Synthesis of Bispecific Antibody Conjugates Using Functionalized Poly-ADP-Ribose Polymers

Abstract

Poly-ADP-ribose (PAR) is a natural type of polymer derived from enzymatic reactions catalyzed by cellular poly(ADP-ribose) polymerases (PARPs). Given its notable solubility and biocompatibility, PAR polymer may function as effective carriers for therapeutics in addition to modulating biomolecular interactions in cells. To explore its therapeutic potential, we herein developed a PAR polymer-based bispecific antibody targeting both human epidermal growth factor receptor 2 (HER2) and T-cell CD3 antigens. This was accomplished by conjugating anti-HER2 and anti-CD3 monoclonal antibodies to azido-functionalized PAR polymers through click chemistry. The generated PAR polymer-anti-HER2/anti-CD3 antibody conjugate could not only bind specifically to both HER2- and CD3-expressing target cells, but also display potent cytotoxicity against HER2-positive breast cancer cells in the presence of non-activated human peripheral blood mononuclear cells (PBMCs). Functionalized PAR polymers provide a new strategy for synthesizing bispecific antibodies and may enable generation of PAR polymer-based conjugates with unique pharmacological activities for biomedical applications.

Graphical Abstract

Introduction

Post-translational poly-ADP-ribosylation (PARylation) is catalyzed by cellular poly(ADP-ribose) polymerases (PARPs), resulting in linear and branched poly-ADP-ribose (PAR) polymers covalently attached to various protein substrates. PAR polymers feature up to 300 units in length of ADP-ribose donated by the nicotinamide adenine dinucleotide (NAD⁺) co-substrate (1,2). These negatively charged polymers are involved in mediating biomolecular interactions and known to play important roles in regulating genome stability, transcriptional activities, protein homeostasis, and many other processes (3–8).

Characterized by high solubility and biocompatibility, PAR represents a natural form of polymer scaffold with valuable pharmacological properties for conjugation with therapeutic agents (9,10). To facilitate orthogonal bioconjugation, we recently synthesized a 3′-azido NAD⁺ with excellent substrate activity for protein PARylation (11). Such clickable PAR polymers enable production of antibody-drug conjugates for targeted delivery (12). Here we asked whether the functionalized PAR polymers allow to generate bispecific antibodies for cancer immunotherapy. By simultaneously engaging both tumors and immune effector cells, genetically or chemically engineered bispecific antibodies promote formation of immunological synapses and induction of tumor-specific immunity, establishing new molecular therapeutics for malignancies (13–22). To test this notion, azido-functionalized PAR polymers were utilized to conjugate with both anti-human CD3 and anti-human epidermal growth factor receptor 2 (HER2) antibodies (Figure 1). The resulting PAR-antibody conjugate shows potent HER2-dependent cytotoxicity in the presence of non-activated human peripheral blood mononuclear cells (PBMCs), demonstrating a new approach for synthesizing bispecific antibodies.

Figure 1.

Schematic of the design and generation of a PAR polymer-based bispecific antibody.

Materials and Methods

Materials.

Unless otherwise specified, all reagents were purchased from common commercial sources and used as received without further purification. Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Corning Inc. Opti-modified Eagle’s medium (Opti-MEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA). BalanCD HEK293 medium and L-glutamine solution (200 mM) were purchased from FUJIFILM Irvine Scientific (Irvine, CA).

Cell lines.

Breast cancer cell lines (SK-BR-3, HCC 1954, MDA-MB-231, and MDA-MB-468) and Jurkat cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and maintained in RPMI 1640 medium supplemented with 10% FBS at 37°C and 5% CO₂. Breast cancer cell line MCF-7 was obtained from ATCC and cultured in DMEM medium with 10% FBS. Expi293F cells were purchased from Thermo Fisher Scientific and cultured in Expi293F expression medium with shaking at a speed of 125 rpm at 37°C and 8% CO₂. Human PBMCs were obtained from HemaCare (Van Nuys, CA).

