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. 2024 Mar 19;33(4):e4924. doi: 10.1002/pro.4924

Synthesis of site‐specific Fab‐drug conjugates using ADP‐ribosyl cyclases

Hyo Sun Kim 1, Kimia Hariri 1, Xiao‐Nan Zhang 1, Liang‐Chieh Chen 1, Benjamin B Katz 2, Hua Pei 3, Stan G Louie 3,4, Yong Zhang 1,4,5,6,✉
PMCID: PMC10949397  PMID: 38501590

Abstract

Targeted delivery of small‐molecule drugs via covalent attachments to monoclonal antibodies has proved successful in clinic. For this purpose, full‐length antibodies are mainly used as drug‐carrying vehicles. Despite their flexible conjugation sites and versatile biological activities, intact immunoglobulins with conjugated drugs, which feature relatively large molecular weights, tend to have restricted tissue distribution and penetration and low fractions of payloads. Linking small‐molecule therapeutics to other formats of antibody may lead to conjugates with optimal properties. Here, we designed and synthesized ADP‐ribosyl cyclase‐enabled fragment antigen‐binding (Fab) drug conjugates (ARC‐FDCs) by utilizing CD38 catalytic activity. Through rapidly forming a stable covalent bond with a nicotinamide adenine dinucleotide (NAD+)‐based drug linker at its active site, CD38 genetically fused with Fab mediates robust site‐specific drug conjugations via enzymatic reactions. Generated ARC‐FDCs with defined drug‐to‐Fab ratios display potent and antigen‐dependent cytotoxicity against breast cancer cells. This work demonstrates a new strategy for developing site‐specific FDCs. It may be applicable to different antibody scaffolds for therapeutic conjugations, leading to novel targeted agents.

Keywords: antibody, antibody‐drug conjugate, CD38, protein engineering, targeted delivery

1. INTRODUCTION

Covalent attachments of small‐molecule agents to immunoglobulin scaffolds enable selective delivery of payloads to diseased tissues, owing to tight and specific binding of antibodies to disease‐associated antigens. Such antibody‐drug conjugates (ADCs) are commonly characterized by high potency and specificity, leading to marked efficacy and safety profiles in clinical applications. To date, 14 ADCs have received approvals from the United States Food and Drug Administration (FDA) for cancer treatment (Fu et al., 2022; Samantasinghar et al., 2023). Furthermore, numerous ADCs are currently at different stages of preclinical and clinical studies for oncology and other diseases (Drake & Rabuka, 2017; Wei et al., 2022). Conventional ADCs are prepared through random conjugation of payloads with cysteine or lysine residues that may vary in number and/or position across different antibody molecules. The resulting ADCs are typically heterogeneous and featured with mixed drug‐to‐antibody ratios (DARs). In contrast, site‐specific drug conjugations produce homogeneous ADCs with defined DARs, giving rise to improved pharmacological properties. Through genetic or chemical engineering of the antibody scaffolds, a variety of approaches have been established for generating ADCs with site‐specifically conjugated drugs (Axup et al., 2012; Hofer et al., 2009; Kularatne et al., 2014; Lim et al., 2015; Shi et al., 2022; Zimmerman et al., 2014).

Most ADCs on market or under development are derived from full‐length immunoglobulin G (IgG) antibodies. In comparison with other antibody formats such as fragment antigen‐binding (Fab) and single‐chain fragment variable (scFv), full‐length IgG offers more choices of position for payload conjugation and carries a fragment crystallizable (Fc) region that may extend serum half‐lives and trigger Fc‐dependent biological activities for ADCs (Belicky et al., 2017; Fu et al., 2022; Mordenti et al., 1999). Conversely, the relatively large size of IgG‐based ADCs could restrict its tissue distribution and penetration (Baart et al., 2023; Chauhan et al., 2005; Graff & Wittrup, 2003; Nelson, 2010; van Dongen, 2021; Yokota et al., 1992), especially for solid tumors with additional physiological barriers, including heterogeneous blood supply, interstitial hypertension, and extended transport path in the interstitium (Jain, 1990; Thurber et al., 2008; van Dongen, 2021). Moreover, higher doses are typically required for the IgG‐derived ADCs in clinic due to low proportions of payloads (Deonarain et al., 2018; Deonarain & Xue, 2020; Kim et al., 2008; Kim et al., 2020; Liu et al., 2019). Therefore, conjugating small‐molecule agents to antibody constructs with reduced sizes allow to generate unique therapeutic candidates (Baart et al., 2023; Badescu et al., 2014; Bauerschlag et al., 2017; Chauhan et al., 2005; Deonarain et al., 2018; Deonarain & Xue, 2020; Hwang & Rader, 2020; Kim et al., 2008; Kim et al., 2013; Nessler et al., 2020; Wang et al., 2007). For example, Fab‐ and scFv‐based ADCs display enhanced tissue penetration in comparison to their full‐length antibody versions (Deonarain et al., 2018; Deonarain & Xue, 2020; Hwang & Rader, 2020; Jager et al., 2021; Liu et al., 2019; Nessler et al., 2020; Wu et al., 2022).

