Targeting CD4+ Cells with Anti-CD4 Conjugated Mertansine Loaded Nanogels

Abstract

CD4⁺ T lymphocytes play an important role in controlling many malignancies. The modulation of CD4⁺ T cells through immunomodulatory or cytotoxic drugs could change the course of disease progression for disorders such as autoimmunity, immunodeficiency, and cancer. Here, we demonstrate that anti-CD4 conjugated polymeric nanogels can deliver a small molecule cargo to primary CD4⁺ T cells and a CD4^high T cell lymphoma. The antibody conjugation not only increased the uptake efficiency of the nanogel (NG) by CD4⁺ T cells, but also decreased the non-specific uptake of NG by CD4^- lymphocytes. For T lymphoma cell lines, the mertansine-loaded conjugate displayed a dose-dependent cell growth inhibition at 17 ng/mL antibody concentration. On the other hand, antibody-drug conjugate (ADC) type formulation of the anti-CD4 reached similar levels of cell growth inhibition only at the significantly higher concentration of 1.8 μg/mL. NG and antibody conjugates have the advantage of carrying a large payload to a defined target in a more efficient manner, as it needs far less antibody to achieve a similar outcome.

Graphical Abstract

INTRODUCTION

Nanomedicine has emerged as a field in cancer treatment to improve chemotherapy by concentrating cytotoxic agents in nano-sized compartments so the pharmacokinetic limitations of the free form of the drug may be avoided.¹ This field gained substantial momentum with Doxil, the nanoparticle formulation of a chemotherapy drug called doxorubicin, as the first FDA-approved nanotherapeutic that outperformed its free drug form in clinical trials.^2–4 The improved drug profile of Doxil is attributed to two points: (i) nanoparticles can penetrate the defective tumor endothelium and accumulate in the tumor environment (also described as enhanced permeability and retention or the EPR effect), (ii) nanoparticles lower the toxic burden on the body because, unlike small molecules, they cannot penetrate the organized and compact endothelium layer of healthy tissue.^5–7 Although, today, much of the research on nanoparticles is focused on targeting solid tumors with leaky vascular structures, nanoparticles also may be used to treat other malignancies to overcome the limitations of small-molecule drugs.

Hematologic malignancies, such as T- or B- cell lymphomas, develop very quickly and have a high rate of relapse and can be refractory to treatment.^8,9 Many of the chemotherapeutic drugs used in the treatment of such diseases have a small therapeutic window. Therefore, a new modality of treatment, in which the drug is selectively delivered to malignant cells, is highly desirable.⁹ Although antibody-drug conjugates have been successful in targeting CD33, CD30 and CD22 markers on malignant cells, the system nevertheless needs improvements to increase the drug load, to increase potency as well as to the lower the cost of the drug.^10–12

The selectivity and potency of nanoparticles can be improved with the introduction of an active targeting unit. This is achieved by decorating nanoparticles with biologically relevant ligands or with antibodies and, thus, generate a more selective nanotherapeutic. In this report, we chose the CD4 glycoprotein found on the T helper cells (CD4⁺ T cells) as the targeting ligand. We show here that a polymer nanoparticle, decorated with anti-CD4 antibody, can achieve selective targeting to primary CD4⁺ T cells as well as CD4 lymphoma cells.

MATERIALS AND METHODS

Polymer synthesis

Random copolymer p(PDS-co-PEG) was synthesized by a reversible addition fragmentation chain transfer (RAFT) polymerization of monomers poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide ethyl methacrylate (PDSMA). After purification of the polymer, monomer composition was calculated to be 30% PEG and 70% PDS according to the NMR results. Detailed procedures for preparation and the characterization of monomer PDSMA, polymer as well as polymer-mertansine (DM1) are available in the Supporting Information.