Chemical synthesis of 3′-azido NAD⁺.

The 3′-azido NAD⁺ was synthesized according to a previously published method (11).

Molecular cloning.

pET-28a (+) vector encoding full-length human PARP1 with a C-terminal His₆ tag was generated in a previous study (11). pFuse vectors encoding anti-HER2 antibody Herceptin heavy chain (HC) and light chain (LC) (pFuse-Herceptin HC and pFuse-Herceptin LC) were gifts from Dr. Peter G. Schultz’s laboratory at The Scripps Research Institute.

Overlap extension polymerase chain reaction (PCR) was adopted to generate DNA fragments encoding the HC and LC of anti-human CD3 UCHT1 antibody by exploiting variable regions of a previously constructed anti-human CD3 UCHT1 single-chain variable fragment (scFv) and constant regions of the Herceptin antibody (23). Primers used for the overlap extension PCRs are listed in Table S1 with indicated restriction enzyme sites of EcoRI and NheI. Amplified fragments were ligated in-frame using T4 DNA ligase in a pFuse vector for the generation of mammalian expression constructs which were confirmed by DNA sequencing.

Protein expression and purification.

The bacterial expression and purification of human full-length PARP1 were carried out by following a previously published protocol (12,24). The purified protein was further passed through an acrodisc unit with mustang E membrane (Pall Corporation, Port Washington, NY) through following the manufacturer’s instructions. The final endotoxin levels (< 0.5 EU mg⁻¹ mL⁻¹) were determined using Pierce LAL chromogenic endotoxin quantitation kits (Thermo Fisher Scientific). Purified PARP1 was analyzed by SDS-PAGE gels, flash frozen in liquid nitrogen, and stored at −80°C.

The anti-HER2 Herceptin antibody and anti-CD3 UCHT1 antibody were expressed through transient transfection into Expi293F cells using polyethylenimine-Max (PEI-MAX) transfection regent (Polysciences, Warrington, PA) by following the manufacturer’s instructions. Culture media of Expi293F cells transfected with the expression constructs were collected at day 3 and day 6 post-transfection and centrifuged at 4,000×g for 30 minutes. The supernatants were loaded on gravity flow columns packed with 2 mL of Protein G resin (GenScript, Piscataway, NJ), followed by washing with PBS. Antibodies was then eluted with elution buffer (100 mM glycine, pH 2.7), neutralized with 1 M Tris buffer (pH 8.0), dialyzed in PBS buffer for overnight and another 6 hours in PBS at 4 °C, and concentrated using 30 kDa-cutoff amicon centrifugal concentrators (EMD Millipore Temecula, CA). Purified antibodies were analyzed by SDS-PAGE gels and stored at −80°C.

Antibody NHS-BCN linker conjugation.

A 20-fold molar excess of endo-BCN-PEG₄-NHS ester linker (BroadPharm, San Diego, CA; dissolved in 100% DMSO) was added into Herceptin or UCHT1 antibody in PBS, respectively. The solutions were mixed gently and allowed to react at room temperature for 2 hours. The reaction mixtures were then buffer exchanged to PBS with dilution factors over 1,000,000 using 30 kDa- cutoff amicon centrifugal concentrators to remove unreacted linkers.

PARP1 automodification.

Purified human PARP1 (3 μM) was incubated with 150 μM of 3′-azido NAD⁺ or NAD⁺ in a reaction buffer containing 30 mM HEPES (pH 8.0), 5 mM MgCl₂, 5 mM CaCl₂, 250 mM NaCl, 1 mM DTT and 100 ng μL⁻¹ activated DNA (Sigma-Aldrich, St. Louis, MO) at 30°C for 8 hours. The reaction mixtures were then buffer exchanged to PBS using 30 kDa-cutoff amicon centrifugal concentrators.

Conjugation of antibodies with PARylated PARP1.

