Identification of the O-Glycan Epitope Targeted by the Anti-Human Carcinoma Monoclonal Antibody (mAb) NEO-201

Simple Summary Glycosylation is an important post-translational modification made on mammalian proteins and lipids. In cancer cells, the disruption of several glycosylation patterns, such as the O-glycosylation, has been observed. The expression of incomplete/truncated O-glycans in cancer cells occurs in both solid and liquid tumors and is correlated with poor prognosis and tumor progression. The employment of monoclonal antibodies (mAbs) targeting truncated O-glycans in cancer cells could serve as an effective strategy to counteract tumor growth. In previous studies, we reported that the IgG1-humanized mAb NEO-201 binds specifically to tumor-associated variants of CEACAM5 and CEACAM6 expressed by colon, ovarian, pancreatic, non-small cell lung, head and neck, cervical, uterine and breast cancers but is not reactive against most normal tissues. Since CEACAMs are highly glycosylated proteins, in this article, we evaluated whether the epitope recognized by NEO-201 is an O-glycan. This study demonstrated that NEO-201 binds to core 1 O-glycans and targets and kills cancer cells expressing core 1 and extended core 1 O-glycans. Usually, GalNAc residue can be added on to threonine and serine to form O-glycans, suggesting that NEO-201 binds to core 1 and extended core 1 O-glycans attached to any protein carrying amino acid regions containing serine and threonine Abstract Truncated O-glycans expressed in cancer cells support tumor progression, and they may serve as potential targets to improve the monitoring and treatment of cancers. Previously, we reported that NEO-201 binds to several tumors expressing tumor-associated CEACAM5 and CEACAM6 variants but does not bind to those expressed in healthy tissues. This specific binding may be associated with the presence of truncated O-glycans attached on the protein sequence of these variants. To evaluate the glycosylation pattern targeted by NEO-201 we performed an O-glycan array consisting of 94 O-glycans. O-glycan profiles were elucidated from the human pancreatic cancer cell line CFPAC-1, human hematological neoplastic cells (HL-60, U937, K562) and human neutrophils. The O-glycan array analysis showed that NEO-201 interacts with core 1-4 O-glycans and that the binding to a specific core 1 O-glycan was the strongest. The O-glycan profiling of the NEO-201-reactive cells CFPAC-1, HL-60, U937 and human neutrophils showed that cells recognized by NEO-201 express mostly core 1 and/or extended core 1 O-glycans. In addition, NEO-201 mediates antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells expressing core 1 or extended core 1 O-glycan profiles. These results demonstrated that NEO-201 binds to core 1 and extended core 1 O-glycans expressed in its target cells. Since GalNAc residue can be added onto threonine and serine to form O-glycans, it is very likely that NEO-201 recognizes these O-glycans attached to any protein with amino acid regions containing serine and threonine.

Reagents
The NEO-201 mAb employed in this study was generated from the Hollinshead vaccine platform, using tumor-associated antigens derived from tumor membrane fractions pooled from surgically resected specimens from patients with colon cancer, as previously described [1,[19][20][21].
The anti-CEACAM6 mAb 9A6 was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Cell Lines and Culture
The following human carcinoma cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA): pancreatic cancer (CFPAC-1), acute myeloid leukemia (AML) (HL-60 and U937), chronic myeloid leukemia (CML) (K562) and human kidney embryo (HEK293T). All human carcinoma cell lines were maintained in a culture medium (Corning Life Science, Manassas, VA, USA) designated by the provider for propagation and maintenance. The culture medium was supplemented with 10% USAsourced and heat-inactivated HyClone fetal bovine serum, defined (GE Healthcare Life Sciences, Issaquah, WA, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Corning Life Science, Manassas, VA, USA).

Isolation of Neutrophils from PBMCs
The negative selection of neutrophils from PBMCs from healthy donors was performed using the EasySep™ Direct Human Neutrophil Isolation Kit (StemCell Technologies, Vancouver, BC, Canada), according to the manufacturer's protocol. Neutrophils isolated from healthy donors were used for O-glycan profiling analysis and for the ADCC assay.

ELISA
To detect NEO-201 binding to the commercially available recombinant human (rh) CEACAM6 expressed in bacterial (E. coli BL21) (Origene, Rockville, MD, USA) and mammalian HEK293T cells (Acro Biosystem, Newark, DE, USA), 96-well ELISA plates were first coated overnight at 4 • C with 100 µL/well of 400 ng/mL recombinant human CEACAM6 (expressed in E. coli BL21 or in HEK293T cells) dissolved in 0.2 M carbonate-bicarbonate buffer pH 9.4. The plates were washed with 1× Tris-buffered saline (TBS) + 0.05% Tween-20 (VWR International, Radnor, PA, USA) (wash buffer) and then blocked with 200 µL/well of 5% milk in 1× TBS for 1 h at 37 • C. The plates were washed three times with wash buffer, and then 100 µL/well of NEO-201 or 9A6 mAb was added in two-fold serial dilution from 20 ng/mL to 0.016 ng/mL and incubated for 1 h at 37 • C.
The plates were washed three times with wash buffer, and 100 µL/well donkey antihuman IgG antibody peroxidase labeled (VWR International, Radnor, PA, USA) was added to the plate at a 1:10,000 dilution and incubated for 1 h at 37 • C. The plates were washed three times with wash buffer, and 100 µL/well of tetramethylbenzidine (TMB) substrate solution (VWR International, Radnor, PA, USA) was added for 10 min at room temperature in the dark. The reaction was stopped by adding 50 µL/well of 2N H 2 SO 4 . Absorbance was measured at 450 nm on a FLUOstar Omega plate reader (BMG Labtech, Ortenmberg, Germany).
The amino acid sequences of the full-length CEACAM5 and CEACAM6 are shown in Figure S1. After staining, the cells were washed twice with cold 1× PBS and examined using an FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). The analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positivity was determined by comparing unstained cells with cells stained with NEO-201. Staining values > 10% were considered positive.

O-Glycan Array to Identify O-Glycan Recognized by NEO-201
An O-glycan array consisting of 94 O-glycans ( Figure S2) was used to identify the O-glycans that bind NEO-201. The array layout is shown in Figure S3. The details of this procedure are described in the Supplementary Materials. The O-glycan array was obtained from Creative Proteomics (Shirley, NY, USA). NEO-201 was used at three concentrations (100 µg/mL, 20 µg/mL, 4 µg/mL) to incubate with the O-glycan array. The binding result was read using an Innopsys InnoScan 710 Microarray Scanner with a high-power laser at 5 PMT. Software was used to detect each spot on the array and calculate the relative fluorescence unit (RFU) intensity for each spot.
Background RFU was subtracted from each spot's RFU value.

O-Glycan Profiling of Cells Reactive with NEO-201
NEO-201-reactive cell lines CFPAC-1, HL-60, U937, human neutrophils and NEO-201 non-reactive cell line K562 were used for O-glycan profile analysis. O-glycan profile analysis was performed by Creative Proteomics (Shirley, NY, USA). Several steps were used in the profiling procedure, including N-glycans removal, O-glycans preparation, permethylation and MS MALDI analysis [22]. The detail of the O-glycan profiling is described in the Supplementary Materials.

Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay
To evaluate the ADCC activity mediated by NEO-201 against HL-60 and human neutrophils, a modification of a previously described radioactive indium (In-111) release ADCC assay was performed [2].
To determine the NEO-201-mediated ADCC activity against HL-60, NK cells were isolated by negative selection from the PBMCs of two healthy donors using the EasySep human NK cell isolation kit (StemCell Technologies, Vancouver, BC, Canada), according to the manufacturer's protocol. Purified NK cells were cultured overnight in RPMI 1640 medium (Corning Life Science, Manassas, VA, USA) supplemented with 10% USA-sourced and heat-inactivated HyClone fetal bovine serum, defined (GE Healthcare Life Sciences, Issaquah, WA, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Corning Life Science, Manassas, VA, USA) prior to being used as effector cells. On the day of the assay, target cells (1 × 10 6 ) were labeled with 60 µCi of In-111. NK cells from two different healthy donors were added to the labeled target cells in the presence of 10 µg/mL of NEO-201 or 10 µg/mL of human IgG1 isotype (as control) at effector:target ratios 20:1 and 10:1 and incubated at 37 • C for 4 h. To determine the NEO-201-mediated ADCC activity against neutrophils, neutrophils were isolated from the whole blood of two healthy donors using the EasySep™ Direct Human Neutrophil Isolation Kit (StemCell Technologies, Vancouver, BC, Canada) and used as target cells. Isolated NK effectors from the same healthy donor were thawed the evening prior to conducting the assay and cultured overnight. On the day of the assay, neutrophils (1 × 10 6 ) were used as the target and labeled with 60 µCi of In-111. Then, neutrophils were incubated at 37 • C for 4 h with 10 µg/mL of NEO-201 or human IgG1 isotype control antibody plus NK as effector cells at effector:target ratios 25:1, 12.5:1 and 6.25:1.
Specific lysis was calculated as % specific lysis = 100 − [{average live target cell count for antibody-treated samples/average live target count for control samples} × 100].

