 pL) was added to the Ab freshly treated with TCEP and mixed well with a pipette. The mixture was then incubated in the Themomixer at 25 °C for 30 minutes. After 30 minutes, C-buffer (300 pL) was added and centrifuged in an Amicon Ultra-0.5 mL, 50 kDa MWCO spin filter (11000 x g, 10 min, room temperature) and the supernatant was discarded. C-buffer (400 pL) was added again and centrifuged again. The process was repeated an additional 2 times.Conjugation of DBCO-modified Ab with Pt polymer.

[0234] A polymer sample labeled withnatPt or196Pt was dissolved in C-buffer (60 pL) and transferred with a pipette to the Amicon Ultra-0.5 mL, 50 kDa MWCO spin filter containing the DBCO-modified Ab. The mixture was pipetted up and down gently to mix well and then incubated at 37 °C in an aluminum heating block for 90 minutes. After 90 minutes, W-buffer (90 pL) was added to the Pt polymer-Ab conjugate, centrifuged (11000 x g, 10 min, room temperature) and the supernatant was discarded. W-buffer (400 pL) was added, and the centrifugation was repeated. This process was repeated a total of 4 times, and in the 4th wash, it was centrifuged at 11000 x g for 15 min at room temperature. Next W-buffer was added until the total volume reached 200 pL and the mixture was mixed bygently pipetting up and down. The mixture was centrifuged again for 10 minutes (11000 x g, 15 min, room temperature).Cell staining experiments with PBMCs

[0235] Preparation of PBMCs for use in staining protocol. PBMCs and CTL anti-aggregate wash solution were thawed in a 37 °C water bath. A buffer solution containing CTL media (500 pL) was added to Gibco RPMI 1640 cell culture media (9.5 mL). PBMCs (ca.10 million cells / mL, each sample vial is calculated to achieve roughly 3 million samples per individual staining experiment) were added to the buffer solution, mixed well, and centrifuged (8 min, 453 x g). The supernatant was carefully removed with a micropipette and the pellet containing the PBMCs was resuspended in 10 mL RPMI containing FBS (10 %). An aliquot (10 pL) was mixed with 10 pL trypan blue and analyzed with a hemocytometer to count the cells in the solution. 20 pL Rh-intercalator (500 pM) was added to the PBMC solution (10 mL in RPMI with FBS) and was incubated at room temperature for 15 minutes. The PBMC solution was centrifuged (5 min, 400 x g) and the supernatant was removed. The PBMC pellet was washed with 10 mL Maxpar PBS and centrifuged again (5 min, 400 x g). The supernatant was removed and the cell pellet was resuspended in desired volume of CSB to achieve ca. 3 million cells per and the mixture was incubated at room temperature for 10 min.Staining protocol of PBMCs.

[0236] PBMC solution (50 pL) was added to a Maxpar cocktail (50 pL) in a centrifuge tube (2 mL) and vortexed gently for 30 minutes at room temperature. The cells were washed with CSB media (1 mL) and centrifuged (5 min, 400 x g). The supernatant was removed and the cell pellet was racked three times to loosen and disperse the cell pellets. CSB media was added to the dispersed cell pellet and the washing procedure was repeated.

[0237] PBMCs were fixed by addition of formaldehyde in PBS (1.6 %, 1 mL) into each tube and incubated for 10 minutes at room temperature. The cells were centrifuged (5 min, 800 x g) and the supernatant was removed. Ir DNA intercalator (500 pM, 2.5 pL) was added to 10 mL fix & perm cell fixation and permeabilization buffer to afford a final concentration of 125 nM. The Ir DNA intercalator buffer solution (1 mL) was added to each tube containing PBMCs and was vortexed for 2 seconds and incubated overnight at4 °C in the fridge. CSB media (1 mL) was added to each sample tube and centrifuged (5 min, 800 x g). The supernatant was removed and the pellet was washed again by repeating the procedure with CSB media (2 mL). The samples were then further washed with Maxpar cell acquisition solution (2 mL), gently vortexed and centrifuged down (5 min, 800 x g). The supernatant was discarded, and the cell pellet was washed once again with Maxpar (2 mL). EQ4 beads (1X, 1.5 mL CAS + beads solution) were added to each sample tube. The cell concentration was adjusted to 1 x 106 cells / mL and then run on the Helios instrument for data acquisition.Tissue stainingPreparation of tissue sections via deparaffination and antigen retrieval

[0238] To prepare tissue sections, the slides were baked for 2 hours at 60 °C, then at 96 °C during the baking step. Prior to dewaxing, 40 mL of antigen retrieval solution (diluted 10X to 1X) was inserted in conical tubes and are heated at 96 °C. The slides were dewaxed in xylene for 20 minutes, then were hydrated with descending grades of ethanol from 100% to 70% for 5 minutes each. The slides were washed with Maxpar Water for 5 minutes in a Copling jar gently agitated on an orbital shaker. The slides with tissues were inserted into preheated antigen retrieval (AR) solution and incubated for 30 minutes. Following incubation, the antigen retrieval solution and slides were cooled to 70 °C by monitoring the temperature of the AR solution. The slides were washed with Maxpar Water for 10 minutes in a Copling jar gently agitated on an orbital shaker. The slides were then washed with Maxpar PBS for 10 minutes with gentle agitation and the sample is encircled with a PAP pen.Staining tissue sections with Pt polymer plus Maxpar antibody cocktail

[0239] A blocking solution with 3% BSA was prepared in Maxpar PBS for 45 minutes at room temperature in a hydration chamber, diluted from a blocking solution of 10% BSA freshly made from powder. An antibody cocktail was prepared using the Pt polymer plus Maxpar antibodies diluted according to the technical data sheets for the recommended dilution ranges for individual antibodies. Pt polymer plus antibodies were spun at 13,000 x g for 2 minutes and top of the tube is pipetted to avoid antibody aggregates. A small volume of individual antibodies was added into a larger volume of 3% BSA in MaxparPBS diluent. Volume of the antibody cocktail was brought to a final volume of 0.5% BDA in Maxpar PBS and the final volume depends on the size and location of the tissue sections and the number of slides determined empirically. The antibody cocktail was stored on ice and used within 1 to 2 hours of preparation. Slides were placed in a hydration chamber and the antibody antibody cocktail was pipetted into the section. The slides with antibody cocktail were incubated overnight at 4 °C in a hydration chamber. The slides were washed in 0.2% Triton™ X-100 in Maxpar PBS for 8 minutes with slow agitation in Coplin jars, repeated as needed. The tissues were stained with Intercalator-lr in Maxpar PBS (300-500 pL / section for a 20 mm2section of 1:400 solution) for 30 minutes at room temperature in a hydration chamber. The slides were washed in Maxpar Water for 5 minutes with gentle agitation and then air-dried for at least 20 minutes at room temperature.Determining the number of Pt per polymer

[0240] To determine the number of Pt atoms per polymer, the mass concentration of Pt metal was measured parts per million (ppm) ICP-OES, denoted as CPt, ppm. A stock solution of Pt polymer of 10 mg / mL in ultrapure water was prepared.

