*In vitro* Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs

*Sara Žigon-Branc, Ariana Barlič and Matjaž Jeras*

### **Abstract**

Human cell-based assays for *in vitro* testing of drugs in preclinical and research studies, as well as in clinical practice, are gaining greater importance especially in view of personalized medicine, which is tailored to the individual needs and benefits of a patient. This chapter begins with an overview of contemporary cell-based assays, routinely used for a comparative *in vitro* potency testing of anti-TNF-α innovator biologics and their biosimilars. In sequel, based on the results of our original work, we will further discuss the establishment and use of 2D normal and osteoarthritic primary chondrocyte monolayer cultures and 3D microspheroidal articular cartilage tissues, prepared in hanging drops from osteoarthritic chondrocytes and chondrogenically differentiated mesenchymal stem cells. Both 2D and 3D cultures will be presented as models for assessing the neutralizing potency of the three wellknown anti-TNF-α biological drugs: adalimumab, etanercept, and infliximab.

**Keywords:** *in vitro* cell-based assays, anti-TNF-α biologics, human articular chondrocytes, mesenchymal stem cells, 2D monolayer cultures, 3D cell cultures, gene expression

#### **1. Introduction**

Following the discovery and characterization of tumor necrosis factor (TNF) in the mid-1980s, this pleiotropic proinflammatory cytokine continues to be the focus of numerous studies and represents an important therapeutic target [1, 2]. The venue of anti-TNF biological drugs has revolutionized treatment of autoimmune and inflammatory diseases like rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriasis, Crohn's disease, ulcerative colitis, and others [2]. Although expensive, biological drugs (biologics) at the moment represent the best-selling group of pharmaceuticals. Nowadays, following the expiry of originators patents, a plethora of less expensive biosimilar drugs (biosimilars) are available to patients. In order to confirm the biocomparability of original and biosimilar products and to prove their quality, safety, and efficacy, the use of reliable and standardized bioassays relevant in assessing their modes of action is of crucial importance.

In this chapter, after a short introductory review of TNF biology, anti-TNF biological drugs and their mechanisms of action, we will present a selection of *in vitro* cell-based tests used either for general or personalized potency testing of anti-TNF biologics and their biosimilars.

#### **2. A short overview of TNF biology**

TNF is produced in various cell types, mainly immune cells such as monocytes and macrophages, microglia, neutrophils, natural killer cells (NK), T lymphocytes, and also in neuronal cells, keratinocytes, and fibroblasts [2, 3]. The cytokine exists in two biologically active forms. The first being a transmembrane protein (tmTNF), which can be cleaved by the metalloproteinase TNF-α-converting enzyme (TACE) (also known as disintegrin and metalloproteinase domain-containing protein 17 (ADAM17)) into its second form, a homotrimeric soluble TNF (sTNF) [2].

There are two TNF-binding homotrimeric transmembrane receptors, namely the TNF receptor 1 (TNFR1 or CD120a) and the TNF receptor 2 (TNFR2 or CD120b) [2]. While the TNFR1 is constitutively expressed on a vast majority of nucleated cells, the TNFR2 expression is inducible and tightly regulated, preferentially on endothelial, hematopoietic, neural, and immune cells [2, 4]. TNFR2 is also expressed on tumor cells where it is supposed to function as a tumor oncogene [5, 6].

Interestingly, tmTNF can induce signals in a bipolar way, as it acts as a ligand of both receptor types and as a receptor itself in cell-to-cell contacts [2, 4]. This means that tmTNF-α-expressing cells transmit signals to cells bearing TNFR1 and/or TNFR2. This phenomenon is called "outside-to-inside" or "reverse signaling," the function of which has not been completely clarified yet [2, 4]. The receptor function of tmTNF has been demonstrated in human monocytes, macrophages, NK cells, and T lymphocytes [4].

adalimumab, golimumab, and infliximab can, after binding to cells expressing tmTNF via their effector Fc regions (IgG1), induce antibody-dependent cytotoxicity (ADCC) of NK cells and activate the classical complement pathway, resulting in a complement-dependent cytotoxicity (CDC) and apoptosis [2, 4]. While ADCC and CDC are also induced by etanercept, which contains a truncated form of IgG1 Fc domain (lacking a CH1 constant region), certolizumab pegol, due to its Fc domain missing structure, acts differently. In treating inflammatory bowel disease with anti-TNF mAbs, another mechanism of their action is based on the interaction between IgG1 Fc domains of therapeutic mAbs and macrophage Fcγ receptors (FcγR), resulting in increased numbers of regulatory M2 macrophages (CD206+

*Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) [2].*

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs*

*DOI: http://dx.doi.org/10.5772/intechopen.85237*

*Structures (A) and mechanisms of action (B) of the five FDA- and EMA-approved anti-TNF biologics; original figure used with the authors' permission under the terms and conditions of the Creative Commons*

in turn inhibit T cell proliferation [2, 7]. Additionally, in rheumatoid arthritis (RA), adalimumab enhances the expression of tmTNF on monocytes, thereby promoting the interaction between tmTNF and TNFR2 present on regulatory T cells (Tregs), which subsequently increase their immunosuppressive activities [2, 8]. Also in RA,

inhibit a proinflammatory cytokine production and replenish a defective pool of these cells, typically found in this autoimmune disease [2, 9]. In RA patients, the adhesion molecules and chemokines are upregulated on their joint vasculature endothelium. Blockage of TNF-α with adalimumab, golimumab, infliximab, or etanercept deactivates inflamed vascular endothelium, thereby decreasing the numbers of inflammatory immune cells entering synovial joints and additionally improving the generation of new synovial blood vessels by increasing the circulating levels of vascular endo-

**4.** *In vitro* **cell-based bioassays for general potency assessment of**

Numerous well-established and standardized cell-based assays are available for assessing and comparing potencies of anti-TNF biologics and their biosimilars. **Table 1** contains some basic information regarding the most frequently used routine TNF-α neutralization (A), ADCC (B), and CDC (C) tests. The majority of data

infliximab promotes the generation of natural Tregs (CD4+

thelial growth factor (VEGF) [10, 11].

**anti-TNF biologics**

**11**

**Figure 1.**

). These cells

), which

CD25highFoxP3+

While TNFR1 is activated by both tmTNF and sTNF, TNFR2 can only be triggered by tmTNF. Both types of membrane-bound receptors are prone to TACE cleavage, resulting in fragments termed soluble TNF receptors (sTNFR) [2]. In turn, sTNFR may contribute to the regulation of cellular TNF responses by capturing and neutralizing circulating TNF (intrinsic TNF inhibitors). Additionally, due to increased receptor shedding, the number of functional signaling membrane TNFRs decreases. Consequently, this leads to a state of transient TNF desensitization [2].

#### **3. Anti-TNF biological drugs and their mechanisms of action**

Among currently available Food and Drug Administration (FDA)- and European Medicines Agency (EMA)-approved originator and biosimilar anti-TNF drugs, there are three full-length monoclonal antibodies (mAbs); these are infliximab (IFX), a chimeric mouse/human mAb (Remicade® and its biosimilars: Remsima®, Inflectra®, Flixabi®, Ixifi®, Renflexis®, and Zessly®), adalimumab (ADA), a fully humanized mAb (Humira® and its biosimilars: Cyltezo®, Imraldi®, Amgevita®, Solymbic®, Hyrimoz®, Hulio®, Halimatoz®, and Heyifa®), and golimumab, another fully humanized mAb (Simponi®) (**Figure 1**) [2, 4]. The additional two anti-TNF biological drugs, which are not mAbs, are etanercept (ETA) (Enbrel® and its biosimilars: Erelzi® and Benepali®), a fusion protein consisting of two extracellular parts of the human TNFR2 and the Fc portion of human IgG1, and certolizumab pegol (Cimzia®) composed of a human Fab' fragment, covalently attached to two cross-linked 20 kDa polyethylene glycol chains (**Figure 1**) [2, 4].

Although all anti-TNF biologics neutralize the same target (sTNF and tmTNF), they are not equally effective in treatment of certain inflammatory pathologies, for example, Crohn's disease. This is due to differences in their characteristics (structure and binding affinities) and mechanisms of action (**Figure 1**) [2, 4]. Besides all of them being efficacious in neutralizing both forms of TNF, infliximab additionally induces "outside-to-inside" signaling via binding to tmTNF, thereby triggering apoptosis of tmTNF-expressing immune cells [2]. Being full-length mAbs,

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*

#### **Figure 1.**

**2. A short overview of TNF biology**

*Cytokines*

cells, and T lymphocytes [4].

**10**

TNF is produced in various cell types, mainly immune cells such as monocytes and macrophages, microglia, neutrophils, natural killer cells (NK), T lymphocytes, and also in neuronal cells, keratinocytes, and fibroblasts [2, 3]. The cytokine exists in two biologically active forms. The first being a transmembrane protein (tmTNF), which can be cleaved by the metalloproteinase TNF-α-converting enzyme (TACE) (also known as disintegrin and metalloproteinase domain-containing protein 17 (ADAM17)) into its second form, a homotrimeric soluble TNF (sTNF) [2].

There are two TNF-binding homotrimeric transmembrane receptors, namely the TNF receptor 1 (TNFR1 or CD120a) and the TNF receptor 2 (TNFR2 or CD120b) [2]. While the TNFR1 is constitutively expressed on a vast majority of nucleated cells, the TNFR2 expression is inducible and tightly regulated, preferentially on endothelial, hematopoietic, neural, and immune cells [2, 4]. TNFR2 is also expressed on tumor

Interestingly, tmTNF can induce signals in a bipolar way, as it acts as a ligand of both receptor types and as a receptor itself in cell-to-cell contacts [2, 4]. This means that tmTNF-α-expressing cells transmit signals to cells bearing TNFR1 and/or TNFR2. This phenomenon is called "outside-to-inside" or "reverse signaling," the function of which has not been completely clarified yet [2, 4]. The receptor function of tmTNF has been demonstrated in human monocytes, macrophages, NK

While TNFR1 is activated by both tmTNF and sTNF, TNFR2 can only be triggered by tmTNF. Both types of membrane-bound receptors are prone to TACE cleavage, resulting in fragments termed soluble TNF receptors (sTNFR) [2]. In turn, sTNFR may contribute to the regulation of cellular TNF responses by capturing and neutralizing circulating TNF (intrinsic TNF inhibitors). Additionally, due to increased receptor shedding, the number of functional signaling membrane TNFRs decreases. Consequently, this leads to a state of transient TNF desensitization [2].

