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. Author manuscript; available in PMC: 2024 Dec 1.
Published in final edited form as: Curr Protoc. 2023 Dec;3(12):e958. doi: 10.1002/cpz1.958

Generation of Bispecific Antibodies by Functionalized Poly-ADP-Ribose Polymers

Hyo Sun Kim 1, Yong Zhang 1,2,3,4,*
PMCID: PMC10754209  NIHMSID: NIHMS1951875  PMID: 38147359

Abstract

Bispecific antibodies have drawn considerate research interests for therapeutic development. Numerous genetic and chemical methods are established to produce bispecific antibodies with varied formats. This protocol describes a novel approach to the synthesis of bispecific antibodies by utilizing chemically functionalized poly-ADP-ribose polymers derived from post-translational poly-ADP-ribosylation. Protocol 1 includes experimental procedures for expressing and purifying recombinant full-length human poly-ADP-ribose polymerase 1 (PARP1) as well as monoclonal antibodies targeting T-cell CD3 and breast cancer tumor-associated human epidermal growth factor receptor 2 (HER2) molecules. Protocol 2 details methods for enzymatic preparation of functionalized poly-ADP-ribose polymers by PARP1 and chemical conjugation of antibody molecules for bispecific antibody production.

Basic Protocol 1: Expression and Purification of PARP1 and Antibodies

Basic Protocol 2: PARP1 Auto-Modification and Antibodies Conjugation

Keywords: Antibody, bispecific antibody, chemical conjugation, immunotherapy, PARP1, poly-ADP-ribose, post-translational modification

INTRODUCTION:

Simultaneously engaging with two distinct types of antigen molecules, bispecific antibodies possess unique and strong potential for treatment of human diseases, especially in cancer immunotherapy (Li, Er Saw, & Song, 2020; Wei, Yang, Wang, & Liu, 2022; Yang, Wen, & Qin, 2017). To date, nine bispecific antibodies are approved for clinical use by FDA, many of which function through recruiting immune effector cells to tumors and activating potent cancer-specific immunity (Esfandiari, Cassidy, & Webster, 2022; Krishnamurthy & Jimeno, 2018). Using different technologies such as quadroma that somatically fuses two types of hybridoma cells, chemical conjugation, and genetic fusion, bispecific antibodies with varied sizes, formats, and pharmacological properties have been generated (Brinkmann & Kontermann, 2017; Dimasi, Kumar, & Gao, 2021; Klausner, 1987).

Protein ADP-ribosylation is an enzymatic post-translational modification featuring additions of ADP-ribose monomer or poly-ADP-ribose (PAR) polymers onto target proteins (Gibson et al., 2016; Morales et al., 2014). Poly-ADP-ribose polymerase 1 (PARP1) is characterized by robust catalytic activity for cellular protein poly-ADP-ribosylation (PARylation) in response to DNA damages (Beck, Robert, Reina-San-Martin, Schreiber, & Dantzer, 2014; Bianchi et al., 2016; De Vos, Schreiber, & Dantzer, 2012; Jeggo, 1998; Lindahl, Satoh, Poirier, & Klungland, 1995; Tan, Krukenberg, & Mitchison, 2012). The resulting linear and branched PAR polymers are highly soluble, providing a biocompatible and polymeric scaffold for biomolecule conjugations and therapeutic applications (Cheng et al., 2023; Cheng et al., 2022; Shi et al., 2020). As nicotinamide adenine dinucleotide (NAD+) molecule participates in the protein ADP-ribosylation by donating the ADP-ribose, use of NAD+ analogues with clickable groups that can serve as strong substrates of PARylation could enable functionalization of PAR polymers for facile bioconjugations (Lam et al., 2021; Zhang et al., 2019; Zhang et al., 2022). An NAD+ analogue with a nicotinamide riboside (NR) 3′-azido was developed with high PARylation activity for human PARP1 enzyme, providing a valuable tool for producing functionalized PAR polymers (Cheng et al., 2023; Zhang et al., 2019). In this protocol, we describe a functionalized PAR polymer-based method for generating bispecific antibodies. By coupling the 3′-azido NAD+ analogue with recombinant full-length human PARP1, clickable PAR polymers derived from auto-PARylated PARP1 were synthesized, allowing subsequent conjugation with bicyclo[6.1.0]nonyne (BCN)-functionalized antibody IgGs specific for human CD3 and human epidermal growth factor receptor 2 (HER2) molecules (Figure 1). The functionalized PAR polymer-mediated antibody conjugation represents a new approach for synthesizing bispecific antibodies.

