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Antibody Therapeutics, 2020, Vol. 3, No. 1 10–17 doi:10.1093/abt/tbaa002 Advance Access Publication on 17 February 2020 Methods Isolation of rabbit single domain antibodies to B7-H3 via protein immunization and phage display 1 2 1,2 3 Ruonan Feng , Ruixue Wang , Jessica Hong , Christopher M. Dower , 3 1,2, Brad St. Croix and Mitchell Ho NCI Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA, Antibody Therapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA and Tumor Angiogenesis Unit, Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD 21702, USA Received: November 2, 2019; Revised: January 4, 2020; Accepted: February 11, 2020 ABSTRACT Single domain antibodies have certain advantages including their small size, high stability and excellent tis- sue penetration, making them attractive drug candidates. Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. In the present study, we established a method to isolate rabbit VH single domain antibodies for potential cancer therapy. We immunized rabbits with recombinant human B7-H3 (CD276) protein, made a phage-displayed rabbit VH single domain library with a diversity of 7 × 10 , and isolated two binders (A1 and B1; also called RFA1 and RFB1) from phage panning. Both rabbit VH single domains exhibited antigen-dependent binding to B7-H3-positive tumor cell lines but not B7-H3 knockout tumor cell lines. Our study shows that protein immunization followed by phage display screening can be used to isolate rabbit single domain antibodies. The two single domain antibodies reported here may have potential applications in cancer immunotherapy. Statement of Signiﬁcance: Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. We established a method to isolate rabbit VH single domain antibodies to B7-H3 by immunization with recombinant protein followed by phage display screening. The rabbit single domain antibodies speciﬁcally bind B7-H3 expressing tumor cells. KEYWORDS: rabbit monoclonal antibody; single domain antibody; phage display library; B7-H3/CD276; Escherichia coli expression INTRODUCTION excellent thermal stability and high tissue penetration as Single domain antibodies (also commonly called ‘nanobod- compared to conventional IgG antibodies, single domain ies’), the smallest antigen-binding fragments of antibod- antibodies have great potential in medical applications. ies, have approximately 120 amino acids with a molecular In 2019, the US FDA approved the first nanobody drug weight of 12–15 kDa . Most single domain antibodies are caplacizumab, a bivalent humanized camelid single domain derived from the variable region of the heavy chain (V ), antibody (V H), for the treatment of acquired thrombotic which naturally occurs in camelids (termed V H) and thrombocytopenic purpura and thrombosis [7, 8]. In sera of Camelidae, both conventional IgG and cartilaginous fishes (V )[3, 4]. They also exist in certain NAR heavy chain only IgG (HCAbs) together account for human heavy chain diseases such as immunoproliferative 45–75% of all serum immunoglobulins . The HCAbs small intestinal disease, Mediterranean lymphoma or Selig- consists of the antigen binding V H fragment and the mann disease [5, 6]. Due to their small size, high solubility, To whom correspondence should be addressed: Mitchell Ho, NCI Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA. Web: https://ostr.ccr.cancer.gov/resources/antibody-engineering-program/. Tel: (240) 760-7848; Fax: (240) 541-4501; Email: email@example.com © The Author(s) 2020. Published by Oxford University Press on behalf of Antibody Therapeutics. Antibody Therapeutics, 2020 11 following CH2 and CH3 constant domains. The recom- (DMEM) medium (Invitrogen, Carlsbad, CA) supple- binant V H domains are the functional entities that are mented with 10% fetal bovine serum (HyClone, Logan, being exploited. Several evolutionarily acquired structural UT), 1% L-glutamine and 1% penicillin-streptomycin features make the naturally occurring camelid V H (Invitrogen) and incubated in 5% CO with a balance H 2 domain highly soluble and stable [9–11]. The first non- of air at 37 C. The neuroblastoma cell line NBEB was V H mammalian domain antibodies were discovered cultured in RPMI-1640 medium with the same supplements in 1989 from a cDNA library made from the spleens as DMEM. The media were refreshed twice