Among currently available Food and Drug Administration (FDA)- and European Medicines Agency (EMA)-approved originator and biosimilar anti-TNF drugs, there are three full-length monoclonal antibodies (mAbs); these are infliximab (IFX), a chimeric mouse/human mAb (Remicade® and its biosimilars: Remsima®, Inflectra®, Flixabi®, Ixifi®, Renflexis®, and Zessly®), adalimumab (ADA), a fully humanized mAb (Humira® and its biosimilars: Cyltezo®, Imraldi®, Amgevita®, Solymbic®, Hyrimoz®, Hulio®, Halimatoz®, and Heyifa®), and golimumab, another fully humanized mAb (Simponi®) (**Figure 1**) [2, 4]. The additional two anti-TNF biological drugs, which are not mAbs, are etanercept (ETA) (Enbrel® and its biosimilars: Erelzi® and Benepali®), a fusion protein consisting of two extracel-

**3. Anti-TNF biological drugs and their mechanisms of action**

lular parts of the human TNFR2 and the Fc portion of human IgG1, and certolizumab pegol (Cimzia®) composed of a human Fab' fragment, covalently attached to two cross-linked 20 kDa polyethylene glycol chains (**Figure 1**) [2, 4]. Although all anti-TNF biologics neutralize the same target (sTNF and tmTNF), they are not equally effective in treatment of certain inflammatory pathologies, for example, Crohn's disease. This is due to differences in their characteristics (structure and binding affinities) and mechanisms of action (**Figure 1**) [2, 4]. Besides all of them being efficacious in neutralizing both forms of TNF, infliximab additionally induces "outside-to-inside" signaling via binding to tmTNF, thereby triggering apoptosis of tmTNF-expressing immune cells [2]. Being full-length mAbs,

cells where it is supposed to function as a tumor oncogene [5, 6].

*Structures (A) and mechanisms of action (B) of the five FDA- and EMA-approved anti-TNF biologics; original figure used with the authors' permission under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) [2].*

adalimumab, golimumab, and infliximab can, after binding to cells expressing tmTNF via their effector Fc regions (IgG1), induce antibody-dependent cytotoxicity (ADCC) of NK cells and activate the classical complement pathway, resulting in a complement-dependent cytotoxicity (CDC) and apoptosis [2, 4]. While ADCC and CDC are also induced by etanercept, which contains a truncated form of IgG1 Fc domain (lacking a CH1 constant region), certolizumab pegol, due to its Fc domain missing structure, acts differently. In treating inflammatory bowel disease with anti-TNF mAbs, another mechanism of their action is based on the interaction between IgG1 Fc domains of therapeutic mAbs and macrophage Fcγ receptors (FcγR), resulting in increased numbers of regulatory M2 macrophages (CD206+ ). These cells in turn inhibit T cell proliferation [2, 7]. Additionally, in rheumatoid arthritis (RA), adalimumab enhances the expression of tmTNF on monocytes, thereby promoting the interaction between tmTNF and TNFR2 present on regulatory T cells (Tregs), which subsequently increase their immunosuppressive activities [2, 8]. Also in RA, infliximab promotes the generation of natural Tregs (CD4+ CD25highFoxP3+ ), which inhibit a proinflammatory cytokine production and replenish a defective pool of these cells, typically found in this autoimmune disease [2, 9]. In RA patients, the adhesion molecules and chemokines are upregulated on their joint vasculature endothelium. Blockage of TNF-α with adalimumab, golimumab, infliximab, or etanercept deactivates inflamed vascular endothelium, thereby decreasing the numbers of inflammatory immune cells entering synovial joints and additionally improving the generation of new synovial blood vessels by increasing the circulating levels of vascular endothelial growth factor (VEGF) [10, 11].

### **4.** *In vitro* **cell-based bioassays for general potency assessment of anti-TNF biologics**

Numerous well-established and standardized cell-based assays are available for assessing and comparing potencies of anti-TNF biologics and their biosimilars. **Table 1** contains some basic information regarding the most frequently used routine TNF-α neutralization (A), ADCC (B), and CDC (C) tests. The majority of data on bioassays presented in **Table 1** (see next page) were summarized from two publications describing the establishment of the first infliximab and etanercept World Health Organization (WHO) International Standards [12, 13]. These were performed within international collaborative studies, confirming their high degree of relevance and analytical laboratory utility.

Additionally, the capability of anti-TNF biological drugs to downregulate Eselectin adhesion molecules expressed on inflamed vascular endothelium can be determined on *in vitro-*cultured human umbilical vein endothelial cells by using appropriately labeled anti-E-selectin detection antibodies [10].

Other bioassay readout approaches, like flow cytometry and measurement of induced endogenous gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR), are also being applied [16–18].

The reason why various human and murine cell lines are used in these assays is that such tests can be standardized and their results can be compared between laboratories. However, the use of different types of primary cells in such general tests is less appropriate due to their high interindividual differences and in certain cases also weak responsiveness to anti-TNF biologics. Therefore, the results obtained in this way can hardly be compared [10].

### **5. Two-dimensional (2D) and three-dimensional (3D) primary cell cultures for personalized** *in vitro* **potency testing of anti-TNF biologics**

Primary cells are indispensable for determining personal responses of patients to a given anti-TNF biologic, thereby generating important information for planning and performing optimal and cost-effective therapies. For this purpose, different cell types, especially those isolated from a patient's disease-affected tissues or *in vitro* differentiated autologous stem cells, can be used. In general, it is well established that in comparison to cells grown in 2D, those cultured in a 3D environment better mimic the scenarios *in vivo*. A number of cellular processes, that is, proliferation, differentiation, morphology, gene, and protein expressions, as well as responsiveness to external stimuli, are significantly affected by the physical aspects of the 3D environment [19–22].

In 2D cell cultures (monolayers), nutrients are evenly accessible to cells, but the communication between cells via secreted soluble molecules is restricted to their diffusion within the fluid, unless the medium is mixed or stirred regularly [23]. On the other hand, in dense multicellular 3D cell constructs prepared and cultured *in vitro*, nutrients and other soluble molecules, as well as oxygen supply, are limited by the mass transport, which is restricted by the construct's thickness/diameter and cell density per volume [23].

*rhTNF-α—recombinant human TNF-α. Cells* ! *CHO-K1: Chinese hamster ovary cells expressing human transmembrane TNF-α (htmTNF-α); HEK 293: human embryonic kidney cell line, transfected with the TNF-α-responsive NFκB-regulated Firefly luciferase reporter gene construct or expressing htmTNF-α; Jurkat: human acute T cell leukemia lymphocytes expressing htmTNF-α, resistant to TACE cleavage, human Fcγ RIIIa or TNF-α-responsive nuclear factor of activated T cells (NFAT) transcription factor-regulated Firefly luciferase reporter gene construct; K2: murine cells expressing the uncleavable htmTNF-α; KD4 Cl21: human rhabdomyosarcoma cell line; KJL: human erythroleukemic K562 cells transfected with the TNF-αresponsive NFκB-regulated Firefly luciferase reporter gene construct, together with the Renilla luciferase reporter gene under the control of a constitutive minimal thymidine kinase promoter; L929: murine fibroblast cell line; NK3.3: human natural killer (NK) cell line cloned from peripheral blood; NK92: NK lymphoblast cells from a malignant non-Hodgkin's lymphoma patient, expressing Fcγ RIIIa; 3T3: murine embryonic fibroblasts expressing htmTNF-α; U937: human histiocytic lymphoma cell line; WEHI-13 VAR and WEHI-164: murine rhabdomyosarcoma cell lines. Readout reagents (absorbance)* ! *CCK-8/ WST-8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; MTT: 3-*

*carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; luminescence detection kits: Bio-Glo™, Caspase-Glo® 3/7, CellTiter-Glo®, CytoTox-Glo®, Dual-Glo®, Steady-Glo® (all from Promega), and Steadylite plus™ luminescence reporter gene system (Perkin Elmer). Assays* ! *TNF-α neutralization (cytotoxicity, apoptosis, reporter gene): measuring the extent of residual TNF-α-induced cytotoxicity and apoptosis in the presence of anti-TNF-α biologics; TNF-α-induced ADCC: measuring the extent of effector cell cytotoxicity on htmTNF-α expressing target cells, in the presence of anti-TNF-biologics; TNF-α-induced CDC: measuring the extent of cytotoxicity in the presence of human serum as a source of complement and anti-*

*Most frequently used routine cell-based bioassays for assessing the TNF neutralization potency (A), ADCC (B),*

*(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-*

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs*

*DOI: http://dx.doi.org/10.5772/intechopen.85237*

*and CDC (C) of anti-TNF biologics and their biosimilars [14, 15].*

*TNF biologics.*

**Table 1.**

**13**

In the following subchapters, we will present our results after establishing 2D and 3D *in vitro* models for potency testing of anti-TNF biologics by using primary normal chondrocytes (NCs) and osteoarthritic chondrocytes (OACs), as well as chondrogenically differentiated bone-marrow-derived MSCs obtained from OA patients, with qRT-PCR gene expression assessment and protein secretion readout measurements.

#### **5.1 Establishment of a 2D primary human chondrocyte-based cell model for** *in vitro* **testing of anti-TNF-α biologicals**

Cartilage, which covers joint surfaces, is one of the most affected tissues in RA and other inflammatory arthritic diseases. Its only living constituents are chondrocytes, which produce and maintain a cartilaginous matrix mainly consisting

#### In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*




*rhTNF-α—recombinant human TNF-α. Cells* ! *CHO-K1: Chinese hamster ovary cells expressing human transmembrane TNF-α (htmTNF-α); HEK 293: human embryonic kidney cell line, transfected with the TNF-α-responsive NFκB-regulated Firefly luciferase reporter gene construct or expressing htmTNF-α; Jurkat: human acute T cell leukemia lymphocytes expressing htmTNF-α, resistant to TACE cleavage, human Fcγ RIIIa or TNF-α-responsive nuclear factor of activated T cells (NFAT) transcription factor-regulated Firefly luciferase reporter gene construct; K2: murine cells expressing the uncleavable htmTNF-α; KD4 Cl21: human rhabdomyosarcoma cell line; KJL: human erythroleukemic K562 cells transfected with the TNF-αresponsive NFκB-regulated Firefly luciferase reporter gene construct, together with the Renilla luciferase reporter gene under the control of a constitutive minimal thymidine kinase promoter; L929: murine fibroblast cell line; NK3.3: human natural killer (NK) cell line cloned from peripheral blood; NK92: NK lymphoblast cells from a malignant non-Hodgkin's lymphoma patient, expressing Fcγ RIIIa; 3T3: murine embryonic fibroblasts expressing htmTNF-α; U937: human histiocytic lymphoma cell line; WEHI-13 VAR and WEHI-164: murine rhabdomyosarcoma cell lines. Readout reagents (absorbance)* ! *CCK-8/ WST-8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; MTT: 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; luminescence detection kits: Bio-Glo™, Caspase-Glo® 3/7, CellTiter-Glo®, CytoTox-Glo®, Dual-Glo®, Steady-Glo® (all from Promega), and Steadylite plus™ luminescence reporter gene system (Perkin Elmer). Assays* ! *TNF-α neutralization (cytotoxicity, apoptosis, reporter gene): measuring the extent of residual TNF-α-induced cytotoxicity and apoptosis in the presence of anti-TNF-α biologics; TNF-α-induced ADCC: measuring the extent of effector cell cytotoxicity on htmTNF-α expressing target cells, in the presence of anti-TNF-biologics; TNF-α-induced CDC: measuring the extent of cytotoxicity in the presence of human serum as a source of complement and anti-TNF biologics.*

#### **Table 1.**

*Most frequently used routine cell-based bioassays for assessing the TNF neutralization potency (A), ADCC (B), and CDC (C) of anti-TNF biologics and their biosimilars [14, 15].*

on bioassays presented in **Table 1** (see next page) were summarized from two publications describing the establishment of the first infliximab and etanercept World Health Organization (WHO) International Standards [12, 13]. These were performed within international collaborative studies, confirming their high degree