Figure 1.

Figure 1.

Schematic diagram of the production of bispecific antibodies by functionalized PAR polymers. Reprinted with permission from Cheng et al., 2023. Copyright 2023 American Chemical Society.

BASIC PROTOCOL 1

Basic protocol title:

Expression and Purification of PARP1 and Antibodies

Introductory paragraph:

In this protocol, plasmids expressing full-length human PARP1 with a C-terminal His6-tag, anti-human CD3 monoclonal antibody IgG (clone: UCHT1), and anti-human HER2 monoclonal antibody IgG (clone: trastuzumab; brand name: Herceptin) are used to express the proteins. cDNA of human PARP1 (GE Healthcare Dharmacon) was cloned into pET-28a(+) vector (Milipore Sigma, cat. no. 69864) for bacterial expression. DNA fragments encoding heavy and light chains of UCHT1 and Herceptin antibodies were cloned into pFuse vector (Invivogen, cat. no. pfuse-hg1fc2) for mammalian cell expression. Please refer to previous studies for molecular cloning and plasmid preparation (Cheng et al., 2023; Zhang et al., 2019). Full-length human PARP1 is expressed via a bacterial expression system, while the antibodies are transiently expressed in mammalian cells. Expressed PARP1 and antibody IgGs are then purified using different chromatographic methods.

Materials:

Bacterial Expression of PARP1

BL21 (DE3) bacterial cells (Invitrogen, cat. no. C600003)

LB broth, Miller (Fisher Bioreagents, cat. no. BP1426–2)

Kanamycin sulfate (VWR, cat. no. 0408–25G)

Incubator shaker (Series 25, New Brunswick Scientific)

Nanodrop 2000c (Thermo Fisher Scientific)

Isopropyl-β-D-thiogalactopyranoside, dioxane free (IPTG) (Chem-IMPEX INT’L INC., cat. no. 00194)

Purification of PARP1

Centrifuge (Beckman J6B Centrifuge with JS-4.2 rotor & JA-17 rotor)

French Press G-M (Fisher Scientific, cat. no. NC0355680)

Lysis buffer (recipe)

Cellulose acetate membranes (0.45 μm) (Advantec, cat. no. C045A047A)

Gravity flow column (Bio-Rad, cat. no. 7321010)

HisPur Ni-NTA resin (Thermo Fisher Scientific, cat. no. 88221)

Equilibrium buffer (recipe)

High-salt buffer (recipe)

Elution buffer (recipe)

No-salt buffer (recipe)

Heparin sepharose 6 fast flow (Cytiva, cat. no. 17099801)

AKTA Pure chromatography system (Cytiva)

Amicon centrifugal filters (EMD Milipore, cat. no. UFC803008)

Superdex 200 Increase 10/300 GL (Cytiva, cat. no. 28990944)

Acrodisc unit with a mustang E membrane (VWR, cat. no. 28140–521)

Pierce LAL chromogenic endotoxin quantitation kits (Fisher Scientific, cat. no. PI88282)

Antibodies Expression and Purification from Mammalian Cells

ZymoPure maxiprep kit (Zymo Research, cat. no. D4203)

Erlenmeyer flasks (Nest Biotechnology, cat. no. 783011)

Expi293F cell line (Gibco, cat. no. A14527)

BalanCD HEK293 medium (Fuji Film, cat. no. 91165)

L-glutamine (Corning, cat. no. 25–005-CI)

Standard analog shaker (VWR)

Trypsin blue stain (gibco, cat. no. 15250–061)

Opti-MEM (Gibco, cat. no. 31985–070)

PEI MAX – transfection grade linear polyethylenimine-hydrochloride (Polysciences Inc., cat. no. 24765–1)

Megafuge 16 (Thermo Fisher Scientific)

Protein G resin (GenScript, cat. no. L00209)

Dialysis tubing 10’ (Science First, cat. no. 2852)

Protocol steps with step annotations:

Bacterial Expression of Full-Length Human PARP1

  • 1

    Electroporate BL21 (DE3) cells with the constructed pET-28a(+) vector encoding full-length human PARP1.

