Binding Properties and Biological Activity of a Novel Therapeutic Monoclonal Antibody Directed toward HER2 Antigen

1Laboratory of Recognition and Biological Activity Assays, Center of Molecular Immunology, Cuba 2Pathology Department, Center of Molecular Immunology, Cuba 3Tumor Bank Department, Instituto Nacional de Enfermedades Neoplásicas (INEN), Perú 4Laboratory of Biochemistry, Center of Molecular Immunology, Cuba 5Tumor Immunology Direction, Center of Molecular Immunology, Cuba 6Innovation Management Direction, Center of Molecular Immunology, Cuba


BACKGROUND
Trastuzumab (Herceptin ® ) is a monoclonal antibody (mAb) directed against the type 2 epidermal growth factor receptor (ERBB2, HER2 or HER-2 / neu), which is overexpressed in approximately 15-30% of invasive breast adenocarcinomas [1]. Herceptin ® is used for the treatment of HER2-positive breast cancer, providing signi icant clinical bene its in patients with advanced or metastatic disease [2,3]. This mAb is also prescribed for the treatment of gastric adenocarcinomas with overexpression of the HER2 receptor [4].
Although effective, Herceptin ® -based therapies remain expensive and inaccessible to meet the needs of many health systems. For these reasons, the development and approval of biosimilars to originator mAb is a necessity to reduce costs, increase health coverage and improve the quality of life of cancer patients. Given that Herceptin ® patents have expired, several biosimilars for this mAb have been developed and approved in recent years (e.g. Herzuma ® , Ogivri TM , Ontruzant ® ) [5][6][7]. In addition, other innovative mAbs are currently in early stages of development or evaluation. In this context, a 5G4 mAb was generated at the Center of Molecular Immunology (Havana, Cuba).
The 5G4 mAb was generated by transferring a plasmid DNA encoding trastuzumab and produced by the non-secretory murine myeloma cells (NSO) expression system [8]. However, trastuzumab is produced using the Chinese Hamster Ovary (CHO) cells. Although similar conformational characteristics of 5G4 mAb and Herceptin ® were previously published [8], differences in the expression system could affect both the binding properties and biological activity of mAb 5G4, due to changes in the glycosylation pattern.
In this study, cross-reactivity of mAb 5G4 in normal adult tissues is reported (this was part of the non-clinical safety assessment of 5G4). In addition, the binding properties of 5G4 mAb are presented in breast tumors, as well as in a variety of human cell lines with different levels of HER2 expression. Preliminary results are also shown regarding the ability of 5G4 to inhibit cell proliferation and induce Antibody-Dependent Cellular Cytotoxicity (ADCC) in vitro.

Monoclonal antibodies
The anti-HER2 mAb 5G4 developed in the Center of Molecular Immunology (Havana, Cuba) was used. This humanized IgG1 mAb was produced by the non-secreting murine myeloma cells (NSO) expression system transfected by plasmid DNA encoding Herceptin ® . In all assays, trastuzumab (Herceptin ® , Roche) was used as reference control. Trastuzumab is produced by means of an expression system in Chinese Hamster Ovary (CHO) cells. Itolizumab, a humanized mAb speci ic against CD6 molecule, served as isotype-matched negative control. For immunohistochemical assays, anti-HER2 mAbs and pooled human immunoglobulins (Intacglobin ® ) were conjugated using the BiotinTag™ Micro Biotinylation Kit (Sigma, 1001976388 BTAG-1KT). For the evaluation of antigenic preservation of frozen tissues, a polyclonal antibody against von Willebrand factor (vWF) (Dako, A0082) was used. An anti-β-actin mAb (Sigma, A2228) was used as a loading control for western blot analysis. committee (INEN number: 151-2017-CRP-DI-DICON/INEN;  number of the Provincial Hospital "Amalia Simoni": 05-04-2000). Tissue samples were stored at -150 °C. Five micrometer sections were obtained on a cryostat and the slides were stored at -70 °C until use.

Immunohistochemical assay
The sections were ixed in acetone for 10 minutes at 2-8 °C and rehydrated in TBS (Dako, S3001). Endogenous peroxidase activity was blocked with a commercial solution (Dako, S2003) for 10 minutes. For kidney, liver and spleen sections, endogenous biotin activity was blocked using the biotin blocking system (Dako, X0590). The tissues were then incubated for 1.5 hours with biotinylated anti-HER2 or Intacglobin ® (100 μg mL-1) at room temperature. After washing with TBS, the sections were incubated with streptavidin/peroxidase (Dako, K0690) for 30 minutes. Sections of breast carcinoma positive for HER2 were used as positive controls. Enzymatic activity was visualized with a DAB solution (Dako, K3467) and the slides were counterstained with Harris hematoxylin, dehydrated and mounted.