Additionally, the capability of anti-TNF biological drugs to downregulate Eselectin adhesion molecules expressed on inflamed vascular endothelium can be determined on *in vitro-*cultured human umbilical vein endothelial cells by using

Other bioassay readout approaches, like flow cytometry and measurement of induced endogenous gene expression by quantitative reverse transcription poly-

The reason why various human and murine cell lines are used in these assays is that such tests can be standardized and their results can be compared between laboratories. However, the use of different types of primary cells in such general tests is less appropriate due to their high interindividual differences and in certain cases also weak responsiveness to anti-TNF biologics. Therefore, the results

**5. Two-dimensional (2D) and three-dimensional (3D) primary cell cultures for personalized** *in vitro* **potency testing of anti-TNF**

Primary cells are indispensable for determining personal responses of patients to a given anti-TNF biologic, thereby generating important information for planning and performing optimal and cost-effective therapies. For this purpose, different cell types, especially those isolated from a patient's disease-affected tissues or *in vitro* differentiated autologous stem cells, can be used. In general, it is well established that in comparison to cells grown in 2D, those cultured in a 3D environment better mimic the scenarios *in vivo*. A number of cellular processes, that is, proliferation, differentiation, morphology, gene, and protein expressions, as well as responsiveness to external stimuli, are significantly affected by the physical aspects of the 3D

In 2D cell cultures (monolayers), nutrients are evenly accessible to cells, but the communication between cells via secreted soluble molecules is restricted to their diffusion within the fluid, unless the medium is mixed or stirred regularly [23]. On the other hand, in dense multicellular 3D cell constructs prepared and cultured *in vitro*, nutrients and other soluble molecules, as well as oxygen supply, are limited by the mass transport, which is restricted by the construct's thickness/diameter and

In the following subchapters, we will present our results after establishing 2D and 3D

*in vitro* models for potency testing of anti-TNF biologics by using primary normal

chondrogenically differentiated bone-marrow-derived MSCs obtained from OA patients, with qRT-PCR gene expression assessment and protein secretion readout

**5.1 Establishment of a 2D primary human chondrocyte-based cell model for**

and other inflammatory arthritic diseases. Its only living constituents are

Cartilage, which covers joint surfaces, is one of the most affected tissues in RA

chondrocytes, which produce and maintain a cartilaginous matrix mainly consisting

chondrocytes (NCs) and osteoarthritic chondrocytes (OACs), as well as

*in vitro* **testing of anti-TNF-α biologicals**

of relevance and analytical laboratory utility.

obtained in this way can hardly be compared [10].

**biologics**

*Cytokines*

environment [19–22].

cell density per volume [23].

measurements.

**12**

appropriately labeled anti-E-selectin detection antibodies [10].

merase chain reaction (qRT-PCR), are also being applied [16–18].

of collagen and proteoglycans [24]. *In vitro*-cultured chondrocytes have already provided useful models to study their response to microenvironment alterations [25]. However, we have extended their *in vitro* use to efficacy testing of anti-TNF-α drugs [26–28]. First, we have established a 2D *in vitro* model by culturing human primary chondrocytes in monolayer cultures and later upgraded it to a 3D cell model, which better mimics the organization of these cells in native cartilage. For this purpose, we chose a combination of physiologically relevant cell sources and a gene expression assessment technique (qRT-PCR), which enables analyses of up- or downregulated genes in comparison to measurable changes in secreted proteins or cell numbers [29]. We have selected and screened 42 genes involved in immune responses, extracellular matrix remodeling, stress response, signaling pathways, expression of adhesion and other molecules, responding to a pathogenic inflammatory environment that was artificially created with the addition of rhTNF-α.

For the establishment of our 2D model, two types of cells were used. Normal, healthy chondrocytes (NCs) were obtained from surplus cartilage biopsies of patients scheduled for an autologous chondrocyte implantation procedure or were acquired postmortem from donors with healthy cartilage, in accordance with National Medical Ethics Committee approvals. On the other hand, osteoarthritic chondrocytes (OACs) were obtained from cartilage samples of patients undergoing total knee replacement surgery, in accordance with National Medical Ethics Committee approval. Following chondrocyte isolation and cultivation, confluent cell cultures were incubated in serum-free conditions with 1 ng/mL of rhTNF-α (PeproTech, USA) 1 μg/mL of each of the two anti-TNF-α biologicals tested, infliximab (IFX; Remicade®, Centocor, Netherlands) and etanercept (ETA; Enbrel®, Wyeth Pharmaceuticals, UK). After 24 h of incubation, chondrocytes and cell culture media were sampled for gene and protein expression analyses, respectively. In experiments using OACs, only the most relevant genes were selected and analyzed. Names and symbols of screened genes are presented in **Table 2**. Data were analyzed by applying the 2ΔΔCq formula (ABI PRISM® 7700 Sequence Detection System User Bulletin #2) with the nontreated chondrocyte samples used for normalization. Results are presented as relative quantities (RQ) or Log2 relative quantity values (Log2 RQ). For protein expression analysis, a custom antibody array (RayBiotech, USA) was designed to detect interleukin-1 receptor antagonist (IL-1Ra), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), monocyte chemoattractant protein-1 (MCP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), and vascular cell adhesion protein 1 (VCAM-1). All protein analysis data were normalized to nontreated controls.

The results of the first set of experiments obtained after stimulation of cultured NCs and OACs with rhTNF-α and after their preincubation with a combination of rhTNF-α and IFX or ETA are presented in **Figure 2** (graphs A and B, respectively). Upon TNF-α stimulation of NCs, the highest gene upregulation was observed for *IL8* and *MMP1* with a >1000-fold change. A very high upregulation (≥80-fold change) was also observed for *IL6*, *IL32*, *MMP3*, *MMP13*,*TLR2*, and *MCP1* genes (**Figure 2**, graph Aa). We considered the differences between treated and nontreated cells as biologically significant whenever the calculated fold change was ≥2, which equals a Log2-fold change of ≥1 unit on a logarithmic scale. Next, we examined the neutralization efficacy of IFX and ETA by monitoring a decrease in TNF-α-induced gene expressions. Although IFX reduced the expression of TNF-αupregulated genes, some of them remained more expressed when compared to nontreated cell samples (**Figure 2**, graph Ab). On the other hand, ETA completely abolished the TNF-α-mediated up- and downregulation of the tested genes (**Figure 2**, graph Ac). Altogether, our results revealed differential sTNF-α

neutralizing potency of IFX and ETA at the level of gene expression patterns. The observed changes in gene expression were then also confirmed with a protein

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs*

*DOI: http://dx.doi.org/10.5772/intechopen.85237*

Because NCs are difficult to obtain, we performed the same IFX and ETA neutralization experiments with rhTNF-α-treated OACs, however, to a lesser extent. A selected group of the most responsive genes were tested using OAC biological samples from four donors (**Figure 2B**). We observed a similar response to NCs when OACs were treated by rhTNF-α alone (**Figure 2**, graph Ba) and after their preincubation with a combination of rhTNF-α and IFX or ETA (**Figure 2**, graphs Bb and Bd, respectively). In **Figure 2**, graphs Bc and Be show the responses of OACs after their exposure to each individual biological drug. The cartilage of OA patients represents a biological waste material, which can be obtained in joint replacement surgeries. Despite changes in gene expression observed during the *in vitro* cultivation of OACs, the stimulation with rhTNF-α reverted them back to their inflammatory phenotype. Our results, employing anti-TNF-α biologics,

expression assay.

*List of genes analyzed in qRT-PCR experiments.*

**Table 2.**

**15**

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*


#### **Table 2.**

of collagen and proteoglycans [24]. *In vitro*-cultured chondrocytes have already provided useful models to study their response to microenvironment alterations [25]. However, we have extended their *in vitro* use to efficacy testing of anti-TNF-α drugs [26–28]. First, we have established a 2D *in vitro* model by culturing human primary chondrocytes in monolayer cultures and later upgraded it to a 3D cell model, which better mimics the organization of these cells in native cartilage. For this purpose, we chose a combination of physiologically relevant cell sources and a gene expression assessment technique (qRT-PCR), which enables analyses of up- or downregulated genes in comparison to measurable changes in secreted proteins or cell numbers [29]. We have selected and screened 42 genes involved in immune responses, extracellular matrix remodeling, stress response, signaling pathways, expression of adhesion and other molecules, responding to a pathogenic inflammatory environment that was artificially created with the addition of rhTNF-α.

For the establishment of our 2D model, two types of cells were used. Normal, healthy chondrocytes (NCs) were obtained from surplus cartilage biopsies of patients scheduled for an autologous chondrocyte implantation procedure or were acquired postmortem from donors with healthy cartilage, in accordance with National Medical Ethics Committee approvals. On the other hand, osteoarthritic chondrocytes (OACs) were obtained from cartilage samples of patients undergoing total knee replacement surgery, in accordance with National Medical Ethics Committee approval. Following chondrocyte isolation and cultivation, confluent cell cultures were incubated in serum-free conditions with 1 ng/mL of rhTNF-α (PeproTech, USA) 1 μg/mL of each of the two anti-TNF-α biologicals tested, infliximab (IFX; Remicade®, Centocor, Netherlands) and etanercept (ETA;

Enbrel®, Wyeth Pharmaceuticals, UK). After 24 h of incubation, chondrocytes and cell culture media were sampled for gene and protein expression analyses, respectively. In experiments using OACs, only the most relevant genes were selected and analyzed. Names and symbols of screened genes are presented in **Table 2**. Data were analyzed by applying the 2ΔΔCq formula (ABI PRISM® 7700 Sequence Detection System User Bulletin #2) with the nontreated chondrocyte samples used for normalization. Results are presented as relative quantities (RQ) or Log2 relative quantity values (Log2 RQ). For protein expression analysis, a custom antibody array (RayBiotech, USA) was designed to detect interleukin-1 receptor antagonist (IL-1Ra), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), monocyte chemoattractant protein-1 (MCP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), and vascular cell adhesion protein 1 (VCAM-1). All protein analysis data were normalized to nontreated controls. The results of the first set of experiments obtained after stimulation of cultured NCs and OACs with rhTNF-α and after their preincubation with a combination of rhTNF-α and IFX or ETA are presented in **Figure 2** (graphs A and B, respectively). Upon TNF-α stimulation of NCs, the highest gene upregulation was observed for *IL8* and *MMP1* with a >1000-fold change. A very high upregulation (≥80-fold change) was also observed for *IL6*, *IL32*, *MMP3*, *MMP13*,*TLR2*, and *MCP1* genes (**Figure 2**, graph Aa). We considered the differences between treated and

nontreated cells as biologically significant whenever the calculated fold change was ≥2, which equals a Log2-fold change of ≥1 unit on a logarithmic scale. Next, we examined the neutralization efficacy of IFX and ETA by monitoring a decrease in TNF-α-induced gene expressions. Although IFX reduced the expression of TNF-αupregulated genes, some of them remained more expressed when compared to nontreated cell samples (**Figure 2**, graph Ab). On the other hand, ETA completely

abolished the TNF-α-mediated up- and downregulation of the tested genes (**Figure 2**, graph Ac). Altogether, our results revealed differential sTNF-α

**14**

*Cytokines*

*List of genes analyzed in qRT-PCR experiments.*

neutralizing potency of IFX and ETA at the level of gene expression patterns. The observed changes in gene expression were then also confirmed with a protein expression assay.