Preparation of dye encapsulated nanogel

Polymer micelles were prepared by dissolving 10 mg of polymer in 1 mL of distilled water. For lipophilic indocarbocyanine dye (DiO and DiI) encapsulation, 20 μL of dye stock solution (5 mg/mL) in acetone was added to the polymer solution and was stirred for six hours with the cap open to allow evaporation of the acetone. Size of the formed micelles was observed by dynamic light scattering (DLS) at room temperature (25 ^oC). 20–30 nm size was achieved by adding Na₂SO₄ to a final concentration of 50 mM as previously reported.¹³ To crosslink the micelle and lock the dye in the nanogel, we added 0.1 molar dithiothreitol (DTT) to crosslink 20 mol% of PDS units. The cross-linking reaction was monitored by UV-Vis spectrum of the released by-product pyridine-2-thiol using molar extinction coefficient of 8080 M⁻¹cm⁻¹ at 343 nm. The UV-Vis spectrum of dye encapsulated nanogel can be found in the Supporting Information (Figure S8c). The nanogel was dialyzed overnight in water using 7 kDa cutoff membrane to remove released the by-product pyridine-2-thiol.

Dynamic Light Scattering Measurements

Size distribution of polymer nanoparticles/nanogels were measured with Malvern Nanozetasizer-ZS. All samples were diluted to 0.1 mg/mL with distilled water and filtered through 0.22-micron syringe filter to remove the non-encapsulated dye prior to measurement. Hydrodynamic diameters, reported here, are intensity averages of three measurements.

Preparation of DM1 conjugated nanogel

The anti-cancer drug mertansine (DM1) conjugated polymer was made by post-polymerization modification. A 10 mg solution of polymer was dissolved in 1 mL distilled water. 100 mM stock solution of DM1 was prepared in DMSO. 20 μL of DM1 stock was mixed with 1 mL of polymer solution to replace 10% of PDS groups. The reaction was stirred for one day under inert conditions. Percentage of DM1 conjugation to polymer was estimated by quantifying the amount of released pyridine-2-thiol. To further secure the drug conjugated micelle, we added DTT to crosslink 20% of the remaining PDS units. The cross-linking reaction can be monitored by UV-Vis spectrum of the released by-product pyridine-2-thiol. The nanogel was dialyzed in PBS to remove the released by-product and unconjugated DM1 as well as to exchange buffer to PBS.

Modification of antibody with PEG linker

Antibody stock (BioXCell, rat anti-mouse CD4, clone GK1.5) was diluted in the reaction buffer composed of 0.2 M Na₂HPO₄ and 0.1 M NaCl at pH 8.5. Solution was spun down at 7500xg for 15 min at 4 °C using Amicon-10 kDa cutoff centrifugal filter unit to exchange the buffer as well as to concentrate in the sodium phosphate buffer. The linker PDS-PEG(1kDa)-NHS was purchased from Creative PEGWorks (Catalog #PHB-994). For antibody modification, 5–10 mg/mL of antibody solution was treated with 1.5–7.5 molar equivalent of linker and stirred at room temperature for 2 to 3 hours. To stop the reaction and quench the unreacted NHS ester on the linker, 1% (v/v) of 1 M Tris-HCl (pH 7) was added into the solution. Unreacted linker was removed from the antibody solution through dialysis with 100 kDa membrane against PBS buffer (pH 6.5) overnight at 4 °C. Antibody concentration at any given time was calculated by measuring the absorbance at 280 nm. Molar extinction coefficient of IgG was taken as 199500 M⁻¹cm⁻¹.

Size Exclusion Chromatography

The Superdex 200 10/300 GL column (purchased from GE) was used for running the antibody samples. The samples were run in 1X PBS buffer (pH 7.2) at 0.5 mL/min for 60 minutes using Agilent 1260 Infinity HPLC system. The absorbance of the UV detector was set at 280 nm for antibody and 214 nm for Nanogel (NG).