    Voltage of 1.8 kV for the electroporation in 1 mm gap cuvettes.

  • 2

    Inoculate the transformed bacterial cells in 10 mL of LB broth media containing 50 μg/mL of kanamycin overnight.

    37 °C and 250 rpm setting for the shaker.

  • 3

    Transfer the 10 mL of LB broth media into 1 L of LB broth media with 50 μg/mL of kanamycin.

  • 4

    Grow the bacterial cells until OD600 reaches at 0.6–0.8.

    Make sure to use the LB broth media for the blank.

  • 5

    Add ZnSO4 with a final concentration of 0.1 mM into the cell media.

  • 6

    Keep growing the bacterial cells until OD600 reaches at 0.8–1.0.

  • 7

    Incubate the flask on ice for 1 hr.

  • 8

    Add IPTG of a final concentration of 0.5 mM in order to induce the protein expression, followed by overnight incubation at 16 °C.

    16 °C and 250 rpm setting for the shaker.

Purification of Full-Length Human PARP1

  • 9

    Centrifuge the bacterial cell culture at 4,550 × g for 30 min.

    Use JS-4.2 rotor.

  • 10

    Discard the supernatant and resuspend the cells in lysis buffer.

  • 11

    Lyse the cells by passing a French Press for three times at 25,000 psi.

  • 12

    Centrifuge the lysates at 27,000 × g for 100 min at 4 °C.

    Use JA-17 rotor.

  • 13

    Collect the supernatant and filter it through a 0.45 μm cellulose acetate membrane.

  • 14

    Pack a gravity flow column with 5 mL of Ni-NTA resins.

    For 5 mL of resin, load 10 mL of the resin with storage buffer into the column and open the column to remove the storage buffer.

  • 15

    Add 10 mL of filtered water, followed by 15 mL of equilibrium buffer to equilibrate the column.

  • 16

    Load the filtered solution from Step 13 onto the column.

  • 17

    Wash the column with 50 mL of high-salt wash buffer.

  • 18

    Elute the protein with 25 mL of elution buffer.

  • 19

    Add 25 mL of no-salt buffer to the eluted proteins then load the mixed solution to a 5 mL heparin column by a low-pressure peristaltic pump at a flow rate of 3 mL/min.

  • 20

    Place the heparin column on an AKTA Pure chromatography system to elute PARP1 using a gradient of 0–100% buffer B in a buffer A at a flow rate of 1 mL/min.

  • 21

    Combine the fractions with eluted PARP1 and spin it down to 500 μL using centrifugal filters with 30 kDa molecular weight cut-off.

  • 22

    Inject the concentrated PARP1 into Superdex 200 Increase 10/300 GL, a size-exclusion chromatography column, and elute it using gel filtration buffer.

  • 23

    Lastly, pass the purified protein through an Acrodisc unit with a mustang E membrane.

  • 24

    Determine the final endotoxin levels using Pierce LAL chromogenic endotoxin quantitation kits.

    < 0.5 EU mg−1 mL −1 of endotoxin is expected.

  • 25

    Use SDS-PAGE gels to evaluate the purified human PARP1 (Figure 2A).

Figure 2.

Figure 2.

Analysis of purified proteins, auto-modified PARP1, and generated bispecific antibodies. A) Coomassie-stained SDS-PAGE analysis of purified PARP1, Herceptin, and UCHT1 antibodies. B) Immunoblot analysis of PARP1 and PARylated PARP1 by 3′-azido NAD+. C) Coomassie-stained SDS-PAGE (left) and immunoblot (right) analysis of PARylated PARP1 and the generated bispecific antibodies. Adapted with permission from Cheng et al., 2023. Copyright 2023 American Chemical Society.

