Isolation of rabbit single domain antibodies to B7-H3 via protein immunization and phage display

Single domain antibodies have certain advantages including their small size, high stability and excellent tissue 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 x 10 9 , 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.

Introduction
Single domain antibodies (also commonly called "nanobodies"), the smallest antigenbinding fragments of antibodies, have approximately 120 amino acids with a molecular weight of 12-15 kDa (1). Most single domain antibodies are derived from the variable region of the heavy chain (V H ), which naturally occurs in camelids (termed V H H) (2) and cartilaginous fishes (V NAR ) (3,4). They also exist in certain human heavy chain diseases such as immunoproliferative small intestinal disease (IPSID), Mediterranean lymphoma, or Seligmann disease (5,6). Due to their small size, high solubility, excellent thermal stability and high tissue penetration as compared to conventional IgG antibodies, single domain antibodies have great potential in medical applications. In 2019, the US FDA approved the first nanobody drug caplacizumab, a bivalent humanized camelid single domain antibody (V H H), for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) and thrombosis (7,8).
In sera of Camelidae, both conventional IgG and heavy chain only IgG (HCAbs) together account for 45% to 75% of all serum immunoglobulins (1). The HCAbs consists of the antigen binding V H H fragment and the following CH2 and CH3 constant domains. The recombinant V H H domains are the functional entities that are being exploited. Several evolutionarily acquired structural features make the naturally occurring camelid V H H domain highly soluble and stable (9)(10)(11). The first non-V H H mammalian domain antibodies were discovered in 1989 from a cDNA library made from the spleens of mice immunized with lysozyme and keyhole-limpet haemocyanin, with two mouse VH domains showing affinities for lysozyme in the 20 nM range (12). Human VH single domain antibodies (13)(14)(15) have been explored with potential advantageous in clinical applications because of their potential Downloaded from https://academic.oup.com/abt/advance-article-abstract/doi/10.1093/abt/tbaa002/5735320 by guest on 19 February 2020 low immunogenicity. Several strategies have been developed to generate human or humanized VH domain antibodies. One strategy involves using phage display on human VH domain libraries (13)(14)(15). Using this approach, we previously reported the human single domain antibody HN3 that targets GPC3, a liver cancer antigen, and showed that the human single domain antibody could inhibit Wnt/Yap signaling by blocking a functional groove on the cell surface protein (13,16,17). Another strategy involves using transgenic animals in which human VH germline genes are introduced in mice (18,19). Single domain antibodies based on shark V NAR s (4,(20)(21)(22) have also been reported . Our laboratory recently described a large phage-displayed shark V NAR library with a size of 1.2 x 10 10 from six naïve adult nurse sharks and isolated a panel of shark V NAR single domain antibodies to tumor and viral antigens (4). The diversity of our shark V NAR library was validated by next-generation sequencing of 1.2 million V NAR sequences. Rabbit antibodies recognize diverse epitopes, including those poorly immunogenic in mice and humans. Our previous work using rabbit hybridomas isolated unique rabbit monoclonal antibodies (e.g. YP218) that recognize a very rare epitope in the C-terminal end (Region III) (23) of mesothelin close to 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) phage display method (26). However, this method resulted in binders that were low in solubility, thermostability, and production yield. We raised the question whether it may be possible to select rabbit VH domain antibodies by first immunizing rabbits and then using the phage display method at physiological temperature (e.g. 37 °C). In the present project, we performed a proof-ofconcept study by immunizing a rabbit with a recombinant protein, followed by phage display screening at 37 °C. We chose to focus on B7-H3 based on its emerging potential as a therapeutic target (27,28), and successfully identified two VH binders that displayed high expression levels in Escherichia coli (E. coli) and specifically bound to cell surface B7-H3.