a week. All of mice immunized with lysozyme and keyhole-limpet the cancer cell lines were described in our previous studies haemocyanin, with two mouse VH domains showing [15, 29]. The B7-H3 knockout lines (IMR32-B7H3 KO affinities for lysozyme in the 20 nM range . Human VH and MC38-B7H3 KO) were engineered via CRISPR/Cas9 single domain antibodies [13–15] have been explored and genome editing technology. have potential advantages in clinical applications because of their potential low immunogenicity. Several strategies Production of recombinant B7-H3 protein have been developed to generate human or humanized VH domain antibodies. One strategy involves using phage The extracellular domain (ECD) of B7-H3 (GenBank display on human VH domain libraries [13–15]. Using this accession number NP_001019907, amino acid 29–466) was approach, we previously reported the human single domain fused with a hFc tag. The B7-H3-hFc as well as a hFc antibody HN3 that targets GPC3, a liver cancer antigen, tag control, IAB-hFc [23, 30] was expressed in HEK-293F and showed that the human single domain antibody could cells. Protein purification was performed using a protein A inhibit Wnt/Yap signaling by blocking a functional groove column (GE Healthcare). A His-Flag was inserted at the on the cell surface protein [13, 16, 17]. Another strategy C-terminus of the VH sequence. The 6xHis tag was used for involves using transgenic animals in which human VH Nickel column (GE Healthcare) affinity purification, and germline genes are introduced in mice [18, 19]. Single the FLAG tag was used to monitor VH binding in ELISAs domain antibodies based on shark V s[4, 20–22]have NAR and flow cytometry. also been reported. Our laboratory recently described a large phage-displayed shark V library with a size of NAR 1.2 × 10 from six naïve adult nurse sharks and isolated Rabbit immunization a panel of shark V single domain antibodies to tumor NAR The rabbit immunization followed our previously published and viral antigens . The diversity of our shark V NAR method . Briefly, two female rabbits were intramuscu- library was validated by next-generation sequencing of larly immunized with 100 μg of purified recombinant B7- 1.2 million V sequences. Rabbit antibodies recognize NAR H3 protein mixed with Freund’s complete adjuvant for the diverse epitopes, including those poorly immunogenic priming. Two boosting immunizations were followed by in mice and humans. Our previous work using rabbit mixing the antigen with Freund’s incomplete adjuvant at hybridomas isolated unique rabbit monoclonal antibodies the same dose at an interval of 14 days. After the third (e.g. YP218) that recognize a very rare epitope in the immunization, the spleens were harvested to make the C-terminal end (Region III)  of mesothelin close to phage library. the tumor cell surface [24, 25]. Recently, it has been shown that rabbits can be used to generate immunized VH domain antibodies using a low temperature (16 C) DNA oligos and construction of the rabbit VH phage library phage display method . However, this method resulted To amplify the rabbit VH cDNA fragment, forward in binders that were low in solubility, thermostability and reverse primers that anneal to the 5 - and 3 -end and production yield. We raised the question whether it of VH cDNA were synthesized as described earlier . may be possible to select rabbit VH domain antibodies The primers are listed as follows, with the underlined by first immunizing rabbits and then using the phage ◦ corresponding to the SfiI restriction enzyme site. display method at physiological temperature (e.g. 37 C). In the present project, we performed a proof-of-concept study by immunizing a rabbit with a recombinant protein, followed by phage display screening at 37 C. We chose VH-F1: 5’-GAGGAGTTGGCCCAGGCGGCCCAGTC to focus on B7-H3 based on its emerging potential as GGTGGAGGAGTCCRGG-3’ a therapeutic target [27,28], and successfully identified VH-F2: 5’-GAGGAGTTGGCCCAGGCGGCCCAGTC two VH binders that displayed high expression levels in AGTGAAGGAGTCCGAG-3’ Escherichia coli and specifically bound to cell surface VH-F3: 5’-GAGGAGTTGGCCCAGGCGGCCCAGTC B7-H3. GYTGGAGGAGTCCGGG-3’ VH-F4: 5’-GAGGAGTTGGCCCAGGCGGCCCAGGA GCAGCTGGAGGAGTCCGGG-3’ MATERIALS AND METHODS VH-F5: 5’-GAGGAGTTGGCCCAGGCGGCCCAGGA Cell lines GCAGCTGAAGGAGTCCGG-3’ Human cancer cell lines (Hep3B, HepG2, IMR32, MC38- VH-F6: 5’-GAGGAGTTGGCCCAGGCGGCCCAGRA B7H3+, A431, IMR32-B7H3 KO and MC38-B7H3 KO) GCAGCTGGTGGAGTCCGG-3’ were cultured in Dulbecco’s Modified Eagle Medium 12 Antibody Therapeutics, 2020 at 37 C for 1 h. Thereafter, the unbound phage solution VH-F7: 5’-GAGGAGTTGGCCCAGGCGGCCCAGG was transferred to the B7-H3-hFc plate and incubated at 37 C for 1 h. B7-H3 specific phage binders were eluted from AGCAGCAGAAGGAGTCCGGG-3’ the plate by pH 2.0 citric acid buffer and were immediately VH-F8: 5’-GAGGAGTTGGCCCAGGCGGCCCAGTC neutralized with pH 8.0 Tris-HCl buffer. The eluted output GCTGGAGGAGTCCAGG-3’ phage was re-amplified by re-infection of fresh TG1 cells, VH-F9: 5’-GAGGAGTTGGCCCAGGCGGCCCAGTC and the re-amplified phage was used as the input for the GCTGGGGGAGTCCAGG-3’ next round of panning. After three rounds of panning, sin- VH-F10: 5’-GAGGAGTTGGCCCAGGCGGCCCAGA gle colonies were randomly picked from the output phage CAGTGAAGGAGTCCGAG-3’ infected TG1 cells and monoclonal phage ELISA was con- VH-F11: 5’-GAGGAGTTGGCCCAGGCGGCCCAGT ducted to identify B7-H3 specific binders. CGCTGGAGGAATTCGGG-3’ VH-R1: 5’-gaggagttTGGCCGGCCTGGCCTGARGA Phage ELISA GAYGGTGACCAGGGTGCC-3’ A 96-well ELISA plate was coated with B7-H3-hFc and VH-R2: 5’-gaggagttTGGCCGGCCTGGCCTGAAGA IAB-hFc tag control. After blocking with PBS buffer con- GACGGTGACGAGGGTCCC-3’ taining 2% BSA, 50 μL of pre-blocked phage solution was added to the plate and incubated at 37 C for 30 min. After the plate was washed twice with PBS buffer con- Rabbit VH cDNA were synthesized from the total RNA taining 0.05% Tween 20, phage binding was detected by TM isolated from the immunized spleen using Invitrogen HRP-conjugated anti-M13 antibody (Sino biological, Cat. TM SuperScript IV First-Strand Synthesis kit according #11973-MM05T-H). to the manufacturer’s instruction (ThermoFisher, Cat. For antibody binding ELISA, various concentrations of #18091050). Each forward primer was paired with one of antibody (1:2 serial dilutions starting from 100 μg/mL) were the two reverse primers (R1 or R2), and 22 combinations incubated on the B7-H3-hFc coated plate as mentioned of forward/reverse primers were used to amplify the whole above. Antibody binding was detected by HRP-conjugated VH cDNA fragments. The PCR products were gel-purified anti-FLAG mouse monoclonal antibody M2 (Sigma, Cat. and digested with the restriction enzyme SfiI (NEB, Cat. #A8592). #R0123S), followed by ligation with the pComb3x plasmid (Scripps Research Institute) that has been pre-digested with Flow cytometry the same enzyme. Ten micrograms of the ligation products were used to transform 0.6 mL of E. coli TG1 competent Cells were harvested by detaching with trypsin-EDTA cells (Lucigen, Cat. #60502-2) by electroporation according (Thermo Fisher, Cat. #25200114), centrifuged to form to the manufacturer’s instruction. The transformed TG1 pellet, resuspended in ice-cold PBS at a density of cells were recovered for 45 min at 37 C, shaking at 150 rpm, 10 cells/mL and incubated with 10 μg/mL of B7-H3 then inoculated into 1 L of 2XYT media and cultured for domain antibodies. The antibody binding was detected additional 1 h at 37 C, shaking at 250 rpm. Thereafter, by allophycocyanin (APC)-conjugated anti-FLAG mouse 1 × 10 helper phage M13KO7 (NEB, Cat. #N0315S) was monoclonal antibody (Biolegend, Cat. #637308). The added to the cell culture, followed by incubation at 37 Cfor fluorescence associated with the live cells was measured 4 h. The cells were centrifuged at 3300 g for 30 min to pellet using a FACS Calibur (BD Biosciences, Franklin Lakes, the cell debris, and the supernatant containing the phage NJ). particles was collected and mixed with 3/10 volume of sterile polyethylene glycol (PEG) 8000/NaCl solution (20% Protein structure modeling PEG in 2.5 M NaCl solution). The phage/PEG solution mixture was incubated on ice for 4 h and centrifuged at Structure modeling of rabbit single domain antibodies is 3300 g for 30 min. The final phage pellet was resuspended visualized using the web tool SWISS-MODEL (https:// in 100 mL of PBS buffer containing 20% glycerol, aliquoted swissmodel.expasy.org/). Auto-models and default param- in 1 mL volume size and stored at −80 C. eters were used to generate modeling. Models were built based on the target-template alignment using ProMod3 Version 1.3.0 algorithm. The SWISS-MODEL template