In this study, we attempted to develop a new form of site‐specific Fab‐drug conjugates (FDCs) by utilizing CD38 and its covalent inhibitor. The type II transmembrane protein CD38 is an ADP‐ribosyl cyclase that catalyzes conversion of nicotinamide adenine dinucleotide (NAD+) into cyclic ADP‐ribose and ADP‐ribose (Dai et al., 2018; Graeff et al., 2009; Mehta et al., 1996; Piedra‐Quintero et al., 2020). 2′‐Cl‐arabinose NAD+ (2′‐Cl‐araNAD+) is a recently identified potent covalent inhibitor that can rapidly form a stable bond with glutamate 226 at CD38 active site during catalysis (PDB ID: 6VUA) (Dai et al., 2021; Dai, Zhang, et al., 2020). By genetically fusing CD38 to full‐length IgGs and chemically derivatizing 2′‐Cl‐araNAD+, we developed IgGs with site‐specifically conjugated cytotoxic agents for selective killing of tumor cells with great potency in vitro and in vivo (Dai et al., 2021; Dai, Zhang, et al., 2020). Here, we extend this technology to synthesize site‐specific FDCs. A Fab antibody targeting human epidermal growth factor receptor 2 (HER2) was fused with single or dual CD38 enzymatic domains, followed by drug conjugations mediated by the 2′‐Cl‐araNAD+ linker. The resulting conjugates, namely ADP‐ribosyl cyclase‐enabled FDCs (ARC‐FDCs), display potent cytotoxicity against breast cancer cells with high levels of HER2 expression, demonstrating a new approach for the generation of site‐specific FDCs with strong therapeutic potential.

2. RESULTS

To develop site‐specific FDCs, we envisioned that the CD38 extracellular domain could be functionally fused to the C‐terminus of Fab light chain and/or heavy chain (Figure 1a) with little effects on antibody binding. The ADP‐ribosyl cyclase activity of the fused CD38 extracellular domain coupled with its covalent inhibitor 2′‐Cl‐araNAD+‐derived linker would enable site‐specific drug conjugation. The anti‐HER2 monoclonal antibody (clone: trastuzumab; brand name: Herceptin) Fab was selected as a model Fab for designing CD38 fusion proteins and generation of FDCs (Bartsch, 2020; Burris et al., 2011; Cheng et al., 2023; Dai, Cheng, & Zhang, 2020; García‐Alonso et al., 2020; Shi, Cheng, & Zhang, 2020; Yu et al., 2023; Zhang, Liu, et al., 2015; Zhang, Zou, et al., 2015). As our previous study indicated that fusion of the CD38 extracellular domain to the N‐terminus of IgG light chain could reduce its antigen‐binding affinity, CD38 was thus placed at the C‐terminus of the anti‐HER2 Fab light chain and/or heavy chain (Figure 1a) (Dai, Zhang, et al., 2020). The generated mammalian expression constructs of anti‐HER2 Fab light chain (LC)‐CD38 fusion and heavy chain (HC)‐CD38 fusion together with vectors encoding anti‐HER2 Fab LC and HC were paired respectively for transient transfection of Expi293F cells. Four different Fab antibodies were expressed and purified via protein G affinity chromatography, including anti‐HER2 Fab wild type (WT), Fab‐CD38 mono fusions (anti‐HER2 Fab HC‐CD38 and anti‐HER2 Fab LC‐CD38), and Fab‐CD38 dual fusion (anti‐HER2 Fab HC/LC‐CD38) (Figure 1). Coomassie blue‐stained sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) analysis reveals molecular weights of approximate 49 kDa for anti‐HER2 Fab WT, 79 kDa for anti‐HER2 Fab HC‐CD38 and anti‐HER2 Fab LC‐CD38, and 110 kDa for anti‐HER2 Fab HC/LC‐CD38 (Figure 1b), consistent with molecule designs. The yields are around 8–10 mg/L for anti‐HER2 Fab WT, anti‐HER2 Fab LC‐CD38, and anti‐HER2 Fab HC/LC‐CD38 and 3 mg/L for anti‐HER2 Fab HC‐CD38. These results show stable expression of Fab‐CD38 mono and dual fusion proteins in mammalian cells.