Conjugation of antibody to nanogel

The conjugation of antibody to NG was carried out in two consecutive steps. In the first step, modified antibody was treated with 2.5 molar excess Tris-(2-carboxyethyl) phosphine (TCEP) for 2 minutes, then it was mixed with NG solution at 1:2 (w:w) ratio in PBS buffer. The solution was stirred overnight at 4 °C. The unconjugated antibody was removed from the solution using size exclusion chromatography (SEC).

Cell culture

mT-ALL cells were kindly provided by Professor Michelle Kelliher (UMass Medical School, Worcester, MA, USA) and grown in RPMI 1640 supplemented with 5% FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin and 2 mM of L-glutamine and incubated in a humidified 37 °C, 5% CO₂ incubator at a starting seeding density of 0.2×10⁶/mL. Splenocytes were isolated from C57BL/6J mouse purchased from the Jackson Laboratory and kept in RDGs media composed of 45% RPMI, 45% DMEM, 10% FBS supplied with 2 mM sodium pyruvate, 2 mM L-glutamine, 100 units/mL of penicillin and 100 μg/mL of streptomycin. All animals were housed in animal facilities as per the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Massachusetts Amherst.

Flow cytometry

Prior to surface staining, cells were treated with a Zombie violet dye (BioLegend, #423113) to stain the dead cells, so they can later be excluded from the analysis. Zombie violet dye staining was performed according to the manufacturer’s protocol. Surface staining of splenocytes was performed with anti-CD4-PerCP (RM4–4, BioLegend), anti-CD8-PE/Cy7 (53–6.7, BioLegend) and anti-CD45R-APC (RA3–6B2, BioLegend) in FACS buffer (1% BSA + PBS). Note that the anti-CD4 conjugated to the nanogel is a GK1.5 clone. We have chosen the surfacing staining antibodies from a non-competing clone to ensure that these antibodies do not bind to the same epitope of CD4 and therefore do not interfere with the functions of anti-CD4 conjugated nanogel on mT-ALL cells. Surface staining of mT-ALL cells was performed with anti-CD4-APC (RM4–4, BioLegend). Flow cytometry data were acquired on a BD LSRFortessa with FACSDiva software (BD Biosciences). Data were analyzed using FlowJo (Tree Star) Software. Imaging flow cytometry data were acquired on Amnis ImageStream MkII (Luminex) and analyzed using IDEAS software (Luminex).

MTT assay

Cells were seeded at a density of 20,000 cells per well (100 μL volume) in 96-well tissue culture plate and treated with serial dilution of polymer or polymer conjugates and incubated for 24 hours at 37 °C in 5% CO₂. Following the treatment, cells were washed twice with PBS and returned to growth media and incubated for another 24 hours. 10 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added into each well followed by a further incubation of 3 hours. The supernatant was removed and formed formazan was solubilized in 100 μL DMSO. The absorbance was measured at 540 nm using The Synergy 2 (BioTek) plate reader.