Statistical Analysis
Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Student's t-test, one-way ANOVA and two-way ANOVA with Bonferroni posttest analysis were performed where indicated, and p < 0.05 was considered statistically significant.

NEO-201 Binds to Mammalian-Expressed rhCEACAM6 but Not to Bacterial-Expressed rhCEACAM6 by ELISA
In previous studies, we showed that NEO-201 specifically recognizes a tumor-associated variant of CEACAM5 and CEACAM6, which is not expressed in normal tissues, and proved that NEO-201 binds to both mammalian-expressed recombinant human CEACAM5 and CEACAM6 but not to CEACAM1 or CEACAM8 by ELISA [2,23].
To evaluate if post-translational modifications made on proteins by mammalian cells are responsible for the NEO-201 binding, we performed an ELISA comparing the binding of NEO-201 and the commercial anti-CEACAM6 mAb 9A6 to HEK293-expressed rhCEACAM6 (mammalian) or E. coli-expressed rhCEACAM6 (bacterial). Figure 1A shows that NEO-201 binds to mammalian-expressed rhCEACAM6 but not to bacterial-expressed rhCEACAM6, while 9A6 mAb binds to both types of rhCEACAM6 ( Figure 1B

NEO-201 Binds to O-Glycans
Since glycosylation is a post-translational modification of proteins affected by oncogenesis and human cancer cells may have several glycan modifications, including the expression of truncated O-glycans, we performed an O-glycan array to test the binding activity of NEO-201 to 94 different O-glycan structures ( Figure S2).
As shown in Figure 2, of these 94 O-glycans tested, 01, 02, 06, 023, 026 and 039 Oglycans showed binding to NEO-201 in a dose-dependent manner. The 06 binding interaction was the strongest of any observed. 01 and 02 are Tn antigens. 06 is core 1, 023 is These results further suggest that NEO-201 binds to a post-translational modification made by mammalian cells on rhCEACAM6.

NEO-201 Binds to O-Glycans
Since glycosylation is a post-translational modification of proteins affected by oncogenesis and human cancer cells may have several glycan modifications, including the expression of truncated O-glycans, we performed an O-glycan array to test the binding activity of NEO-201 to 94 different O-glycan structures ( Figure S2).

NEO-201 Binds to O-Glycans
Since glycosylation is a post-translational modification of proteins affected by oncogenesis and human cancer cells may have several glycan modifications, including the expression of truncated O-glycans, we performed an O-glycan array to test the binding activity of NEO-201 to 94 different O-glycan structures ( Figure S2).

Human Cancer Cell Lines and Human Neutrophils Recognized by NEO-201 Express Core 1 and Extended Core 1 O-Glycans
To further confirm that O-glycans are the real target of NEO-201, we profiled different human carcinoma cell lines for NEO-201 binding using flow cytometry. The assessment of the binding activity of NEO-201 revealed that CFPAC-1, HL-60 and U937 cancer cell lines show a strong reactivity for NEO-201, while K562 is negative ( Figure 3).

Human Cancer Cell Lines and Human Neutrophils Recognized by NEO-201 Express Core 1 and Extended Core 1 O-Glycans
To further confirm that O-glycans are the real target of NEO-201, we profiled different human carcinoma cell lines for NEO-201 binding using flow cytometry. The assessment of the binding activity of NEO-201 revealed that CFPAC-1, HL-60 and U937 cancer cell lines show a strong reactivity for NEO-201, while K562 is negative ( Figure 3). These cell lines were then screened to evaluate if they express O-glycans recognized by NEO-201. We also included neutrophils for the O-glycan profiling, since we observed in previous studies that they are targeted by NEO-201 [1,4] O-glycan profiles for these cells, including m/z, compositions, proposed structures and the relative abundance of the most expressed O-glycans are reported in Tables 1 and  2 Figure S4.

. The MS spectrum of the O-glycans of all cells screened is reported in
The O-glycan profile of the human pancreatic cancer cell line CFPAC-1 shows that this cell line expresses mostly sialylated core 1 O-glycans ( Table 1).
The O-glycan profile of human neutrophils showed that these cells have a significant number of O-glycans in common with CFPAC-1, but the relative abundance varies (Table  1).  These cell lines were then screened to evaluate if they express O-glycans recognized by NEO-201. We also included neutrophils for the O-glycan profiling, since we observed in previous studies that they are targeted by NEO-201 [1,4] O-glycan profiles for these cells, including m/z, compositions, proposed structures and the relative abundance of the most expressed O-glycans are reported in Tables 1 and 2. The MS spectrum of the O-glycans of all cells screened is reported in Figure S4.
The O-glycan profile of the human pancreatic cancer cell line CFPAC-1 shows that this cell line expresses mostly sialylated core 1 O-glycans ( Table 1).
The O-glycan profile of human neutrophils showed that these cells have a significant number of O-glycans in common with CFPAC-1, but the relative abundance varies ( Table 1).
The O-glycan profile of the AML cell lines HL-60 and U937 and the CML cell line K562 showed that HL-60 only expresses the extended core 1 profile, U937 mostly expresses the extended core 1 and core 2 profiles and K562 only expresses the extended core 2 profile ( Table 2).
The K562 cell line only expresses the extended core 2 glycans and is negative for the binding with NEO-201, while all the other cells positive for NEO-201 in flow cytometry mostly express core 1 and/or extended core 1 O-glycans. These data suggest that core 1 or extended core 1 O-glycans are epitopes recognized by NEO-201.
In this study, to further prove the concept that NEO-201 can kill target cells expressing core 1 and/or extended core 1 O-glycans through ADCC, we used HL-60 and human neutrophils as target cells in an in vitro ADCC assay. Human isolated NK cells from two different healthy donors were used as effector cells. The reason for not using U937 and K562 as a target is that both U937 and K562 are highly susceptible to NK cells killing and are thus not suitable to be used as a target for the ADCC assay. Figure 4 shows that NEO-201 can mediate ADCC activity against HL-60 and human neutrophils with isolated NK cells as effectors at all E:T ratios.   Table 1).
The O-glycan profile of human neutrophils showed that these cells hav number of O-glycans in common with CFPAC-1, but the relative abundanc 1). O-glycan profiles for these cells, including m/z, compositions, propo and the relative abundance of the most expressed O-glycans are reported i 2. The MS spectrum of the O-glycans of all cells screened is reported in Fig  The O-glycan profile of the human pancreatic cancer cell line CFPAC this cell line expresses mostly sialylated core 1 O-glycans ( Table 1).
The O-glycan profile of human neutrophils showed that these cells ha number of O-glycans in common with CFPAC-1, but the relative abundanc 1). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U9 presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2). The O-glycan profile of the AML cell lines HL-60 and U937 and the CML ce K562 showed that HL-60 only expresses the extended core 1 profile, U937 mos presses the extended core 1 and core 2 profiles and K562 only expresses the extende 2 profile (Table 2).  The O-glycan profile of the AML cell lines HL-60 and U937 and the K562 showed that HL-60 only expresses the extended core 1 profile, U presses the extended core 1 and core 2 profiles and K562 only expresses the 2 profile (Table 2).   Cancers 2022, 14, x FOR PEER REVIEW 6 of 6
In this study, to further prove the concept that NEO-201 can kill target cells expressing core 1 and/or extended core 1 O-glycans through ADCC, we used HL-60 and human neutrophils as target cells in an in vitro ADCC assay. Human isolated NK cells from two different healthy donors were used as effector cells. The reason for not using U937 and K562 as a target is that both U937 and K562 are highly susceptible to NK cells killing and are thus not suitable to be used as a target for the ADCC assay. Figure 4 shows that NEO-201 can mediate ADCC activity against HL-60 and human neutrophils with isolated NK cells as effectors at all E:T ratios.
These data suggest that NEO-201 is able to target and kill cancer cells expressing core 1 and/or extended core 1 O-glycans through ADCC.  These data suggest that NEO-201 is able to target and kill cancer cells expressing core 1 and/or extended core 1 O-glycans through ADCC.