FIGURE 1.

FIGURE 1

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Anti‐HER2 Fab‐CD38 fusion proteins. (a) Schematic diagram of engineered anti‐HER2 Fab antibody constructs fused with the CD38 extracellular domain at c‐termini of either the light chain or the heavy chain for mono‐fusions, or both chains for the double‐fusion. (b) A sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel with Coomassie blue staining for purified Fab antibody constructs. Anti‐HER2 Fab HC/LC, anti‐HER2 Fab heavy chain and light chain; Anti‐HER2 Fab WT, anti‐HER2 Fab wild type; Fab, fragment antigen‐binding; HER2, human epidermal growth factor receptor 2; IgG, immunoglobulin G.

The enzymatic activities of the fused CD38 domains were then examined using nicotinamide guanine dinucleotide (NGD+)‐based fluorescence assays. CD38 catalyzes production of fluorescent cyclic GDP‐ribose from NGD+ (Figure S1) (Dai et al., 2018; de Oliveira et al., 2018; Graeff et al., 1994). The fluorescence assays indicate that anti‐HER2 Fab LC‐CD38 exhibits higher catalytic activity than anti‐HER2 HC‐CD38 (Figure 2). Compared with Fab‐CD38 mono fusions, the Fab‐CD38 dual fusion (anti‐HER2 Fab HC/LC‐CD38) shows increased fluorescence signals, consistent with the incorporation of two CD38 extracellular domains. As controls, reactions with phosphate‐buffered saline (PBS) only or anti‐HER2 Fab WT give no increases of fluorescence intensity. These results support generation of anti‐HER2 Fab‐CD38 fusions with robust catalytic activities.

FIGURE 2.

FIGURE 2

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Enzymatic activity of recombinant CD38 and anti‐HER2 Fab‐CD38 fusions as measured by NGD+ fluorescence assays. PBS, phosphate‐buffered saline; Anti‐HER2 Fab HC/LC, anti‐HER2 Fab heavy chain and light chain; Anti‐HER2 Fab WT, anti‐HER2 Fab wild type; Fab, fragment antigen‐binding; HER2, human epidermal growth factor receptor 2; NGD+, nicotinamide guanine dinucleotide.

Next, binding of the Fab‐CD38 fusions to the HER2 receptor was analyzed by enzyme‐linked immunosorbent assay (ELISA) (Figure 3). Similar to anti‐HER2 Fab WT with a half‐maximal effective concentration (EC50) of 2.50 nM, the anti‐HER2 Fab‐CD38 mono and dual fusions reveal tight binding to the HER2 antigen with EC50 values in a range of 0.75–2.60 nM. As a control, recombinant CD38 displays no binding to the HER2 molecule at a concentration up to 100 nM. These binding data indicate that genetic fusions of the CD38 enzymatic domain onto anti‐HER2 Fab C‐termini cause no reduction in its binding affinity. In contrast to anti‐HER2 Fab HC‐CD38, anti‐HER2 Fab LC‐CD38 shows higher levels of expression yield, catalytic activity, and binding affinity and was therefore selected along with anti‐HER2 Fab HC/LC‐CD38 for subsequent production of FDCs.

FIGURE 3.

FIGURE 3

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Binding affinity of anti‐HER2 Fab antibody fusion constructs to human recombinant HER2 as examined by ELISA. Anti‐HER2 Fab HC/LC, anti‐HER2 Fab heavy chain and light chain; Anti‐HER2 Fab WT, anti‐HER2 Fab wild type; EC50, half‐maximal effective concentration; ELISA, enzyme‐linked immunosorbent assay; Fab, fragment antigen‐binding; HER2, human epidermal growth factor receptor 2.