[0241] In the Pt-polymer, one chloride ligand per Pt metal was assumed. The total molar mass of the sample (MPt-Pol) (eq. S1) was estimated by calculating the sum of the chelated Pt metal part, which was calculated by the number of Pt atoms per polymer chain (NPt) multiplied by the sum of the molar mass of Pt (195 g / mol) and the molar mass of chloride (35.45 g / mol), plus the molar mass of the polymer (MnPol, 33,000 g / mol).MPt-poi= Nptx (195 + 35.45)(^ / moZ) + Mnpol(eq. S1)

[0242] The total moles of Pt-polymer (Molpt-Poi) was obtained by dividing the mass of the polymer containing Pt (masspt-poi, 3 mg) by the total molar mass of the sample Mpt-poi. The total mass of Pt metal in the sample was calculated by multiplying Mol pt-Poi by Npt and the molar mass of Pt (195 g / mol). This value should be equal to the total mass of Pt measured from ICP-OES in the stock solution (300 pL), denoted as Cpt (ppm) multiplied by the volume of the stock solution (volwater, 300 pL) (eq. S2).CPt(ppm) x Volwater= Nptx MolPt-poiX 195 g / mol = NptXm°SSpt po1X 195 g / mol =K K MPt- polNvPtx - NptX -( -195+35.45) g / m -ol+33,000 g / mol x 195 g / mol ( \eq ~i. S2) / Rearranging the equation:M'1'ipol'’'Cpt ppm xVolwaterLmassPt_pOimgx 195 g / mol-Cptppmx (195+35.45) g / molxVolwaterL Determining the number of azides per polymer

[0243] Concentration of the measured Cy5 dye in the DBCO-Cy5 modified polymer was determined UV-vis spectroscopy at A649nm and from the Beer-Lambert equation:[dye] = (eq. S4)I X C649nmThe concentration of the polymer was determined by the following equation:[polymer] =(eq S5)^n, polymerBy combining eq. S4 and eq. S5, the number of dyes per polymer can be expressed with the following equation:^649 nmdye_ Myel _ I XS649 nm / QQ gg\ polymer [polymer]cpoiymer(m3 / mL^ \ / Mn, polymerwhere A649nm is the measured absorbance of the DBCO-Cy5 modified polymer, scys is the molar extinction coefficient of Cy5 (271,000 M’1cnr1, Lumiprobe), cPoiymer(mg / rnL) is the concentration of polymer used for the UV-vis measurement, I is the path length of 1 cm, and Mn, polymer is the number average molecular weight of the polymer and was calculated using the degree of polymerization (determined from1H NMR) of the polymer multiplied by the molar mass of each pendant repeat unit. The polymer carries multiple Pt ions per polymer chain that contributes to the number average molecular weight of the polymer. The number of Pt metal ions per polymer is determined by ICP-OES, and assuming 1 ligand (all Cl or all GSH after GSH treatment), an accurate estimate of the polymer average molecular weight can be determined.The polymer concentration was determined by accurately weighing (1.00-2.00 mg) on a microbalance and preparing a stock solution by dissolving the polymer in a known volume of ultrapure water. This provided a polymer stock solution in units of mg / mL.

[0244] The stock solution was diluted to achieve an absorbance at A649nm to be under a value of 1.0 a.u. Using the dilution factor, the polymer concentration used to measure the UV-vis absorbance could be calculated. This value in mg / mL could be converted to mmol / mL by dividing the mass by the average molecular weight of the polymer. The average molecular weight was accurately calculated using the degree of polymerization (determined from1H NMR) of the polymer multiplied by the molar mass of each pendant repeat unit. The polymer carried multiple Pt ions per polymer chain that contributes to the number average molecular weight of the polymer. The number of Pt metal ions per polymer was determined by ICP-OES, and assuming one ligand (all Cl or all GSH after GSH treatment), an accurate estimate of the polymer average molecular weight could be determined.Table 14. Maxpar Antibodies and their metal isotopes that made up the 10-plex assay for SMC.Antibody (anti-) Clone MetalCD3 UCHT1170ErCD14 RMO52160GdCD33 WM53158GdCD4 RPA-T4145NdCD66b 80H3152SmCD16 3G8148NdCD56 NCAM16.2163DyCD45 HI3089YCD20 2H7147SmCD8a RPA-T8162DyExample 6

[0245] A polymer mass tag with Hg2+bound to the pendant DPA chelators was prepared. Ligand exchange of the chlorides in the DPA-HgCl2 complex with a series of monothiol or vicinal dithiol ligands was examined. The monothiol derivatives led to a loss of H2,3-dimercapto-1 -propanol (DMP) and 2, 3-dimercapto-1 -propanesulfonic acid sodium salt monohydrate (DMPS) led to polymers II-5 and II-7 respectively in which most of the Hg atoms were retained. Evaluation of the NSB of the Hg polymers showed that with asuspension of PBMCs there was a low level of NSB for polymer 11-5 and very low NSB for polymer 11-7. After conjugation of both polymers to anti-CD8a Abs, the conjugates were incorporated with Maxpar reagents into an 11 -plex assay with PBMCs. Polymer ll-5-CD8a showed specific binding to CD8+T-cells, generating a202Hg signal with high specificity. Polymer ll-7-CD8a showed no202Hg signal, reflecting a potential loss of Hg2+during the Ab conjugation step. These results represent an important step forward toward the creation of useful Hg mass tags for MC.Preparation of a Hg metal-chelating polymerPolymer synthesis.

[0246] The synthesis of the polymer backbone was followed by pendant group modification. The overall strategy and early synthesis details followed the same route as in Examples 1 -5.9First, poly(pentafluorophenyl acrylate) (PPFPA) was prepared by RAFT polymerization. The degree of polymerization (DP) of the polymer was determined to be 22 using1H NMR end-group analysis (FIG. 32). Subsequently, a lysine-substituted DPA was grafted onto the PPFPA backbone via aminolysis of the pendant pentafluorophenyl esters with the primary amine of the lysine-DPA, forming amide bonds (FIG. 33 for1H NMR). A water-soluble polymer was attached to the -COOH group of the lysine-DPA pendant group to enhance solubility.9A mixture of PEG24-amine and azido-PEG23-amine was coupled to the carboxylic acid groups on the lysine-DPA, yielding PEGylated polymer 1-1, whose structure is presented in Scheme 11. The azide group at the end of the PEG23-pendants enable antibody (Ab) conjugation using Abs bearing reactive dibenzocyclooctyne (DBCO) groups.

[0247] The extent of PEG grafting was determined by1H NMR (FIG. 34. ca. 0.96 total PEG [PEG24 and N3-PEG23] per polymer), while the dispersity (D = 1.4) of the polymer was assessed by size-exclusion chromatography (SEC). To determine the mean number of azide groups on polymer 1-1, they were reacted with DBCO-Cy5 via strain promoted azide-alkyne chemistry (SPAAC). Unreacted DBCO-Cy5 was removed by spin filtration. The azide content was determined indirectly by measuring the Cy5 absorbance by UV-vis spectroscopy. By end group analysis (1H NMR), a mean degree of polymerization DPn= 22 ( / WnNMR= 33 000) was determined. The polymer was treated with excess DBCO-Cy5, purified by spin filtration, and analyzed by UV-vis spectroscopy to determine the mean number of Cy5 dyes per polymer ( max = 649 nm, £649 = 271,000 M’1crrr1, Lumiprobe). Assuming that each pendant azide group reacted with one DBCO-Cy5, it was calculated that there were three pendant azide groups per polymer.Scheme 12. Synthesis of polymer derivatives prepared by treatment of Hg polymer 11 with different thiol-containing ligands that are either monodentate or bidentate ligands, i. HgCl2, water, room temperature, 24 h. ii. Thiol-containing ligand, water, 2 h.Introducing Hg2+to obtain Polymer 11.