Herceptin antibody-BCN (2 mg/mL) and UCHT1 antibody-BCN (2 mg/mL) were added into PARylated PARP1 (0.5 mg/mL) in 2 mL PBS with a molar ratio of 3:3:1. The conjugations were allowed to react for 3 days at room temperature. PARylated PARP1 conjugates were purified through size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare, Princeton, NJ) and eluted with PBS. The first peak eluted was collected and concentrated using amicon centrifugal concentrators with 30 kDa cutoff. Purified PARylated PARP1-Herceptin/UCHT1 conjugates were examined by SDS-PAGE.

Immunoblot analysis.

PARP1, PARylated PARP1, and PARylated PARP1-Herceptin/UCHT1 (2 μg of protein) were boiled with 10 mM DTT in NuPAGE LDS sample buffer (Thermo Fisher Scientific) at 98°C for 5 minutes. Samples were then run on 4–20% ExpressPlus-PAGE gels (GenScript, Piscataway, NJ), transferred to immun-blot PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were subsequently blocked with 5% bovine serum albumin (BSA) in PBS with 0.1% Tween-20 (PBST) for 1 hour at room temperature, followed by incubation with anti-poly-ADP-ribose (PAR) monoclonal antibody (clone: 10H, Santa Cruz Biotechnology) and anti-mouse IgG-HRP (catalog: 62-6520, Thermo Fisher Scientific) or anti-human IgG (H+L)-HRP (catalog: 5220-0277, SeraCare). The immunoblots were developed by additions of supersignal west pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and imaged with a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc).

Flow cytometric analysis.

HER2 expression levels of SK-BR-3, HCC 1954, MCF-7, MDA-MB-231, and MDA-MB-468 cells were evaluated by flow cytometry. Cells were incubated with the Herceptin in PBS with 2% FBS at 4°C for 30 minutes. Following three-time washing with PBS containing 2% FBS, cells were then incubated with Alexa Fluor-488 goat anti-human IgG (H+L) (catalog: A11013, Thermo Fisher Scientific) in PBS with 2% FBS at 4°C for 30 minutes. After washing three times with PBS containing 2% FBS, cells were analyzed using a Fortessa X20 flow cytometer (BD Biosciences, San Jose, CA). Data were processed by FlowJo software (Tree Star Inc., Ashland, OR).

The binding of PARylated PARP1 and PARylated PARP1-Herceptin/UCHT1 conjugates to HER2⁺ cell line HCC 1954, CD3⁺ cell line Jurkat, and HER2⁻ CD3⁻ cell line MDA-MB-468 were evaluated by flow cytometry. PARylated PARP1 and PARylated PARP1-Herceptin/UCHT1 conjugates were first labeled with NHS-fluorescein (Thermo Fisher Scientific) at a 1:20 molar ratio according to the manufacturers’ instructions. Free dyes were removed by buffer exchanged to PBS with dilution factors over 1,000,000 using 30 kDa-cutoff amicon centrifugal concentrators. Cells were incubated with fluorescein-labeled PARylated PARP1 or fluorescein-labeled PARylated PARP1-Herceptin/UCHT1 conjugates at 100 μg mL⁻¹ for 30 minutes at 4°C and washed three times with PBS containing 2% FBS. Samples were analyzed using the Fortessa X20 flow cytometer and data were processed by FlowJo software.

Confocal microscopy of cell-cell crosslinking.

HCC 1954 cells and MDA-MB-468 cells were stained with MitoSpy Red (BioLegend) and Jurkat cells were stained with CFSE (BioLegend) by following the manufacturer’s instructions. Jurkat cells (6×10⁴) were incubated with PARylated PARP1 or PARylated PARP1-Herceptin/UCHT1 conjugates (0.1 mg mL⁻¹) in 100 μL PBS for 30 minutes at 4°C. Following washing with 1 mL of cold PBS, Jurkat cells were resuspended in 500 μL RPMI-1640 medium with 10% FBS, then mixed with HCC1954 or MDA-MB-468 cells (2×10⁴) in the same medium. The cell mixtures were subsequently added into clear bottoms of 24-well plates and incubated for 5 hours at 37°C with 5% CO₂. The cells were then gently washed four times with PBS and imaged with a Leica SP8 confocal laser scanning microscope (Leica Microsystems Inc., Buffalo Grove, IL) equipped with a 40×, 1.3 NA PLAPO oil immersion objective lens using FITC (for CFSE) and rhodamine (for MitroSpy Red) filters. Images were processed using LAS X software (Leica Microsystems Inc., Buffalo Grove, IL).