To make ARC‐FDCs, monomethyl auristatin F (MMAF), a tubulin inhibitor, was used as model payload (Chen et al., 2017; Hingorani et al., 2022; Shi, Zhang, et al., 2020). According to previous studies, MMAF was first functionalized with a terminal alkyne and then attached to azido‐derivatized 2′‐Cl‐araNAD+ (Figure 4a) via click chemistry (Dai et al., 2021; Dai, Zhang, et al., 2020). The resulting drug‐linker conjugate was incubated with Fab‐CD38 mono or dual fusion proteins overnight to afford anti‐HER2 ARC‐FDCs with a drug‐to‐Fab ratio (DFR) of 1 or 2, respectively (Figure 4b). The conjugation levels were examined through the measurements of remaining CD38 catalytic activity with NGD+‐based fluorescence assays. Following overnight conjugation with 2′‐Cl‐araNAD+‐MMAF, anti‐HER2 Fab LC‐CD38 and anti‐HER2 Fab HC/LC‐CD38 show no detectable enzymatic activities (Figure S2). The resulting ARC‐FDCs were purified via buffer exchange for removals of free drug‐linker conjugates. Furthermore, purified anti‐HER2 ARC‐FDCs together with unconjugated Fab‐CD38 fusion proteins were analyzed by mass spectrometry. The observed mass shifts support successful conjugation of one or two MMAF payloads to the anti‐HER2 Fab LC‐CD38 or anti‐HER2 Fab HC/LC‐CD38, respectively (Figures S3 and S4).

FIGURE 4.

FIGURE 4

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Anti‐HER2 ARC‐FDCs with variable DFRs. (a) Schematic diagram of the 2′‐Cl‐araNAD+ linker‐mediated site‐specific Fab‐drug conjugation. (b) Schematic diagram depicting ARC‐FDCs with DFR of 1 and 2. ARC, ADP‐ribosyl cyclase; DFR, drug‐to‐Fab ratio; Fab, fragment antigen‐binding; FDC, Fab‐drug conjugates; HER2, human epidermal growth factor receptor 2; MMAF, monomethyl auristatin F; 2'‐Cl‐araNAD+, 2'‐Cl‐arabinose nicotinamide adenine dinucleotide.

The generated anti‐HER2 ARC‐FDCs were evaluated for cytotoxicity using two breast cancer cell lines with varied HER2 expression levels, including HCC1954 (HER2+++) and MDA‐MB‐231 (HER2+). Cell viability assays indicate that both anti‐HER2 ARC‐FDCs could potently kill HCC1954 cells in a dose‐dependent manner (Figure 5a). The EC50 values are 1.39 ± 0.44 nM for anti‐HER2 ARC‐FDC with a DFR of 2 and 2.30 ± 0.37 nM for anti‐HER2 ARC‐FDC with a DFR of 1, slightly lower than that of free MMAF‐alkyne (EC50: 5.79 ± 2.81 nM). Relative to the anti‐HER2 ARC‐FDC carrying one payload, the anti‐HER2 ARC‐FDC with two payloads shows modestly higher potency for HCC1954 cells. In contrast to free MMAF‐alkyne with an EC50 of 2.55 nM for MDA‐MB‐231 cells, both anti‐HER2 ARC‐FDCs reveal no significant killing of this low‐HER2‐expressing cell line at concentrations below 100 nM (Figure 5b). As controls, anti‐HER2 Fab WT and anti‐HER2 Fab‐CD38 fusions have little or weak cytotoxic effects on HCC1954 and MDA‐MB‐231 cell lines at concentrations up to 300 nM. We also examined binding of anti‐HER2 ARC‐FDCs against CD31 that is a known ligand of CD38 (Horenstein et al., 1998). ELISA analysis revealed no detectable signals of binding to human CD31 for up to 100 nM of anti‐HER2 ARC‐FDCs, but tight binding toward human HER2 antigens (Figure S5). Moreover, anti‐HER2 ARC‐FDCs below 300 nM lack cytotoxicity for CD31‐expressing U937 cells (Figure S6) (Horenstein et al., 1998). Taken together, these results support potent anti‐HER2 ARC‐FDCs with HER2‐dependent and DFR‐correlated cytotoxicity.