Because NCs are difficult to obtain, we performed the same IFX and ETA neutralization experiments with rhTNF-α-treated OACs, however, to a lesser extent. A selected group of the most responsive genes were tested using OAC biological samples from four donors (**Figure 2B**). We observed a similar response to NCs when OACs were treated by rhTNF-α alone (**Figure 2**, graph Ba) and after their preincubation with a combination of rhTNF-α and IFX or ETA (**Figure 2**, graphs Bb and Bd, respectively). In **Figure 2**, graphs Bc and Be show the responses of OACs after their exposure to each individual biological drug. The cartilage of OA patients represents a biological waste material, which can be obtained in joint replacement surgeries. Despite changes in gene expression observed during the *in vitro* cultivation of OACs, the stimulation with rhTNF-α reverted them back to their inflammatory phenotype. Our results, employing anti-TNF-α biologics,

confirmed that OACs and NCs can be interchangeably used for obtaining valuable preliminary information regarding the neutralization efficacy of these drugs [27]. With the data obtained, we were able to establish a statistical model for the evaluation of IFX and ETA TNF-α neutralization efficacy. Expressions of the nine most representative genes were chosen for a graphical presentation of results. Geometrical means of RQ values were plotted on radial axes of radar graphs and connected by a polygon, forming a distinctive shape. A comparison of shapes obtained with IFX and ETA revealed differences in their inhibition of gene expressions. Value 0, depicted in the center of graphs, represents total gene inhibition. For easier comparisons of results, shaded areas of twofold changes were plotted as well. Arbitrary fold-change cutoffs >2 (0.5 for down- and 2 for upregulated genes) were considered biologically significant. In our experimental conditions, the twofold change rims only overlapped in case of *VCAM1* and *MMP3* gene expressions, indicating that both IFX and ETA inhibit these two genes to a similar extent. However, in the case of *MMP13*, *IL32*, *MCP1*, *IL6*, *MMP1*,*TLR2*, and *IL8*, the inhibition efficacy of ETA was significantly more pronounced. Altogether, in our 2D NC-based model, ETA exhibited higher sTNF-α neutralization efficacy than IFX (**Figure 3**).

*Graphical representation of results obtained with the statistical model for evaluation of sTNF-α neutralization efficacy of biological drugs. Radar graphs for IFX (a), ETA (b), and both of them (IFX and ETA) (c) present geometrical means of RQ values obtained with six NC biological samples. Corresponding twofold area changes are presented. The overlap of twofold rims is only seen in two (MMP3 and VCAM-1) out of nine genes, indicating higher sTNF-α neutralization efficacy of ETA. Value 0 in the center of the radar graphs denotes total inhibition of gene expression. Please note the difference in scales: (b) intervals of 0.2 units, (a) and (c) intervals of 2 units. Original figure used with authors' permission under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) [28].*

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs*

*DOI: http://dx.doi.org/10.5772/intechopen.85237*

The presented statistical model is also suitable for a comparative neutralization efficacy determination of new bioactive molecules and biosimilars relative to wellestablished and approved biologics, according to effective criteria for the assess-

As discussed in the introduction, 2D and 3D cell culture conditions have different impacts on cell phenotype and biological behavior, which were also confirmed for primary chondrocytes and chondrogenically differentiated MSCs [30–35]. In the last decade, cell-based research shifted toward 3D tissue/organ models, providing more physiologically realistic biochemical and biomechanical microenvironments. However, besides their biological relevance, in order to meet the expectations of the pharmaceutical industry, drug screening assays should be high-throughput, widely applicable, and low cost. With this in mind, we established a new *in vitro* 3D

**5.2 Establishment of a 3D human osteoarthritic model for** *in vitro* **efficacy testing of anti-TNF-α biologicals, using primary human osteoarthritic**

ment of biosimilarity, nonsimilarity, and incomparability.

**chondrocytes and mesenchymal stem cells**

**Figure 3.**

**17**

#### **Figure 2.**

*Exposure of* in vitro*-cultured normal healthy chondrocytes (NCs) (A) and osteoarthritic chondrocytes (OACs) (B) to rhTNF-α markedly increased the expression of various genes coding for interleukins, matrix metalloproteinases, and other factors involved in inflammation and stress response (graphs Aa and Ba). TNF-α neutralizing effect of IFX (graphs Ab and Bb) and ETA (graphs Ac and Bd) is reflected in significantly decreased gene expressions. Graphs Bc and Be show the responses of OACs following their exposure to each individual biological drug tested. All gene expressions were calibrated to nontreated cells. Log2 RQ values of individual biological samples (*○*) and their corresponding geometrical means (*●*) are shown. Original figures used with authors' permission under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) [27, 28].*

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*

#### **Figure 3.**

*Graphical representation of results obtained with the statistical model for evaluation of sTNF-α neutralization efficacy of biological drugs. Radar graphs for IFX (a), ETA (b), and both of them (IFX and ETA) (c) present geometrical means of RQ values obtained with six NC biological samples. Corresponding twofold area changes are presented. The overlap of twofold rims is only seen in two (MMP3 and VCAM-1) out of nine genes, indicating higher sTNF-α neutralization efficacy of ETA. Value 0 in the center of the radar graphs denotes total inhibition of gene expression. Please note the difference in scales: (b) intervals of 0.2 units, (a) and (c) intervals of 2 units. Original figure used with authors' permission under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) [28].*

confirmed that OACs and NCs can be interchangeably used for obtaining valuable preliminary information regarding the neutralization efficacy of these drugs [27].

With the data obtained, we were able to establish a statistical model for the evaluation of IFX and ETA TNF-α neutralization efficacy. Expressions of the nine most representative genes were chosen for a graphical presentation of results. Geometrical means of RQ values were plotted on radial axes of radar graphs and connected by a polygon, forming a distinctive shape. A comparison of shapes obtained with IFX and ETA revealed differences in their inhibition of gene expressions. Value 0, depicted in the center of graphs, represents total gene inhibition. For easier comparisons of results, shaded areas of twofold changes were plotted as well. Arbitrary fold-change cutoffs >2 (0.5 for down- and 2 for upregulated genes) were considered biologically significant. In our experimental conditions, the twofold change rims only overlapped in case of *VCAM1* and *MMP3* gene expressions, indicating that both IFX and ETA inhibit these two genes to a similar extent. However, in the case of *MMP13*, *IL32*, *MCP1*, *IL6*, *MMP1*,*TLR2*, and *IL8*, the inhibition efficacy of ETA was significantly more pronounced. Altogether, in our 2D NC-based model, ETA exhibited higher sTNF-α neutralization efficacy than IFX (**Figure 3**).

The presented statistical model is also suitable for a comparative neutralization efficacy determination of new bioactive molecules and biosimilars relative to wellestablished and approved biologics, according to effective criteria for the assessment of biosimilarity, nonsimilarity, and incomparability.

#### **5.2 Establishment of a 3D human osteoarthritic model for** *in vitro* **efficacy testing of anti-TNF-α biologicals, using primary human osteoarthritic chondrocytes and mesenchymal stem cells**

As discussed in the introduction, 2D and 3D cell culture conditions have different impacts on cell phenotype and biological behavior, which were also confirmed for primary chondrocytes and chondrogenically differentiated MSCs [30–35]. In the last decade, cell-based research shifted toward 3D tissue/organ models, providing more physiologically realistic biochemical and biomechanical microenvironments. However, besides their biological relevance, in order to meet the expectations of the pharmaceutical industry, drug screening assays should be high-throughput, widely applicable, and low cost. With this in mind, we established a new *in vitro* 3D

**Figure 2.**

*Cytokines*

**16**

*Exposure of* in vitro*-cultured normal healthy chondrocytes (NCs) (A) and osteoarthritic chondrocytes (OACs)*

*metalloproteinases, and other factors involved in inflammation and stress response (graphs Aa and Ba). TNF-α neutralizing effect of IFX (graphs Ab and Bb) and ETA (graphs Ac and Bd) is reflected in significantly decreased gene expressions. Graphs Bc and Be show the responses of OACs following their exposure to each individual biological drug tested. All gene expressions were calibrated to nontreated cells. Log2 RQ values of individual biological samples (*○*) and their corresponding geometrical means (*●*) are shown. Original figures used with authors' permission under the terms and conditions of the Creative Commons Attribution (CC BY)*

*(B) to rhTNF-α markedly increased the expression of various genes coding for interleukins, matrix*

*license (http://creativecommons.org/licenses/by/4.0/) [27, 28].*

chondrogenic tissue model which, combined with the qRT-PCR readout method, can be used for preclinical or patient-specific potency assessment of anti-TNF-α and anti-interleukin-1β biological drugs (anti-IL-1β) [26]. For establishing this model, we used human OACs and chondrogenically differentiated MSCs.

As already stated, OACs represent an attractive source of cells for cell-based models as besides being rather easily accessible and free of ethical concerns, they are also genetically stable during their long-term *in vitro* expansion [36, 37]. Reports show that MSCs isolated from bone marrow of OA patients are capable of producing hyaline cartilage suitable for tissue repair. MSCs obtained from OA and RA patients possess similar chondrogenic potential as those from healthy individuals [38–41]. Therefore, we used paired samples of MSCs and OACs from two donors and a set of genetically mismatched biological samples of patient's OACs and commercially available MSCs. The paired cell sampling approach allowed us to reduce the high patient-to-patient variability, which influences the chondrogenic potential of both OACs and MSCs [42].

Among the numerous commercially available 3D cell culture systems, we have chosen Perfecta 3D® scaffolds (3D Biomatrix Inc., USA) to create tissues in hanging drops. Generation of scaffold-free spheroids of micrometric dimensions (microspheroids) by gravity-enforced self-assembly in hanging drops allows cell aggregation and tissue formation in a natural manner, without interference from the scaffold material [19, 32]. This technique has important advantages, especially the drop size control and consequent uniformity of formed microspheroids. Moreover, it is compatible with automated liquid handling systems, a prerequisite for high-throughput screening in drug discovery. The microspheroid formation in hanging drops mimics the condensation process of MSCs, which is one of the earliest phases of *in vivo* cartilage development [32].