[0248] Polymer 1-1 was treated with HgCl2 (natural abundance, 3 eqv. per pendant DPA) to obtain polymer 11, (Scheme 12) containing pendant Hg-DPA complexes. The Hg content of polymer 11 was quantified using ICP-OES. Calculations show that approximately 19 out of 22 DPA units were coordinated with Hg2+ions (Table 15). Polymer 11 was characterized by1H NMR (FIG. 35) and a downfield chemical shift of the aromatic protons of the pyridyl groups of DPA was observed, from (5 6.8 - 8.6 ppm) before metalation to (5 6.9 - 9.2 ppm) after metalation with HgCl2. The peaks in the spectrum of polymer 11 also exhibit noticeable peak broadening compared with the well- defined peaks in the spectrum for polymers II-2 to II-7. Further characterization by SEC(FIG. 36) shows polymer I-3 has a similar retention volume as that of polymer I-1, however a slight shift toward lower retention volume can be observed. Lastly, the polymer was characterized with electrophoretic mobility studies (FIG. 37). Electrophoretic mobility provides a measure of the charge of DPA polymers, and enables us to see if metalation of the polymer and subsequent ligand exchange affect the overall charge of the polymer. These measurements show that the Hg polymer 11 has an electrophoretic mobility ca. +3 pm.cm / V.s, indicating that it is a positively charged species, compared with polymer I-1, which has an electrophoretic mobility ca. 0 pm.cm / V.s.

[0249] Anderegg et al.28identified a metal complex with a high stability constant (log K = 22.3) of the form HgL2, where L represents DPA. If two DPA were required to bind each Hg2+, then the polymer with DP = 22 would bind only 10 or 11 metal ions. The fact that 19 Hg / polymer were found suggests a 1:1 Hg / DPA complex in each pendant group. There are other reports of triamine-DPA complexes prepared with HgCl2.28–31For instance, it was reported that a DPA derivative metalated with HgCl2formed a [Hg(DPA)Cl2HgCl2] complex, where the Hg bound to DPA contains two chloride ligands bridged by another HgCl2molecule.31It was demonstrated that Hg-DPA complexes with unsymmetrical bis(pyridyl) ligands can adopt the structure [Hg(DPA)Cl2]n, with bridging interactions between adjacent Hg2+ions forming a supramolecular network.24These studies support that the polymer contains a 1:1 Hg / DPA complex with two chloride ligands. This is the structure depicted for polymer 11 in Scheme 11.Ligand exchange with thiol ligandsLigand exchange reaction of Hg polymer 1 with GSH.

[0250] In previous examples with a Pt / DPA polymer based on polymer I-1, a nucleophilic substitution reaction was used with the thiol of glutathione (GSH) to replace the Pt-CI bond of the metal complex. It was found that this treatment led to a strong reduction in non-specific binding (NSB) interactions with PBMCs as assessed by MC. The Hg-CI bonds in polymer 11 should also be susceptible to this type of reaction. Hg(ll) is highly thiophilic and the thermodynamic stability of mercury-thiol complexes are high.3233Thus the same approach was used with polymer 11. The ligand exchange was performed by reacting Hg polymer 11 with GSH (2 eqv.) in water for 2 hours at room temperature,followed by purification using spin filtration with an Amicon Ultra-4 10 kDa MWCO spin filter (2700 x g, 15 min, 5 times) to remove low molecular weight products.

[0251] To assess the impact of ligand exchange on the Hg content in the Hg-DPA polymer 11, ICP-OES was used to measure the Hg content before (polymer 11) and after ligand exchange (polymer II-2). The Hg metal content, expressed as the number of Hg ions per polymer, was determined using the following equations. The Hg content in the polymer expressed in units of mg Hg per mg polymer was also calculated. The results are presented in Table 15. Treatment of polymer 11 with GSH led to a significant loss of Hg from polymer II-2, with approximately five Hg2+ions remaining per polymer after ligand exchange.

[0252] The ratio of mg Hg per mg polymer was calculated from ICP-OES measurements from a known concentration of polymer. The concentration of Hg measured in the polymer (CHg, ppm) was determined by ICP-OES on a stock solution diluted by a factor of zdil. The concentration of polymer stock solution (Cpolymer(mg / mL)) was determined by accurately weighing the mass of polymer on a microbalance and dissolving in a known volume of ultrapure water with a micropipette.(mg Hg per mg polymer) = (eq. S7)Or similarly for other metals, such as PtCPt,ppm(mg Pt per mg polymer) =Cpolymer(mg / mL)(eq. S8)

[0253] The mean number of Hg ions per polymer (NHg) was calculated from ICP-OES measurements on a known quantity of polymer. The total molar mass of the Hg-polymer (MnHg+Pol) as a function of NHg.MnHg+pol(g / mol) = Mnpol+ NHg× (MHg+ X × Mligand) (eq. S9)where / WnPoi is the molar mass of the polymer without Hg (33,000 g / mol), MHgis the molar mass of Hg (202 g / mol), X refers to the number of ligands per Hg-DPA complex (for chloride and monodentate ligands, it was assumed that there are two per Hg-DPA complex, for bidentate ligands, it was assumed that there is one ligand with two thiol groups bound to the each Hg-DPA complex) and Mgand is the molar mass of the ligand.The mass fraction of Hg in the polymer can be expressed as:fHg= (NHg× MHg) / MnHg+pol(eq. S10)This mass fraction is equal to the measured concentration from ICP-OES (CHg, ppm) divided by the stock solution concentration:-massHg+pol / volwaterBy combining these equations and solving for / Hg:NHg= (Mnpol× CHg× volwater) / (massHg+pol× MHg− CHg× volwater× (MHg+ X × Mligand)) (eq. S12)massHg+pol× MHg− CHg× volwater(MHg+ X × Mligand)To achieve a concentration (ppm) of Hg within the calibration curve, the stock solutions were diluted by a factor of zdil. This gives the following equation:NHg= (Mnpol× (CHg× zdil) × volwater) -massHg+pol× MHg− (CHg× Cdil× volwater) (MHg+ X × Mligand)

[0254] In a typical experiment, ca. 3 mg of Hg-polymer (massHg+poi) were weighed on a microbalance and dissolved in 300 pL of ultrapure water (volwater). This created a stock solution with a concentration of 10 mg / mL. The stock solution was diluted by a factor of 2000 to achieve a concentration between 0.2 to 1.5 ppm. The mass concentration of Hg metal of the diluted sample was determined by ICP-OES in parts per million (CHg, ppm).

[0255] For example, for chloride ligand (MCl= 35.45 g / mol), it is assumed that there were X = two Cl atoms per Hg-DPA complex. For a value of CHg = 0.5 ppm measured by ICP-OES, it can be calculated thatNHg= (33,000 g / mol × 0.5 ppm × 2000 × 300 μL) / (3.0 mg × 202 g / mol − 0.5 ppm × 2000 × 300 μL(202 g / mol + 2 × 35.45 g / mol)) ≈ 19 (eq. S14)In this way it was calculated that there are on average 19 Hg ions per polymer molecule.Ligand Exchange with cysteine and thioglycolic Acid.