FIGURE 5.

FIGURE 5

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In vitro cytotoxicity of anti‐HER2 ARC‐FDCs. Breast cancer cells with varied levels of HER2 expression were incubated for 72 h with ARC‐FDCs, antibody constructs, or free MMAF‐alkyne at different concentrations, followed by MTT assays. (a) Percent cell viability of HCC1954 cell line (HER2+++). (b) Percent cell viability of MDA‐MB‐231 cell line (HER2+). Anti‐HER2 Fab HC/LC, anti‐HER2 Fab heavy chain and light chain; Anti‐HER2 Fab WT, anti‐HER2 Fab wild type; ARC, ADP‐ribosyl cyclase; DFR, drug‐to‐Fab ratio; EC50, half‐maximal effective concentration; Fab, fragment antigen‐binding; FDC, Fab‐drug conjugate; HER2, human epidermal growth factor receptor 2; MMAF, monomethyl auristatin F.

3. DISCUSSION

Site‐specific FDCs with varied DFRs are successfully generated through coupling bifunctional Fab‐CD38 fusions with the 2′‐Cl‐araNAD+ drug linker. The robust catalytic activity of CD38 extracellular domain enables rapid and single‐step conjugation of cytotoxic agents to Glu226 at the active site. As revealed in previous studies, the dinucleotide‐based drug linker is capable of stably carrying the payload and facilitating its release upon cellular uptake (Dai et al., 2021; Dai, Zhang, et al., 2020). CD38 could be functionally fused to C‐terminus of HC and/or LC, giving no significant impact on antigen binding and producing FDCs with customized DFRs of 1 or 2. Compared with IgG‐derived ADCs, the FDCs are much smaller, potentially enhancing tissue penetration and distribution. The fractions of small‐molecule payloads for FDCs are higher than those of IgG‐based ADCs, providing more flexibility for dosage selection. Despite reduced half‐lives for FDCs due to the decreased size and lack of Fc domain (Hwang & Rader, 2020; Liu et al., 2019), genetic fusion of one or two CD38 molecules in ARC‐FDCs increases overall molecular weights and could help prolong serum half‐lives. In addition to Fab constructs, the CD38‐fusion method is likely applicable to other antibody formats for generating site‐specific conjugates.

Compared with anti‐HER2 Fab LC‐CD38, the anti‐HER2 Fab HC‐CD38 shows a lower expression yield and slightly decreased CD38 enzymatic activity and HER2‐binding affinity, suggesting that fusing CD38 to the C‐terminus of anti‐HER2 Fab HC causes destabilization of the resulting fusion protein. Interestingly, combining the LC‐CD38 with the HC‐CD38 results in a Fab‐CD38 dual fusion with the yield and antigen‐binding affinity comparable to those of anti‐HER2 Fab WT and anti‐HER2 Fab LC‐CD38. These results suggest that the fused CD38 catalytic domains on LC and HC have little effects on Fab chain pairing and the presence of LC‐CD38 fusion stabilizes the associated HC‐CD38 fusion. The generated anti‐HER2 ARC‐FDCs with DFRs of 1 or 2 need to be further evaluated for therapeutic efficacy and potential toxicity in animal models of breast cancer. Future studies should also include comparative analysis of pharmacokinetics and tissue distribution and penetration between ARC‐FDCs and IgG‐derived ADCs. Additionally, Fab antibodies specific for other antigens or different forms of antibody scaffold could be utilized together with CD38 for developing site‐specific drug conjugates.

In conclusion, this proof‐of‐concept study demonstrates a facile approach for the synthesis of site‐specific FDCs with defined DFRs by utilizing CD38 catalytic activity and an NAD+‐based drug linker. The synthesized anti‐HER2 ARC‐FDCs show excellent and HER2‐dependent cytotoxicity against breast cancer cells, providing new candidates for targeted therapy.