Phage panning library (SMTL version 2019-04-11, PDB release 2019-04- Phage panning was carried out using immobilized B7-H3- 05) was searched with BLAST. Overall 16 597 templates hFc protein using our lab protocol [13, 32]. To exclude were found, and the models were built on the template hFc tag binders, an irrelevant IAB-hFc control was also 5DUB, which had a high sequence identity of 78.76 for A1 immobilized in parallel. Ninety six-well ELISA plates were binder and 78.26 for B1 binder. The GMQE scores were coated with 50 μL/well B7-H3-hFc and IAB-hFc proteins 0.96 for both binders, and the QMEAN scores were 0.74 (100 μg/mL in PBS) and incubated at 37 Cfor 1h.After and 2.09 for A1 and B1 binders, respectively. removing the coated protein solution, the plate and phage solution were pre-blocked using PBS buffer containing 2% Statistical analysis BSA and incubated at 37 C for 30 min. After removing the blocking buffer, pre-blocked phage solution was added to All statistical analyses were conducted using GraphPad the IAB-hFc plate to deplete hFc binders by incubating Prism (GraphPad Software, Inc., La Jolla, CA). Antibody Therapeutics, 2020 13 antigen IAB-hFc [23, 30] as a control (Fig. 2B). The serum from the second (M2) and third (M3) immunization showed increased binding to B7-H3-hFc compared to IAB-hFc, although both M2 and M3 showed weak hFc tag binding. Cell binding activity of the polyclonal serum was assessed by flow cytometry (Fig. 2C). The polyclonal serum of the final immunization showed clear cell binding to hepatocellular carcinoma cell lines Hep3B and HepG2 [33, 34]. The results of both ELISA and flow cytometry indicated that B7-H3 is immunogenic in rabbits, at least in the hFc-fusion format, even though this protein is highly conserved across species. Rabbit and human B7-H3 share 90.94% amino acid identity while the overall similarity of mouse, rabbit and human B7-H3 is 98.6%. Phage screening of B7-H3 specific binders After confirming a successful immunization, the spleens of Figure 1. Schematic of rabbit VH antibody production. Infographic demonstrating the procedure used to generate rabbit VH antibodies. the two immunized rabbits were harvested separately, and the VH gene fragments from each rabbit were amplified using degenerate primers  and ligated into the phage dis- RESULTS play vector pComb3x. Ten micrograms of the ligations were used to transform TG1 competent cells by electroporation, Schematic of the rabbit VH single domain antibody resulting in a VH library containing 7 × 10 clones. Phage production panning was carried out at 37 C. To generate rabbit single domain antibodies, as shown The panning was performed on immobilized B7-H3- in Fig. 1, we immunized rabbits with the recombinant hFc. We randomly picked 96 clones and 41 were found ECD of B7-H3. After the rabbits developed immune to specifically bind B7-H3, but not hFc alone. Sequencing responses, mRNA was isolated from lymphocytes, and a analysis identified two highly enriched binders, A1 and B1 cDNA library of variable heavy chain domains was created (also called RFA1 and RFB1) (Fig. 3A). These two binders by reverse transcription. The cDNA was then used to shared very similar germline sequences that were also sim- express VH as fusions containing the phage coat proteins ilar to a VH from rabbit anti-hypusine mAb (deposited in (phage display) and the VH binders that were enriched by GeneBank structure database, protein data bank (PDB) # three rounds of panning against the immobilized antigen. 5DUB). The selected binders were expressed in E. coli with a Structure modeling of A1, B1 using SWISS-MODEL, C-terminal hexahistidine tag to allow purification by showed that A1 and B1 share similar CDR1 and CDR2 nickel-nitrilotriacetic acid affinity chromatography, and loop conformations, with anti-hypusine VH (PDB# contained a N-terminal secretion signal sequence to direct 5DUB), but the CDR3 loops of A1 and B1 were apparently the expressed protein to the periplasm for disulfide bond different (Fig. 3B). formation. Binding properties of the B7-H3 VH single domains Expression and purification of recombinant B7-H3-hFc and The VH domain of A1 and B1 were fused with a tandem immunization of rabbits with recombinant B7-H3-hFc His-FLAG tag at their C-termini, expressed in E.coli, The ECD of human B7-H3 (NCBI Reference Sequence: and soluble VH domains were