Western blot
The protein lysates of SK-BR-3 (cells that overexpress HER2) and Ramos (HER2 negative cells) were obtained following standard procedures. Protein samples were electrophoresed in 7.5% Bis-Tris gels and transferred by electroblotting to nitrocellulose membranes using the TE 70 Semi-Dry Transfer Unit (Amersham, United Kingdom). The transfers were blocked with 5% BSA/ 0.1% Tween-20 in TBS for 1 hour and then incubated for 1.5 hours at room temperature with respective anti-HER2 (10 μg mL-1) or anti-β-actin (1 μg mL-1) mAbs. Then, the transfers were washed and incubated with corresponding horseradish peroxidase (Sigma, A4416) or Alkaline Phosphatase (AP) (Sigma, A1543) secondary antibody bound at 1: 1000 dilutions for 1 h. Finally, the spots were washed and the enzymatic activity was visualized using DAB (Sigma, D8001) or SIGMAFASTTM BCIP®/ NBT substrate (Sigma, B5655). The molecular weight of the protein was estimated based on the values obtained for the bands in the protein standard. The images were analyzed with GeneTools ™ 4.3.8.0 software (Synoptics Ltd, United Kingdom).

Immunocytochemical assay
Cells monolayers were ixed in absolute acetone during 1.5 minutes at 2-8 ºC and rehydrated in PBS (Capricorn, PBS-1A) for 10 minutes. Samples were incubated with anti-HER2 antibodies at 10 μg mL -1 during 30 minutes at room temperature. After that, cells were washed with PBS for 5 minutes and incubated with FITC-conjugated rabbit anti human IgG (Sigma, F1641) 1:50 dilution for 30 minutes. Finally, cells were contrasted with propidium iodide (5 μg mL -1 ) for 1 minute. For microscopic analysis, an Olympus inverted microscope CKX41 (Tokyo, Japan) was used for analyses.

ADCC assay
The ADCC assay was performed using the lactate dehydrogenase (LDH) detection kit (Roche, 11644793001) according to manufacturer´s instructions. Brie ly, 5 000 SK-BR-3 cells (target cells, T) were incubated with 3 concentrations (1.00, 0.037 and 0.0013 μg mL -1 ) of 5G4 mAb and Herceptin ® for 30 minutes at 37 °C, 5 % CO 2 in McCoy´5A containing 1% FBS. Then, 50 μL of effector cells (peripheral mononuclear cells from a healthy volunteer donor, E) (E: T ratio 1:100) was added to each well and plates were incubated for 4 hours. Then, supernatant (100 μL) was collected and incubated with equal volume of LDH reaction mixture for 30 minutes at room temperature protected from light. Sample absorbances were measured at 490 nm, and absorbance at 620 nm was used as reference. Adequate controls for spontaneous and maximal lysis values were included to calculate the percentage of speci ic cytotoxicity.

Statistical analysis
The Mean Of Fluorescence Intensity (MFI) for each mAb and concentration was determined using Kaluza software version 1.2 (Beckman Coulter). Curves were constructed using GraphPad Prism 4.0 (GraphPad Software Inc., USA) and PLA 2.0 (Stegmann Systems, Germany). The value of EC50 was calculated and the relative binding activity of 5G4 mAb using Herceptin ® as standard was determined as follows: (1/EC50 Sample ) / (1/ EC50 Standard ) x 100. For anti-proliferative assays, EC50 values and relative potencies were calculated using SoftMax Pro 5.4.1 program (Molecular Devices), and for ADCC assays data were analyzed using GraphPad Prism 4.0 software. Mann Whitney U Test was used to compare the reactivity of mAbs in different cell lines. Kruskal-Wallis followed by Dunn's multiple comparison tests were used to compare the ADCC activity of mAbs at different concentrations. The criterion for statistical signi icance was p < 0.05.

Validation of tissue samples preservation
No signi icant alterations in histoarchitecture, based on hematoxylin and eosin staining, was observed in the frozen tissues panel. Moreover, an intense reactivity of the anti-vWF polyclonal antibody in the cytoplasm of vascular endothelial cells was evidenced, verifying the antigenic preservation of tissues ( Figure 1).