Production of recombinant B7-H3 protein.
The extracellular domain of B7-H3 (GenBank accession number NP_001019907, amino acid 29-466) was fused with a hFc tag. The B7-H3-hFc as well as a hFc tag control, IAB-hFc (23,30) was expressed in HEK-293F cells. Protein purification was performed using a protein A column (GE Healthcare). A His-Flag was inserted at the C-terminus of the VH sequence. The 6x His tag was used for Nickel column (GE Healthcare) affinity purification, and the FLAG tag was used to monitor VH binding in ELISAs and flow cytometry.
Rabbit immunization. The rabbit immunization followed our previously published method (24). Briefly, two female rabbits were intramuscularly immunized with 100 μg of purified recombinant B7-H3 protein mixed with Freund's complete adjuvant for the priming. Two boosting immunizations were followed by mixing the antigen with Freund's incomplete adjuvant at the same dose at an interval of 14 days. After the third immunization, the spleens were harvested to make the phage library.
DNA oligos and construction of the rabbit VH phage library. To amplify the rabbit VH cDNA fragment, forward and reverse primers that anneal to the 5'-and 3'-end of VH cDNA were synthesized as described earlier (31). The primers are listed as follows, with the underlined corresponding to the SfiI restriction enzyme site.
Rabbit VH cDNA were synthesized from the total RNA isolated from the immunized spleen using Invitrogen™ SuperScript™ IV First-Strand Synthesis kit according to the manufacturer's instruction (ThermoFisher, Cat. #18091050). Each forward primer was paired with one of the two reverse primers (R1 or R2), and 22 combinations of forward/reverse primers were used to amplify the whole VH cDNA fragments. The PCR products were gelpurified and digested with the restriction enzyme SfiI (NEB, Cat. #R0123S), followed by ligation with the pComb3x plasmid (Scripps Research Institute) that has been pre-digested with the same enzyme. Ten micrograms of the ligation products were used to transform 0. Phage panning. Phage panning was carried out using immobilized B7-H3-hFc protein using our lab protocol (13,32). To exclude hFc tag binders, an irrelevant IAB-hFc control was also immobilized in parallel. Ninety six-well ELISA plates were coated with 50 μL/well B7-H3-hFc and IAB-hFc proteins (100 μg/mL in PBS) and incubated at 37°C for 1 hour. After removing the coated protein solution, the plate and phage solution were pre-blocked using PBS buffer containing 2% BSA and incubated at 37°C for 30 min. After removing the blocking buffer, pre-blocked phage solution was added to the IAB-hFc plate to deplete hFc binders by incubating at 37 °C for 1 hour. Thereafter, the unbound phage solution was transferred to the B7-H3-hFc plate and incubated at 37 °C for 1 hour. B7-H3 specific phage binders were eluted from the plate by pH 2.0 citric acid buffer and were immediately neutralized with pH 8.0 Tris-HCl buffer. The eluted output phage was re-amplified by reinfection of fresh TG1 cells, and the re-amplified phage was used as the input for the next round of panning. After three rounds of panning, single colonies were randomly picked from the output phage infected TG1 cells and monoclonal phage ELISA was conducted to identify B7-H3 specific binders.

Schematic of the rabbit VH single domain antibody production.
To generate rabbit single domain antibodies, as shown in Figure 1, we immunized rabbits with the recombinant extracellular domain of B7-H3. After the rabbits developed immune responses, mRNA was isolated from lymphocytes, and a cDNA library of variable heavy chain domains was created by reverse transcription. The cDNA was then used to express VH as fusions containing the phage coat proteins (phage display) and the VH binders which were enriched by three rounds of panning against the immobilized antigen. The selected binders were expressed in E. coli with a C-terminal hexahistidine tag to allow purification by nickel-nitrilotriacetic acid affinity chromatography, and contained a N-terminal secretion signal sequence to direct the expressed protein to the periplasm for disulfide bond formation. After three immunizations with an interval of 14 days between doses, the titer of anti-B7-H3-hFc antibodies was measured at about 1:10,000 by ELISA, using hFc tagged irrelevant 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 the two immunized rabbits were harvested separately, and the VH gene fragments from each rabbit were amplified using degenerate primers (31) and ligated into the phage display vector pComb3x. Ten micrograms of the ligations were used to transform TG1 competent cells by electroporation, resulting in a VH library containing 7 x 10 9 clones. Phage panning was carried out at 37 °C.
These two binders shared very similar germline sequences that were also similar to a VH from rabbit anti-hypusine mAb (deposited in GeneBank structure database, protein data bank (PDB) # 5DUB).
Binding properties of the B7-H3 VH single domains. The VH domain of A1 and B1 were fused with a tandem His-FLAG tag at their C-termini, expressed in E.coli., and soluble VH domains were purified by Nickel affinity chromatography (4). The purification yields for A1 and B1 was 2 mg/L and 10 mg/L, respectively while the rabbit single domain antibodies previously selected at a low temperature had extremely low expression levels (0.003 and 0.08 mg/L) (26). The purity was high as shown by SDS-PAGE (Fig. 4A). The theoretical size of a reduced rabbit VH single domain is about 15 kDa, with an apparent migration position of a single protein band for A1 or B1 under the non-reducing condition. Biophysics analysis using size exclusion chromatography and nano-differential scanning fluorimetry (nanoDSF) may be useful to more accurately check the molecular weight (or oligomerization state) and thermostability of rabbit single domain antibodies in the future when developing rabbit single domains as drugs. Protein binding was validated by ELISA (Fig. 4B). Next, flow cytometry was performed to assess cell binding capability (Fig. 4C). In this assay, A1 and B1 specifically bound B7-H3-positive human neuroblastoma cell lines IMR32 and NBEB, murine colon adenocarcinoma MC38B7H3+, and epidermoid carcinoma A431, but not their B7-H3 knockout counterparts IMR32-B7H3 KO and MC38-B7H3 KO. Therefore, we were able to successfully isolate two rabbit VH domain antibodies that bind cell surface B7-H3 in multiple cancer lines