4. EXPERIMENTAL METHODS

4.1. Materials

Synthetic DNA encoding human CD38 extracellular domains and Herceptin heavy chain and light chain were obtained from the previous study (Dai, Zhang, et al., 2020). AccuPrime Pfx DNA polymerase kit (12344024), electroporation cuvettes plus with 1 mm gap (FB101), Opti‐MEM I reduced serum medium (31985070), goat anti‐human kappa light chain secondary antibody with horseradish peroxidase (HRP) (18853), QuantaBlu fluorogenic peroxidase substrate kit (15169), fetal bovine serum (FBS) (26140079), 0.25% trypsin–EDTA with phenol red (25200056), 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reagent (M6494), and penicillin–streptomycin (15140122) were purchased from Thermo Fisher Scientific (MA, USA). EcoRI DNA restriction enzyme (R3101S), NheI DNA restriction enzyme (R3131S), and T4 DNA ligase (M0202S) were purchased from New England Biolabs (MA, USA). Zymoclean gel DNA recovery kit (D4001), ZymoPURE plasmid miniprep kit, and ZymoPURE II plasmid maxiprep kit were purchased from Zymo Research (CA, USA). Zeocin solution (ant‐zn‐05) was purchased from InvivoGen (CA, USA). Protein G resin (L00209) was purchased from GenScript (NJ, USA). PEI MAX transfection grade linear polyethylenimine hydrochloride (24765–1) was purchased from Polysciences Inc. (PA, USA). BalanCD HEK293 medium (91165) and L‐glutamine (200 mM) were purchased from Irvine Scientific (CA, USA). NGD+ (sc‐215563) was purchased from Santa Cruz Biotechnology (TX, USA). Tissue culture‐treated vented flasks (250 mL) (25–209) were purchased from Genesee Scientific (CA, USA). Black 96‐well microplates (655209) and black 96‐well microplates with high‐binding base (655077) were purchased from Greiner Bio‐One (Kremsmünster, Austria). Recombinant human ErbB2/Her2‐Fc chimera protein (1129‐ER‐050) was purchased from R&D Systems (MN, USA). Paclitaxel, 99 + % (AC328420010), amicon ultra‐15 centrifugal filter units with 10‐kDa molecular weight cutoff (UFC901024) and 30‐kDa molecular weight cutoff (UFC903024), and bovine serum albumin (BSA) (1265925GM) were purchased from MilliporeSigma (MA, USA). Dulbecco's modified Eagle's medium (DMEM) (10‐017‐CV), RPMI 1640 medium (10‐040‐CV), and Dulbecco's PBS (DPBS) (21‐031‐CM) were purchased from Corning Inc. (NY, USA). Erlenmeyer flasks (250–500 mL) (89095–278), U‐100 BD micro‐fine IV insulin syringes (BD‐329424), and tissue culture (TC)‐treated 96‐well plates (10062–900) were purchased from VWR International (PA, USA). Recombinant human CD31 protein (150–06) was purchased from PeproTech (NJ, USA).

4.2. Cell lines

Breast cancer cell lines HCC1954 and MDA‐MB‐231 and the U937 cell line were purchased from the American Type Culture Collection (VA, USA). Expi293F cells were purchased from Thermo Fisher Scientific (Waltham, MA).

4.3. Chemical synthesis

The linker 2′‐Cl‐araNAD+ and 2′‐Cl‐araNAD+‐MMAF conjugate were prepared according to the previously published work (Dai, Zhang, et al., 2020).

4.4. Molecular cloning for Fab‐CD38 fusion constructs

To produce DNA fragments for Herceptin light chain and heavy chain Fab fusions with human CD38, overlap extension polymerase chain reactions were performed using DNA fragments encoding for human CD38 extracellular domain and Herceptin light chain or heavy chain Fab. Primers for the overlap PCR were designed based on pFUSE vector (Table S1). The fusion constructs were designed with a GGS linker between the C terminus of Herceptin light chain or heavy chain Fab and human CD38. The human CD38 DNA encodes four asparagine mutations (N100D, N164A, N129D, and N209D) to avoid N‐glycosylation.

Amplified DNA fragments were then analyzed by agarose DNA gel electrophoresis and purified with DNA recovery kits (Zymo Research, CA). Purified DNA fragments and the pFUSE vector backbone were digested for 3 h at 37°C with NheI and EcoRI restriction enzymes (New England Biolabs, MA), purified, and ligated using DNA T4 ligase (New England Biolabs, MA) at room temperature for 3 h. Lastly, ligated products were transformed into electrocompetent DH10B Escherichia coli, followed by zeocin selection on agar plates (InvivoGen, CA). Positive colonies with zeocin resistance were verified by DNA sequencing.