Transient Transfection of Mammalian Cells for Antibody Expression and Purification

  • 26

    Amplify the sequence-verified plasmids using maxiprep kits.

  • 27

    Grow Expi293F cells in BalanCD HEK293 medium supplemented with L-glutamine for more than 3 passages in a 37 °C incubator with 5% CO2

  • 28

    Count the cells and passage them to reach at the final density of 5 million cells/mL in 120 mL of the medium.

  • 29

    Prepare the transfection reagent by mixing 12 mL of Opti-MEM medium, 120 μg of plasmid encoding antibody IgG heavy chain, 120 μg of plasmid encoding antibody IgG light chain, and 960 μL of transfection grade PEI-MAX (1 mg/mL).

    Add the reagents in order and make sure to thoroughly mix the solution by vortexing and inverting in-between each addition.

  • 30

    Incubate the solution at room temperature for 20 minutes.

    This allows for the plasmid-reagent complexes to form for efficient transfection.

  • 31

    Add the transfection complexes to the cell media and incubate the cell media in the incubator for 2 hours.

    37 °C with 5% CO2

  • 32

    Add an additional 120 mL of BalanCD HEK293 medium into the cell flasks to have the final density of 2.5 million cells/mL in 240 mL media total.

  • 33

    Incubate the cells in the incubator for five days.

  • 34

    On day 5, centrifuge the cells at 700 rpm for 10 minutes in order to collect the supernatant with secreted antibodies.

  • 35

    Centrifuge the collected supernatant at 4,000 × g for 30 min to further remove cell debris or impurities, then collect the supernatant.

  • 36

    Pack a gravity flow column with 2 mL of protein G resin and equilibrate the column with 10 mL of PBS.

    For 2 mL of protein G resin, initially load 4 mL of the resin and storage buffer mixture and remove the storage buffer by opening the column.

  • 37

    Load the supernatant collected in step 34 onto the column.

  • 38

    Wash the column with 15 mL of PBS and elute the protein with the elution buffer (100 mM glycine, pH 2.7).

    Collect the first 10 fractions of 1 mL eluate and measure the protein concentration using nanodrop. Select only the fractions with significant protein concentrations for the following steps.

  • 39

    Neutralize the fractions with significant protein concentrations with 100 μL of 1 M Tris buffer (pH 8.0).

    Add 100 μL of 1 M Tris to 1 mL fractions then combine them.

  • 40

    Dialyze the collected fractions against PBS two times (one overnight and one 6-hour dialysis) using 10 kDa molecular weight cutoff dialysis bags at 4 °C.

  • 41

    Use SDS-PAGE gels to evaluate each of the purified antibodies (Figure 2A).

BASIC PROTOCOL 2:

Basic protocol title:

PARP1 Auto-Modification and Antibodies Conjugation

Introductory paragraph:

In this protocol, purified PARP1 from above protocol undergoes auto-modification with 3′-azido NAD+ in order to generate azido-functionalized PAR polymers. Please refer to a previous study for the synthesis protocol of 3′-azido NAD+ (Zhang et al., 2019). Purified antibodies from the above protocol are functionalized with BCN groups for conjugation with auto-PARylated PARP1 by 3′-azido NAD+ via copper-free click chemistry to form bispecific antibodies.

Materials:

Endo-BCN-PEG4-N-hydroxysuccinimide (NHS) ester linker (BroadPharm, cat. no. BP-22851)

Amicon centrifugal filters (EMD Milipore, cat. no. UFC803008)

Activated DNA (Sigma-Aldrich, D4522)

Superdex 200 Increase 10/300 GL (Cytiva, cat. no. 28990944)

Protocol steps with step annotations:

Antibody NHS-BCN Linker Conjugation

  • 1

    Add endo-BCN-PEG4-NHS ester linker to purified Herceptin or UCHT1 antibody suspended in PBS at a molar ratio of 20 linker to 1 protein.

    Dissolve the linker in 100% DMSO before addition.

  • 2

    Gently mix the solution and incubate at room temperature for 2 hours

    BCN is added onto the antibodies via NHS ester-mediated amine coupling reactions.

  • 3

    Buffer exchange the mixture against PBS using 30 kDa molecular weight cut-off Amicon centrifugal filters.