Isolated OACs were first expanded in 2D monolayer cultures and then, from passage 2 and on, 10,000 cells were transferred into each hanging drop. In this way, the loss of chondrogenic phenotype of OACs in 2D was restored in 3D conditions, as already reported [30, 43]. Similarly as in our previously described 2D primary chondrocyte model, the TNF-α neutralizing efficiencies of ADA (Humira®, Abbott Laboratories, USA), ETA (Enbrel®, Immunex Corp., USA), IFX (Remicade®, Janssen Biotech, USA), and the anti-IL-1β drug anakinra (ANA; Kineret®, Swedish Orphan Biovitrum AB, Sweden) were assessed with both cell types by determining the extent of downregulation of six selected genes (*IL6*, *IL8*, *MCP1*, *MMP1*, *MMP13*, and *VCAM1*) [27, 28]. Gene expression was determined after a 24 h incubation of microspheroids in a medium supplemented with 1 ng/mL of an appropriate inflammatory cytokine (rhTNF-α or rhIL-1β; both from PeproTech, USA) or working macrophage conditioned medium (MCM) solution, combined with 1 μg/mL of each individual biological drug tested (**Figure 4**).

inflammation process in microspheroids. However, when microspheroids were incubated with MCM, none of the three tested anti-TNF-α drugs were successful in diminishing its inflammatory effect. Conversely, ANA could downregulate the expression of *IL6*, *IL8*, and *MMP1* genes. The described changes at the gene level were accompanied by significant differences in the expression of IL6, IL8, and MCP1 proteins, detected in supernatants of microspheroid cultures, 24 h after their incubation with a given inflammatory agent selected anti-inflammatory biological drug [26]. Moreover, these results were additionally supported by the amount of glycosaminoglycans present in chondral spheroids composed of 100,000 OACs treated with various combinations of a particular inflammatory agent a given

*microspheroids. Value 0 in the center of each radar graph represents total inhibition of gene expression. Original figure used with authors' permission under the terms and conditions of the Creative Commons Attribution*

*(a) Gene expression profiles following the addition of inflammatory mediators TNF-α, IL-1β, or MCM working solution and anti-inflammatory biological drugs ADA, IFX, ETA, and ANA. Blue and green dots represent values obtained in microspheroidal chondral tissues made of MSCs and OACs (three donors), respectively. Statistically significant changes, that is, Log2 RQ* ≥*1 and* ≤ *1 are outlined with median values for all groups. (b) Radar graphs representing anti-TNF-α neutralization efficacies of ADA (blue), IFX (red), and*

*ETA (green). Mean RQ values of three biological samples are shown for OAC- and MSC-derived*

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs*

*DOI: http://dx.doi.org/10.5772/intechopen.85237*

When microspheroids were incubated with MCM, a superior anti-IL-1β neutral-

ization capacity of ANA compared to the three tested anti-TNF-α biologics was observed. This difference was probably due to the fact that MCM contained a much

anti-inflammatory biologic, for a period of 3 weeks.

*(CC BY) license (http://creativecommons.org/licenses/by/4.0/) [26].*

**Figure 4.**

**19**

According to our criteria, Log2 RQ ≥1 and ≤ 1, TNF-α significantly upregulated the expression of *IL6*, *IL8*, *MCP1*, *MMP1*, *MMP13*, and *VCAM1* genes in the 3D microspheroidal model as well (**Figure 4a**). The same was true when IL-1β or MCM was added to microspheroids. MCM was obtained from cell cultures of the human monocytic cell line THP-1 (ATCC, USA) and represented a rich source of inflammatory cytokines with 0.05 ng/mL TNF-α and 0.45 ng/mL IL-1β, and numerous other growth factors. In terms of influencing gene expression, IL-1β was the most potent inflammation inducer, followed by MCM and then TNF-α. The inflammatory process triggered by each of these three inducers could always be reversed by ADA, IFX, or ETA, as well as ANA (**Figure 4a**). When inflammation was triggered by TNF-α, all tested anti-TNF-α biologics extraordinarily suppressed the expression of monitored genes, sometimes even reaching their constitutively expressed levels (log2 RQ = 0). Similarly, in the presence of IL-1β, ANA markedly reversed the

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*

#### **Figure 4.**

chondrogenic tissue model which, combined with the qRT-PCR readout method, can be used for preclinical or patient-specific potency assessment of anti-TNF-α and anti-interleukin-1β biological drugs (anti-IL-1β) [26]. For establishing this model,

As already stated, OACs represent an attractive source of cells for cell-based models as besides being rather easily accessible and free of ethical concerns, they are also genetically stable during their long-term *in vitro* expansion [36, 37]. Reports show that MSCs isolated from bone marrow of OA patients are capable of producing hyaline cartilage suitable for tissue repair. MSCs obtained from OA and RA patients possess similar chondrogenic potential as those from healthy individuals [38–41]. Therefore, we used paired samples of MSCs and OACs from two donors and a set of genetically mismatched biological samples of patient's OACs and commercially available MSCs. The paired cell sampling approach allowed us to reduce the high patient-to-patient variability, which influences the chondrogenic potential

Among the numerous commercially available 3D cell culture systems, we have chosen Perfecta 3D® scaffolds (3D Biomatrix Inc., USA) to create tissues in hanging

(microspheroids) by gravity-enforced self-assembly in hanging drops allows cell aggregation and tissue formation in a natural manner, without interference from the scaffold material [19, 32]. This technique has important advantages, especially the drop size control and consequent uniformity of formed microspheroids. Moreover, it is compatible with automated liquid handling systems, a prerequisite for high-throughput screening in drug discovery. The microspheroid formation in hanging drops mimics the condensation process of MSCs, which is one of the

Isolated OACs were first expanded in 2D monolayer cultures and then, from passage 2 and on, 10,000 cells were transferred into each hanging drop. In this way, the loss of chondrogenic phenotype of OACs in 2D was restored in 3D conditions, as already reported [30, 43]. Similarly as in our previously described 2D primary chondrocyte model, the TNF-α neutralizing efficiencies of ADA (Humira®, Abbott Laboratories, USA), ETA (Enbrel®, Immunex Corp., USA), IFX (Remicade®, Janssen Biotech, USA), and the anti-IL-1β drug anakinra (ANA; Kineret®, Swedish Orphan Biovitrum AB, Sweden) were assessed with both cell types by determining the extent of downregulation of six selected genes (*IL6*, *IL8*, *MCP1*, *MMP1*, *MMP13*, and *VCAM1*) [27, 28]. Gene expression was determined after a 24 h incubation of microspheroids in a medium supplemented with 1 ng/mL of an appropriate inflammatory cytokine (rhTNF-α or rhIL-1β; both from PeproTech, USA) or working macrophage conditioned medium (MCM) solution, combined with 1 μg/mL of each

According to our criteria, Log2 RQ ≥1 and ≤ 1, TNF-α significantly upregulated the expression of *IL6*, *IL8*, *MCP1*, *MMP1*, *MMP13*, and *VCAM1* genes in the 3D microspheroidal model as well (**Figure 4a**). The same was true when IL-1β or MCM was added to microspheroids. MCM was obtained from cell cultures of the human monocytic cell line THP-1 (ATCC, USA) and represented a rich source of inflammatory cytokines with 0.05 ng/mL TNF-α and 0.45 ng/mL IL-1β, and numerous other growth factors. In terms of influencing gene expression, IL-1β was the most potent inflammation inducer, followed by MCM and then TNF-α. The inflammatory process triggered by each of these three inducers could always be reversed by ADA, IFX, or ETA, as well as ANA (**Figure 4a**). When inflammation was triggered by TNF-α, all tested anti-TNF-α biologics extraordinarily suppressed the expression of monitored genes, sometimes even reaching their constitutively expressed levels (log2 RQ = 0). Similarly, in the presence of IL-1β, ANA markedly reversed the

drops. Generation of scaffold-free spheroids of micrometric dimensions

earliest phases of *in vivo* cartilage development [32].

individual biological drug tested (**Figure 4**).

**18**

we used human OACs and chondrogenically differentiated MSCs.

of both OACs and MSCs [42].

*Cytokines*

*(a) Gene expression profiles following the addition of inflammatory mediators TNF-α, IL-1β, or MCM working solution and anti-inflammatory biological drugs ADA, IFX, ETA, and ANA. Blue and green dots represent values obtained in microspheroidal chondral tissues made of MSCs and OACs (three donors), respectively. Statistically significant changes, that is, Log2 RQ* ≥*1 and* ≤ *1 are outlined with median values for all groups. (b) Radar graphs representing anti-TNF-α neutralization efficacies of ADA (blue), IFX (red), and ETA (green). Mean RQ values of three biological samples are shown for OAC- and MSC-derived microspheroids. Value 0 in the center of each radar graph represents total inhibition of gene expression. Original figure used with authors' permission under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) [26].*

inflammation process in microspheroids. However, when microspheroids were incubated with MCM, none of the three tested anti-TNF-α drugs were successful in diminishing its inflammatory effect. Conversely, ANA could downregulate the expression of *IL6*, *IL8*, and *MMP1* genes. The described changes at the gene level were accompanied by significant differences in the expression of IL6, IL8, and MCP1 proteins, detected in supernatants of microspheroid cultures, 24 h after their incubation with a given inflammatory agent selected anti-inflammatory biological drug [26]. Moreover, these results were additionally supported by the amount of glycosaminoglycans present in chondral spheroids composed of 100,000 OACs treated with various combinations of a particular inflammatory agent a given anti-inflammatory biologic, for a period of 3 weeks.

When microspheroids were incubated with MCM, a superior anti-IL-1β neutralization capacity of ANA compared to the three tested anti-TNF-α biologics was observed. This difference was probably due to the fact that MCM contained a much higher concentration of IL-1β (0.45 ng/mL) than TNF-α (0.05 ng/mL). Nevertheless, these concentrations of both cytokines are much higher than those measured in synovial fluids of OA and RA patients (0.028 ng/mL TNF-α and 0.1 ng/mL IL-1β) [44]. Although MCM proved to be an excellent *in vitro* inducer of inflammation, its use for potency testing of anti-inflammatory biologicals targeting a specific cytokine is questionable. In fact, from the multiple synergistic proinflammatory effects evoked by different biogenic factors present in MCM, it is very hard to define the potency of a biological targeting a single inflammatory factor.

and well standardized so that the results are reproducible and can be compared among different laboratories. When used for drug potency testing, such assays usually rely on the use of reference standards. Recently, the WHO has prepared two international standards for the two anti-TNF-α biologics, etanercept and infliximab. These have been tested by several laboratories within an international collaborative study using a number of different cell-based assays [12, 13]. In this chapter, we have presented an overview of the most routinely used tests for potency testing of anti-TNF-α biologics, which measure *in vitro* responses of nonmanipulated or genetically

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs*

*DOI: http://dx.doi.org/10.5772/intechopen.85237*

engineered human and animal cell lines, with various readout systems.

a readout system for assessing differences in selected gene expressions. The obtained data led us to the establishment of an original statistical method, which

The authors wish to express their gratitude to Prof. Andrej Blejec, Dr. Miomir

Knežević, Asst. Prof. Nevenka Kregar Velikonja, and Prof. Gordana Vunjak-Novakovic for their contribution to our common, published original research work,

Vandenbroucke for approving the use of a picture presented in **Figure 1** and to

partly presented in this chapter. We are also grateful to Rosmarijn E.

The authors declared that no competing interests exist.

was used for the evaluation and comparison of results.

Andrej Branc for proofreading the manuscript.