RESULTS AND DISCUSSION

The nanogel (NG) used in this report is, by definition, a chemically cross-linked, water-soluble polymeric nanoparticle. The polymer p(PDS-co-PEG) contains two randomly substituted monomeric units that vary only in the nature of the substituents on the side chains (Figure S8a). One of these monomer units contains a short hydrophilic polyethyleneglycol (PEG), whereas the other one bears a relatively hydrophobic pyridyl disulfide (PDS) unit. The composition of PEG and PDS units on the polymer has been optimized in our previous work.¹⁴ In regard to encapsulation stability and cell uptake efficiency, the polymer with PEG:PDS molar ratio of 3:7 showed superiority. The amphiphilic nature of this polymer causes them to form nano-sized self-assemblies which are consistently of spherical morphology.¹⁴ The hydrophobic core of this aggregate favors physical encapsulation of hydrophobic molecules. The self-assembly of this polymer in aqueous solution is dictated by hydrophobicity of the PDS group so the size of the nanogel can be tuned by adding salts in accordance with ions from the Hofmeister series.¹³ Salting-out behavior of sulfate ions results in aggregates of larger sizes that improve the loading capacity of the nanogel (Figure S8b). NG is the cross-linked form of this nanoaggregate. The PDS units of the polymer chain can be triggered to cross-link the polymer chains, which stabilizes guest encapsulation by entrapping the molecules in a matrix like structure. This cross-linking is a two-step process where DTT first reacts with PDS to generate a free thiol. The free thiol can either react with a neighboring disulfide in the second step or remain free. Because disulfide bonds are part of the crosslinking unit, the NG disassembles and releases the guest molecules in the presence of glutathione (GSH), a disulfide reducing agent present at millimolar concentrations in the cytoplasm.¹⁴ On the other hand, NG is quite stable in the extracellular environment since the glutathione levels are at micromolar concentrations in the extracellular environment. Pharmacokinetics of our NGs has been studied in triple negative human mammary carcinoma models and mice induced with inflammation.^15,16 In both cases, we have observed that NG with 30% PEG composition can survive under in vivo conditions for up to 3 days which is comparable to IgG since its half-life in mouse models can range from 4 to 8 days depending on the isotype.¹⁷

Conjugation of proteins to nanoparticles is commonly achieved by decorating the nanoparticle surface with a biorthogonal click group.^18–20 These groups can be introduced to the polymer during the synthesis or after NG formation through post-polymerization modifications. Both approaches have the disadvantage of altering the hydrophilic-hydrophobic balance, the nanoaggregate size, surface charge and loading capability. For example, introducing amine reactive groups on p(PDS-co-PEG) induced the formation of nanoclusters at physiological pH which then reverted back to original state at lower pH.²¹ Therefore, we decided to modify the antibody with a functional handle bearing a free thiol such that the latter could be reacted with the PDS groups on the nanogel. PDS groups are an excellent handle for modification as they exhibit robust reactivity towards free thiols.

A heterobifunctional PEG linker with an average molecular weight of 1 kDa was used to modify the anti-CD4 (Figure 1a). This linker contains an amine reactive N-hydroxysuccinimide ester (NHS ester) group at one end and a PDS group on the other. The primary amines on the lysine residue are surface accessible for reaction with NHS group on the linker. The contour length of a 1 kDa PEG linker is around 6 nm.²² Shorter linkers were also tested for conjugation of the antibody to nanogel but were found to be unsuccessful (data not shown). Failings of short linkers can be attributed to their inability to reach the core of NG where the PDS groups reside. Additionally, the long PEG units on NG suppress any non-specific adsorption of the antibody on the nanogel.

Figure 1.

(a) Structure of the NHS-PEG-PDS linker, its reaction scheme with the primary amine on antibody and the reduction reaction schemes of modified antibody with TCEP. (b) Absorption spectrum of unmodified anti-CD4 and NHS-PEG-PDS modified anti-CD4 after the addition of TCEP. (c) Average number of linkers conjugated to antibody upon its reaction with various concentrations of linker. The data is representative of two independent experiments. (d) SEC analysis of native anti-CD4 and anti-CD4^PEG with 1:1 linker to antibody ratio. The samples were run through Superdex 200 Increase (GE) column in PBS buffer with 0.5 mL/min flow rate. The wavelength detector was set at 280nm.

Various concentrations of the linker were reacted with anti-CD4 for 2 hours and the unreacted linker was removed through dialysis overnight. The extent of linker incorporation was assessed by the release of pyridine-2-thiol after the addition of TCEP (Figure 1b). Absorbances at 343 nm and 280 nm were used to calculate linker-to-antibody ratio using molar extinction coefficients of the PDS and antibody (Table S1). The average linker to antibody ratio was found to be approximately 1, when anti-CD4 was treated with 6 molar excess of linker (Figure 1c). Moreover, size exclusion chromatography (SEC) analysis of the PEG modified anti-CD4 demonstrated the formation of two peaks (Figure 1d). This peak is likely due to dimerization of the antibody or another form of aggregation caused by PEGylation and was calculated to be approximately 10% of the total. Moreover, CD4 recognition of anti-CD4 antibody was not affected by PEGylation as demonstrated by an Enzyme-linked Immunosorbent Assay (ELISA) (Figure S12b).