Discussion
Glycosylation is an important post-translational modification made on mammalian proteins and lipids. Glycosylation is essential in various cellular processes such as cellmatrix interaction, cell-to-cell recognition and intracellular signaling, and glycosylation status is strongly affected by oncogenesis [24].
One glycosylation pattern disrupted in cancer cells is the O-glycosylation. The expression of incomplete/truncated O-glycans in cancer cells, especially those of epithelial origin such as breast, ovarian, gastric, pancreatic and colon cancers, is correlated with poor prognosis and tumor progression [14][15][16][17][18].
The aberrant expression of truncated O-glycans at the level of cancer cell proteins occurs when different saccharide components are added to the N-acetyl galactosamine (O-GalNAc) attached to the amino acids serine and threonine. In addition, the sialylation of the sugar of the glycan chain introduces additional diversity in the O-glycan repertoire expressed by cancer cells. All these aberrant O-glycans may serve as potential targets to improve the diagnosis and treatment of tumors and provide the molecular probes for their specific recognition [17,[24][25][26][27]. The appearance of truncated O-glycans on the cancer cell surface is widely accepted as one of the characteristics of cancer [28]. Understanding how the immune system interacts with tumor-associated O-glycans will help in developing novel effective cancer immunotherapies.
The hyper sialylation of O-glycans made by tumor cells could be one of the mechanisms used by tumors to escape from immune surveillance [29].
One promising strategy to subvert this mechanism and improve cancer immunotherapy efficacy is the employment of monoclonal antibodies that specifically recognize truncated O-glycans expressed in cancer cells.
Several antibodies have been developed against O-glycans to counteract tumor growth. For example, in vivo experiments showed that mAbs against Tn-antigen use different mechanisms to kill tumor cells, including ADCC, CDC and the direct blocking of receptor signaling [30]. Examples of other anti-glycan antibodies used in pre-clinical studies and in clinical trials are the anti-GD3 ganglioside mAb R24 used to treat melanoma [31], antibodies against M2, fucosyl GM1, globo H and polysialic acid used to treat small cell lung cancers [32], the humanized mAb Hu3s193 for the treatment of patients with advanced epithelial cancers expressing the Lewis-Y antigen [33] and human monoclonal antibodies against the sialyl-Lewis (CA19.9) antigen for the treatment of different types of tumors [34]. Antibodies against CA19.9 can also be used for the detection and monitoring of advanced pancreatic cancer [35,36].
The first monoclonal antibody approved by the U.S. Food and Drug Administration (FDA) for the treatment of high-risk neuroblastoma was dinutuximab in 2015, a chimeric mAb recognizing the glycolipid GD2, a tumor-associated carbohydrate antigen (TACA) [37]. Furthermore, the National Cancer Institute has developed the Database of Anti-Glycan Reagents, where researchers reported the activity of more than 1100 anti-glycan antibodies [38]. In the context of anti-glycan mAbs, NEO-201 can represent a novel promising approach to treating cancer cells expressing O-glycans.
In previous studies, we demonstrated that NEO-201 reacts to colon, ovarian, pancreatic, non-small cell lung, head and neck, cervical, uterine and breast cancers expressing tumor-associated variants of CEACAM5 and CEACAM6 but does not react to normal tissues [1,2,23]. NEO-201 can also bind to human regulatory T cells as well as human neutrophils, AML and MM cell lines in vitro [1][2][3][4][5][6].
In vivo, we demonstrated that NEO-201 attenuates the growth of human tumor xenografts in pancreatic and ovarian cancer murine models and shows safety/tolerability in non-human primates, with a transient decrease in neutrophils being the only adverse effect observed [1,2].
In addition, in a NEO-201 pharmacokinetics study performed in purpose-bred cynomolgus monkeys, we injected NEO-201 at dose levels of 5 mg/kg, 20 mg/kg and 49 mg/kg, and we observed quantifiable and dose-dependent serum concentrations of NEO-201 until 14 days post-dose. The mean half-life (HL) was 167 (20 mg/kg) or 170 (49 mg/kg) hours at higher doses [1]. Furthermore, NEO-201 was manufactured for clinical use (GMP), and stability tests performed on the NEO-201 drug product showed that NEO-201 has a long-term stability of 48 months.
A first-in-human clinical trial using NEO-201 for the treatment of patients with metastatic colon cancer, pancreatic cancer, breast cancer, non-small cell lung cancer (NSCLC) and mucinous ovarian cancer, who were no longer eligible for standard therapy, has completed accrual and is currently undergoing data analysis [23].
Since NEO-201 binds to tumor-associated variants of CEACAM5 and CEACAM6 and CEACAM proteins are highly glycosylated proteins involved in cell migration, metastasis and drug resistance [7], in this study, we evaluated if NEO-201 can recognize specific glycan patterns attached on the protein structure of CEACAM5 and CEACAM6 during post-translational modifications made by mammalian cells. In this regard, we compared the binding of NEO-201 and of a commercially available anti-CEACAM6 mAb 9A6 to both mammalian and bacterial rhCEACAM6. As shown in Figure 1, NEO-201 binds to HEK293T cells (post-translational modification-competent mammalian cells) but not to rhCEACAM6 expressed by E. coli (post-translational modification-incompetent bacterial cells), while the 9A6 mAb binds to both types of rhCEACAM6.
As described above, truncated O-glycans are highly expressed in cancer cells. Since NEO-201 binds only to mammalian rhCEACAM6 containing glycan patterns attached on the protein structure, we then evaluated the capacity of NEO-201 to recognize O-glycans expressed by cancer cells using different O-glycan arrays.
Several methods have been used to evaluate antibodies specificity for glycans, such as ELISA, surface plasmon resonance (SPR) and glycan microarrays. Of these methods, glycan microarrays offer the highest throughput since they are composed of numerous glycan determinants or fragments of determinants, immobilized in a spatially defined arrangement [39].
In this study, using an O-glycan array consisting of 94 O-glycans, we demonstrated that NEO-201 recognizes six different O-glycans and that the strongest interaction is with a core 1 O-glycan (06) (Figure 2). These data were confirmed in the O-glycan array performed on cancer cell lines and human neutrophils positive for NEO-201 in flow cytometry. As shown in Tables 1 and 2 Our data confirm observations made in other studies. For example, Babu and colleagues reported that the human neutrophil glycome profile of the O-glycans consists of both core 1 and core 2 O-glycans with sialyl Lewis x and Lewis x as terminal epitopes [40]. In addition, Blöchl et al. showed that HL-60 and U937 AML cell lines predominantly express core 1 O-glycans [41]. Furthermore, both HL-60 and U937 do not express CEACAM5 and CEACAM6, suggesting that NEO-201 can bind to core 1 and extended core 1 O-glycans attached to any tumor-associated protein containing the amino acids serine and threonine.
Despite the available information regarding antiglycan antibodies, the identification and characterization of antiglycan antibodies remains a major challenge.
One of the main limitations of antibodies targeting glycans is their low affinity for their target, which, in turn, reduces their antitumor efficacy.
The enhancement of the antibody affinity for glycans is determined by conformational changes induced by the glycan in the peptide backbone. This phenomenon could partially affect the binding affinity of antiglycan antibodies targeting mixed epitopes composed of both glycan and the carrier molecule to which the glycan is attached, such as the peptide or lipid portions of the glycoproteins or glycolipids. An example of this phenomenon has been provided by Martínez-Sáez et al., who demonstrated that the binding of the antibody SM3 to glycopeptides bearing the α-O-GalNAc-Thr and α-O-GalNAc-Ser antigens is affected by distinct atomic presentations of the Tn-carrying serine (α-GalNAc-Ser) and threonine (α-GalNAc-Thr) antigens when bound to SM3 [42]. Furthermore, it has been demonstrated that antiglycan antibody binding between a single binding site and a single glycan determinant is usually weak, with equilibrium dissociation constants (KD) in the micromolar to low millimolar range. The multivalent binding of antibodies engaging two or more binding sites with two or more glycan determinants can achieve a much higher functional affinity [43][44][45].
The KD of NEO-201 is 5.49 × 10 −9 with Rmax = 99.3 and Chi2 = 3.47 (unpublished data), allowing NEO-201 to have a strong binding affinity for its target. In this regard, here, we have demonstrated that NEO-201 binds to core 1 and/or core 1 extended O-glycans expressed in its target cells. O-glycans can be attached to amino acid regions containing serine and threonine on proteins. This study suggests that NEO-201 could be a good anti-glycan mAb for the treatment of different types of cancers not only expressing core 1 and/or extended core 1 O-glycans attached to the tumor-associated variant of CEACAM5 and CEACAM6 but also to other tumor-associated proteins. Further studies will evaluate if core-1 and/or extended core-1 O-glycans recognized by NEO-201 are attached to other specific tumor-associated proteins (beyond CEACAM5 and CEACAM6) expressed by the different types of tumors targeted by NEO-201.