MCM macrophage conditioned medium

NCs normal human articular chondrocytes OACs osteoarthritic human articular chondrocytes

RQ relative quantity of gene expression

MSCs mesenchymal stem cells

**21**

**Acknowledgements**

**Conflict of interest**

**Appendices and nomenclature**

2D two-dimensional 3D three-dimensional ADA adalimumab ANA anakinra ETA etanercept IFX infliximab IL-1β interleukin 1β

Nowadays, with an expanding personal medicine approach, laboratory assayguided pharmacotherapeutical strategies are becoming more and more important. In order to obtain relevant data on drug potencies for a particular patient, these kinds of tests should be based on the patient's own, that is, autologous primary cells, as these can significantly reduce costs and enable safer and more effective therapies. Therefore, we dedicated a part of this chapter to our experience in establishing *in vitro* 2D monolayer cultures consisting of normal and OA chondrocytes and 3D microspheroidal chondral tissues, formed from OACs or chondrogenically differentiated bone-marrow-derived MSCs, and their use for testing anti-TNF-α efficacy of adalimumab, etanercept, and infliximab. The qRT-PCR technique was applied as

In our 3D microspheroidal rhTNF-α-induced inflammation model, the neutralization capacity of ADA was superior over that of ETA and the even weaker IFX (**Figure 4b**). Similar results were obtained with both microspheroids, regardless of whether they were made of OACs or chondrogenically differentiated MSCs. The observed differences in neutralizing efficiencies of ADA, ETA, and IFX can be attributed to differences in their molecular structures and sTNF-α-binding affinities [45]. The superior anti-TNF-α efficacy of ADA over ETA and IFX has already been reported together with data, showing that the sTNF-α-binding affinity of ADA is higher for ADA (Kd = 7.05 <sup>10</sup>11) than ETA (Kd = 2.35 <sup>10</sup>11) and IFX (Kd = 1.17 <sup>10</sup>10) [46–48]. However, according to our criterion, a particular biologic would be statistically more efficient than the compared one if it would cause a ≥2-fold decrease in a selected gene expression. This was not the case in any of our 3D microspheroidal model experiments. Consequently, we assumed that the observed differences in TNF-α neutralizing potency of ADA, ETA, and IFX were comparable (**Figure 4b**). Interestingly, although we showed in our 2D OACs model that ETA was significantly more efficient than IFX, the same kind of experiments carried out in a 3D microspheroidal model did not confirm this finding [26, 27]. We assume that compared to the 2D model, the diffusion of tested biologics in our 3D microspheroidal model was much slower and limited. Undoubtedly, the 3D model better resembles *in vivo* conditions and therefore has a higher relevance. Thus, we concluded that 2D cell culture models may be useful for obtaining preliminary data regarding the anti-inflammatory effects of a particular biological drug, while 3D microtissue models enable more relevant insights in drug-tissue interactions and possible outcomes *in vivo*. The results obtained with our 3D microspheroidal model are also supported by the outcomes of clinical studies conducted on patients with RA, where the efficacies of anti-TNF-α biologics proved to be comparable [49].

We found that OACs and chondrogenically differentiated MSCs are suitable sources for hanging drop chondral 3D microspheroid cultures formation, which are useful for the assessment of neutralization potencies of anti-inflammatory biologics [26]. Although the use of these two types of microspheroids resulted in different gene expression profiles following their incubation with tested combinations of rhTNF-α, and each of the three tested anti-TNF-α biological drugs (**Figure 4b**), these differences were rather small. Therefore, we concluded that MSCs can be used as an alternative and probably even more accessible cell source for *in vitro* testing of neutralization potency of anti-TNF-α biologics. The main advantages of our 3D model are the use of small amounts of human cells and cytokines, personalized testing approach, and the possibility of automation. In addition, the presented approach can also be used as a platform for testing other anti-inflammatory biologics with different mechanisms of action, as shown for ANA, the antagonist of IL-1β.

#### **6. Conclusion**

Cell-based assays are complex analytical tools, susceptible to multiple variables that are virtually impossible to control. Therefore, they have to be precise, reliable,

#### In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*

and well standardized so that the results are reproducible and can be compared among different laboratories. When used for drug potency testing, such assays usually rely on the use of reference standards. Recently, the WHO has prepared two international standards for the two anti-TNF-α biologics, etanercept and infliximab. These have been tested by several laboratories within an international collaborative study using a number of different cell-based assays [12, 13]. In this chapter, we have presented an overview of the most routinely used tests for potency testing of anti-TNF-α biologics, which measure *in vitro* responses of nonmanipulated or genetically engineered human and animal cell lines, with various readout systems.

Nowadays, with an expanding personal medicine approach, laboratory assayguided pharmacotherapeutical strategies are becoming more and more important. In order to obtain relevant data on drug potencies for a particular patient, these kinds of tests should be based on the patient's own, that is, autologous primary cells, as these can significantly reduce costs and enable safer and more effective therapies. Therefore, we dedicated a part of this chapter to our experience in establishing *in vitro* 2D monolayer cultures consisting of normal and OA chondrocytes and 3D microspheroidal chondral tissues, formed from OACs or chondrogenically differentiated bone-marrow-derived MSCs, and their use for testing anti-TNF-α efficacy of adalimumab, etanercept, and infliximab. The qRT-PCR technique was applied as a readout system for assessing differences in selected gene expressions. The obtained data led us to the establishment of an original statistical method, which was used for the evaluation and comparison of results.

## **Acknowledgements**

higher concentration of IL-1β (0.45 ng/mL) than TNF-α (0.05 ng/mL). Nevertheless, these concentrations of both cytokines are much higher than those measured in synovial fluids of OA and RA patients (0.028 ng/mL TNF-α and 0.1 ng/mL IL-1β) [44]. Although MCM proved to be an excellent *in vitro* inducer of inflammation, its use for potency testing of anti-inflammatory biologicals targeting a specific cytokine is questionable. In fact, from the multiple synergistic proinflammatory effects evoked by different biogenic factors present in MCM, it is very hard to define the

In our 3D microspheroidal rhTNF-α-induced inflammation model, the neutralization capacity of ADA was superior over that of ETA and the even weaker IFX (**Figure 4b**). Similar results were obtained with both microspheroids, regardless of whether they were made of OACs or chondrogenically differentiated MSCs. The observed differences in neutralizing efficiencies of ADA, ETA, and IFX can be attributed to differences in their molecular structures and sTNF-α-binding affinities [45]. The superior anti-TNF-α efficacy of ADA over ETA and IFX has already been reported together with data, showing that the sTNF-α-binding affinity of ADA is higher for ADA (Kd = 7.05 <sup>10</sup>11) than ETA (Kd = 2.35 <sup>10</sup>11) and IFX (Kd = 1.17 <sup>10</sup>10) [46–48]. However, according to our criterion, a particular biologic would be statistically more efficient than the compared one if it would cause a ≥2-fold decrease in a selected gene expression. This was not the case in any of our 3D microspheroidal model experiments. Consequently, we assumed that the observed differences in TNF-α neutralizing potency of ADA, ETA, and IFX were comparable (**Figure 4b**). Interestingly, although we showed in our 2D OACs model that ETA was significantly more efficient than IFX, the same kind of experiments carried out in a 3D microspheroidal model did not confirm this finding [26, 27]. We assume that compared to the 2D model, the diffusion of tested biologics in our 3D microspheroidal model was much slower and limited. Undoubtedly, the 3D model better resembles *in vivo* conditions and therefore has a higher relevance. Thus, we concluded that 2D cell culture models may be useful for obtaining preliminary data regarding the anti-inflammatory effects of a particular biological drug, while 3D microtissue models enable more relevant insights in drug-tissue interactions and possible outcomes *in vivo*. The results obtained with our 3D microspheroidal model are also supported by the outcomes of clinical studies conducted on patients with RA, where the efficacies of anti-TNF-α biologics proved to be comparable [49]. We found that OACs and chondrogenically differentiated MSCs are suitable sources for hanging drop chondral 3D microspheroid cultures formation, which are useful for the assessment of neutralization potencies of anti-inflammatory biologics [26]. Although the use of these two types of microspheroids resulted in different gene expression profiles following their incubation with tested combinations of rhTNF-α, and each of the three tested anti-TNF-α biological drugs (**Figure 4b**), these differences were rather small. Therefore, we concluded that MSCs can be used as an alternative and probably even more accessible cell source for *in vitro* testing of neutralization potency of anti-TNF-α biologics. The main advantages of our 3D model are the use of small amounts of human cells and cytokines, personalized testing approach, and the possibility of automation. In addition, the presented approach can also be used as a platform for testing other anti-inflammatory biologics with different mechanisms of action, as shown for ANA, the antagonist of IL-1β.

Cell-based assays are complex analytical tools, susceptible to multiple variables that are virtually impossible to control. Therefore, they have to be precise, reliable,

potency of a biological targeting a single inflammatory factor.

*Cytokines*

**6. Conclusion**

**20**

The authors wish to express their gratitude to Prof. Andrej Blejec, Dr. Miomir Knežević, Asst. Prof. Nevenka Kregar Velikonja, and Prof. Gordana Vunjak-Novakovic for their contribution to our common, published original research work, partly presented in this chapter. We are also grateful to Rosmarijn E. Vandenbroucke for approving the use of a picture presented in **Figure 1** and to Andrej Branc for proofreading the manuscript.

### **Conflict of interest**

The authors declared that no competing interests exist.

#### **Appendices and nomenclature**



**References**

2011-04-325225

ijms19051442

20065-20071

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*DOI: http://dx.doi.org/10.5772/intechopen.85237*

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Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α. MAbs.

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## **Author details**

Sara Žigon-Branc1†, Ariana Barlič2† and Matjaž Jeras<sup>3</sup> \*

1 Institute of Materials Science and Technology, Technische Universität Wien, Vienna, Austria

2 Educell Cell Therapy Service Ltd., Trzin, Slovenia

3 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

\*Address all correspondence to: matjaz.jeras@ffa.uni-lj.si

† These authors contributed equally to this work

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*

## **References**

rhTNF-α recombinant human tumor necrosis factor α sTNF-α soluble form of tumor necrosis factor α

**Author details**

*Cytokines*

Vienna, Austria

**22**

Sara Žigon-Branc1†, Ariana Barlič2† and Matjaž Jeras<sup>3</sup>

2 Educell Cell Therapy Service Ltd., Trzin, Slovenia

† These authors contributed equally to this work

provided the original work is properly cited.

\*Address all correspondence to: matjaz.jeras@ffa.uni-lj.si

1 Institute of Materials Science and Technology, Technische Universität Wien,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

3 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

\*

tmTNF-α transmembrane form of tumor necrosis factor α

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[3] Yan L, Hu R, Tu S, Cheng W-J, Zheng Q, Wang J-W, et al. Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC. International Journal of Clinical and Experimental Medicine. 2015;**8**: 20065-20071

[4] Mitoma H, Horiuchi T, Tsukamoto H, Ueda N. Molecular mechanisms of action of anti-TNF-α agents— Comparison among therapeutic TNF-α antagonists. Cytokine. 2018;**101**:56-63. DOI: 10.1016/j.cyto.2016.08.014

[5] Chen X, Oppenheim JJ. Targeting TNFR2, an immune checkpoint stimulator and oncoprotein, is a promising treatment for cancer. Science Signaling. 2017;**10**:eaal2328. DOI: 10.1126/scisignal.aal2328

[6] Torrey H, Butterworth J, Mera T, Okubo Y, Wang L, Baum D, et al. Targeting TNFR2 with antagonistic antibodies inhibits proliferation of ovarian cancer cells and tumorassociated Tregs. Science Signaling. 2017; **10**:eaaf8608. DOI: 10.1126/scisignal. aaf8608

[7] Vos ACW, Wildenberg ME, Duijvestein M, Verhaar AP, van den Brink GR, Hommes DW. Anti-tumor necrosis factor-α antibodies induce regulatory macrophages in an Fc regiondependent manner. Gastroenterology.