    This step removes unreacted linkers.

PARP1 Auto-Modification

  • 4

    Prepare the reaction buffer.

  • 5

    Incubate the purified human PARP1 (3 μM) in the reaction buffer for 20 minutes for activation.

  • 6

    Add 150 μM of 3′-azido NAD+ in the reaction buffer and incubate the mixture at 30 °C for 8 hours.

    This step produces PARylated PARP1 by 3′-azido NAD+.

  • 7

    Buffer exchange the mixture against PBS using 30 kDa molecular weight cut-off Amicon centrifugal filters.

    This step removes unreacted NAD+ molecules.

  • 8

    Perform immunoblot analysis with an anti-PAR antibody (clone: 10H) to evaluate the formation of PAR polymers following auto-modification (Figure 2B).

Generation of Bispecific Antibodies

  • 9

    Mix Herceptin-BCN and UCHT1-BCN antibodies and PARylated PARP1 with a molar ratio of 3:3:1, respectively, in PBS.

    Use a 2 mL of reaction volume with antibodies at a concentration of 2 mg/mL and PARylated PARP1 at a concentration of 0.5 mg/mL for optimal condition.

  • 10

    Incubate the reaction mixture at room temperature for 3 days.

  • 11

    Load the reaction mixture to Superdex 200 Increase 10/300 GL column for size exclusion chromatography purification.

    This step separates the generated bispecific antibodies from unconjugated mono-specific antibodies present in the reaction mix. Each unconjugated antibody is expected to have an approximate molecular weight of 180 kDa (Figure 2A). Therefore, only fractions of the generated bispecific antibodies (> 180 kDa) should be collected to avoid unconjugated mono-specific antibodies in the samples.

  • 12

    Elute the fractions with PBS.

  • 13

    Collect the first peak of eluate and concentrate it using Amicon centrifugal filters with 30 kDa molecular weight cut-off.

  • 14

    Evaluate antibody conjugation using both Coomassie-stained SDS-PAGE gels and immunoblot (Figure 2C).

REAGENTS AND SOLUTIONS:

PARP1 Purification

Lysis Buffer (should be stored in 4 °C)

  • 25 mM HEPES pH 8.0

  • 500 mM NaCl

  • 1 mM phenylmethylsulfonyl fluoride (PMSF)

Equilibrium Buffer

  • 25 mM HEPES pH 8.0

  • 500 mM NaCl

  • 20 mM imidazole

High-salt Wash Buffer

  • 25 mM HEPES pH 8.0

  • 1 M NaCl

  • 20 mM imidazole

Elution Buffer

  • 25 mM HEPES pH 8.0

  • 500 mM NaCl

  • 400 mM imidazole

No-salt Buffer

  • 50 mM Tris pH 7.0

  • 1 mM EDTA

  • 0.1 mM DTT

Buffer A

  • 50 mM Tris pH 7.0

  • 1 mM EDTA

  • 0.1 mM DTT

  • 250 mM NaCl

Buffer B

  • 50 mM Tris pH 7.0

  • 1 mM EDTA

  • 0.1 mM DTT

  • 1 M NaCl

Gel Filtration Buffer

  • 25 mM HEPES pH 8.0

  • 150 mM NaCl

  • 1 mM EDTA

  • 0.1 mM DTT

Antibody Purification

Elution Buffer

  • 100 mM glycine pH 2.7

PARP1 Auto-Modification

Reaction Buffer (Make fresh buffer prior to each reaction)

  • 30 mM HEPES pH 8.0

  • 5 mM MgCl2

  • 5 mM CaCl2

  • 250 mM NaCl

  • 1 mM DTT

  • 100 ng/μL activated DNA

COMMENTARY:

Background Information:

Protein PARylation is involved in a variety of cellular events (Beck et al., 2014; Gibson et al., 2016; Jeggo, 1998). The resulting ADP-ribose-based polymers provide a valuable scaffold with high solubility and biocompatibility for therapeutic conjugation. Human PARP1 is characterized by robust auto-PARylation (Bianchi et al., 2016; Mendoza-Alvarez & Alvarez-Gonzalez, 1993) and facilitates the preparation of functionalized PAR polymers via its catalyzed auto-modification with NAD+ analogues. PARylation reactions with 3’-azido NAD+, an excellent substrate for PARP1, lead to rapid generation of azido-functionalized PAR polymers. Through copper-free click chemistry, different types of monoclonal antibodies could then be conjugated to generate bispecific antibodies. The PAR polymer-based bispecific antibodies feature high molecular weights and increased valency, which may result in improved pharmacological properties such as extended half-lives and enhanced binding affinity. Despite abundant conjugation sites on functionalized PAR polymers, the molar ratio for conjugation is set at 3:3:1 for anti-CD3 antibody, anti-HER2 antibody, and PARylated PARP1, respectively, to avoid potential steric hindrance between IgG molecules. Given the nature of the functionalized PAR polymers, additional types of antibodies or different format of antibodies could be included for production.

Critical Parameters:

PARP1 Expression and Purification

To ensure proper folding of full-length human PARP1 in bacteria, IPTG-induced expression of PARP1 needs to be conducted at 16 °C. As PARP1 is sensitive to proteolysis during purification, freshly prepared PMSF should be added prior to the lysis. French Press apparatus need to be kept at 4 °C before use. Buffers, cell lysates, and the collected supernatants for Ni-NTA affinity chromatography column should be stored on ice.

Expression and Purification of Antibodies

The purity of the plasmids used for transient transfection of Expi293F cell is critical for antibody expression in mammalian cells. Make sure to sterilize the plasmids by filtering it through 0.2 μm filters before use. Purified antibodies need to be buffer exchanged against PBS for long-term storage at −80 °C.

PARP1 Auto-Modification

Additions of activated DNA is critical for PARP1 catalytic activity. Concentration of PARP1 is also important for PARP1-catalyzed auto-modification. Eight hours or overnight reactions are needed to ensure generation of high levels of functionalized PAR polymers on PARP1. To prevent antibody conjugation with unreacted 3′-azido NAD+ molecules, the reaction mixtures should be buffer exchanged into PBS using 30 kDa molecular weight cut-off centrifugal concentrators following completion of the reactions.

Antibody NHS-BCN Linker Conjugation

The removal of the unreacted linkers in reactions is critical. Excess amounts of unreacted BCN linkers can react with the azide groups on PAR polymers, reducing antibody conjugation efficiency. Make sure to extensively dialyze or buffer exchange the reactions against PBS to remove unreacted linkers.

Generation of Bispecific Antibodies

The molar ratio of each antibody to PARP1 can be critical to the production of bispecific antibodies. Even though PARylated PARP1 has abundant azido groups for conjugation, a large number of antibody molecules conjugated to each PARylated PARP1 may result in steric hindrance to each other, reducing binding affinity.

Troubleshooting:

Understanding Results:

Basic Protocol 1 describes protein expression and purification. Following this protocol, human full-length PARP1, anti-CD3 antibody (UCHT1), and anti-HER2 antibody (Herceptin) are expected to be purified, which can be examined with Coomassie stained SDS-PAGE gels. As shown in Figure 2A, purified full-length human PARP1 migrates slightly below 140 kDa together with two cleaved fragments due to proteolysis (Chaitanya, Alexander, & Babu, 2010; Gobeil, Boucher, Nadeau, & Poirier, 2001). The two purified antibodies, Herceptin and UCHT1, migrate as intact bands above 150 kDa in the absence of DTT and are reduced to heavy and light chains at 50 kDa and 25 kDa, respectively, in the presence of DTT.

Basic Protocol 2 covers antibody BCN linker conjugation, PARP1 auto-PARylation, and bispecific antibodies generation. Following auto-modification reactions with 3′-azido NAD+, the resulting PARylated PARP1 is anticipated to carry functionalized PAR polymers that can be specifically detected by an anti-PAR monoclonal antibody via immunoblots as shown in Figure 2B. The formed PARylated PARP1 feature smeared bands across the regions for size of 100 kDa and higher, while unmodified PARP1 has no detectable signals. The azido-functionalized PAR polymers mediate generation of bispecific antibodies through copper-free click chemistry, resulting in smeared signals at around 140 kDa and higher as revealed by immunoblots using an anti-human IgG antibody (Figure 2C). Coomassie stained SDS-PAGE gels also show the generated bispecific antibodies at the size higher than 180 kDa, but little signals for PARylated PARP1, likely lower than the detection threshold of Coomassie stain.