Conclusions
This study demonstrated that NEO-201 recognizes different types of O-glycans, with the strongest binding to core 1 and extended core 1 O-glycans. NEO-201-reactive cells such as the pancreatic cell line CFPAC-1, human neutrophils and the human hematological neoplastic cells HL60 and U937 express mostly core 1 and/or extended core 1 O-glycan profiles. These cells can be killed by NEO-201 through NK-mediated ADCC.
The aberrant expression of O-glycans is associated with cancer development, progression, invasion and metastasis in both solid tumors of epithelial origin [14][15][16][17][18] and hematological malignancies such as AML and multiple myeloma [46,47]. The employment of NEO-201, which has a strong binding affinity for tumor-associated proteins carrying core 1 and/or extended core 1 O-glycans, could represent a novel and promising tool in the immunotherapy treating both solid and liquid tumors.


Construction of a Humanized Artificial VHH Library Reproducing Structural Features of Camelid VHHs for Therapeutics

A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important. Thus, To this end, a synthetic VHH library that reproduces the structural properties of camelid VHHs was constructed. First, the characteristics of VHHs were classified according to the paratope formation based on crystal structure analyses of the complex structures of VHHs and antigens. Then, we classified 330 complementarity-determining region 3 (CDR3) structures of VHHs from the Protein Data Bank (PDB) into three loop structures: Upright, Half-Roll, and Roll. Moreover, these structures depended on the number of amino acid residues within CDR3. Furthermore, in the Upright loops, several amino acid residues in the FR2 are involved in the paratope formation, along with CDR3, suggesting that the FR2 design in the synthetic library is important. A humanized synthetic VHH library, comprising two sub-libraries, Upright and Roll, was constructed and named PharmaLogical. A validation study confirmed that our PharmaLogical library reproduces VHHs with the characteristics of the paratope formation of the camelid VHHs, and shows good performance in VHH screening.

Introduction
In 1993, it was reported that camelids produced unconventional heavy-chain-only antibodies that bind to antigens solely by the variable domain of their heavy chain. The variable domain of heavy chain of heavy chain antibody (VHH) was expected to be the next generation of therapeutic antibodies [1]. In those days, however, fierce competition in the development of conventional human or humanized antibodies had only begun, and thus, a few pharmaceutical companies were seriously looking at camelid VHHs. Among them, Ablynx (a subsidiary of Sanofi since 2018) generated several VHH clinical candidates, including caplacizumab. Therefore, many were surprised when caplacizumab, the world's first VHH drug, was approved as a treatment for thrombotic thrombocytopenia by the European Medicines Agency in 2018, and by the Food and Drug Administration (FDA) in 2019 [2,3]; after that, many pharmaceutical companies turned their attention to VHH research and development. In March 2021, in Japan, a second VHH drug, ozoralizumab, was submitted to the Pharmaceuticals and Medical Devices Agency for approval against rheumatoid arthritis.

1.
Chain IDs for heavy chains included, but light chains IDs not included; 2.
Tertiary structures determined using X-ray crystallography with a resolution better than 2.8 Å; 3.
Exclude structures in a complex with a hapten-antigen.
Criteria for human VH data: 1.
Chain IDs for both heavy and light chains; 2.
Tertiary structures determined using X-ray crystallography with a resolution better than 2.8 Å; 3.
Heavy and light antibody chains were both of the species homo sapiens; 4.
Exclude structures in a complex with a hapten-antigen.
The amino acid sequences of each chain were derived from the SEQRES records in the PDB files. The residue numbers were renumbered automatically by the Kabat numbering scheme, using the ANARCI tool [31] (http://opig.stats.ox.ac.uk/webapps/newsabdab/ sabpred/anarci/ (accessed on 31 May 2021)). The residue number was used to identify the variable domain region and the CDR. Clustering of variable domain region, based on these amino acid sequences, was conducted using CD-HIT (http://weizhong-lab.ucsd.edu/cdhit/ (version 4.8.1, accessed on 31 May 2021)) to eliminate redundancies by removing perfect matches for which the % id was 100% [32]. Data curation resulted in a VHH dataset of 330 and a human VH dataset of 926.

VHH and Human VH Datasets
The structure of CDR3 was classified into three groups (Upright, Roll, and Half-Roll) according to the structural feature using the following index based on Shirai [33] and Gray [34] (the classification does not apply to short CDR3s less than five amino acids. For those short CDR3s, the structures were visually classified from the X-ray structures): 1.
Distance between Cα atoms: distance between the H46 residue and the (n −5)th residue, with H102 (endpoint of CDR3) as the (n)th residue; 2.

Design of PharmaLogical Library
First, it was necessary to set the boundary between CDRs and FRs. We used the Kabat numbering rule [35] and structural classifications from Chothia [36]. Consequently, they Humanized FRs for each Upright and Roll type are designed based on the human framework sequence IGHV3-23 * 01 (DP- 47), and VHHs on the market or under clinical development. We carefully determined the Vernier zone, which is the framework region interacting with the CDRs, based on the structural data of the VHHs. Additionally, the amino acid sequences of the FRs, especially FR2, were specialized for each Upright and Roll type.
Four sub-libraries were designed based on the features of the CDR3 structures, and named Upright 6, Upright 12, Roll 12, and Roll 15. CDR2, with 17 amino acid residues, prefers to form the Roll type, whereas CDR2, with 16 amino acid residues, prefers to form the Upright type. For CDR1, 150 varieties of amino acid sequences were selected; for CDR2, 69 and 71 varieties of sequences were selected in the Roll and Upright libraries, respectively. Alternatively, CDR3s were randomly designed with 17 amino acid residues, excluding methionine, proline, and cysteine. Finally, the CDR1s, CDR2s, and CDR3s were combined with humanized FRs.

Library Construction
First, we synthesized a DNA fragment encoding the FR1 including the 5 UTR, the FR3 including a restriction enzyme BtgZI site at the 3 terminus, and a DNA fragment encoding the FR4 including a His tag sequence and the 3 UTR (Eurofins Genomics Inc., Eurofins Scientific SE, Luxembourg, Luxembourg). Next, 150 oligonucleotides, including CDR1, FR2, and a restriction enzyme BsmBI site at the 3 terminus, were synthesized. In this construct, two types of oligonucleotides were prepared for each Upright and Roll types. For the Upright type, 71 oligonucleotides, including the CDR2, the FR2, and a restriction enzyme BsmBI site at the 5 terminus, were synthesized, and for the Roll type, 69 oligonucleotides, including CDR2, FR2, and a restriction enzyme BsmBI site at the 5 terminus, were synthesized (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). One hundred fifty oligonucleotides for the CDR1, 71 for the CDR2, and 69 for the CDR2 were mixed. Finally, oligonucleotides, including a randomized CDR3 region and a restriction enzyme BsmBI site at the 5 terminus, were synthesized (Ella Biotech, Fürstenfeldbruck, Germany). The randomized regions included amino acid residues of lengths of 6, 12, or 15, and were synthesized using 17 types of trimer phosphoramidites (excluding cysteine, methionine and proline). Notably, these 17 types of trimer phosphoramidites were equally mixed.
Overlapping extension polymerase chain reactions (PCRs) were conducted with the mixed oligonucleotides (FR1 and CDR1), mixed oligonucleotides 2 (CDR2 and FR3), and mixed oligonucleotides 3 (CDR3 and FR4), 100 pmol per mixture. The extension PCRs synthesized strands of FR1-CDR1, CDR2-FR3, and CDR3-FR4. The FR1-CDR1 and the CDR2-FR3 were purified with AMPure XP (Beckman Coulter, Inc., Brea, CA, USA) and digested with a restriction enzyme BsmBI (New England Biolabs, Ipswich, MA, USA). After purification with AMPure XP, the FR1-CDR1 and the CDR2-FR3 fragments were ligated together with T4 DNA ligase (Takara Bio Inc., Shiga, Japan) at 16 • C overnight. The ligated products were purified by an 8 M urea denaturing polyacrylamide gel electrophoresis (PAGE) and amplified by PCR using the outer primers. The amplified DNA fragments of FR1-CDR1-FR2-CDR2-FR3 were purified. The purified products were digested with a restriction enzyme BtgZI (New England Biolabs), and DNA fragments of CDR3-FR4 were digested with a restriction enzyme BsmBI after purification with AMPure XP. The DNA fragments of FR1-CDR1-FR2-CDR2-FR3 and CDR3-FR4 were ligated together with T4 DNA ligase at 16 • C overnight. The ligated products were purified by an 8 M urea denaturing PAGE. The DNA library from our PharmaLogical library is composed of 5 UTR (T7 promoter, Shine-Dalgarno sequence), VHH genes, the His-tag region, and 3 UTR linker hybridization region.