[0256] Ligand exchange reactions with monodentate thiol ligands including cysteine and thioglycolic acid were performed on the Hg-DPA complex. These ligands were chosen based on their reported use in the literature for biological applications, Hg chelation therapy, and environmental remediation.20-22The ligand exchange reactions were carried out following the same procedure as described for GSH, and the resulting polymers (II-3to 11-7) were characterized by ICP-OES to determine the Hg content (Table 15). After treating polymer 11 with cysteine, the resulting polymer (polymer II-3) had a metal content of ca. one Hg2+ion per polymer, whereas treatment with thioglycolic acid (polymer II-4) had ca. two Hg2+ions per polymer. These two reactants led to even more loss of metal from the polymer than GSH. These results are consistent with a report by Wang et al.34who found that GSH showed slower kinetics in displacement reactions of Cl-Hg bonds compared to smaller monothiol species at thiol ligand to Hg(ll) ratios of 10:1. It is of note that Hg complexes are susceptible to ligand substitution with thiols.Table 15. Metal content determined by ICP-OES of Hg polymers before and after treatment with monothiol ligands. The values are calculated using equations in Example 6 to determine Hg content in the polymer with units of mg of Hg per mg of polymer, and to express the metal content in terms of Hg ions per polymer.Metal content by ICP- Metal per polymerOESSample Ligand (mg Hg / mg(# Hg ions / polymer of polymer)DP=22) Polymer 11aChloride 0.103 19 / 22 Polymer ll-2bGlutathione 0.03 5 / 22 Polymer ll-3bCysteine 0.007 1 / 22 Polymer ll-4bThioglycolic acid 0.01 2 / 22aChloride ligand is from the metalation with HgCl2bPolymers were modified by treatment with the corresponding ligandLigand exchange with bidentate thiol ligands.

[0257] In this section, dithiol ligands were examined to provide better kinetic stabilization of the Hg-DPA chelate on polymer 11.43-45Three bidentate ligands were studied, 2,3-dimercapto-1 -propanol (DMP), meso-2,3-dimercapto-1 -succinic acid (DMSA), and 2,3-dimercapto-1 -propanesulfonic acid sodium salt monohydrate (DMPS). These ligands were selected based on their reported use in biomedical applications, such as Hg chelators for the treatment of Hg poisoning.18’19

[0258] The ligand exchange reaction was carried out in the same way (2 eqv. of ligand thiol per Hg) as described for the monothiol ligands. However, due to the poor solubility of DMSA in water, the reaction between polymer 11 and DMSA was performed in phosphate buffer (pH 8.0). The metal content was analyzed by ICP-OES and calculated using equations in Example 6. The results are tabulated in Table 16. The polymer treated with DMSA (polymer II -6) in phosphate buffer showed very low levels of Hg2+remaining, with a value of 0.5 Hg per polymer. In contrast, two of the polymer samples treated with the bidentate ligands, DMP (polymer II-5), and DMPS (polymer II-7) retained most of the Hg, with values of 14 / 22 and 18 / 22 Hg ions per polymer, respectively.1H NMR of polymer II-5 (FIG. 38) and polymer II-7 (FIG. 39) both show a narrowing of the aromatic peaks (ca.δ 6.8 – 8.7 ppm) compared with polymer 11 (FIG. 35, δ 6.9 – 9.2 ppm). The polymers II-5 and II-7 were further characterized by SEC (FIG. 36) and show very slight shifts to lower retention volume compared with polymer I-1 and polymer 11. Electrophoretic mobility measurements (FIG. 37) showed a shift upon ligand exchange for both polymers II-5 and II-7 to ca -1 pm.cm / V.s and 0 pm.cm / V.s, respectively, from the +3 pm.cm / V.s value for polymer 11. Thus, polymers II-5 and II-7, after ligand exchange, are both close to no net electrical charge in aqueous solution.

[0259] Based on the results, the improvement in Hg2+ion retention of the polymer after treatment with DMP and DMPS was noted. The metal content was still sufficient for MC applications. Thus, it was proceeded to test both polymer II-5, and II-7 to evaluate their NSB to PBMC samples studied by SMC.Table 16. Metal content determined by ICP-OES of Hg polymers after treatment with dithiol ligands. The values are calculated using equation S7 to determine Hg content in the polymer with units of mg of Hg per mg of polymer, and with equation S14 to express the metal content in terms of Hg ions per polymer.Metal content by ICP- Metal per polymerOESSample Ligand (mg Hg / mg(# Hg ions / polymer of polymer)DP=22) Polymer ll-5aDMP 0.07 14 / 22 Polymer ll-6a bDMSA 0.003 0.5 / 22Polymer ll-7aDMPS 0.08 18 / 22aPolymers were modified by treatment with the corresponding ligandbLigand was not soluble in water and 0.2 M phosphate buffer (pH 8.0) was used insteadEvaluating the non-specific binding of Hg polymersStaining of PBMCs with Hg polymers.

[0260] In these experiments, a suspension of PBMCs is treated with a polymer solution (polymer II-5 or II-7) along with a cocktail of Abs labeled with Maxpar reagents. Here the Hg polymers have no targeting groups to bind to the cells, and thus any observed signal corresponding to an Hg isotope (e.g,202Hg) is indicative that the polymer is binding or sticking non-specifically to the cells. The Maxpar reagents provide a 10-plex assay that allows one to identify B-cells (CD45+; CD20+), all T-cells (CD45+; CD3+), and non-B, T-cells (CD20’; CD3_) in the sample, as well as associate these cell types with any m / z 202 signal detected. These Abs and their corresponding metal isotope identifier are described in Table 17. It is also important that a new mass tag reagent be compatible with other reagents in the assay and not interfere (i.e. by transmetalation) with their performance. A control sample of PBMCs stained with only the Maxpar Ab cocktail (10-plex assay) was used as to determine background signal for the202Hg signal as none of the Maxpar reagents contain any Hg and should not exhibit any Hg signal. As a general metric, any metal counts above five, is indicative of NSB. This experiment also provides information about whether the new MCP interferes in any way with the ability of the Abs in the Maxpar cocktail to recognize their target antigens.Table 17. Maxpar Antibodies and their metal isotopes that made up the 10-plex assay for SMC. Table reproduced from Ref Y.Antibody (anti-) Clone MetalCD3 UCHT1170ErCD14 RMO52160GdCD33 WM53158GdCD4 RPA-T4145NdCD66b 80H3152SmCD16 3G8148NdCD56 NCAM16.2163DyCD45 HI3089YCD20 2H7147SmCD8a RPA-T8162Dy

[0261] The results of the NSB experiments at a titer of 5 pg / mL of MCP are shown as histograms of Hg signal in FIG. 40 and the corresponding Hg counts detected by the mass cytometer are tabulated in Table 18. Polymer II-5 shows greater Hg signal with median Hg counts were ca. 6, while polymer II-7 shows low signal with Hg counts ca. 1. From these results, it was noted that there were some levels of NSB (>5 counts) for polymer II-5, and low NSB for polymer II-7, with specific binding tests for both II-5 and II-7, anticipating that the NSB of II-5 would not significantly hinder cell population discrimination once conjugated to anti-CD8a Abs.Table 18.202Hg signal counts from PBMCs stained with polymer 2d and 2f in a nonspecific binding study at titer of 5 pg / mL.MedianMean202Hg signal202HgSample SignalAll All T CD8a T CD4 T AllB cellsPBMCs cells cells cells PBMCs Polymer7.7 8.8 7.4 7.5 7.3 5.6 II-5Polymer6.0 1.2 8.3 10.0 7.7 0.3 II-7Maxpar0.3 0.3 0.3 0.3 0.3 0.0ControlAb conjugation and specific binding tests with Hg polymer-anti-CD8a conjugatesConjugation of Hg polymers to Abs.