In vitro cytotoxicity assays.

Breast cancer cells (SK-BR-3, HCC 1954, MCF-7, MDA-MB-231, and MDA-MB-468) (1×10⁴ cells) were mixed with human PBMCs (effector cells) (1×10⁵ cells) and incubated for 40 hours in the presence of various concentrations of PARylated PARP1 or PARylated PARP1-Herceptin/UCHT1 conjugates at 37°C. Cells were then washed twice with PBS to remove PBMC suspensions, followed by additions of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution. Following 3-hour incubation at 37°C and subsequent additions of 100 μL of lysis buffer (20% SDS in 50% dimethylformamide, 0.5% (v:v) 80% acetic acid, 0.4% (v:v) 1 N HCl, pH 4.7), plates were incubated for 4 hours at 37°C and measured for absorbance at 570 nm using a BioTek Synergy H1 Hybrid Multi-Mode Microplate reader (BioTek, Winooski, VT). Cell viability was calculated as: % cell viability = [(absorbance_experimental – absorbance_{spontaneous average})/(absorbance_{maximal viability average} – absorbance_{spontaneous average})]×100.

Results and Discussion

Post-translational PAR is a natural hydrophilic form of polymer that feature unique pharmacological properties. We envisioned that functionalized PAR polymers may enable facile conjugation of two different types of monoclonal antibodies for generating synthetic bispecific antibodies with immunotherapeutic potential. To explore this concept, we exploited recombinant full-length human PARP1 and the 3′-azido NAD⁺ molecule to produce functionalized PAR polymers. PARP1 catalyzes robust auto-modification upon activation by damaged DNA (8,25), resulting in PARylated PARP1. The 3′-azido substitution for the nicotinamide riboside moiety of NAD⁺ affords an analogue with high substrate activity for protein PARylation (11). Full-length monoclonal anti-human HER2 IgG and anti-human CD3 IgG functionalized with bicyclo[6.1.0]nonyne (BCN) via N-hydroxysuccinimide (NHS) ester-mediated amine coupling were selected as model antibodies for conjugation with the 3′-azido PARylated PARP1 via copper-free click chemistry to generate a PAR polymer-based bispecific antibody (Figure 1).

Full-length human PARP1 with a C-terminal His₆ tag was expressed and purified from bacteria using a previously established protocol (8,11). Coomassie-stained SDS-PAGE gels revealed both intact and cleaved human PARP1 following three-step chromatographic purification (Figure 2A). The anti-human HER2 IgG (clone: trastuzumab; brand name: Herceptin) and anti-human CD3 IgG (clone: UCHT1) were transiently expressed in mammalian cells and purified through single-step affinity chromatography (23,26–29). SDS-PAGE gels indicated that under the reducing condition, light and heavy chains of both Herceptin and UCHT1 antibodies migrate at approximately 25 kDa and 50 kDa, respectively (Figure 2A). Auto-modification of the purified human PARP1 with the 3′-azido NAD⁺ was then carried out by following previously optimized conditions (11,12). Immunoblot analysis using an anti-PAR monoclonal antibody showed strong smeared signals for the PARylated PARP1 but no signals for non-modified PARP1 (Figure 2B), supporting generation of PAR polymers.

Figure 2.