purified by Nickel affinity NP_001019907, amino acids 29–466) was fused with chromatography . The purification yields for A1 and B1 human IgG1 Fc and expressed in HEK293 cells. After was 2 mg/L and 10 mg/L, respectively, while the rabbit purification on protein A columns, the purity was assessed single domain antibodies previously selected at a low by sodium dodecyl sulfate polyacrylamide gel electrophore- temperature had extremely low expression levels (0.003 and sis (SDS-PAGE) under both reducing and non-reducing 0.08 mg/L) . The purity was high as shown by SDS- conditions (Fig. 2A). The theoretical size of the reduced PAGE (Fig. 4A). The theoretical size of a reduced rabbit B7-H3-hFc is about 75 kDa, with an apparent migration VH single domain is about 15 kDa, with an apparent migra- position of about 100 kDa on the gel, likely due to tion position of a single protein band for A1 or B1 under glycosylation since B7-H3 contains six N-glycosylation the non-reducing condition. Biophysics analysis using size sites. Two female rabbits were intramuscularly immunized exclusion chromatography and nano-differential scanning with 100 micrograms of B7-H3-hFc in PBS buffer mixed fluorimetry (nanoDSF) may be useful to more accurately with an equal volume of Freund’s adjuvant. After three check the molecular weight (or oligomerization state) and immunizations with an interval of 14 days between doses, thermostability of rabbit single domain antibodies in the the titer of anti-B7-H3-hFc antibodies was measured at future when developing rabbit single domains as drugs. about 1:10 000 by ELISA, using hFc tagged irrelevant Protein binding was validated by ELISA (Fig. 4B). Next, 14 Antibody Therapeutics, 2020 Figure 2. SDS-PAGE analysis of purified B7-H3-hFc, titer of B7-H3-hFc immunized rabbit sera and confirmation of its cell binding. (A) One or five micrograms of purified B7-H3-hFc, non-reduced (Non.) or reduced (Red.) by β -mercaptoethanol were separated on 8% SDS-PAGE gel. Protein bands were visualized by Coomassie Blue R-250 staining. (B) Titering of B7-H3-hFc immunized rabbit sera by protein binding ELISA. IAB-hFc, which is derived from an N-terminal fragment of mesothelin, served as a hFc tag control. R31M0, R31M1, R31M2 and R31M3 represented the sera of pre-immunization, 1st, 2nd and 3rd immunization. (C) Cell binding assay of the immunized sera. Shaded area represents cells stained with fluorescein isothiocyanate (FITC)- conjugated 2nd (goat anti-rabbit) antibody only; blue curve represents cells stained with pre-immunization sera; red curve represents cells stained with immunized sera R31M3. flow cytometry was performed to assess cell binding capa- fore those conserved proteins that are poorly immunogenic bility (Fig. 4C). In this assay, A1 and B1 specifically bound in mice may evoke better immunogenicity in rabbits [24, B7-H3-positive human neuroblastoma cell lines IMR32 36]. We previously isolated a unique high affinity rabbit and NBEB, murine colon adenocarcinoma MC38B7H3+ monoclonal antibody (YP218) from almost 8000 rabbit and epidermoid carcinoma A431, but not their B7-H3 hybridoma clones, which binds a poorly immunogenic site knockout counterparts IMR32-B7H3 KO and MC38- in the C-terminal region of mesothelin . Second, rabbit B7H3 KO. Therefore, we were able to successfully isolate monoclonal antibodies generally have high affinity and two rabbit VH domain antibodies that bind cell surface specificity, with the affinity range of 20–200 pM [35, 37]. B7-H3 in multiple cancer lines. Third, our previous work showed that rabbit monoclonal antibodies can be successfully humanized without losing their affinity and specificity , therefore immunogenicity should not be a barrier for therapeutic applications. Despite DISCUSSION their superiority, unlike mouse monoclonal antibodies, only Rabbits are a unique source for generating monoclonal a few rabbit monoclonal antibodies have been investigated antibodies that can be used as research tools, diagnostics for clinical applications likely due to the lag in develop- and therapeutics . There are several major advantages ment of rabbit hybridoma technology  and rabbit anti- of using rabbit antibodies. First, rabbits are phylogenet- body phage display libraries  at major institutes and ically more distant from humans than mice, and there- companies. Antibody Therapeutics, 2020 15 