[0262] Polymers II-5 and II-7 were conjugated to an anti-CD8a Ab. In these experiments, the anti-CD8a Abs were labeled with DBCO by tris(2-carboxyethyl)phosphine reduction of disulfide bonds on the Ab, followed by thiol-maleimide coupling. The DBCO groupserves as the reactive functional group for attaching the Ab to the Hg polymers by a strain-promoted alkyne-azide coupling (SPAAC) reaction with the pendant azide groups on the polymer (See Scheme 11 for structure of the polymer). The Ab conjugates are denoted as polymer ll-5-CD8a and ll-7-CD8a, respectively.Staining of PBMCs with Hg polymer-Ab conjugates.

[0263] Polymer ll-5-CD8a and ll-7-CD8a were independently mixed with Maxpar Ab cocktail to prepare two solutions for an 11 -plex MC experiment and used to stain separate PBMC samples. In this set of experiments, the Maxpar Ab cocktail control sample also has162Dy-CD8a in place ofnatHg-CD8a to allow direct comparisons on the separation of CD8 T cells (CD3+; CD8+). PBMCs were stained in a similar manner as described above in the NSB experiments in Example 3. The results of these functional tests are presented below.

[0264] The MC results were gated to separate and distinguish cell types in PBMCs. The biaxial scatter plots were first gated to identify all PBMCs (CD45+; CD66’). Further gating of the cells was performed by separating B cells (CD20+), all T cells (CD45+; CD3+), and non-B, T cells (CD45+; CD20−; CD3−). Gating on all T cells led to the biaxial scatter plots (FIG. 41A-G) plotted as145Nd_CD4 (y-axis) vs202Hg_CD8a (x-axis) to distinguish between CD4 T cells (CD3+; CD4+) and CD8 T cells (CD3+; CD8+). The histograms showing the mean202Hg counts for CD8a T cells, CD4 T cells, and non-CD4, non-CD8 T cells are shown in FIG. 41 H and 411 and are tabulated in Table 19.Table 19. Metal signal counts obtained from PBMCs stained with Maxpar 10-plex antibody cocktail plusnatHg-polymer-anti-CD8a Abs. The Maxpar control is used to stain PBMCs in a similar manner, however thenatHg-polymer-anti-CD8a Ab was replaced with162Dy-anti-CD20 Ab. The concentrations for cell staining are low titer: 0.56 pg / mL, med titer: 1.67 pg / mL, and high titer: 5.0 pg / mL.Mean202Hg Signal Signal-to- Signal-to- Sample CD8a CD4 Non- noise (CD8 noise (CD8TiterName T T T T cells / CD4 T cells / Non- Cells cells cells T cells) T cellsLow 24.3 0.6 4.5 40.1 5.4 Polymer ll-5aMed 22.3 0.8 4.4 27.4 5.1High 25.4 1.7 6.3 14.8 4.1Low 6.0 0.3 0.4 18.3 15.0 Polymer ll-7aMed 6.6 0.4 0.4 18.1 15.9High 6.3 0.4 0.4 15.8 14.7 MaxparControl ** 1325 2.4 15 549 86 (162Dy-anti- CD8a)ba.202Hg channelb.162Dy channel** Maxpar titer according to the protocol optimized by Standard BioTools Inc.

[0265] From these biaxial scatter plots, 4 separate quadrants are distinguished based on the separation of the cells. These quadrants provide information on the percent population of cells (comers of each plot) and the values are also tabulated in Table 20. In the upper left quadrant, CD4 percent populations T cells are identified by the145Nd signal. In the lower right quadrant, CD8a is identified by162Dy in the Maxpar control sample (FIG. 41 A), and by202Hg for the rest of the samples (FIG. 41 B-G). The lower left quadrant shows non-CD4 and non-CD8 T cells with low signal from both145Nd (y-axis) and162Dy or202Hg (x-axis), and lastly the upper right quadrant shows double positive signal characterized by high levels of145Nd and162Dy or202Hg.Table 20. Population percentages of different cell types, T cells, B cells, and non-B / T cells of PBMCs stained with the Maxpar 10-plex Ab cocktail plusnatHg-anti-CD8a analyzed by SMC. The values are obtained from the 4 separated quadrants in the biaxial scatter plots of145Nd_CD4 vs202Hg (FIG. 2, main text). For the Maxpar control, CD8 T cells were detected with162Dy-anti-CD8a Ab. The concentrations for cell staining are low titer: 0.56 pg / mL, med titer: 1.67 pg / mL, and high titer: 5.0 pg / mLDoubleCD8 T cells CD4 T cells Non-T cells positiveSample Titer (Lower-right (Upper left (Lower left signal quadrant) quadrant) quadrant) (Upper-right quadrant) Polymer 11-5 Low 30.1 64.9 4.0 1.0Med 29.4 64.5 4.4 1.8High 29.6 62.3 4.6 3.6 Low 0.3 65.6 34.1 0.1 Polymer 11-7 Med 0.4 65.8 33.7 0.1High 0.3 66.3 33.4 0.1 Maxpar**Control32.8 65.0 1.4 0.9(162Dy-anti- CD8a)a.202Hg channelb.162Dy channel** Maxpar titer according to the protocol optimized by Standard BioTools Inc.

[0266] From these results, it was seen that polymer ll-5-CD8a was able to separate the CD8a T cells and CD4 T cells (FIG. 41 B-D). The binding of polymer ll-5-CD8a also shows good specificity to the target CD8a T cells, with relatively low levels of non-specific binding to other cell types in the PBMCs, as indicated by the histograms in FIG. 41 H. A look at the T-cell percent populations identified by polymer ll-5-CD8a shows 30.1 % CD8 T cells, 64.9 % CD4 T cells, and 4.0 % non-CD4, non-CD8 T cells. For the Maxpar control these values were 32.8 % CD8 T cells, 65.0 % CD4 T cells, and 1.4 % non-CD4, non-CD8 T cells. Comparing the percent populations detected by polymer ll-5-CD8a shows that the signal for CD4 T cells is similar, whereas CD8 T cells is slightly lower, and smaller for non-CD4, non CD8 T cells when compared to the values from the Maxpar control sample. This difference in percent populations can be attributed to the low202Hg signal intensity detected for polymer ll-5-CD8a at all titers (FIG. 41 B-D), resulting in a less than desirable separation of CD8a T cells with non-CD8, non-CD4 T cells. It was further noted that the signal also did not increase with an increase in titer, suggesting that the202Hg signal had already reached a maximum even at the lowest titer of 0.56 pg / mL.

[0267] Polymer ll-7-CD8a, prepared with the ligand DMSA, showed no202Hg signal in the functional tests (FIG. 41 F) despite the higher Hg content of the unconjugated polymer II-7 compared to 11 -5.

[0268] Overall, this work shows the development of a Hg mass tag probe for MC, and also demonstrates the importance of ligands on the metal complex to achieve both low levels of NSB and high specificity to PBMCs.Example 7

[0269] In addition to other ligands presented above, other smaller sulfur-containing small molecules were considered as potential ligands to replace the Pt— Cl bond in Pt polymers. Sulfur-containing ligands are known to bind readily with Pt(ll) complexes and form stable Pt-S bonds. Chemoprotective agents with strong S-containing nucleophiles such as GSH, thiourea, sodium thiosulfate (thiosulfate), and diethyldithiocarbamate (DDTC) are capable of displacing these strong Pt-S bonds. Greater nucleophilic substitution rate constants were reported for thiourea, thiosulfate, and diethyldithiocarbamate in comparison to GSH. Greater nucleophilic substitution rate constants of these ligands suggest that they would offer more kinetic stability to the metal complex. Without being bound to theory, it was hypothesized that increased kinetic stability will deter other sulfur donors inside of the cell from binding to the polymer, thus minimizing NSB. Also smaller molecules may result in less stearic hindrance and increased Ab binding affinity of conjugates..