Immunohistochemistry-based
tissue cross-reactivity study of 5G4 mAb Table 2 shows the cross-reactivity study for 5G4 mAb in a panel of normal human tissues. 5G4 mAb reacted with breast ductal cells (1/2) (Figure 2A,2B), glandular cells from stomach (1/3) ( Figure 2C,2D) and renal tubes (2/3) showing a weak to moderate cytoplasmic staining. In general, the reaction pattern of 5G4 was similar, but slightly inferior in intensity compared to Herceptin ® . No immunostaining with both anti-HER2 mAbs was detected in the rest of evaluated tissue samples. No reactivity was also observed in tissues samples incubated with Intacglobin ® , used as negative control.
A speci ic, intense and linear staining of cell membrane was evidenced with 5G4 mAb in breast adenocarcinomas ( Figure  2E,2F) and SK-BR-3 cells previously classi ied as HER2-positive (3+) (Figure 3A,3C). This pattern of staining was comparable to that obtained with Herceptin ® . No reaction was detected in HER2negative breast adenocarcinoma sections neither in Ramos cell monolayers ( Figure 3B,3D). Moreover, the capacity of 5G4 mAb to speci ically bind to HER2 molecule (~ 180 kDa for both) was also con irmed by western blots using cell lysates obtaining from SK-BR-3 breast cancer cell line ( Figure 2E).

Reactivity of 5G4 mAb in human cell lines by fl ow cytometry
The binding of HER2 molecule by 5G4 mAb was also evaluated by low cytometry in a series of tumor cell lines (Table  3). This mAb displayed similar recognition pro ile in HER2overexpressing cells (SK-BR-3 and SKOV3) to Herceptin ® as well as in some cells with low expression of this molecule (H292, A431 and HEK-293) ( Figure 3F,3K). But, statistically signi icant differences in NCI-H125 (p = 0.0022) and HEp-2 (p = 0.0152) cells (that express HER2 at low levels) were observed when compared with Herceptin ® . No recognition with 5G4 mAb or Herceptin ® was achieved in the HER2-negative cells (Ramos).

Relative binding activity of 5G4 mAb in SK-BR-3 cells
5G4 mAb was able to binds HER2 molecule in a dosedependence manner using SK-BR-3 breast cancer cells. The binding saturation was achieved at 10 μg mL-1 for both 5G4 mAb and Herceptin ® ( Figure 4A). In all low cytometric assays, the coef icients of variation were ≤ 16% for each tested concentration and mAb. The EC50 range of MFI was 1.765-1.821 and 1.649-2.100 μg mL-1 for 5G4 mAb and Herceptin ® batches, respectively. In consequence, the relative binding activity range of 5G4 mAb was 91-115% using Herceptin® as standard (Table  4). No reaction was obtained with the isotype negative control.

Capacity of 5G4 to inhibit cell proliferation
The anti-proliferative effect of 5G4 mAb and Herceptin ® on SK-BR-3 cells is shown in igure 4B. In all experiments, the coef icients of variation were ≤ 13% for each tested concentration and mAb. The maximum inhibition of cell growth of 74% and 77% was obtained with 3 μg mL -1 of 5G4 mAb and Herceptin ® , respectively. The EC50 values for 5G4 mAb ranged from 0.078-0.295 μg mL -1 , similar to those obtained with Herceptin ® used as control (0.077-0.316 μg mL -1 ). The relative potency range of 5G4 mAb was 91-106% using Herceptin ® as reference.

ADCC activity of 5G4 mAb
The capacity of 5G4 mAb to induce ADCC activity was evidenced in SK-BR-3 cells using peripheral mononuclear cells from healthy volunteer donors. Two batches of this mAb (2 and 3) showed non-inferior activity when compared with Herceptin ® ( Figure 4C). The batch 1 of 5G4 mAb showed decreased ADCC activity when compared with batch 2 of Herceptin ® in all evaluated concentrations (p = 0.0091, p = 0.0192 and p = 0.0144, respectively). Coef icients of variation were ≤ 15% for each tested concentration and mAb. No ADCC activity was detected with negative controls.   Observe the similar dosesresponse curves for this mAb (blue color) and Herceptin® (red color). B: Potency curves of 5G4 mAb obtained from the anti-proliferative assay. The capacity of 5G4 mAb (blue color) was similar to that of Herceptin® (red color). C: ADCC activity of 5G4 mAb. In general, the cytotoxicity of this mAb was non-inferior when compared with Herceptin®, except for batch 1 of 5G4 mAb that exhibited a decreased ADCC activity when compared with batch 2 of Herceptin®.