Obtaining VHHs against Target Molecules
We built a high-throughput screening platform called "The Month" and conducted a verification study using that platform. The combination of the cDNA display method and the Corynebacterium glutamicum secretion expression system enabled the rapid isolation of many target-binding VHHs.
2.5.1. In Vitro Selection Using cDNA Display Each sub-library, according to DNA form Upright 6, Upright 12, Roll 12, and Roll 15, was transcribed by T7 RNA polymerase in a RiboMAX Express Large Scale RNA Production System (Promega Corp., Madison, WI, USA). The RNA products were purified with RNAClean XP (Beckman Coulter, Brea, CA, USA), and then hybridized with a puromycin linker using cnvK. The cnvK puromycin linkers were individually photo-cross-linked with the 3 -terminal region of the mRNA by ultraviolet light at 365 nm for 30 s.
The photo-cross-linked products were mixed and translated in vitro using PUREflex1.0 (GeneFrontier Corporation, Chiba, Japan) at 30 • C for 30 min. KCl and MgCl 2 were added to the reaction mixture at a final concentration of 800 and 80 mM, respectively, and incubated at 37 • C for 60 min, and then ethylenediaminetetraacetic acid (EDTA) was added to the reaction mixture at a final concentration of 100 mM, and the mixture was further incubated at 4 • C for 15 min. A 2× binding buffer (20 mM Tris-HCl pH 7.5, 2 M NaCl, 25 mM EDTA, 0.1% Tween20) was added to the translation reaction mixture. The reaction mixture was incubated with streptavidin-coated magnetic beads (SA beads, Dynabeads TM , MyOne TM Streptavidin C1; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 25 • C for 60 min to immobilize on SA beads. After washing with 1× binding buffer three times, the immobilized library was reverse-transcribed by GeneAce Reverse Transcriptase (Nippon Gene, 200 U/µL GeneAce Reverse Transcriptase in 200 µL buffer) at 42 • C for 30 min. After washing twice with a His-tag wash buffer (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 5 mM imidazole, 0.05% Tween20), the His-tag wash buffer with 5 U/µL RNase T1 (Thermo Fisher) was mixed with the SA beads, and the mixture was incubated at 37 • C for 15 min to release the cDNA display molecules from the SA beads. The supernatant containing the cDNA display was captured with Ni-NTA magnetic beads (His Mag Sepharose Ni ® , Cytiva, Marlborough, MA, USA) at 25 • C for 60 min. The Ni-NTA magnetic beads were then collected and washed twice with a His-tag wash buffer. The cDNA display molecules bound to the beads were eluted by incubation at 25 • C for 10 min in a His-tag elution buffer (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 250 mM imidazole, 0.05% Tween20) at 25 • C for 15 min.
At the first round of selection, cDNA display libraries were mixed with 100 pmol biotinylated HER2 or 100 pmol biotinylated GPC3, and incubated at 4 • C for 30 min. Then, 240 µL SA beads was added to the reaction mixtures and incubated at 4 • C for 30 min to immobilize the cDNA display molecules bound to the biotinylated target proteins. The supernatant was removed, and the beads were washed three times with PBS-T. The cDNA display molecules remaining on the beads were eluted and purified by AMPure XP. The purified products were subjected to PCR amplification. The amplified DNAs were purified and used for the next round of selection. For the second and third rounds of selection, a smaller amount of mRNA linker (12.5 pmol and 6.25 pmol, respectively), and a smaller amount of biotinylated target proteins (10 pmol HER2 and GPC3) were used.

Preparation of Selected VHHs
After selection, cDNA fragments from the selected clones were PCR-amplified using specific primers, including restriction enzymes BamHI and SfiI restriction enzyme sites, and transferred into an expression vector for Corynebacterium glutamicum. Approximately 200 transformants were picked up and these were cultured in a protein-expression medium at 25 • C for 72 h. The culture supernatants were centrifuged and filtered through a 0. 22-µm membrane filter. VHHs with binding activity against the target molecules were determined by using a biolayer interferometry (BLI), Octet ® RED384 (Sartorius AG, Göttingen, Germany). A solution of 70 µL was added to a 384-well black plate (Sartorius AG), and the measurements were conducted as described below. The loading baseline was measured in kinetic buffer for 30 s. The VHHs were immobilized on a His1K biosensor. The measurement baseline was measured in kinetic buffer for 30 s. The loaded sensors were dipped into 400 nM HER2-Fc (ACROBiosystems, Newark, DE, USA) and GPC3-Fc (ACROBiosystems) to measure a 100-s specific binding at the association step. The dissociation was obtained by dipping the biosensors one more time into the kinetic buffer for 100 s. VHH clones with a binding response of >0.1 were identified as a binder, and sequence analysis was conducted on those clones.

VHH Expression and Purification
The VHH candidates identified by screening were expressed in Corynebacterium, and purified by His tag at the C-terminus of VHHs. The bacterial cells were cultured in CM2G medium at 30 • C overnight. The cells were inoculated in a protein-expression medium and cultured at 25 • C for 72 h. The culture supernatants obtained by centrifugation were filtrated by a 0.22-µm membrane filter and subjected to Ni-NTA HP (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in a spin-column. The resin was washed with the Tris buffer (50 mM Tris-HCl pH7.5, 300 mM NaCl) containing 30 mM Imidazole, and then VHHs were eluted with the Tris buffer containing 500 mM Imidazole.

Binding Affinity Analysis
Binding affinities of VHHs were determined using BLI, Octet ® RED384. A solution of 70 µL was added to a 384-well black plateand the measurements were performed as described below. The loading baseline was measured in the kinetic buffer for 30 s. The purified VHHs with C-terminal His tag were immobilized on a His1K biosensor. The measurement baseline was measured in the kinetic buffer for 30 s. The loaded sensors were dipped into two-fold serial dilutions from 100 nM of the HER2-Fc and GPC3-Fc to measure a 120-s specific binding at the association step. The dissociation was obtained by dipping the biosensors into the kinetic buffer for 300 s. The data were analyzed using ForteBio Octet Date Analysis HT 11.1.2.48, and kinetic parameters were determined.

Thermal Stability Analysis
The purified VHHs were analyzed for their thermal stability using differential scanning fluorimetry (DSF) and static light-scattering (SLS) assays. A heating rate of 1 • C/min was monitored from 25 to 95 • C. The SLS assays were measured at 266 nm. Additionally, T m and T agg from the DSF assay were analyzed and calculated using UNCLE Analysis Software (Unchained Labs, Pleasanton, CA, USA).

Flow Cytometry
The purified VHHs were analyzed for cell binding using a flow cytometer. Cells were cultured in dishes at 80% confluence and then harvested with 5 mM EDTA in PBS. Collected cell samples were washed with 1% BSA/PBS, resuspended with the VHH solution in 1% Antibodies 2022, 11, 10 7 of 18 BSA/PBS (1:100 dilution), and incubated at 4 • C for 1 h. After being washed three times with 1% BSA/PBS, the cells were incubated with an anti-His-tag antibody conjugated with Alexa Fluor 488 (1:1000 dilution, Medical & Biological Laboratory, Tokyo, Japan) at 4 • C for 1 h. After incubation with the detection antibody, cells were washed with 1% BSA/PBS, resuspended with 1% BSA/PBS, and subjected to a flow cytometry (FCM) analysis (SH800; Sony, Tokyo, Japan).

Molecular Dynamics Simulations of VHHs
Although our libraries were carefully designed on the basis of the bioinformatics explained above, we needed to check whether the VHHs in the libraries had the expected CDR3 structures. To verify whether the VHHs had the expected CDR3 structural types, we conducted molecular dynamics (MD) simulations of representative sequences from the libraries. The initial structure was created by homology modeling, using the SWISS-MODEL web server [37]. Generally, loop structures such as CDR3 require a very long simulation time due to their slow relaxation, which is difficult to access with conventional MD simulations. Thus, to efficiently sample CDR3 structures, we used the generalized replica-exchange solute-tempering method (gREST) [38], a variant of replica-exchange MD [39]. In the replica-exchange MD, temperatures of whole replicated systems are exchanged to accelerate structural sampling. In the gREST, we could select a local region of the molecule and a part of the potential energy terms for temperature exchange. We found that the CDR3 structure can be efficiently sampled when selecting the CDR3 region and its dihedral angle potential energy terms for the temperature exchange (Higashida and Matsunaga, submitted). Following this procedure, we conducted 100-ns gREST simulations with eight replicas (i.e., 800 ns aggregation time). Finally, we obtained an ensemble of the VHH structure at 1 atm and 300 K, which corresponds to the lowest temperature of the eight replicas. The simulations were conducted with GENESIS software [40] using the CHARMM36 force field [41] and the TIP3P water molecule. Electrostatic interactions were treated using the smoothed particle mesh Ewald (S-PME) [42], and the bonds involving hydrogen atoms were constrained using SHAKE [43] and SETTLE [44] algorithms.