The conjugation of anti-CD4^PEG-PDS to NG was carried out in two consecutive steps. In the first step, anti-CD4^PEG-PDS was treated with 2.5 molar excess TCEP to release pyridine-2-thione and generate a free thiol on the linker (anti-CD4^PEG-SH) (Figure 2a). Next, the NG was added to the solution and the mixture was incubated overnight for conjugation to yield anti-CD4 conjugated nanogel (anti-CD4^PEG/NG) (Figure 2a). To test selectivity of our nanogels, we used a mouse T-cell acute lymphoblastic leukemia (mT-ALL).^23,24 These cells were CD4⁺ when freshly isolated from the animal, however the cells lose CD4 expression over time in culture. We took advantage of the loss of CD4 and established two lines of mT-ALL, one which displayed low levels of CD4 (CD4^low) and another line, created by the introduction of a CD4 expression construct followed by cell sorting for high levels of CD4 expression (CD4^high). To determine selectivity, the anti-CD4^PEG/NG was incubated with co-cultures of our CD4^low and CD4^high mT-ALL cell lines. A lipophilic fluorescent dye, DiO, was encapsulated in the NG prior to anti-CD4 conjugation to detect the selective uptake in cells (Figure S8c). Incubation of anti-CD4^PEG-PDS and NG(DiO) mixture with mT-ALL co-culture did not demonstrate NG(DiO) uptake at 4 °C, as both CD4^low and CD4^high population displayed background levels of DiO signal (Figure 2b,c). However, at 37 °C incubation cells had taken up NG(DiO) independent of CD4 levels on the cell surface, because both cell types demonstrated similar levels of DiO fluorescence (Figure 2b,c). As predicted, anti-CD4^PEG/NG treated sample displayed selectivity towards CD4^high cells indicating successful conjugation. At 4 °C, DiO fluorescence correlated with anti-CD4 surface staining, meaning cells displaying high CD4 receptor had stronger DiO fluorescence in comparison to cells displaying medium CD4 receptor. Moreover, at 37 °C, DiO intensity of CD4^low mT-ALL cells was at the background level and very similar to control sample, whereas DiO intensity of CD4^high cells was substantially higher (Figure 2b, c). These data support the success of the conjugation, but it does not necessarily confirm that conjugation took place through a linker as antibody harbors several disulfide bonds that could be reduced during TCEP treatment to free thiols and potentially be conjugated to the nanogel directly. Although this is unlikely due to (i) the low concentration of TCEP that has been used in the reaction and (ii) the inaccessibility of the native thiols in the antibody to the PDS moieties in the core of the NG, a control experiment was carried out to validate the effect of linker. Native anti-CD4 was first treated with TCEP then mixed with NG(DiO) solution. The subsequent product was incubated with the co-culture of CD4^low and CD4^high mT-ALL cells but as expected did not display any selectivity towards the CD4 receptor (Figure S9). In addition, anti-CD4^PEG-PDS was prepared under four different linker concentrations to show the correlation between average linker number per antibody and conjugation efficiency. When these samples were incubated with co-culture of CD4^low and CD4^high mT-ALL cells, CD4^low cells displayed similar levels of DiO intensity independent of the modification levels on anti-CD4. On the other hand, we observed that DiO intensity on CD4^high cells correlated well with the average linker number per antibody (Figure S9). This data demonstrated that it is, in fact, the linker that reacts with NG under these conditions.

Figure 2.