Figure 3. Sequences and structural modeling of the B7-H3 single domain antibodies. (A) Sequence alignment of the B7-H3 binders, along with a similar VH from a rabbit anti-hypusine mAb (PDB# 5DUB). CDR regions were defined by IMGT delineating system. (B) Structure modeling of A1 and B1 binders using online software SWISS-MODEL (https://swissmodel.expasy.org/). The crystal structure of 5DUB is also shown fo r comparison. With the advantages of small size, high solubility and two representative binders that bound B7-H3 protein and stability, single domain antibodies are garnering increased B7-H3 positive cancer cells. Importantly, the rabbit single attention. New therapeutic applications for V Hs are con- domain antibodies previosuly isolated by using a low tem- tinuously being identified in many diseases. A recently study perature phage panning had extremely low expression (less demonstrated that high affinity rabbit VH domain anti- than 0.1 mg/L) in E. coli; in contrast, the purification yields bodies could be generated by a low temperature (16 C) for the A1 and B1 rabbit single domain antibodies isolated phage display method . However, the low temperature in the physiological temperature (37 C) were 10–100 fold phage display method tends to enrich binders that are higher (2–10 mg/L). Furthermore, the A1 and B1 binders unstable and difficult to express, bolstering the need for had positive cell binding for B7-H3-positive human tumor additional efforts to improve the physicochemical proper- cells, but not B7-H3 knockout tumor cells. ties of the binders, especially their expression . In the Phage panning is an artificial selection system. The fact present study, we sought to test the possibility of screening that we can isolate rabbit single domain antibodies by phage well-expressed binders using a physiological temperature display does not establish whether rabbits naturally can phage display method. have heavy chain antibodies. It has been reported that some As a proof-of-concept, we chose B7-H3 as a target, humans with certain heavy chain diseases may naturally because it is overexpressed in many cancer types, inhibits produce heavy chain antibodies [5, 6]. T-cell activation and is regarded as an important immune Our study demonstrates that rabbit VH domain anti- check point member of the B7 and CD28 families [28, 40]. bodies can be generated by phage display. Given the It is also overexpressed in many solid tumors, making it important role of B7-H3 in regulating T-cell functions, an attractive therapeutic target . Here,wewereableto the two B7-H3 domain antibodies generated here may successfully express the ECD of B7-H3 in HEK-293 cells. have some unique potential applications, especially for We then used a physiological temperature (37 C) phage cancer immunotherapy where single domain antibodies display method to make the rabbit VH phage library par- can be used for development of CAR-T cells or bispecific ticles and performed phage panning. We finally obtained antibodies. 16 Antibody Therapeutics, 2020 Figure 4. Binding properties of the B7-H3 single domain antibodies. (A) SDS-PAGE analysis of the purified A1 and B1 binders (VH-His-FLAG fusion) from E. coli. Two micrograms of purified rabbit VH single domain antibody (A1 and B1), non-reduced (Non.) or reduced (Red.) by β-mercaptoethanol were separated on 8% SDS-PAGE gel. Protein bands were visualized by Coomassie Blue R-250 staining. (B) Antigen binding by ELISA. Five micrograms of B7-H3-hFc were coated on the ELISA plate, and different concentrations of rabbit single domain antibodies were incubated. Binding was detected by HRP conjugated anti-FLAG mouse monoclonal antibody M2. The binding curves were plotted using the software GraphPad Prism and the calculated EC values were determined by the software’s algorism of hyperbola one site binding. (C) Cell binding determination by flow cytometry. Ten microgram per mL of rabbit single domain antibodies was co-incubated with 1 million cells. Antibody binding was visualized by APC conjugated anti-FLAG monoclonal antibody. 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Antibody Therapeutics – Oxford University Press
Published: Feb 17, 2020
Keywords: rabbit monoclonal antibody; single domain antibody; phage display library; B7-H3/CD276; Escherichia coli expression
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