[0270] Three sulfur-containing small molecules were tested: thiourea, thiosulfate, and DDTC as potential ligands to replace the Pt— Cl bond in these MCPs. The ligand exchange with thiourea led to an insoluble polymer that was not suitable for cell studies in biological media. Ligand exchange with diethyldithiocarbamate resulted in significant metal leaching. In contrast, polymers after ligand exchange with thiosulfate remained water-soluble, and minimum metal leaching was detected. The thiosulfate polymers were incubated with PBMCs, and ultralow NSB was obtained in the SMC measurements. After Ab conjugation, specific binding studies were carried out by SMC. The thiosulfate-exchanged polymer demonstrated good signal resolution even at low titers. More importantly, an increased signal-to-noise (S / N) ratio was observed for the thiosulfate-exchanged polymer compared to the GSH-exchanged polymer. Similarly, in IMC experiments, the thiosulfate-exchanged polymer displayed a strong image contrast that has the potential to offer a stronger signal than the GSH-exchanged polymer.Ligand exchange with sodium thiosulfate, thiourea and DDTC

[0271] Polymer synthesis and metal loading with Pt was conducted as previously described to lead to intermediate polymer 10. Ligand exchange was then conducted as follows, as shown in Scheme 12Scheme 12: Synthetic Scheme for DPA Polymers Modified with PEG and Metalation of Polymer with Pt with Various Sulfur-Based Ligands

[0272] Polymer 10 (3 mg) was dissolved in 300 pL water in a 4 mL vial. Sodium Thiosulfate (STS) (1.77 mg, 5.0 equiv per pendant) was added to the solution and stirred at room temperature for 2 h. The reaction mixture was washed via spin filtration over Amicon Ultra-4 mL 10 kDa MWCO spin filters (5 times, 2700g, 15 min). The retentate was lyophilized overnight to afford a light-yellow polymer (polymer ) (3.2 mg). The procedure was adapted by changing the identity of the ligand to prepare samples with glutathione (polymer 11-1), thiourea and DDTC ligand exchange.Polymer-Antibody Conjugation and Cell / Tissue Staining Experiments

[0273] Modification of antibodies with a DBCO linker for coupling with polymers 11-1 and II-8 followed the standard protocol provided above in Example 4. The protocol for thepreparation of PBMC suspensions employed here and for cell staining experiments are also described above in Example 3. Tissue staining of FFPE sections was performed using a protocol that involves six main steps in the workflow: (1) dewaxing in xylene, (2) hydration in ethanol, (3) antigen retrieval (AR) in AR buffer, (4) blocking with BSA (3%), (5) primary incubation via dilution of Abs in PBS / BSA followed by staining overnight, and (6) secondary incubation using an iridium (lr) intercalator stain.Suspension Mass Cytometry (SMC) and Imaging Mass Cytometry (IMC)

[0274] Mass cytometry studies were conducted in a Standard BioTools Helios instrument in Markham, Ontario, Canada. SMC experiments were done using a Helios CyTOF (cytometry by time-of-flight) system. The data were collected in the dual counting mode and processed with Cytobank software. IMC experiments were run by using a Hyperion Imaging system. Data were collected in dual counting mode and processed with MCD Viewer software.Results

[0275] For the new polymer system to be applicable in SMC and IMC, it must have minimal NSB and to have non-sterically hindered and fast reacting functional groups for conjugation to its target antibody. To evaluate NSB of polymer II-8, PBMC samples were first stained with a Maxpar antibody cocktail along with polymer II-8. The NSB signal of polymer II-8 measured at m / z was compared to that of the Maxpar control (treated with Maxpar antibody cocktail shown in Table 2), where no signal would be expected. Results from NSB cell study tests showed that polymer II-8 had a mean Pt signal of 0.29. A sample displaying a Pt signal of less than 5 is considered to have low NSB. Such low NSB exhibited by the thiosulfate-exchanged polymer is comparable to the performance of the GSH polymer 11-1.

[0276] Electrophoretic mobility tests of the polymer were conducted before and after ligand exchange. As seen in FIG.42, polymer 10 has an electrophoretic mobility of +1.0 ± 0.2 pm cm / (V s) after metal loading. This implies that the polymer is positively charged. After ligand exchange with GSH, the electrophoretic mobility shifted to -1.5 ± 0.2 pm cm / (V s). After thiosulfate ligand exchange, the electrophoretic mobility was -1.3 ± 0.1 pm cm / (V s). Both sulfur ligands provide negative charge to the Pt complex,generating an anionic complex as a result. The electrophoretic mobility data indicate that the anionic charge is important for suppressing NSB.

[0277] Cell staining experiments with polymers 11-1 and II-8 were conducted. The Pt polymers were conjugated to a dibenzocyclooctyne (DBCO)-modified CD20 antibody via a SPAAC reaction. The polymer-Ab conjugates are referred as polymer ll-1-anti-CD20 and polymer ll-8-anti-CD20. PBMC samples were stained with a cocktail of antibodies along with polymer ll-1-anti-CD20 or polymer ll-8-anti-CD20. The ability for the Pt conjugates to distinguish T cells, B cells, and non-B, T cells is used to gauge the effectiveness of these MCPs in MC (FIG.43).

[0278] As shown in FIG.43, the signals for polymer ll-8-anti-CD20 at low (1.11 pg / mL), medium (3.33 pg / mL), and high titers (10.0 pg / mL) displayed good resolution and are comparable to the Maxpar control sample. The good separation even at low titer and using natural abundance Pt demonstrate the high specificity of the polymer II-8 anti-CD20.

[0279] To better analyze the data quantitatively, the ratio of specific binding Pt signals of B cells to the nonspecific binding Pt signals to non-BT cells was defined as S / N to illustrate the signal resolution. Polymer ll-8-anti-CD20 displayed improved resolution of signal compared to polymer ll-1-anti-CD20. It is worth noting that polymer 11-1 is loaded with monoisotopic196Pt, so in theory, it should depict higher signals in the 196-mass channel than the polymer II-8 with natural abundance Pt. For polymer ll-1-anti-CD20 and polymer ll-8-anti-CD20, the S / N ratio for the medium titer sample was 122.6 and 218.9, respectively. Although polymer ll-8-anti-CD20 showed lower absolute Pt counts than polymer ll-1-anti-CD20 due to the use of natural abundance Pt, it exhibits a significant increase in the S / N ratio due to less background noise. The same trend was also observed in S / N: (B cells / T cells) and S / N: (B cells / all CD3+cells)

[0280] A new batch of STS and GSH polymers with isotopically enriched Pt was prepared (195Pt and196Pt). The resulting polymers are denoted as polymer ll-1195Pt, polymer II-1196Pt, polymer ll-8195Pt, and polymer ll-8196Pt. In this set of experiments,195Pt polymers were conjugated to anti-CD4Ab and196Pt polymers were conjugated to anti-CD19Ab. Theresulting polymer-Ab conjugates were stored as solutions in Ab stabilizer medium at 4 °C for 3-4 weeks before being used for cell surface staining.