(a) Conjugation scheme of anti-CD4^PEG-SH to NG. (b) Flow cytometry dot plots showing CD4 surface receptor staining intensity (on x-axis) and DiO intensity (y-axis). Co-cultured mT-ALL cells were incubated with anti-CD4^PEG-PDS + NG(DiO) or anti-CD4^PEG/NG(DiO) at 4 °C for 30 minutes (upper panel) or 37 °C for 3 hours (lower panel) at a concentration of 50 μg/mL of NG. CD4^low cells are shown in red, CD4^high cells are shown in blue color. (c) Bar plot shows the DiO mean fluorescence intensity of CD4^low and CD4^high mT-ALL cells. Error bars indicative of three replicates (S.D.)

Next, we wanted to test the conjugates with primary CD4⁺ T cells. These cells are known for their poor uptake of nano-sized materials. For the CD4 selective uptake assay, we used isolated splenocytes where the main cell types are CD4⁺ T cells, CD8⁺ T cells and B cells. For this assay, a lipophilic fluorescent dye, DiI, was encapsulated in the NG prior to anti-CD4 conjugation. The splenocytes were treated with NG(DiI) or anti-CD4^PEG/NG(DiI) at 4 °C for 30 minutes or at 37 °C for 1 hour. Afterward, they were stained with anti-CD4 (PerCP), anti-CD8 (PE-Cy7) and anti-CD45 (APC) to analyze subpopulations of CD4⁺ T, CD8⁺ T and B cells, respectively. The percentage of subpopulations are shown in Figure 3a, left panel. NG(DiI) alone did not show significant amount of uptake at 37 °C, in fact DiI fluorescence intensity was low for all populations (Figure 3a, left panel). DiI-positive population percentage was determined as 15%, 18% and 59% for CD4⁺ T cells, CD8⁺ T cells and CD45R⁺ B cells, respectively (Figure 3b). Curiously, B cells showed higher DiI fluorescence—compared to T cells—which might be related to the innate phagocytotic ability of B cells. As expected, anti-CD4^PEG/NG(DiI) displayed high selectivity towards CD4⁺ T cells at 4 °C and at 37 °C (Figure 3a,b). The DiI positive population of CD4⁺ T cells reached 100%. Additionally the non-specific uptake of NG(DiI) by CD8⁺ T cells and B cells disappeared upon anti-CD4 conjugation to NG demonstrating the strength of active targeting (Figure 3a,b).

Figure 3.

(a) The bar graph on the left shows the percentage of CD4⁺, CD8⁺, CD45R⁺ cells isolated from mouse spleen. Graph on the right shows the mean fluorescence intensity of DiI for each cell type after splenocytes were incubated with NG(DiI) or anti-CD4^PEG/NG(DiI) for 30 minutes at 37 °C. Error bars are indicative of S.D. of two independent experiments. (b) Representative histogram plots of CD4⁺, CD8⁺ T cells and B cells. Numbers indicate the percentage of DiI positive population for each cell type. Dashed line indicates the applied gating settings. (c) Five representative images of anti-CD4^PEG/NG(DiI) treated CD4⁺ T cells. DiI fluorescence is shown in red. Upper panel shows the images of cells that were treated at 4 °C for 30 minutes, lower panel at 37 °C for 2 hours.

Because flow cytometry does not provide information about the location of the fluorescence signal, next we tested the internalization of DiI in CD4⁺ T lymphocytes. Anti-CD4 antibody targets the surface protein CD4, so NG(DiI) should be internalized along with anti-CD4 antibody through receptor-mediated endocytosis. To visualize the internalization of anti-CD4/NG(DiI), Amnis imaging flow cytometry was conducted. This instrument captures the image—brightfield and fluorescent—of each cell in addition to scatter plots of conventional flow cytometry. The splenocytes were incubated with anti-CD4/NG(DiI) at 4 °C for cell surface staining (Figure 3c, upper panel) and 37 °C for internalization (Figure 3c, lower panel). As expected, DiI fluorescence was localized on the surface when incubation took place at 4 °C due to anti-CD4 binding to CD4 receptor on the cell surface. However, when incubation took place at 37 °C, DiI was not only found at the cell surface but inside the cell as well. Interestingly, the signal was concentrated in specific spots which might indicate localization in endosomes or lysosomes.