DISCUSSION
The introduction of novel therapeutic mAbs into clinical practice requires careful characterization, including binding properties to both normal and malignant tissues during preclinical studies. Some techniques are commonly used to evaluate the antigen binding attributes of mAbs such as, Immunohistochemistry (IHC), western blot with tissue lysates and low cytometry using cell lines that expresses the target molecule.
Tissue cross-reactivity permits the identi ication of both, previously unknown sites of the antigen expression and binding of mAbs to unexpected targets, such as cross-reactive epitopes, Scientifi c Journal of Immunology & Immunotherapy alerting oncologists to potential toxicity to certain organs that could be observed clinical trials [9]. These studies are an essential part of the preclinical safety assessment package of therapeutic mAbs [10,11]. The preservation of both tissue morphology and molecular antigenic determinants has to be irstly demonstrate in IHC studies [9]. Here, both morphological and antigenic tissue conservation was evidenced in all tissue samples, permitting to consider these specimens adequate for the cross-reactivity study.
In general, the expression of HER2 molecule was limited in normal adult tissues. These results are in line with previous reports concerning the low expression of HER2 molecule in normal tissues when compared with malignant tumors [1,12,13]. The pattern of recognition of 5G4 mAb was similar to those obtained with Herceptin ® , although a slightly diminished in intensity, suggesting no dramatic differences in the af inity and/ or in the speci icity of this new anti-HER2 mAb. However, in this panel of frozen tissues no immunostaining with 5G4 mAb was observed in other normal epithelial cells previously reported positive to HER2 molecule (e.g. small intestine, tonsil, uterus) [12,13]. Therefore, experiments with a larger number of these tissue samples should be performed to con irm these results.
Similar to Herceptin ® , an intense membrane staining with 5G4 mAb was evident in breast carcinomas that overexpress HER2 but not in HER2-negative breast tumors, which support the speci icity of this mAb. This fact was also con irmed by Western blots using lysates of SK-BR-3 cells (cells that overexpress HER2). In addition, mAb 5G4 was able to react with a variety of epithelial cells positive for HER2. However, in two low-expression cell lines, the reactivity of 5G4 mAb decreased compared to Herceptin ® , in concordance with the previous results obtained by IHC. More studies are needed, such as the af inity of mAb by surface plasmon resonance and/or homologous displacement curves to con irm this result.
Bioassays are key tools for product characterization and depend on the use of reference standard. The innovator (reference product) is necessary to demonstrate biosimilarity [14] and here was used as standard. The biological activity related to Fab fragments was determined both by in-vitro binding and by antiproliferative activity of candidate mAb and the potency estimate for each bioactivity was calculated. In the present study, the relative binding activity of mAb 5G4 using EC50 calculation ranged between 91-115% with respect to Herceptin ® . These values met the acceptance criteria (80-120%) commonly used for this type of assays [15] and support the ability of 5G4 mAb to bind to HER2 antigen as well as the reference product. Similarly, the antiproliferative activity of mAb 5G4 ranged between 91 and 106 % compared to Herceptin ® , values that are also considered comparable [15].
It is known, ADCC is a primary mechanism of action for Herceptin ® [16]. Here, preliminary data on the ability of 5G4 mAb to also induce this mechanism of action is shown. However, a decrease in the capacity of this mAb when compared with Herceptin ® was observed, although only statistically signi icant differences were detected for a batch of 5G4 mAb. This result could be related to differences in the glycosylation pattern of 5G4 mAb due to differences in expression systems [8,17], which affect this Fc-dependent mechanism. In fact, a decrease in both % of fucosylation and galactose-terminal was found in 5G4 mAb (unpublished data). These changes were previously related with a decrease in Herceptin ® ADCC activity [16].
However, a reduction in the % of fucosylation and galactosylation was unable to affect the binding and inhibition of cell proliferation activity of Herceptin ® [16], similar to the present study. In addition, differences in ADCC activities could not affect the effectiveness of Herceptin ® [16]. Interestingly, the anti-tumor effects of mAb 5G4 on athymic nude mice with SKOV3 ovarian cancer xenografts were not inferior to Herceptin ® (unpublished data). In general, these results suggest the potential use of 5G4 mAb for the treatment of tumors that overexpress HER2. However, the development of a new anti-HER2 mAb using CHO cells as expression system is recommended.

CONCLUSION
In summary, this study reported the binding properties and biological activity of 5G4, a novel anti-HER2 mAb. The limited reactivity of mAb 5G4 against normal adult tissues provided information to clinical oncologists about the possible low toxicity to normal organs as observed in the irst clinical trials in humans. Our data allow us to consider that mAb 5G4 has binding properties similar to the HER2 antigen and biological activity compared to Herceptin ® . However, other determinations such as af inity and FcgRIIIa binding activity of mAb 5G4 could be useful for con irming these results.

Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information iles. The histological slides are archived in the Laboratory of Recognition and Biological Activity Assays (Center of Molecular Immunology, Havana, Cuba). Archived stained slides are available from the corresponding author on reasonable request.

Authors' contributions
RB performed and analyzed the data from almost all experiments included in the study, discussed the results and was the major contributor in writing the manuscript. LAA and MR performed the tissue processing for histology and the bio repository management. EL, LC, DY and MC performed additional experiments. SC and MC performed the histological examination of normal and malignant tissues. BS designed and supervised ADCC assays. NL and MC revised and corrected the manuscript. All authors read and approved the inal manuscript.