[0281] The195Pt polymer-anti-CD4 and196Pt polymer-anti-CD19 polymer-Ab conjugates were mixed with 10 other Maxpar reagents (shown in Table 2, but without CD4 conjugate) to make a 12-plex cocktail assay.

[0282] As shown in FIG.44 a-d, the signals for polymer II-8196Pt-anti-CD19 at all three titers displayed good resolution for identifying B cells (CD19+; CD3-) from T cells (CD19-; CD3+). The results are comparable to the Maxpar control sample. The CD19 signal intensity increased with higher titer but the S / N decreased. Notably, the highest S / N ratio (136.3), observed at the lowest titer, was comparable to that of the Maxpar reagent (167.8).

[0283] Moreover, the CD19 mean value for polymer ll-8196Pt-anti-CD19 was higher than that of polymer 11-1196Pt-anti-CD19 at the matched titers. Polymer ll-8195Pt-anti-CD4 also demonstrated a clear separation of CD4+ T cells from the CD8a+ Tcells, comparable to the Maxpar CD4 conjugates (FIG.44 e-h).

[0284] Quantitative analysis suggested that both CD4 counts and S / N values of polymer II-8195Pt-anti-CD4 were higher than those of polymer 11-1195Pt-anti-CD4 at matched titers.

[0285] This new set of experiments indicates the excellent stability of the materials for long-term storage as well as compatibility of the isotopically enriched Pt polymers with other Pt isotopes. Notably, these Pt polymers are also compatible with Hg polymers reported above. The ability to conjugate Pt polymers with three different antibodies underscores their wide-ranging utility across different targets.

[0286] MCPs can also be used in IMC to stain tissue samples. Polymers 11-1 and II-8 were conjugated to anti-CD45RA via the SPAAC reaction to make polymer ll-1-anti-CD45RA and polymer ll-8-anti-CD45RA, respectively. The Maxpar reagent166Er-anti-CD45RA was used for the control sample. Human tonsil tissue samples were used for IMC, and three regions of interest (ROI) were examined. All of the human tonsil tissue samples were stained with a Maxpar reagent antibody cocktail in a 5-plex assay (see Table 12) alongwith the respective anti-CD45RA mass tag reagents. The images obtained from IMC are shown in FIG.45, for three ROI images of the tissue section.

[0287] One method of qualitatively analyzing signal intensity to compare image quality is to calculate the maximum threshold values. This value is determined from the pixel of the image that contains the highest metal count and is calculated to be the 98th percentile of the metal count per pixel. The max threshold values for ROI1 FIG.45 show that polymer ll-8-anti-CD45RA produces signal 48% of Maxpar control while polymer ll-1-anti-CD45RA produces signal 62% of the control. In ROI2 and ROI3, the max threshold values are relatively lower, at 39 and 40% of the Maxpar control, respectively.

[0288] This result indicates that polymer ll-1-anti-CD45RA produces greater image contrast in comparison to polymer ll-8-anti-CD45RA. However, a natural abundance of Pt was used for polymer ll-8-anti-CD45RA. Mass tag would show an increased signal with monoisotopic Pt.

[0289] The image area and mean signal intensity were measured for each high and low signal region. The ratio of high-to-low signal in the grayscale image was calculated. A larger signal intensity ratio indicates a more distinct separation between regions of high and low intensity, resulting in greater image contrast. For ROI1, the Maxpar control sample had a signal intensity ratio of 1.40. Polymer ll-1-anti-CD45RA and polymer II-8-anti-CD45RA had similar signal intensity ratios of 1.29 and 1.27, respectively. In ROI2, the signal intensity ratio for polymer ll-8-anti-CD45RA was 1.0 compared to 1.3 for Polymer ll-1-anti-CD45RA and the Maxpar control. In ROI3, the signal intensity ratios were comparable to those observed in ROI2. While polymer ll-8-anti-CD45RA generally exhibits a lower signal intensity ratio, with monoisotopic Pt, the signal intensity was expected to increase. This demonstrates the ability of polymer ll-8-anti-CD45RA to produce good image contrast in an IMC.

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Claims

CLAIMS1. A compound of Formula Iwhereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci- Csalkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci- Cealkylphosphonate;n is an integer from 15 to 75, from which in 2 to 14 repetitions, each R is independently a solubility modifier selected fromwherein:y is an integer from 20 to 48,R3is a reactive functional group,and the remaining repetitions of R are each independently selected from:y'y’ is an integer from 20 to 48;owherein z is an integer from 10 to 75,andR1and R2are each independently a terminal group,t is an integer from 0 to 6, andone or more A are configured to coordinate to a soft metal selected from Pt, Re, Pd and Hg.

2. The compound of claim 1, wherein each A is independently a pyridine, a thiazole or an imidazole, each of which being substituted or unsubstituted.

3. The compound of claim 1 or 2, wherein each reactive functional group is independently a maleimide, a maleimide-thiol conjugate, an azide, a carboxylic acid, an amine, a fluorophenyl ester, a sulfotetrafluorophenyl electrophile, an aldehyde, an isothiocyanate derivative, a tetrazine, a trans-cyclooctyne or a combination thereof.

4. The compound of any one of claims 1 to 3, wherein one or more of the reactive functional groups is conjugated to a biomolecule.

5. The compound of claim 4, wherein the biomolecule is an affinity reagent.

6. The compound of claim 5, wherein the affinity reagent is an antibody.

7. The compound of any one of claims 1 to 6, for use as a metal polymer chelator (MPC).

8. The compound of any one of claims 1 to 7, wherein the one or more A are configured to chelate a soft metal selected from Re and Pt.

9. The compound of any one of claims 1 to 8, wherein the soft metal is a naturally occurring isotope or isotopically enriched.

10. The compound of any one of claims 1 to 9, wherein the soft metal is configured to be substituted with one or more solubility ligands being a thiol-based ligand.

11. The compound of claim 10, wherein the thiol-based ligand is glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2, 3-dimercapto-1 -propanesulfonate, or a combination thereof.

12. The compound of any one of claims 1 to 11, wherein each terminal group comprises, a linker, a spacer, a capping group, a reactive group, or a combination thereof.

13. The compound of claim 6, wherein the compound conjugated to the antibody is for use as a mass tag for targeting specific cells for detection in suspension mass cytometry (SMC) or imaging mass cytometry (IMC).

14. The compound of claim 13, wherein the mass tag is for use with further mass cytometry mass tags in a multiplex mass cytometry assay.

15. Acompound of Formula II:whereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci-Csalkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci-Cealkylphosphonate;n is an integer from 15 to 75, from which in 2 to 14 repetitions, each R is independently a solubility modifier selected fromywherein:y is an integer from 20 to 48,R3is a reactive functional group,and the remaining repetitions of R are each independently selected from:y’ is an integer from 20 to 48,iOwherein z is an integer from 10 to 75,andR1and R2are each independently a terminal group,n is an integer from 15 to 75,t is an integer from 0 to 12,v is an integer from 1 to 3;each M is a soft metal selected from Pt, Re, Pd and Hg, andeach Q is independently absent or a thiol-based solubility ligand.

16. The compound of claim 15, wherein each A is independently a pyridine, a thiazole or an imidazole, each of which being unsubstituted or substituted.

17. The compound of claim 15 or 16, wherein each reactive functional group is independently a maleimide, a maleimide-thiol conjugate, an azide, a carboxylic acid, an amine, a fluorophenyl ester, a sulfotetrafluorophenyl electrophile, an aldehyde, an isothiocyanate derivative, a tetrazine, a trans-cyclooctyne or a combination thereof.