After successfully targeting CD4^high mT-ALL cells as well as CD4⁺ T cells with anti-CD4^PEG/NG, we asked if this conjugate can be used to selectively deliver cytotoxic drugs. To minimalize the leakage, a cytotoxic drug molecule, DM1, was covalently conjugated to the polymer p(PDS-co-PEG) prior to NG formation. DM1, also called mertansine, is a microtubule inhibitor from maytansine drug class, which has been used in ADC therapies.^25–28 It contains a free thiol group and thus simplifies conjugation and linker chemistry designs for ADCs. The disulfide bonds between the polymer and the drug will only be reduced in intracellular conditions, where the glutathione concentrations are high, thereby releasing the drug in its native form.

By taking advantage of the free thiol group on DM1, polymer-DM1 was prepared by reacting the polymer solution, one DM1 molecule was added into the solution (Figure 4a). The reaction efficiency was approximately 90%, meaning for every 10 PDS groups on the polymer, one was converted to DM1. DM1 on the polymer-DM1 conjugate could be seen on the absorbance spectrum as it holds a strong absorbance at 252 nm (Figure S10a). This absorbance peak has been used to calculate the drug to antibody ratio (DAR) of ADC conjugates in the past.²⁹ The peak intensity at 252 nm did not change after NG formation (Figure S10a). However, addition of highly hydrophobic mertansine units on the polymer caused an increase in the size of nanogel (Figure S10b).

Figure 4.

(a) Structure of the polymer-DM1. (b, c, d) CD4^high and CD4^low mT-ALL cells were incubated with Polymer-DM1, NG-DM1 or anti-CD4^PEG/NG-DM1 for 24 hours. After 24 hours of treatment, cells were washed and incubated for 24 more hours with fresh media. MTT assay was performed to measure the cell proliferation and activity. Untreated cells were used as control (not shown in the graphs) to normalize the absorbance of MTT formazan. Non-linear regression curve with a variable slope was drawn to calculate IC₅₀ values. (e) The plot shows the IC₅₀ values of 4 independent experiment in which each dot represents one experiment. Paired t-test analysis was performed to calculate p value. (f) Comparison of anti-CD4^PEG/NG-DM1 and anti-CD4-DM1 in regard to antibody concentration.

Anti-CD4 was conjugated to NG-DM1 following the optimized protocol stated earlier. Conjugated samples were run on SEC to remove the free (unreacted) anti-CD4 from the solution (Figure S11a). Amount of antibody conjugated to NG-DM1 was assessed by ELISA and calculated to be 2 μg of antibody per 125 μg of NG-DM1 (Figure S12a). DLS analysis of SEC purified anti-CD4^PEG/NG-DM1 conjugates demonstrated that the morphology of the NG did not change after anti-CD4 conjugation as the size of the particle remained unaffected (Figure S11b).

To determine whether DM1-conjugated NG does have intrinsic selectivity towards any of the cell lines as well as to observe the effective concentration, mT-ALL cells lines were incubated with polymer, polymer-DM1, NG or NG-DM1 (Figure 4b,c and S13a,b). In addition, cells were incubated with anti-CD4 alone to see at what concentration antibody was toxic to these cells (Figure S13a). After 24 hours of treatment, cells were washed and incubated for 24 more hours with a fresh media. As expected, anti-CD4 antibody showed slight toxicity starting from 0.5 μg/mL concentration for CD4^high cells, while CD4^low mT-ALL cells were indifferent to anti-CD4 concentration. Polymer and NG displayed toxicity only at relatively high concentrations and never reached 100% cell growth inhibition (Figure S13b, c). Polymer-DM1 and NG-DM1 also did not show any selectivity. The IC₅₀ (50% cell growth inhibition) was calculated to be approximately 1 μg/mL for polymer-DM1 and 0.1 μg/mL for NG-DM1. These values indicate that NG-DM1 is almost 10 times more toxic than polymer-DM1.