Classification of CDR3 Structure
To analyze the structural features of camelid VHH, we created structural datasets of VHH and human VH. First, we obtained the structural data of VHH and human VH available in PDB, and then extracted the data with a resolution better than 2.8 Å by X-ray crystallography and excluding the perfect match sequences. As a result, we constructed a dataset of 330 sequences of VHH and a dataset of 926 sequences of human VH.
We analyzed the constructed VHH structure dataset and found that the structure of the CDR3 loop can be roughly classified into three groups. Based on the characteristics of the CDR3 structure, each classification was named "Upright," "Half-Roll," and "Roll" ( Figure 1A). In the Upright type, the CDR3 stands upright and the FR2 is exposed to the outside. This Upright loop of VHH stab into an epitope of the target antigen, or binds to the target with FR2. The Roll type of VHHs has the formation that the CDR3 loop covers the FR2 region. For this formation, large hydrophobic residues (H37, H44, H45, H47) are directly related. Half-roll is categorized as the intermediate CDR3 type between Roll and Upright. Compared with other types of CDR3, this type does not have a clear propensity in its sequence. Next, for those CDR3 loop structure classifications (Upright, Half-Roll, and Roll), the classification was defined by combining the CDR3 classification methods in past studies [32,33]. Figure 1B shows a scatter plot of the pseudo-dihedral angles of CDR3 C-terminal and the distances between the Cα of the CDR3 loop and the closest residue in FR2 for the structures of VHH. The structures with pseudo-dihedral angles more than 140 • , distances more than 15 Å, and an elongated-type CDR3 conformation were classified as Upright (pink dots). Structures with a pseudo-dihedral angle less than 140 • , distances less than 10 Å, and a CDR3 conformation with a curved shape relative to FR2 were classified as Roll (green dots). The group of VHHs that do not belong to Upright and Roll was classified as Half-Roll (blue dots) because the CDR3 conformation was intermediate between Upright and Roll. We determined the regions of CDR3 in human VH and classified the structures by applying the same scheme as we did for VHH. In human VH, there were no Roll types and few Upright types for CDR3 ( Figure 1C). Although most of the structures were Half-Roll type, few showed pseudo-dihedral angles more than 140 • and less than 15 Å in distance. The presence of a light chain is a steric hindrance and will prevent folding, as with the Roll-type structures of CDR3. The above suggests that Upright and Roll are unique CDR3 structures of VHH that are rarely found in human VH.   Figure 2A,B shows a histogram of the length of CDR3 in the three structure types of VHHs and human VH from the PDB. In VHH, CDR3s with 12 or more amino acids tend to form a Roll-type structure. Alternatively, shorter a CDR3 tends to form an Upright conformation in both VHH and human VH. The Half-Roll type is widely distributed throughout and independently forms the length of the CDR3. Moreover, CDR3s with 16 or more amino acids tend to have a cysteine residue in the CDR3 that forms a disulfide bond with a cysteine in the CDR1 or CDR2 to stabilize the CDR3 structure. This additional disulfide bond is unwanted in the process development of biologics. Therefore, the maximum length of CDR3 was determined to be 15 amino acids. Furthermore, we found a correlation between CDR2 length and CDR3 structural classification. A total of 87% of the Roll-type VHHs in the dataset had 17 amino acid CDR2s, and 62% of the Upright-type VHHs had 16 amino acid CDR2s. This suggests that the length of CDR2 affects the structure determination of the CDR3 loop. The distances between Cα atoms of CDR3 loop and FR2 (distance between H46 residue and (n−5)th residue with H102 as (n)th residue) and pseudo-dihedral angles of CDR3 Cterminal (dihedral angle consisting of the four Cα atoms of the (n+1)th, (n)th, (n−1)th, and (n−2)th residues, with H102 as the (n)th residue) were calculated from structures and the value was plotted. (B) The structures of CDR3 were classified into the three groups according to type (Upright, Roll, and Half-roll) based on the distance and pseudo-dihedral angle. The VHHs belonging in the Upright group (pink dots) had a distance of 15Å or more and an angle of 140° to 360°, the VHHs in the Roll group (green dots) had a distance of 10Å or more and an angle of less than 140°, and the other VHHs were classified as the Half-Roll type (blue dots). (C) The CDR3 of human VH (gray dots) from a conventional VH-VL complex and VHH (yellow dots) are shown. In particular, the FR2 of the Upright type (highlighted in yellow) shows that many amino acid residues directly contribute to the paratope formation.

Construction of the PharmaLogical Library
We designed a rational artificial library of VHHs based on the analysis of the paratope, residue length, and structural type of CDR3, and, based on a huge number of camelid VHH amino acid sequences accumulated in our laboratory, the frequency of the appearance of all amino acid residues on VHH was analyzed. Four sub-libraries were designed and designated Upright 6, Upright 12, Roll 12, and Roll 15 according to the structure type and the length of CDR3 loops. First, the humanization framework area of the VHH library was designed for the Upright and Roll types, respectively. The humanized framework sequence was determined based on the human framework sequence IGHV3-23 * 01 (DP-47) and the humanized sequence of VHH currently on the market ( Figure 3A). Because humanization often reduces the binding activity, it is advantageous to have alreadyhumanized VHHs in the library. The CDR3 of the synthetic library was designed to have Many VHHs with CDR3 lengths of 12 amino acids or more belong to Roll (green), and VHHs or human VHs with short CDR3 tend to belong to Upright (purple). Those belonging to the Half-Roll group (blue) are widely distributed. (C) Comparison of amino acid residues between Upright and Roll types involved in the paratope and surroundings. Amino acid residues of VHH close to the antigen are highlighted in red in the crystallography of VHH-antigen complexes listed in PDB. In particular, the FR2 of the Upright type (highlighted in yellow) shows that many amino acid residues directly contribute to the paratope formation.
Detailed information on the paratope structure was obtained from the X-ray crystallographic analysis of VHHs bound to the antigen ( Figure 2C). Initially, we precisely defined CDRs using the Kabat numbering scheme. From the paratope analysis, Kabat numbering of CDR2 (H50-H65) and CDR3 (H95-H102) contained a sufficient region of the paratope. However, since the paratope of the CDR1 region is wider than that defined by the Kabat numbering system, we defined CDR1 (H26-H35) as the combined region of Kabat numbering (H31-H35) and the Chothia-aligned CDR1 (H26-H32). In the Upright type, FR2 plays a major role in paratope formation. The FR2 interacts with the L chain in a conventional VL-VH format, but is exposed in the VHH with an Upright CDR3 loop. Alternatively, in the Roll type, the FR2 and CDR3 are close to each other, stabilize as a Roll structure, and the entire CDR3 recognizes the antigen.

Construction of the PharmaLogical Library
We designed a rational artificial library of VHHs based on the analysis of the paratope, residue length, and structural type of CDR3, and, based on a huge number of camelid VHH amino acid sequences accumulated in our laboratory, the frequency of the appearance of all amino acid residues on VHH was analyzed. Four sub-libraries were designed and designated Upright 6, Upright 12, Roll 12, and Roll 15 according to the structure type and the length of CDR3 loops. First, the humanization framework area of the VHH library was designed for the Upright and Roll types, respectively. The humanized framework sequence was determined based on the human framework sequence IGHV3-23 * 01 (DP-47) and the humanized sequence of VHH currently on the market ( Figure 3A). Because humanization often reduces the binding activity, it is advantageous to have already-humanized VHHs in the library. The CDR3 of the synthetic library was designed to have 6 and 12 amino acids in the Upright type sub-library, and 12 and 15 amino acids in the Roll type sub-library, based on the analysis of the structural classification and length distribution of CDR3. Furthermore, because some amino acids become modified during the development process, our library was designated to reduce the frequency of appearance of potentially risky amino acids in CDR3. For CDR1 and CDR2, sequences were selected from the alpaca germline and the alpaca sequence data obtained in our laboratory. In this artificial VHH library, CDR1 consists of 150 sequences, and CDR2 consists of 71 sequences of 16 amino acids in the Upright type and 69 sequences of 17 amino acids in the Roll type. Importantly, the structural properties of camelid VHH were kept in the design of the CDRs.
Based on the library design, four types of sub-libraries, Upright 6, Upright 12, Roll 12, and Roll 15, were constructed. Each constructed library had a diversity of more than 10 13 and was synthesized by adding a UTR sequence for the cDNA display method ( Figure 3B). The constructed library was analyzed by next-generation sequencing (NGS) and verified. As a result of NGS analysis, the four sub-libraries had the sequences as designed, and the mixing ratio of the amino acids of CDR3 was almost the same as designed.