4.5. Mammalian cell expression and purification

Expi293F cells (Thermo Fisher Scientific, MA) were transiently transfected with the sequence‐verified expression vectors using PEI MAX reagent (Polysciences, PA). To express different Fab‐CD38 fusions, Herceptin light chain plasmids with or without CD38 fusion and heavy chain Fab plasmids with or without CD38 fusion were incubated at a molar ratio of 1:1 in Opti‐MEM medium with PEI MAX transfection reagent for 20 min and added to Expi293F cells cultured at 2.5 million cells/mL. The expressed antibody or fusion constructs include wild‐type Herceptin Fab (anti‐HER2 Fab WT), Herceptin Fab with CD38 fused on the heavy chain (anti‐HER2 Fab HC‐CD38), Herceptin Fab with CD38 fused on the light chain (anti‐HER2 Fab LC‐CD38), and Herceptin Fab with CD38 fused on both heavy and light chains (anti‐HER2 Fab HC/LC‐CD38).

Following incubation on an orbital shaker at 37°C with 5% CO2 for 5 days, media were collected and loaded through Protein G resins (GenScript, NJ) for purification. Eluted antibodies in 100 mM glycine (pH 2.7) were neutralized with 1 M Tris (pH 8) and dialyzed against PBS (pH 7.4). Purified antibodies were examined with SDS‐PAGE gels stained with Coomassie blue. Protein concentrations were measured with a Nanodrop 2000C spectrometer.

4.6. Enzymatic activity assays

Catalytic activity of CD38 was measured via nicotinamide guanine dinucleotide (NGD+) fluorescence‐based assays. (de Oliveira et al., 2018; Graeff et al., 1994; Graeff et al., 2009) In a 96‐well plate, 50 nM of each purified antibody in PBS was mixed with 100 uM of NGD+ in a total volume of 100 uL. The fluorescent intensity with excitation at 300 nm and emission at 410 nm was monitored using a Synergy H1 plate reader (BioTek, VT).

4.7. Binding affinity analysis

The binding affinity of each purified antibody or ARC‐FDC to recombinant HER2 or CD31 was measured via ELISA. A 96‐well ELISA plate (Greiner Bio‐One, NC) was coated overnight with recombinant HER2‐Fc fusion protein (R&D Systems, MN) or CD31 protein (PeproTech, NJ) (2.5 μg/mL) at 4°C in 80 μL of PBS (pH 7.4). Coated wells were then blocked with 3% bovine serum albumin (BSA) (MilliporeSigma, MA) in PBS with 0.05% tween‐20 solution (0.05% PBST) for 2 h at room temperature, followed by three times of wash with 200 μL of 0.05% PBST solution. Purified antibodies or ARC‐FDCs with 3‐fold serial dilution (100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.13, and 0.04 nM) were prepared and added to the wells for 1‐hour incubation. After three times of wash with 0.05% PBST, 80 μL of HRP‐conjugated anti‐human IgG kappa antibody (Thermo Fisher Scientific, MA) was added and incubated at room temperature for another hour. After three times of wash with 0.05% PBST, 80 μL of QuantaBlu fluorogenic substrates (Thermo Fisher Scientific, MA) were then added for fluorescence measurements with excitation at 325 nm and emission at 420 nm by a Synergy H1 plate reader (Bio Tek, VT). The three‐parameter nonlinear regression fitting in GraphPad Prism Version 9.4.0 (GraphPad software, CA) was used to calculate EC50 for different antibody constructs against the recombinant HER2 antigen.

4.8. Synthesis of ARC‐FDCs

Antibody Fab‐CD38 fusion construct was incubated with the 2′‐Cl‐araNAD+‐MMAF conjugate at a molar ratio of 1:100 in 50 mM Tris buffer (pH 8.5) at 4°C overnight. NGD+ assays were then carried out to confirm completed conjugation based on remaining CD38 enzymatic activity. The resulting ARC‐FDCs were buffer exchanged against PBS using 10 kDa molecular weight cutoff centrifugal concentrators.