Discussion
Rabbits are a unique source for generating monoclonal antibodies that can be used as research tools, diagnostics, and therapeutics (35). There are several major advantages of using rabbit antibodies. First, rabbits are phylogenetically more distant from humans than mice, and therefore those conserved proteins that are poorly immunogenic in mice may evoke better immunogenicity in rabbits (24,36). We previously isolated a unique high affinity rabbit monoclonal antibody (YP218) from almost 8000 rabbit hybridoma clones, which binds a poorly immunogenic site in the C-terminal region of mesothelin (24). Second, rabbit monoclonal antibodies generally have high affinity and specificity, with the affinity range of 20-200 pM (35,37). Third, our previous work showed that rabbit monoclonal antibodies can be successfully humanized without losing their affinity and specificity (25), therefore immunogenicity should not be a barrier for therapeutic applications. Despite their superiority, unlike mouse monoclonal antibodies, only a few rabbit monoclonal antibodies have been investigated for clinical applications likely due to the lag in development of rabbit hybridoma technology (38) and rabbit antibody phage display libraries (39) at major institutes and companies.
With the advantages of small size, high solubility and stability, single domain antibodies are garnering increased attention. New therapeutic applications for V H Hs are continuously being identified in many diseases. A recently study demonstrated that high affinity rabbit VH domain antibodies could be generated by a low temperature (16 °C) phage display method (26). However, the low temperature phage display method tends to enrich binders that are unstable and difficult to express, bolstering the need for additional efforts to improve the physicochemical properties of the binders, especially their expression (10). In the present study, we sought to test the possibility of screening well-expressed binders using a physiological temperature phage display method.
As a proof-of-concept, we chose B7-H3 as a target, because it is over-expressed in many cancer types, inhibits T-cell activation and is regarded as an important immune check point member of the B7 and CD28 families (28,40). It is also overexpressed in many solid tumors, making it an attractive therapeutic target (27). Here, we were able to successfully express the extracellular domain of B7-H3 in HEK-293 cells. We then used a physiological temperature (37 °C) phage display method to make the rabbit VH phage library particles and performed phage panning. We finally obtained two representative binders that bound B7-H3 protein and B7-H3 positive cancer cells. Importantly, the rabbit single domain antibodies previosuly isolated by using a low temperature phage panning had extremely low expression (less than 0.1 mg/L) in E. coli; in contrast, the purification yields for the A1 and B1 rabbit single domain antibodies isolated in the physiological temperature (37 o C) were 10-100 fold higher (2-10 mg/L). Furthermore, the A1 and B1 binders had positive cell binding for B7-H3positive human tumor cells, but not B7-H3 knockout tumor cells.
Phage panning is an artificial selection system. The fact that we can isolate rabbit single domain antibodies by phage display does not establish whether rabbits naturally can have heavy chain antibodies. It has been reported that some humans with certain heavy chain diseases may naturally produce heavy chain antibodies (5,6).
Our study demonstrates that rabbit VH domain antibodies can be generated by phage