Generation and characterization of the PARylated PARP1-Herceptin/UCHT1 conjugate. (A) Coomassie stain of purified human PARP1, Herceptin, and UCHT1 antibody. (B) PARylated PARP1 by 3′-azido NAD⁺ as revealed through immunoblotting using an anti-PAR antibody. (C) Conjugation of Herceptin and UCHT1 antibody with PARylated PARP1 by 3′-azido NAD⁺ as revealed by Coomassie stain (left) and immunoblotting (right) using an anti-human IgG antibody-HRP. (D) Flow cytometric analysis of the binding of fluorescein-labeled PARylated PARP1-Herceptin/UCHT1 conjugate to HCC 1954 cells (HER2⁺), Jurkat cells (CD3⁺), and MDA-MB-468 cells (HER2⁻ CD3⁻). Fluorescein-labeled PARylated PARP1 was used as a control. (E) Confocal microscopic analysis of cell-cell crosslinking induced by PARylated PARP1-Herceptin/UCHT1 conjugate. PARylated PARP1 was used as a control. Red: HCC 1954 and MDA-MB-468 cells. Green: Jurkat cells. Scale bars: 50 μm.

Following functionalization of the purified Herceptin and UCHT1 IgGs with BCN groups (Figure 1), 3′-azido PARylated PARP1 was incubated with the Herceptin-BCN and UCHT1-BCN at a molar ratio of 1:3:3 for three days at room temperature. The resulting PARylated PARP1-Herceptin/UCHT1 conjugate was then purified through size-exclusion chromatography with an estimated molecular weight of 410–1010 kDa (Figure S1). Coomassie-stained SDS-PAGE gels revealed that the PARylated PARP1-Herceptin/UCHT1 conjugate is characterized by molecular weights above 180 kDa (Figure 2C). In comparison, the PARylated PARP1 features heterogeneous PAR polymers below the detection limit of Coomassie stain and three major cleaved fragments in a range of 35 to 65 kDa, likely due to high sensitivity to proteolysis during auto-PARylation (30,31). Furthermore, anti-human IgG-based immunoblot indicated that unlike PARylated PARP1 giving no signals, the PARylated PARP1-Herceptin/UCHT1 conjugate has significant levels of signal in regions over 140 kDa (Figure 2C). These results support successful conjugation of IgG antibodies to the functionalized PAR polymers.

Figure 3.

In vitro cytotoxicity of the PARylated PARP1-Herceptin/UCHT1 conjugate. Human PBMCs (effector cells) were incubated with breast cancer cells (target cells) at an E:T ratio of 10 in the presence of various concentrations of the PARylated PARP1-Herceptin/UCHT1 conjugate. PARylated PARP1 was used as a control. Data are shown as mean ± SD of triplicates.

The binding specificity of the generated PARylated PARP1-Herceptin/UCHT1 conjugate toward HER2 and CD3 cognate antigens was examined by flow cytometry using HCC 1954 (HER2⁺), Jurkat (CD3⁺), and MDA-MB-468 (HER2⁻ CD3⁻) cell lines (32,33). Flow cytometric analysis showed that the PARylated PARP1-Herceptin/UCHT1 conjugate can not only bind to HCC 1954 cells but also Jurkat cells, whereas the PARylated PARP1 displays no significant binding to both cell lines (Figure 2D). Both the PARylated PARP1-Herceptin/UCHT1 conjugate and PARylated PARP1 show little or no binding to MDA-MB-468 cells. These data indicate generation of the anti-human HER2/anti-human CD3 bispecific antibody through functionalized PAR polymer-mediated conjugation.

To further demonstrate dual targeting capability of the PARylated PARP1-Herceptin/UCHT1 conjugate, confocal microscopy studies were performed for fluorescently labeled Jurkat cells (CD3⁺) and breast cancer cells (HER2⁺ or HER2⁻) following incubation with the PARylated PARP1-Herceptin/UCHT1 conjugate. Confocal imaging analysis revealed crosslinking of HCC 1954 and Jurkat cells in the presence of the PARylated PARP1-Herceptin/UCHT1 conjugate (Figure 2E). As a control, PARylated PARP1 induces no formation of cell clusters for HCC 1954 and Jurkat cells. Moreover, the PARylated PARP1-Herceptin/UCHT1 conjugate gives rise to no crosslinking of MDA-MB-468 and Jurkat cells under the same conditions. The confocal microscopic results indicate that the PAR polymer-based bispecific antibody could engage CD3-expressing Jurkat cells to HER2-positive breast cancer cells through simultaneous binding to both cell-surface antigens.