4.9. Mass spectrometry analysis

Anti‐HER2 Fab LC‐CD38, anti‐HER2 Fab HC/LC‐CD38, and anti‐HER2 ARC‐FDCs were reduced by dithiothreitol (DTT) (1:1 (v/v) with 18 mg/mL DTT in 50 mM ammonium bicarbonate) and analyzed by LC/MS. (ACQUITY UPLC H‐class system, Xevo G2‐XS QTof, Waters corporation). Protein samples were separated away from storage buffer and salt using a phenyl guard column at 45°C (ACQUITY UPLC BEH Phenyl VanGuard Pre‐column, 130 Å, 1.7 μm, 2.1 mm × 5 mm, Waters corporation). The 5‐min method used 0.2 mL/min flow rate of a gradient of buffer A consisting of 0.1% formic acid (#85178, Thermo Fisher Scientific) in water (#9831–02, J.T. Baker) and buffer B acetonitrile (#A956, Thermo Fisher Scientific). The gradient was run (flow rate set at 0.2 mL/min and curve set as 6) as follows: maintaining 100% buffer A from 0 to 30 s, reaching to 10% buffer A and 90% buffer B from 30 to 120 s as a gradient, maintaining 10% buffer A and 90% buffer B from 120 to 150 s, reaching to 100% buffer A from 150 to 240 s as a gradient, and maintaining 100% buffer A from 240 to 300 s.

The Xevo Z‐spray source was operated in a positive MS resolution mode, 400–4000 Da, with a capillary voltage of 3000 V and a cone voltage of 40 V (NaCsI calibration, Leu‐enkephalin lock‐mass). Nitrogen was used as the desolvation gas at 350°C and a total flow of 800 L/h. Total average mass spectra were reconstructed from the charge state ion series using the MaxEnt1 algorithm from Waters MassLynx software V4.1 SCN949 according to the manufacturer's instructions. To obtain the ion series described, the major peak of the chromatogram was selected for integration before further analysis.

5. CYTOTOXICITY ASSAYS

Human breast cancer cells in passage 4 or 5 or U937 cells in passage 3 were seeded in 96‐well culture plates at a density of 15,000 cells/well in 90 uL culture media. Cells were then treated in duplicates with anti‐HER2 ARC‐FDCs, anti‐HER2 Fab, anti‐HER2 Fab‐CD38 fusions, or MMAF‐alkyne that were prepared in 3‐fold serial dilution starting from 300 nM. Cells treated with 5 uM paclitaxel (MilliporeSigma, MA) and PBS were included for 0% and 100% viability controls, respectively. Following incubation at 37°C in an incubator with 5% CO2 for 72 h, cells were added with MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide) reagent (Thermo Fisher Scientific, MA), incubated for 3 h, and lysed with 20% SDS in 50% dimethylformamide. Absorbance at 580 nm was then measured with a Synergy H1 plate reader after 1‐hour incubation at 37°C with 5% CO2. EC50 was calculated by the three‐parameter nonlinear regression fitting in GraphPad Prism Version 9.4.0 (GraphPad software, CA).

AUTHOR CONTRIBUTIONS

Yong Zhang: Conceptualization; formal analysis; funding acquisition; writing – original draft; writing – review and editing. Hyo Sun Kim: Investigation; formal analysis; writing – original draft. Kimia Hariri: Investigation; formal analysis. Xiao‐Nan Zhang: Investigation; formal analysis. Liang‐Chieh Chen: Investigation; formal analysis. Benjamin B. Katz: Investigation; formal analysis. Hua Pei: Resources. Stan G. Louie: Resources.

Supporting information

Data S1: Supporting information.

PRO-33-e4924-s001.docx (452.2KB, docx)

ACKNOWLEDGMENTS

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 NIH grant R01EB031830 (to Y. Z.), and National Cancer Institute (NCI) of NIH grant R01CA276240 (to Y. Z.).

Kim HS, Hariri K, Zhang X‐N, Chen L‐C, Katz BB, Pei H, et al. Synthesis of site‐specific Fab‐drug conjugates using ADP‐ribosyl cyclases. Protein Science. 2024;33(4):e4924. 10.1002/pro.4924

Hyo Sun Kim, Kimia Hariri, and Xiao‐Nan Zhang contributed equally to this work.

Review Editor: Aitziber L. Cortajarena

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Supplementary Materials

Data S1: Supporting information.

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