The bispecific antibodies generated from this protocol are expected to display specific binding for T-cell CD3 as well as cancer cell-associated HER2 antigens. As shown in Figure 3, the resulting bispecific antibodies can tightly bind to both HER2-positive HCC 1954 breast cancer cells and CD3-positive Jurkat cells but show little binding to MDA-MB-468 cancer cells that lack HER2 and CD3 expression. In vitro cytotoxicity assays indicate that in the presence of non-activated human peripheral blood mononuclear cells (PBMCs), the generated bispecific antibodies induce potent killing of HER2-positive HCC 1954 breast cancer cells in a dose-dependent manner (Figure 4). No significant cytotoxicity is observed for HER2-negative MDA-MB-468 cells treated with human PBMCs and bispecific antibodies.

Figure 3.

Figure 3.

Binding of the generated bispecific antibodies to HCC 1954 (HER2 positive), Jurkat (CD3 positive), and MDA-MB-468 (HER2 and CD3 negative) cells as analyzed by flow cytometry. Adapted with permission from Cheng et al., 2023. Copyright 2023 American Chemical Society.

Figure 4.

Figure 4.

Cytotoxicity of the generated bispecific antibodies for HCC 1954 (HER2 positive) and MDA-MB-468 (HER2 negative) cells in the presence of non-activated human PBMCs. Adapted with permission from Cheng et al., 2023. Copyright 2023 American Chemical Society.

Time Considerations:

Bacterial expression of full-length human PARP1 takes 2 days and subsequent three-step chromatographic purification requires 2–3 days. Expression of antibody IgG molecules in mammalian cells needs 6 days following transient transfection. To ensure production of antibodies in high yields, it is recommended to use Expi293F cells in passages of 3–8, which adds about one week to thaw and culture the cells from frozen stocks. Antibody purification can be completed within 1–2 days. Reactions for human PARP1-catalyzed auto-modification are incubated for 8 hours or overnight. Functionalization of antibodies with BCN groups is a 2-hour reaction, followed by a 2-hour buffer exchange process. 3′-azido PARylated PARP1 is then incubated with Herceptin-BCN and UCHT1-BCN for 3 days to generate bispecific antibodies that can be purified by size exclusion chromatography within 4–6 hours. In case that the expression and purification of human PARP1 and the antibody IgGs can be carried out simultaneously, the total time needed to generate bispecific antibodies is anticipated to be about 3 weeks.

Table 1.

Troubleshooting Guide for Production of Bispecific Antibodies by Functionalized PAR Polymers.

Problem Possible Cause Solution
Low yield or catalytic activity of PARP1 Proteolysis during purification Use freshly prepared PMSF inhibitor, keep all reagents and samples at 4 °C, and minimize the time for all purification steps.
Low yield of antibodies Impurity in the plasmids Avoid the use of overgrown bacterial culture for maxiprep, ensure complete lysis and neutralization for maxiprep, and sterilize plasmids using 0.2 μm syringe filters before use.
Weak signals on immunoblots after PARP1 auto-modification Incomplete auto-modification Increase PARP1 concentration, extend the incubation time to overnight, and ensure 20-min activation time.
Failure of antibody conjugation Free BCN linkers in the samples Perform extensive dialysis or buffer exchange to remove free linkers.

Acknowledgement:

This work was supported in part by Sharon L. Cockrell Cancer Research Fund, National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) grant R35GM137901 (to Y. Z.), National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the NIH grant R01EB031830 (to Y. Z.), National Cancer Institute (NCI) of NIH grant R01CA276240 (to Y. Z.), and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the NIH grant P30DK048522 (to USC Research Center for Liver Diseases).

Footnotes

CONFLICT OF INTEREST STATEMENT:

The authors declare no competing interest to disclose.

DATA AVAILABILITY STATEMENT:

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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