In vitro cytotoxicity of the PARylated PARP1-Herceptin/UCHT1 conjugate was next evaluated using non-activated human PBMCs (including T cells, B cells, natural killer cells, monocytes, and dendritic cells) and breast cancer cell lines with varied levels of HER2 expression (Figure S2) (32,33). Following treatments of the mixtures of PBMCs (effector cells) and breast cancer cells (target cells) with various concentrations of the PARylated PARP1-Herceptin/UCHT1 conjugate or PARylated PARP1, viabilities of target cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Figure 3). Notably, the bispecific PARylated PARP1-Herceptin/UCHT1 conjugate exhibits potent cytotoxicity (EC₅₀ of 9.02 ± 0.12 ng mL⁻¹) against SK-BR-3 breast cancer cells in the presence of human PBMCs. Furthermore, the PARylated PARP1-Herceptin/UCHT1 conjugate could induce killing of HCC 1954 and MCF-7 cells by human PBMCs in an EC₅₀ of 22.86 and 51.07 ng mL⁻¹, respectively. In comparison, PARylated PARP1 shows little or no killing activity under the same conditions. The cytotoxicity of the PAR polymer-based bispecific antibody is correlated with HER expression levels of target cells. Unlike those breast cancer cells with high to moderate levels of HER2 expression, MDA-MB-231 and MDA-MB-468 cells with low or no HER2 expression (Figure S2) are less sensitive to the treatment of PARylated PARP1-Herceptin/UCHT1 conjugate, supporting HER2-dependent cytotoxicity. These results indicate excellent in vitro potency and specificity for the PARylated PARP1-Herceptin/UCHT1 conjugate against HER2-positive breast cancer cells.

This study demonstrates facile synthesis of bispecific antibodies with functionalized PAR polymers. Each PARylated PARP1 may carry 2–6 antibody molecules based on the conjugation molar ratio and size-exclusion chromatography. The resulting bispecific antibody displays marked cytotoxicity against HER2-expressing cancer cells in the presence of non-activated human PBMCs. Its EC₅₀ for HCC 1954 breast cancer cells is 22.86 ± 0.13 ng/mL, comparable to that of a previously generated anti-CD3/HER2 bispecific scFv for the same cell line (EC₅₀: 1.92 ± 0.32 ng/mL) considering large difference for their molecular weights (28). In comparison with established genetic and chemical approaches for generating bispecific antibodies, functionalized PAR polymers facilitate rapid conjugation of two or more types of monoclonal antibodies in different formats and may improve plasma half-lives due to higher molecular weights and possibly increased resistance to proteolysis. Through this PAR polymer-based scaffold, bi- or multi-specific antibodies with distinct combinations could be readily created for functional assessment. Moreover, the polymeric PAR may increase avidity of conjugated antibodies for improved binding as well as allow conjugation of additional immune-modulating molecules for enhanced efficacy. Future studies include quantitative analysis of conjugated antibodies, in-depth characterization of bispecific antibody-mediated T-cell activation, evaluation of in vivo pharmacological activities, development of new functionalized PAR polymers for orthogonal conjugation of distinct antibodies, and generation of different types of PAR polymer-based bi- and multi-specific antibodies for cancer immunotherapy.

In conclusion, an anti-human HER2/anti-human CD3 bispecific antibody was synthesized by utilizing functionalized PAR polymers. In the presence of human PBMCs, this bispecific antibody could induce potent cytotoxicity for HER2-positive breast cancer cells. The functionalized PAR polymers provide a valuable approach for generating bispecific antibodies with immunotherapeutic potential.