18. The compound of any one of claims 15 to 17, wherein one or more of the reactive functional groups is conjugated to a biomolecule.

19. The compound of claim 18, wherein the biomolecule is an affinity reagent.

20. The compound of claim 19, wherein the affinity reagent is an antibody.

21. The compound of any one of claims 15 to 20, wherein the soft metal is selected from Re and Pt.

22. The compound of any one of claims 15 to 21, wherein the soft metal is a naturally occurring isotope or isotopically enriched.

23. The compound of any one of claims 15 to 22, wherein the thiol-based ligand is glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1- propanesulfonate, or a combination thereof.

24. The compound of claim 20, wherein the compound conjugated to the antibody is a mass tag for targeting specific cells for detection in suspension mass cytometry (SMC) or imaging mass cytometry (IMC).

25. The compound of claim 24, wherein the mass tag is for use with further mass cytometry mass tags in a multiplex mass cytometry assay.

26. A compound of Formula IIIwhereineach A is independently a nitrogen-containing 5-membered or 6-membered heteroaryl, unsubstituted or substituted with one or more groups selected from Ci-Csalkyl, C2-Cealkenyl, C2-Cealkynyl, C1-C6alkylCO2H, C1-C6alkoxy, and Ci-Cealkylphosphonate;n is an integer from in 2 to 14, and each R is independently a groupywherein:y is an integer from 20 to 48,R3is a reactive functional group,m is an integer from 13 to 73, and each R’ is independently selected from: / CLy'y’ is an integer from 20 to 48,owherein z is an integer from 10 to 75,andwherein n + m is an integer from 15 to 75,R1and R2are each independently a terminal group,n is an integer from 15 to 75,stat indicates that the n and m repetitions of each monomer are statistically distributed to form a polymer of Formula III;t is an integer from 0 to 12,v is an integer from 1 to 3;each M is a soft metal selected from Pt, Re, Pd and Hg, andeach Q is independently absent or a thiol-based solubility ligand.

27. The compound of claim 26, wherein each A is independently a pyridine, a thiazole or an imidazole, each of which being unsubstituted or substituted.

28. The compound of claim 26 or 27, wherein each reactive functional group is independently a maleimide, a maleimide-thiol conjugate, an azide, a carboxylic acid, an amine, a fluorophenyl ester, a sulfotetrafluorophenyl electrophile, an aldehyde, an isothiocyanate derivative, a tetrazine, a trans-cyclooctyne or a combination thereof.

29. The compound of any one of claims 26 to 28, wherein one or more of the reactive functional groups is conjugated to a biomolecule.

30. The compound of claim 29, wherein the biomolecule is an affinity reagent.

31. The compound of claim 30, wherein the affinity reagent is an antibody.

32. The compound of any one of claims 36 to 31, wherein the soft metal is selected from Re and Pt.

33. The compound of any one of claims 26 to 32, wherein the soft metal is a naturally occurring isotope or isotopically enriched.

34. The compound of any one of claims 26 to 33, wherein the thiol-based ligand is glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1- propanesulfonate, or a combination thereof.

35. The compound of claim 30, wherein the compound conjugated to the antibody is a mass tag for targeting specific cells for detection in suspension mass cytometry (SMC) or imaging mass cytometry (IMC).

36. The compound of claim 35, wherein the mass tag is for use with further mass cytometry mass tags in a multiplex mass cytometry assay.

37. A compound selected from the compounds of Table 1.

38. A kit comprisingan isotopic composition comprising multiple soft metal atoms of a single isotope of a soft metal; andan element tag comprising a compound as defined in any one of claims 1 to 37, the compound comprising a plurality of chelating groups capable of binding at least one soft metal atom of the isotopic composition, and wherein the at least one soft metal atom is bound to a thiol-based solubility ligand;optionally wherein the kit does not comprise any radioactive soft metal selected from Pt, Re, Pd and Hg.

39. The kit of claim 38, wherein the soft metal is Re or Pt.

40. The kit of claim 38 or 39, wherein the thiol-based ligand is glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2, 3-dimercapto-1 -propanesulfonate, or a combination thereof.

41. The kit of any one of claims 38 to 40, wherein the element tag is functionalized to bind a biomolecule and / or is attached to a biomolecule.

42. The kit of claim 41, wherein the biomolecule is an oligonucleotide or an antibody.

43. The kit of any one of claims 38 to 42, further comprising a second isotopic composition, wherein the second isotopic composition comprises multiple additional soft metal atoms of a second single isotope of a soft metal that is different from the single isotope of the soft metal of the isotopic composition.

44. A method for preparing an element tagged reagent comprising:providing an isotopic composition comprising multiple soft metal atoms of a single isotope of a soft metal selected from Re, Pt, Pd and Hg;providing an element tag comprising a compound as defined in any one of claims 1 to 37, comprising a plurality of chelating groups wherein each chelating group is capable of binding at least one of the soft metal atom of the isotopic composition; binding the soft metal atoms of the isotopic composition to the one or more chelating groups of the element tag;binding the soft metal atoms bound to the one or more chelating groups of the element tag, to a thiol-based solubility ligand to provide the element tagged reagent; andwherein the soft metal atoms are non-radioactive.

45. The method of claim 44, wherein the isotopic composition does not comprise a natural mixture of isotopes.

46. The method of claim 44 or 45, further comprising providing a second isotopic composition wherein the second isotopic composition comprises multiple second soft metal atoms of a second single isotope of a non-radioactive soft metal that is different from the single isotope of the non-radioactive soft metal of the isotopic composition.

47. The method of any one of claims 44 to 46, wherein the soft metal is Re or Pt.

48. The method of any one of claims 44 to 47, wherein the thiol-based ligand is glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1- propanesulfonate, or a combination thereof.

49. A method for analyzing an analyte in a biological sample, comprising:(i) incubating an element tagged affinity reagent with the analyte, the element tagged affinity reagent comprising an affinity reagent tagged with an element tag, the element tag comprising a compound of any one of claims 1 to 36 multiple soft metal atoms of a single isotope of a soft metal selected from Pt, Re, Pd and Hg, and comprising chelating groups binding at least one of the soft metal atoms, wherein the soft metal atoms are bound to a thiol-based solubility ligand, the soft metal atoms are non-radioactive, and the affinity reagent specifically binds the analyte,(ii) separating unbound element tagged affinity reagent from bound element tagged affinity reagent; and(iii) analyzing the element tag bound to the affinity reagent attached to the analyte by mass spectrometric atomic spectroscopy.

50. The method of claim 49, wherein incubating the element tagged affinity reagent with the analyte comprises: incubating two or more differential element tagged affinity reagents with two or more analytes, wherein the element tagged affinity reagents specifically bind with the two or more analytes to produce two or more differentially tagged analytes, wherein analyzing the element tag bound to the affinity reagent comprises analyzing the differential element tags bound to the two or more analytes by mass spectrometric atomic spectroscopy.

51. The method of claim 49 or 50, wherein the element tagged affinity reagent is configured to bind to an analyte in a biological sample, and the biological sample comprises cells.

52. The method of any one of claims 49 to 51, wherein the soft metal is selected from Re and Pt.

53. The method of any one of claims 49 to 52, wherein the thiol-based ligand is glutathione, cysteine, thiosulfate, thioglycolic acid, mercaptosuccinic acid, methyl thioglycolate, dimercaprol, dimercaptosuccinic acid, 2,3-dimercapto-1- propanesulfonate, or a combination thereof.

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