2011;**140**:221-230.e3. DOI: 10.1053/j. gastro.2010.10.008

[8] Nguyen DX, Ehrenstein MR. Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF–TNF-RII binding in rheumatoid arthritis. The Journal of Experimental Medicine. 2016;**213**:1241-1253. DOI: 10.1084/jem.20151255

[9] Nadkarni S, Mauri C, Ehrenstein MR. Anti-TNF-α therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-β. The Journal of Experimental Medicine. 2007;**204**:33-39. DOI: 10.1084/ jem.20061531

[10] Shealy DJ, Cai A, Staquet K, Baker A, Lacy ER, Johns L, et al. Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α. MAbs. 2010;**2**:428-439

[11] Paleolog E. Target effector role of vascular endothelium in the inflammatory response: Insights from the clinical trial of anti-TNF alpha antibody in rheumatoid arthritis. Molecular Pathology. 1997;**50**:225-233

[12] Wadhwa M, Bird C, Dilger P, Rigsby P, Jia H, Gross MEB. Establishment of the first WHO International Standard for etanercept, a TNF receptor II Fc fusion protein: Report of an international collaborative study. Journal of Immunological Methods. 2017;**447**:14-22. DOI: 10.1016/j. jim.2017.03.007

[13] Metcalfe C, Dougall T, Bird C, Rigsby P, Behr-Gross M-E, Wadhwa M, et al. The first World Health Organization International Standard for infliximab products: A step towards maintaining harmonized biological activity. MAbs. 2019;**11**:13-25. DOI: 10.1080/19420862.2018.1532766

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[15] Lallemand C, Kavrochorianou N, Steenholdt C, Bendtzen K, Ainsworth MA, Meritet J-F, et al. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists. Journal of Immunological Methods. 2011;**373**: 229-239. DOI: 10.1016/j.jim.2011.08.022

[16] Mitoma H, Horiuchi T, Tsukamoto H, Tamimoto Y, Kimoto Y, Uchino A, et al. Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor αexpressing cells: Comparison among infliximab, etanercept, and adalimumab. Arthritis and Rheumatism. 2008;**58**:1248-1257. DOI: 10.1002/ art.23447

[17] Ueda N, Tsukamoto H, Mitoma H, Ayano M, Tanaka A, Ohta S, et al. The cytotoxic effects of certolizumab pegol and golimumab mediated by transmembrane tumor necrosis factor α. Inflammatory Bowel Diseases. 2013;**19**: 1224-1231. DOI: 10.1097/ MIB.0b013e318280b169

[18] Moore M, Ferguson J, Burns C. Applications of cell-based bioassays measuring the induced expression of endogenous genes. Bioanalysis. 2014;**6**: 1563-1574. DOI: 10.4155/bio.14.98

[19] Rimann M, Graf-Hausner U. Synthetic 3D multicellular systems for drug development. Current Opinion in Biotechnology. 2012;**23**:803-809. DOI: 10.1016/j.copbio.2012.01.011

[20] Baker BM, Chen CS. Deconstructing the third dimension—How 3D culture

microenvironments alter cellular cues. Journal of Cell Science. 2012;**125**: 3015-3024. DOI: 10.1242/jcs.079509

efficacy testing of anti-TNFα drugs. Biologicals. 2017;**45**:96-101. DOI: 10.1016/j.biologicals.2016.09.013

*DOI: http://dx.doi.org/10.5772/intechopen.85237*

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[35] Sart S, Tsai A-C, Li Y, Ma T. Threedimensional aggregates of mesenchymal

stem cells: Cellular mechanisms, biological properties, and applications. Tissue Engineering. Part B, Reviews. 2014;**20**:365-380. DOI: 10.1089/ten.

[36] Laganà M, Arrigoni C, Lopa S, Sansone V, Zagra L, Moretti M, et al.

Characterization of articular chondrocytes isolated from 211 osteoarthritic patients. Cell and Tissue Banking. 2014;**15**:59-66. DOI: 10.1007/

[37] Neri S, Mariani E, Cattini L, Facchini A. Long-term in vitro expansion of osteoarthritic human articular chondrocytes do not alter genetic stability: A microsatellite instability analysis. Journal of Cellular Physiology. 2011;**226**:2579-2585. DOI:

[38] Tallheden T, Bengtsson C, Brantsing C, Sjogren-Jansson E, Carlsson L, Peterson L, et al. Proliferation and

[39] Kafienah W, Mistry S, Dickinson SC, Sims TJ, Learmonth I, Hollander AP. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis and Rheumatism. 2007;**56**:177-187. DOI:

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dedifferentiated human articular

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GJ. Morphological, genetic and

culture: The way to enhance

**25**

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phenotypic comparison between human articular chondrocytes and cultured chondrocytes. Histochemistry and Cell Biology. 2016;**146**(2):183-189. DOI: 10.1007/s00418-016-1437-4

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[23] Alépée N. State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. ALTEX. 2014;**31**(4):441-477. DOI: 10.14573/ altex1406111

[24] Sophia Fox AJ, Bedi A, Rodeo SA. The basic science of articular cartilage: Structure, composition, and function. Sports Health: Multidisciplinary Approach. 2009;**1**:461-468. DOI: 10.1177/1941738109350438

[25] Otero M, Favero M, Dragomir C, Hachem KE, Hashimoto K, Plumb DA, et al. Human chondrocyte cultures as models of cartilage-specific gene regulation. In: Mitry RR, Hughes RD, editors. Human Cell Culture Protocols. Vol. 806. Totowa, NJ: Humana Press; 2012. pp. 301-336

[26] Žigon-Branc S, Barlič A, Knežević M, Jeras M, Vunjak-Novakovic G. Testing the potency of anti-TNF-α and anti-IL-1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor-matched chondrogenically differentiated mesenchymal stem cells. Biotechnology Progress. 2018;**34**: 1045-1058. DOI: 10.1002/btpr.2629

[27] Žigon-Branc S, Jeras M, Blejec A, Barlič A. Applicability of human osteoarthritic chondrocytes for in vitro In vitro *Cell-Based Assays for Potency Testing of Anti-TNF-α Biological Drugs DOI: http://dx.doi.org/10.5772/intechopen.85237*

efficacy testing of anti-TNFα drugs. Biologicals. 2017;**45**:96-101. DOI: 10.1016/j.biologicals.2016.09.013

[14] Hofmann H-P, Kronthaler U, Fritsch C, Grau R, Müller SO, Mayer R, et al. Characterization and non-clinical assessment of the proposed etanercept biosimilar GP2015 with originator etanercept (Enbrel(®)). Expert Opinion

*Cytokines*

microenvironments alter cellular cues. Journal of Cell Science. 2012;**125**: 3015-3024. DOI: 10.1242/jcs.079509

[21] Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in

Development Technologies. 2014;**12**: 207-218. DOI: 10.1089/adt.2014.573

[22] Duval K, Grover H, Han L-H, Mou Y, Pegoraro AF, Fredberg J, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology. 2017;**32**:266-277. DOI:

[23] Alépée N. State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. ALTEX. 2014;**31**(4):441-477. DOI: 10.14573/

[24] Sophia Fox AJ, Bedi A, Rodeo SA. The basic science of articular cartilage: Structure, composition, and function. Sports Health: Multidisciplinary Approach. 2009;**1**:461-468. DOI: 10.1177/1941738109350438

[25] Otero M, Favero M, Dragomir C, Hachem KE, Hashimoto K, Plumb DA, et al. Human chondrocyte cultures as models of cartilage-specific gene regulation. In: Mitry RR, Hughes RD, editors. Human Cell Culture Protocols. Vol. 806. Totowa, NJ: Humana Press;

[26] Žigon-Branc S, Barlič A, Knežević M, Jeras M, Vunjak-Novakovic G. Testing the potency of anti-TNF-α and anti-IL-1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor-matched chondrogenically differentiated mesenchymal stem cells. Biotechnology Progress. 2018;**34**: 1045-1058. DOI: 10.1002/btpr.2629

[27] Žigon-Branc S, Jeras M, Blejec A, Barlič A. Applicability of human osteoarthritic chondrocytes for in vitro

drug discovery and cell-based biosensors. Assay and Drug

10.1152/physiol.00036.2016

altex1406111

2012. pp. 301-336

on Biological Therapy. 2016;**16**: 1185-1195. DOI: 10.1080/ 14712598.2016.1217329

[15] Lallemand C, Kavrochorianou N, Steenholdt C, Bendtzen K, Ainsworth MA, Meritet J-F, et al. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists. Journal of Immunological Methods. 2011;**373**: 229-239. DOI: 10.1016/j.jim.2011.08.022

[16] Mitoma H, Horiuchi T, Tsukamoto H, Tamimoto Y, Kimoto Y, Uchino A, et al. Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor αexpressing cells: Comparison among

adalimumab. Arthritis and Rheumatism. 2008;**58**:1248-1257. DOI: 10.1002/

[17] Ueda N, Tsukamoto H, Mitoma H, Ayano M, Tanaka A, Ohta S, et al. The cytotoxic effects of certolizumab pegol

transmembrane tumor necrosis factor α. Inflammatory Bowel Diseases. 2013;**19**:

[18] Moore M, Ferguson J, Burns C. Applications of cell-based bioassays measuring the induced expression of endogenous genes. Bioanalysis. 2014;**6**: 1563-1574. DOI: 10.4155/bio.14.98

[19] Rimann M, Graf-Hausner U. Synthetic 3D multicellular systems for drug development. Current Opinion in Biotechnology. 2012;**23**:803-809. DOI:

10.1016/j.copbio.2012.01.011

**24**

[20] Baker BM, Chen CS. Deconstructing the third dimension—How 3D culture

infliximab, etanercept, and

and golimumab mediated by

1224-1231. DOI: 10.1097/ MIB.0b013e318280b169

art.23447

[28] Barlič A, Žigon S, Blejec A, Kregar Velikonja N. Gene expression of cultured human chondrocytes as a model for assessing neutralization efficacy of soluble TNFα by TNFα antagonists. Biologicals. 2015;**43**: 171-180. DOI: 10.1016/j. biologicals.2015.03.001

[29] Burns CJ, Silva MMCG, Gray E, Robinson CJ. Quantitative RT-PCR as an alternative to late-stage bioassays for vascular endothelial growth factor. Journal of Pharmaceutical and Biomedical Analysis. 2008;**47**:460-468. DOI: 10.1016/j.jpba.2008.02.011

[30] Caron MMJ, Emans PJ, Coolsen MME, Voss L, Surtel DAM, Cremers A, et al. Redifferentiation of dedifferentiated human articular chondrocytes: Comparison of 2D and 3D cultures. Osteoarthritis and Cartilage. 2012;**20**:1170-1178. DOI: 10.1016/j. joca.2012.06.016

[31] Mata-Miranda MM, Martinez-Martinez CM, Noriega-Gonzalez JE, Paredes-Gonzalez LE, Vázquez-Zapién GJ. Morphological, genetic and phenotypic comparison between human articular chondrocytes and cultured chondrocytes. Histochemistry and Cell Biology. 2016;**146**(2):183-189. DOI: 10.1007/s00418-016-1437-4

[32] Lehmann M, Martin F, Mannigel K, Kaltschmidt K, Sack U, Anderer U. Three-dimensional scaffold-free fusion culture: The way to enhance chondrogenesis of in vitro propagated human articular chondrocytes. European Journal of Histochemistry. 2013;**57**:31. DOI: 10.4081/ejh.2013.e31

[33] Bhumiratana S, Vunjak-Novakovic G. Engineering physiologically stiff and stratified human cartilage by fusing condensed mesenchymal stem cells.