Validation of PharmaLogical Library
The combination of the PharmaLogical library with the most current cDNA robust screening method showed robust VHH screening. Various VHHs were quickly obtained from the cDNA display based on our VHH screening platform, "The Month" (Figure 4A). A mixture of the four types of sub-libraries from the PharmaLogical library was used for screening against human HER2 and human GPC3. In both validation studies, various binders with high affinity were efficiently isolated for each antigen within a month's time. Figure 4B shows the outcome of validation studies using the PharmaLogical library with our VHH screening platform "The Month". It was confirmed that a wide variety of VHHs was obtained by combining the synthetic VHH library and our VHH screening platform based on the cDNA display method. For GPC3, 104 clones were isolated and, among them, 84 were unique clones. For HER2, 52 clones were obtained and 38 were unique clones. Very interestingly, for HER2, 70% of clones were derived from the Upright 12 sub-library. The Upright VHHs are most likely to bind to the epitopes, such as crevices and clefts on the antigen, to which conventional antibodies cannot bind. Therefore, the upright VHHs are expected to have new binding characteristics compared to those of conventional antibodies. For GPC3, 50% were from the Upright 6 and Upright 12 sub-libraries.

Validation of PharmaLogical Library
The combination of the PharmaLogical library with the most current cDNA robust screening method showed robust VHH screening. Various VHHs were quickly obtained from the cDNA display based on our VHH screening platform, "The Month" (Figure 4A). A mixture of the four types of sub-libraries from the PharmaLogical library was used for screening against human HER2 and human GPC3. In both validation studies, various binders with high affinity were efficiently isolated for each antigen within a month's time. Each unique clone was expressed in Corynebacterium glutamicum, which secrete recombinant protein into a culture supernatant. The supernatants were filtered through a 0.22-µm membrane filter, and subjected to analysis of K D , T m , and T agg by BLI (Octet RED 384, Pall Life Sciences) and the UNCLE system (Uncle Analysis Software, Unchained Labs, Pleasanton, CA, USA), respectively. As shown in Figure 5A, many VHHs obtained from the synthetic VHH library showed high T m and T agg values. Moreover, a T m -T agg plot shows a good correlation, especially in GPC3. Binding affinity analysis confirmed that VHHs obtained from the synthetic VHH library have a binding affinity of approximately 10 7 to 10 −12 M for each antigen ( Figure 5B). A K D -T m plot is very useful for selecting candidates for further development (K D < 1.0 × 10 −9 and T m > 60 • C) ( Figure 5B, red-colored box). The Month" platform. The cDNA display selection strategy is used to isolate VHHs that bind to target molecules from the PharmaLogical library. The PharmaLogical Library, with > 1.0 × 10 13 diversity, was made into a cDNA display molecule with genotype and phenotype correspondence. Only the cDNA display molecules that bind to the target protein are immobilized on the magnetic bead via the target protein, and the unbound cDNA display molecules were washed away. The DNA of the recovered cDNA display molecule was amplified by PCR, and the PCR product was used as a template in the next cycle. Three-cycle selection was conducted with these series of flows as one cycle. The DNA Library subjected to the selection cycle was cloned into a protein-expression vector, and each clone was expressed. VHHs in the culture supernatant were collected and the binder was identified using Octet384. (B) The number of binding VHHs detected as hit clones in the primary binding assay is shown. Unique VHHs are clones detected as hit clones but that do not have duplicate sequences. Unique VHHs were derived from four libraries: Upright 6, Upright 12, Roll 12, and Roll 15. VHH clones whose origin could not be determined due to mutations in the screening process were designated other. Figure 4B shows the outcome of validation studies using the PharmaLogical library with our VHH screening platform "The Month". It was confirmed that a wide variety of VHHs was obtained by combining the synthetic VHH library and our VHH screening platform based on the cDNA display method. For GPC3, 104 clones were isolated and, among them, 84 were unique clones. For HER2, 52 clones were obtained and 38 were unique clones. Very interestingly, for HER2, 70% of clones were derived from the Upright 12 sub-library. The Upright VHHs are most likely to bind to the epitopes, such as crevices and clefts on the antigen, to which conventional antibodies cannot bind. Therefore, the upright VHHs are expected to have new binding characteristics compared to those of conventional antibodies. For GPC3, 50% were from the Upright 6 and Upright 12 sub-libraries.
Each unique clone was expressed in Corynebacterium glutamicum, which secrete recombinant protein into a culture supernatant. The supernatants were filtered through a 0.22-μm membrane filter, and subjected to analysis of KD, Tm, and Tagg by BLI (Octet RED 384, Pall Life Sciences) and the UNCLE system (Uncle Analysis Software, Unchained Labs, Pleasanton, CA, USA), respectively. As shown in Figure 5A, many VHHs obtained from the synthetic VHH library showed high Tm and Tagg values. Moreover, a Tm-Tagg plot shows a good correlation, especially in GPC3. Binding affinity analysis confirmed that VHHs obtained from the synthetic VHH library have a binding affinity of approximately 10 7 to 10 −12 M for each antigen ( Figure 5B). A KD-Tm plot is very useful for selecting candidates for further development (KD < 1.0 × 10 −9 and Tm > 60 °C) ( Figure 5B, red-colored box). The Month" platform. The cDNA display selection strategy is used to isolate VHHs that bind to target molecules from the PharmaLogical library. The PharmaLogical Library, with > 1.0 × 10 13 diversity, was made into a cDNA display molecule with genotype and phenotype correspondence. Only the cDNA display molecules that bind to the target protein are immobilized on the magnetic bead via the target protein, and the unbound cDNA display molecules were washed away. The DNA of the recovered cDNA display molecule was amplified by PCR, and the PCR product was used as a template in the next cycle. Three-cycle selection was conducted with these series of flows as one cycle. The DNA Library subjected to the selection cycle was cloned into a protein-expression vector, and each clone was expressed. VHHs in the culture supernatant were collected and the binder was identified using Octet384. (B) The number of binding VHHs detected as hit clones in the primary binding assay is shown. Unique VHHs are clones detected as hit clones but that do not have duplicate sequences. Unique VHHs were derived from four libraries: Upright 6, Upright 12, Roll 12, and Roll 15. VHH clones whose origin could not be determined due to mutations in the screening process were designated other.
Flow cytometry (FCM) analysis confirmed a cell-binding activity of VHHs against target antigens. When screening VHHs for peripheral membrane proteins, soluble recombinant proteins were usually used, and thus, sometimes isolated VHHs cannot bind to the antigen on living cells. For clinical applications, therefore, an FCM evaluation needs to select VHHs that bind to target molecules on living cells. Several VHHs isolated by screening were analyzed for cell-binding to SK-BR-3 (highly expressed HER2) and HepG2 (highly expressed GPC3). In this FCM analysis, cell-binding was detected by recognition of the His tag at the C-terminus of VHHs by the Anti-His-tag mAbs conjugated with Alexa Fluor 488. As shown in Figure 6, some VHHs for both GPC3 and HER2 bind to their target liver cancer cell hepG2 and breast cancer cell SK-BR-3, respectively. Therefore, it was confirmed that VHHs obtained from this screening platform using recombinant proteins also showed the binding ability to target molecules on the cell surface.
ing were analyzed for cell-binding to SK-BR-3 (highly expressed HER2) and HepG2 (highly expressed GPC3). In this FCM analysis, cell-binding was detected by recognition of the His tag at the C-terminus of VHHs by the Anti-His-tag mAbs conjugated with Alexa Fluor 488. As shown in Figure 6, some VHHs for both GPC3 and HER2 bind to their target liver cancer cell hepG2 and breast cancer cell SK-BR-3, respectively. Therefore, it was confirmed that VHHs obtained from this screening platform using recombinant proteins also showed the binding ability to target molecules on the cell surface.

Confirmation of Reproduction of Paratopes by Molecular Dynamics Simulation
To verify whether the VHHs have the expected CDR3 structural types, we conducted MD simulations of representative sequences from the sub-libraries. Figure 7 shows the results of gREST simulations of the four representative VHHs from sub-libraries Upright 6, Upright 12, Roll 12, and Roll 15, respectively. The obtained structures were projected onto the same distance and pseudo-angle space as the bioinformatics analysis. It was confirmed that the structural ensembles of the VHHs were distributed in the expected Upright or Roll regions defined in the bioinformatics analysis. Particularly, clones from the Upright Figure 6. FCM analysis of cell-binding on SK-BR-3 (highly expressed HER2) and HepG2 (highly expressed GPC3). Anti-His-tag mAbs conjugated with Alexa Fluor 488 were used as the secondary antibody, cell binding was detected by the secondary antibody recognizing the C-terminal His tag of VHHs. Negative control (NC) was stained with secondary antibody only.