As anticipated, the anti-CD4^PEG/NG-DM1 conjugate displayed dose-dependent cell growth inhibition for CD4^high mT-ALL cells. It was five times more potent against CD4^high cells. The IC₅₀ values for these cells were calculated to be 1.09 μg/mL for polymer and 17 ng/mL for antibody (Table 1 and Table S2). To compare the efficiency of our antibody-NG conjugate with the conventional ADC, DM1 was directly conjugated to anti-CD4 antibody using a maleimide linker. The DAR was calculated to be ~3 for anti-CD4-DM1 conjugate which is consistent with previously reported antibody-DM1 conjugates.²⁶ Anti-CD4-DM1 inhibited the growth of CD4^high mT-ALL cells in a similar fashion but required significantly more antibody to do it (IC₅₀ = 1.8 μg/mL antibody). Antibody is a powerful tool when it comes to selectivity, but it is not an efficient vehicle for drug delivery. NG, on the other hand, has been developed to carry large quantities of drug molecules and when conjugated to antibody can deliver its cargo to its target cell, while requiring much less antibody.

Table 1 –

Calculated IC₅₀ values

	IC50 for Polymer (μg/mL)		IC50 for anti-CD4 (μg/mL)
mT-ALL cell	CD4low	CD4high	CD4low	CD4high
Polymer-DM1	1.04 ± 0.18	0.98 ± 0.16	-	-
NG-DM1	0.1 ± 0.01	0.12 ± 0.01	-	-
Anti-CD4PEG/NG-DM1	5.06 ± 0.33	1.09 ± 0.12	0.079 ± 0.004	0.017 ± 0.002
Anti-CD4-DM1	-	-	12.33 ± 2.032	1.807 ± 0.774

CONCLUSIONS

In conclusion, we demonstrate here that by conjugating an antibody to polymeric nanogels, we can deliver cytotoxic drug molecules to target cells. Anti-CD4 conjugated nanogels selectively accumulated in cells expressing the CD4 glycoprotein on their surface. The capability of CD4 targeting was tested with mixed splenocyte population as well as mT-ALL cell lines bearing either high or low levels of the CD4 antigen. The advantage of this delivery platform can be summarized by two points: (i) the payload amount per nanogel is many folds higher than ADCs, whose potency is limited by the low drug to antibody ratio, (ii) the amount of antibody required to achieve 50% cell growth inhibition is 100-fold less for nanogel conjugates compared to conventional ADCs. These results show that antibody-nanoparticle conjugates may prove to be an effective drug delivery platform and need to be further studied in more complex systems.

ABBREVIATIONS

NG

nanogel

CD4

cluster of differentiation 4

ADC

antibody drug conjugate

mT-ALL

mouse T cell acute lymphoblastic leukemia

FDA

Food and Drug Administration

PEG

Polyethylene Glycol

PDS

Pyridyl Disulfide

MA

methacrylate

DTT

Dithiothreitol

PBS

Phosphate Buffer Saline

NHS

N-hydroxysuccinimide

RAFT

Reversible Addition Fragmentation Chain Transfer

TCEP

Tris-(2-carboxyethyl) phosphine

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

BSA

Bovine Serum Albumin

FBS

Fetal Bovine Serum

DMSO

Dimethyl sulfoxide

GSH

Glutathione

SEC

Size Exclusion Chromatography

DAR

Drug to Antibody ratio

IC

Inhibitory Concentration

CD

Cluster of Differentiation

DLS

Dynamic Light Scattering

ELISA

Enzyme-linked Immunosorbent Assay

DM1

Mertansine