Methods. 2015;**84**:109-114. DOI: 10.1016/j.ymeth.2015.03.016

[34] Baraniak PR, McDevitt TC. Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell and Tissue Research. 2012;**347**:701-711. DOI: 10.1007/s00441-011-1215-5

[35] Sart S, Tsai A-C, Li Y, Ma T. Threedimensional aggregates of mesenchymal stem cells: Cellular mechanisms, biological properties, and applications. Tissue Engineering. Part B, Reviews. 2014;**20**:365-380. DOI: 10.1089/ten. teb.2013.0537

[36] Laganà M, Arrigoni C, Lopa S, Sansone V, Zagra L, Moretti M, et al. Characterization of articular chondrocytes isolated from 211 osteoarthritic patients. Cell and Tissue Banking. 2014;**15**:59-66. DOI: 10.1007/ s10561-013-9371-3

[37] Neri S, Mariani E, Cattini L, Facchini A. Long-term in vitro expansion of osteoarthritic human articular chondrocytes do not alter genetic stability: A microsatellite instability analysis. Journal of Cellular Physiology. 2011;**226**:2579-2585. DOI: 10.1002/jcp.22603

[38] Tallheden T, Bengtsson C, Brantsing C, Sjogren-Jansson E, Carlsson L, Peterson L, et al. Proliferation and differentiation potential of chondrocytes from osteoarthritic patients. Arthritis Research & Therapy. 2005;**7**:R560-R568

[39] Kafienah W, Mistry S, Dickinson SC, Sims TJ, Learmonth I, Hollander AP. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis and Rheumatism. 2007;**56**:177-187. DOI: 10.1002/art.22285

[40] Jagielski M, Wolf J, Marzahn U, Völker A, Lemke M, Meier C, et al. The influence of IL-10 and TNFα on chondrogenesis of human mesenchymal stromal cells in three-dimensional cultures. International Journal of Molecular Sciences. 2014;**15**: 15821-15844. DOI: 10.3390/ ijms150915821

[41] Dudics V, Kunstár A, Kovács J, Lakatos T, Géher P, Gömör B, et al. Chondrogenic potential of mesenchymal stem cells from patients with rheumatoid arthritis and osteoarthritis: Measurements in a microculture system. Cells, Tissues, Organs. 2009;**189**:307-316. DOI: 10.1159/000140679

[42] Agar G, Blumenstein S, Bar-Ziv Y, Kardosh R, Schrift-Tzadok M, Gal-Levy R, et al. The chondrogenic potential of mesenchymal cells and chondrocytes from osteoarthritic subjects: A comparative analysis. Cartilage. 2011;**2**: 40-49. DOI: 10.1177/1947603510380899

[43] Schulze-Tanzil G, de Souza P, Villegas Castrejon H, John T, Merker H-J, Scheid A, et al. Redifferentiation of dedifferentiated human chondrocytes in high-density cultures. Cell and Tissue Research. 2002;**308**:371-379. DOI: 10.1007/s00441-002-0562-7

[44] Westacott CI, Whicher JT, Barnes IC, Thompson D, Swan AJ, Dieppe PA. Synovial fluid concentration of five different cytokines in rheumatic diseases. Annals of the Rheumatic Diseases. 1990;**49**:676-681

[45] Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP. Tumor necrosis factor antagonist mechanisms of action: A comprehensive review. Pharmacology & Therapeutics. 2008;**117**:244-279. DOI: 10.1016/j.pharmthera.2007.10.001

[46] Hu S, Liang S, Guo H, Zhang D, Li H, Wang X, et al. Comparison of the inhibition mechanisms of adalimumab and infliximab in treating tumor necrosis factor α-associated diseases

from a molecular view. The Journal of Biological Chemistry. 2013;**288**: 27059-27067. DOI: 10.1074/jbc. M113.491530

**Chapter 3**

**Abstract**

Tumor Necrosis Factor Alpha:

Tumor necrosis factor (TNF) is one of the most extensively studied cytokine with about 19 distinct superfamily members and many more to be found. Prominent among these members is tumor necrosis factor alpha (TNF-α) that is known to be a potent promoter of inflammation, as well as many normal physiological functions in homeostasis and health and antimicrobial immunity. Nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) is one of the most important transcription factors that activate transcription of many proinflammatory genes, and the unraveling of TNF-α induced NFκB activation forms the foundation of TNF-α as major cytokine of neuroinflammation. This review discusses summary of literature on unique role of TNF-α in neuroinflammation and various agents that

**Keywords:** tumor necrosis factor, tumor necrosis factor alpha, neuroinflammation,

Tumor necrosis factor (TNF) alpha is one of the first discovered cytokines shown by Carswell [1] in 1975 and was named for tumor regression activity induced in the serum of mice treated with *Serratia marcescens* polysaccharide [2]. Cytokines are low-molecular-weight peptides secreted by activated immune cells as well as stromal cells and exerting biological activities through binding to cognate receptors on cell surface. Cytokines are produced by a number of cell types, predominantly leukocytes that regulate a number of physiological and pathological functions including innate immunity, acquired immunity, and a plethora of inflammatory responses [3]. Cytokines excite or hinder the generation, propagation, and differentiation of different associated target cells positive on antigen induction, thus leading to mediation in the activity of diverse other cells involved in the immune response especially the more pronounced macrophages, mast cells, B cells, T cells, and natural killer (NK) cells. Thus, cytokine is regarded as secreted proteins with growth, differentiation, and activation functions that regulate and determine the nature of immune responses [4]. The broad classification of cytokines are termed in a group as follows: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony stimulating factor (CSF), and chemokine and growth factor (GF), and these exerts biological functions through action mode and characteristics as paracrine,

A Major Cytokine of Brain

Neuroinflammation

mediate neuroinflammation via TNF-α modulation.

*Mubarak Muhammad*

cytokine, brain, inflammation

**1. Introduction**

**27**

[47] Scallon B, Cai A, Solowski N, Rosenberg A, Song X-Y, Shealy D, et al. Binding and functional comparisons of two types of tumor necrosis factor antagonists. The Journal of Pharmacology and Experimental Therapeutics. 2002;**301**:418-426

[48] Granneman RG, Zhang YM, Noertersheuser PA, Velagapudi RB, Awni WM, Locke CS, et al. Pharmacokinetic/pharmacodynamic (PK/PD) relationships of adalimumab (HUMIRA™, Abbott) in rheumatoid arthritis (RA) patients during phase II/ III clinical trials. In: Arthritis and Rheumatism. Vol. 48. Div John Wiley & Sons Inc, 605 Third Ave, New York, NY 10158–0012 USA: Wiley-Liss; 2003. pp. S140-S141

[49] Hyrich KL, Lunt M, Watson KD, Symmons DPM, Silman AJ. British Society for Rheumatology Biologics Register. Outcomes after switching from one anti-tumor necrosis factor alpha agent to a second anti-tumor necrosis factor alpha agent in patients with rheumatoid arthritis: Results from a large UK national cohort study. Arthritis and Rheumatism. 2007;**56**:13-20. DOI: 10.1002/art.22331

## **Chapter 3**

influence of IL-10 and TNFα on

[41] Dudics V, Kunstár A, Kovács J, Lakatos T, Géher P, Gömör B, et al.

mesenchymal stem cells from patients

[42] Agar G, Blumenstein S, Bar-Ziv Y, Kardosh R, Schrift-Tzadok M, Gal-Levy R, et al. The chondrogenic potential of mesenchymal cells and chondrocytes from osteoarthritic subjects: A

comparative analysis. Cartilage. 2011;**2**: 40-49. DOI: 10.1177/1947603510380899

[44] Westacott CI, Whicher JT, Barnes IC, Thompson D, Swan AJ, Dieppe PA. Synovial fluid concentration of five different cytokines in rheumatic diseases. Annals of the Rheumatic

[45] Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP. Tumor necrosis factor antagonist mechanisms of action: A comprehensive review. Pharmacology & Therapeutics. 2008;**117**:244-279. DOI: 10.1016/j.pharmthera.2007.10.001

[46] Hu S, Liang S, Guo H, Zhang D, Li H, Wang X, et al. Comparison of the inhibition mechanisms of adalimumab and infliximab in treating tumor necrosis factor α-associated diseases

Diseases. 1990;**49**:676-681

**26**

[43] Schulze-Tanzil G, de Souza P, Villegas Castrejon H, John T, Merker H-J, Scheid A, et al. Redifferentiation of dedifferentiated human chondrocytes in high-density cultures. Cell and Tissue Research. 2002;**308**:371-379. DOI: 10.1007/s00441-002-0562-7

Chondrogenic potential of

10.1159/000140679

with rheumatoid arthritis and osteoarthritis: Measurements in a microculture system. Cells, Tissues, Organs. 2009;**189**:307-316. DOI:

ijms150915821

*Cytokines*

chondrogenesis of human mesenchymal stromal cells in three-dimensional cultures. International Journal of Molecular Sciences. 2014;**15**: 15821-15844. DOI: 10.3390/

from a molecular view. The Journal of Biological Chemistry. 2013;**288**: 27059-27067. DOI: 10.1074/jbc.

[47] Scallon B, Cai A, Solowski N, Rosenberg A, Song X-Y, Shealy D, et al. Binding and functional comparisons of two types of tumor necrosis factor

[48] Granneman RG, Zhang YM, Noertersheuser PA, Velagapudi RB,

Pharmacokinetic/pharmacodynamic (PK/PD) relationships of adalimumab (HUMIRA™, Abbott) in rheumatoid arthritis (RA) patients during phase II/ III clinical trials. In: Arthritis and Rheumatism. Vol. 48. Div John Wiley & Sons Inc, 605 Third Ave, New York, NY 10158–0012 USA: Wiley-Liss; 2003. pp.

[49] Hyrich KL, Lunt M, Watson KD, Symmons DPM, Silman AJ. British Society for Rheumatology Biologics Register. Outcomes after switching from one anti-tumor necrosis factor alpha agent to a second anti-tumor necrosis factor alpha agent in patients with rheumatoid arthritis: Results from a large UK national cohort study. Arthritis and Rheumatism. 2007;**56**:13-20. DOI:

antagonists. The Journal of Pharmacology and Experimental Therapeutics. 2002;**301**:418-426

Awni WM, Locke CS, et al.

M113.491530

S140-S141

10.1002/art.22331