Confirmation of Reproduction of Paratopes by Molecular Dynamics Simulation
To verify whether the VHHs have the expected CDR3 structural types, we conducted MD simulations of representative sequences from the sub-libraries. Figure 7 shows the results of gREST simulations of the four representative VHHs from sub-libraries Upright 6, Upright 12, Roll 12, and Roll 15, respectively. The obtained structures were projected onto the same distance and pseudo-angle space as the bioinformatics analysis. It was confirmed that the structural ensembles of the VHHs were distributed in the expected Upright or Roll regions defined in the bioinformatics analysis. Particularly, clones from the Upright 6, Roll 12, and Roll 15 are spatially located in typical Upright and Roll regions. Although a clone from Upright 12 showed a broad distribution which included the Half-Roll region as a metastable state, most stable states correspond to the upright region. In fact, the average value of Upright 12 (black diamonds) in this 2D space located in the typical upright region was confirmed by the 3D structural view.

Discussion
There are several approaches to the construction of human VHH libraries. Binders for the COVID-19 spike protein were isolated from a human VHH library, indicating that the human VH could be used in the VHH format [45]. However, it is unclear whether human VH reproduces the structural properties of camelid CDR3 that make a paratope in a single-domain antibody. A comparison between VHH and human VH revealed that the camelid VHH has three types of CDR3 structures, whereas for human VH, the Half-Roll type is dominant. This is due to the presence of VL in the conventional antibody and may influence the paratope formation. Since the VHH in our PharmaLogical library retains the paratope-forming ability of VHH, it is expected that the VHH will reproduce the binding characteristics of camelid VHH. Therefore, PharmaLogical is a logical library that will greatly contribute to antibody drug discovery.
Detailed structural data of the paratope structures were obtained from the X-ray structural analysis of antigen-bound VHH. Compared to computer structure analysis based on the amino acid sequence of CDR, this analyzes the state of binding to the actual antigen, and so more accurate structure data can be obtained. We found that the VHH CDR3 produced by camelids followed three major structural pattern forms. The relationship between CDR2 and CDR3 is particularly important, and there are two types of CDR2 with lengths of 16 or 17 amino acid residues, which defines the possible structural patterns of CDR3. We believe that our artificial library that retains the expected basic structural relationship of VHH is extremely important for reproducing various structures to take advantage of the inherent characteristics of the CDR3 of VHH antibodies.
A synthetic library construction based on universal FRs has been reported [26,27]. However, we found that the Upright and Roll types interact structurally differently be-

Discussion
There are several approaches to the construction of human VHH libraries. Binders for the COVID-19 spike protein were isolated from a human VHH library, indicating that the human VH could be used in the VHH format [45]. However, it is unclear whether human VH reproduces the structural properties of camelid CDR3 that make a paratope in a single-domain antibody. A comparison between VHH and human VH revealed that the camelid VHH has three types of CDR3 structures, whereas for human VH, the Half-Roll type is dominant. This is due to the presence of VL in the conventional antibody and may influence the paratope formation. Since the VHH in our PharmaLogical library retains the paratope-forming ability of VHH, it is expected that the VHH will reproduce the binding characteristics of camelid VHH. Therefore, PharmaLogical is a logical library that will greatly contribute to antibody drug discovery.
Detailed structural data of the paratope structures were obtained from the X-ray structural analysis of antigen-bound VHH. Compared to computer structure analysis based on the amino acid sequence of CDR, this analyzes the state of binding to the actual antigen, and so more accurate structure data can be obtained. We found that the VHH CDR3 produced by camelids followed three major structural pattern forms. The relationship between CDR2 and CDR3 is particularly important, and there are two types of CDR2 with lengths of 16 or 17 amino acid residues, which defines the possible structural patterns of CDR3. We believe that our artificial library that retains the expected basic structural relationship of VHH is extremely important for reproducing various structures to take advantage of the inherent characteristics of the CDR3 of VHH antibodies.
A synthetic library construction based on universal FRs has been reported [26,27]. However, we found that the Upright and Roll types interact structurally differently between CDR3 and FR2, and thus, FR2 sequences need to be optimized for each type. That is, a universal FR in the same structure type makes sense, but not for all structure types. Amino acid residues in the FRs generally contribute to direct binding and/or a paratope formation. As shown in Figure 2C, amino acid residues in the FR2, especially in the Upright type, contributed to the paratope formation. Therefore, for synthetic libraries, the FR2 should be carefully designed and introduced into the library ( Figure 3A).
The usefulness of VHHs has been widely recognized in drug discovery. Not only are the characteristics of VHHs attractive, but also the speed at which drug candidates can be obtained using VHHs, especially in an emergency, is advantageous. Currently, the world is amid a pandemic caused by the coronavirus disease, COVID-19. The time required for developing therapeutic drugs against COVID-19 is a serious issue, and thus, it is expected that VHHs that can be obtained more rapidly from the generation of mutant strains will increase the speed of progress. Wu et al. reported that a yeast surface-display library of synthetic VHHs (>2 × 10 9 ) provides neutralizing binders to the spike ectodomain [46]. In addition to designing our library, we built "The Month," a VHH-screening platform that quickly selects development candidates by combining an artificial humanized library with >10 13 diversity and a cDNA display technology. Thus, our screening platform, named "The Month", is a powerful tool for a rapid response to the outbreak of coronavirus mutants.
As a model case of drug discovery, HER2 and GPC3 were subjected to a VHH drugdiscovery process. We used "The Month," our cDNA display screening platform that allows the selection of 50-100 positive clones and analyzes their K D , T m , and T agg . This process was completed within one month, and was, thus, named "The Month." Two-dimensional plotting with some parameters can be created. For example, when plotting T m against T agg , a high correlation is obtained in GPC3 ( Figure 5A). A correlation also can be seen in HER2. In other words, the selection of a clone population with high T m and T agg , which determines protein stability, as a development candidate is confirmed. Additionally, when viewed in terms of K D and T m , development candidate VHHs with high affinity and high stability can be selected. Determining the window setting (in this study, K D < 1.0 × 10 −9 M, T m > 65 • C) quickly narrows down the number of candidates for development. After that, various cell assays can be applied to further obtain candidates to develop. It is useful to quickly select candidates by using "The Month" and two-dimensional plot analysis. The purpose of this FACS evaluation study is to evaluate whether VHHs obtained using a soluble antigen can bind firmly to the true target membrane-binding antigen. Therefore, control cells are not always necessary. Of course, it is very important to verify the characteristics of VHH in clinical development. At the next step, therefore, we will investigate the cross-reactivity of VHHs to wide variety of cells. This is an important process that forms the basis of safety assessments in animal and human clinical trials. We will report the results of cellular specificity in the near future.
The most important feature of the PharmaLogical library is that it reproduces the paratope formation of camelid VHHs. That is, a selected VHH should reflect the concept of the structural design of the library. To confirm this, the CDR3 structures of selected VHHs were subjected to computer modeling, which is less time-consuming than crystal structure analysis. As expected, the CDR3 structure of the clone derived from the Roll sub-libraries clearly showed the Roll type structure. Therefore, our PharmaLogical library is guaranteed to be composed of the designed structural subtypes. Many VHH libraries have been reported, but our PharmaLogical library is the first library to deploy the original structural characteristics for camelid VHHs. Reproducing the original structural properties will lead to more success in the drug-discovery application of VHHs. Furthermore, Zimmermann et al. also reported a synthetic VHH library [47]. The idea of this paper is similar in that it, as our research does, features a structure-based design. However, they specialize for complex membrane proteins such as transporters and ion channels and focus on epitope structure. On the other hand, we are trying to reproduce the binding properties of camelid antibodies in an artificial humanized library for any kind of targets, focusing on paratope structure. There are structural relationships between epitopes and paratopes, and thus, we expect that this interesting structural relationship could be useful in a structure-based drug design.

Conclusions
The library we logically designed based on structural analysis makes it possible to secure VHHs that reproduce the attractive paratope of the camelid VHH. Currently, although we have completed the construction of the Half-Roll type sub-libraries, we are in the process of undergoing validation. Once this is complete, we will be able to provide an artificial library that covers about 95% of the original camelid VHHs. PharmaLogical is an in vitro antibody production system that completely mimics the VHH production of an in vivo system. Furthermore, in the future, it may be useful to select the appropriate sub-library according to the antigen.

Data Availability Statement:
The data presented in this study are available within this article.