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

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. Basic 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. Basic 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. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.


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 et al., 2020;Wei et al., 2022;Yang et al., 2017).To date, nine bispecific antibodies are approved for clinical use by the FDA, many of which function through recruiting immune effector cells to tumors and activating potent cancer-specific immunity (Esfandiari et al., 2022;Krishnamurthy & Jimeno, 2018).Using different technologies (e.g., 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 et al., 2021;Klausner, 1987).

EXPRESSION AND PURIFICATION OF PARP1 AND ANTIBODIES
In this protocol, plasmids expressing full-length human PARP1 with a C-terminal His 6tag, 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 the 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 the 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 25.Use SDS-PAGE gels to evaluate the purified human PARP1 (Fig. 2A).

Transient transfection of mammalian cells for antibody expression and purification
26. Amplify the sequence-verified plasmids using maxiprep kits.

Grow Expi293F cells in BalanCD HEK293 medium supplemented with L-glutamine
for >3 passages in a 37°C incubator with 5% CO 2 .
28. Count the cells using trypan blue stain and passage them to reach at the final density of 5 million cells/ml in 120 ml medium.
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 min.
This allows for the plasmid-reagent complexes to form for efficient transfection.
31.Add the transfection complexes to the cell medium and incubate the cell medium in the incubator for 2 hr at 37°C with 5% CO 2 .
32. Add an additional 120 ml BalanCD HEK293 medium into the cell flasks for a final density of 2.5 million cells/ml in 240 ml medium total.
33. Incubate the cells in the incubator for 5 days.
34.On day 5, centrifuge the cells 10 min at 100 × g, room temperature, to collect the supernatant with secreted antibodies.
35.Centrifuge the collected supernatant 30 min at 4000 × g, 4°C, to further remove cell debris or impurities, then collect the supernatant.
36.Pack a gravity flow column with 2 ml protein G resin and equilibrate the column with 10 ml PBS.
For 2 ml of protein G resin, initially load 4 ml resin and storage buffer mixture and remove the storage buffer by opening the column.

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Current Protocols 37. Load the supernatant collected in step 34 onto the column.
38. Wash the column with 15 ml PBS and elute the protein with Protein G column elution buffer (100 mM glycine, pH 2.7).
Collect the first 10 fractions of 1 ml eluate and measure the protein concentration using a 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-hr dialysis) using 10 kDa molecular weight cutoff dialysis bags at 4°C.

41.
Use SDS-PAGE gels to evaluate each of the purified antibodies (Fig. 2A).

PARP1 AUTO-MODIFICATION AND ANTIBODY CONJUGATION
In this protocol, purified PARP1 from Basic Protocol 1 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 Basic Protocol 1 are functionalized with BCN groups for conjugation with auto-PARylated PARP1 by 3 -azido NAD + via copper-free click chemistry to form bispecific antibodies.

Antibody NHS-BCN linker conjugation
1. Add endo-BCN-PEG 4 -NHS ester linker to purified Herceptin or UCHT1 antibody suspended in PBS at a molar ratio of 20 linkers to 1 protein.
Dissolve the linker in 100% DMSO before addition.
2. Gently mix the solution and incubate at room temperature for 2 hr.
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.
5. Incubate the purified human PARP1 (3 μM) in the reaction buffer for 20 min for activation.
6. Add 150 μM of 3 -azido NAD + in the reaction buffer and incubate the mixture at 30°C for 8 hr.
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 (Fig. 2B).
Use a 2 ml reaction volume with antibodies at a concentration of 2 mg/ml and PARylated PARP1 at a concentration of 0.5 mg/ml for the optimal condition.
10. Incubate the reaction mixture at room temperature for 3 days.
11. Load the reaction mixture to a 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 molecular weight of ∼180 kDa (Fig. 2A).Therefore, only fractions of the generated bispecific antibodies (>180 kDa) should be collected to avoid unconjugated mono-specific antibodies in the samples.
13. Collect the first peak of eluate and concentrate it using Amicon centrifugal filters with 30 kDa molecular weight cut-off.

REAGENTS AND SOLUTIONS
Use ddH 2 O for all solutions.Unless otherwise stated, all buffers can be stored up to 6 months at room temperature.For commonly used reagents, please see Current Protocols (2006).

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 ADPribose-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, e.g., extended halflives 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 a different format of antibodies could be included for production.

PARP1 expression and purification
To ensure proper folding of full-length human PARP1 in bacteria, IPTG-induced Current Protocols  1).
The French Press apparatus needs 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 them 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 (Table 1).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 (Table 1).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.

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 et al., 2010;Gobeil et al., 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 antibody 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 a size of ≥100 kDa, 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 ∼140 kDa and higher as revealed by immunoblots using an anti-human IgG antibody (Fig. 2C).Coomassie stained SDS-PAGE gels also show the generated bispecific antibodies at the size >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 HCC1954 breast cancer cells and CD3positive 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 nonactivated human peripheral blood mononuclear cells (PBMCs), the generated bispecific antibodies induce potent killing of HER2positive HCC1954 breast cancer cells in a dose-dependent manner (Fig. 4).No significant cytotoxicity is observed for HER2negative MDA-MB-468 cells treated with human PBMCs and bispecific antibodies.

Time Considerations
Bacterial expression of full-length human PARP1 takes 2 days and subsequent three-step chromatographic purification requires 2 to 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 to 8, which adds ∼1 week to thaw and culture the cells from frozen stocks.Antibody purification can be completed within 1 to 2 days.Reactions for human PARP1catalyzed auto-modification are incubated for 8 hr or overnight.Functionalization of antibodies with BCN groups is a 2-hr reaction, followed by a 2-hr 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 to 6 hr.In the 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 ∼3 weeks.
Current Protocols

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.

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.

Table 1
Troubleshooting Guide for Production of Bispecific Antibodies by Functionalized PAR Polymers