Monoclonal Antibodies for Cancer Treatment

*Annemeri Livinalli and Taís Freire Galvão*

### **Abstract**

Therapeutic monoclonal antibodies have emerged in the 1990 decade as an important option for cancer treatment. These molecules have a diverse set of clinically relevant antitumor mechanisms, directly targeting tumor cells. It has been established as "standard of care" for several human cancers. This chapter reviews the use of monoclonal antibodies in oncology and introduces available biosimilars. The requirements for biosimilar antibody development, mechanisms of action and current clinical applications for cancer treatment is also presented.

**Keywords:** biosimilar, equivalence trial, efficacy, monoclonal antibodies, cancer, extrapolation of indication

### **1. Introduction**

Since the development of monoclonal antibodies by hybridoma technology in 1975 [1] over 80 molecules were developed and approved for therapeutic use in immunological, oncological, and infectious diseases [2]. Over time, these molecules have revolutionized the treatment of main autoimmune diseases and cancer that previously had a bleak prognosis. These molecules are usually administered by subcutaneous or intramuscular routes due to poor oral bioavailability (less than 1%) caused by large size, polarity, limited membrane permeability, and poor gastrointestinal stability [3].

In oncology, the approach in the use of monoclonal antibodies consists in targeting tumor antigens and killing cancer cells [4]. Growth factor receptors that are overexpressed in tumor cells are recognized as main target by monoclonal antibodies [4, 5]. Blocking ligand binding/signaling result in decrease growth rate of cancer cells, which in turn, induce apoptosis and sensitize tumors cells to chemotherapy [6, 7].

As of the first semester of 2021, the arsenal of monoclonal antibodies in oncology counts on more than 30 molecules [8]. Among the first molecules, we have: bevacizumab, cetuximab, rituximab, trastuzumab, indicated for treating solid tumors and hematological malignancies (**Table 1**). From all monoclonal antibodies, there are only three biosimilar products marketed (bevacizumab, rituximab, trastuzumab; **Table 2**).


*Legend: EMA, European of Medicines Agency; FDA, United States Food and Drug Administration; INN, international nonproprietary name.*

*a Available at: www.ema.europe.eu.*

*b Available at: www.accessdata.fda.gov.*

### **Table 1.**

*First monoclonal antibodies used in oncology.*



*a Available at: www.ema.europe.eu.*

*b Available at: www.fda.gov/drugs/biosimilars/biosimilar-product-information.*

*\* The product received the recommendation of the granting of marketing authorization.*

### **Table 2.**

*Biosimilar monoclonal antibodies with marketing approval for cancer treatment (until February 2021).*

### **2. Development of monoclonal antibodies**

Monoclonal antibodies consist in homogenous preparations of antibodies – or fragments of antibodies – in which every antibody in the product is identical in its protein sequence. All antibodies should have the same antigen recognition site, affinity, biological interactions, and downstream biological effects [2].

There are four types of monoclonal antibodies [9]:


In summary, the traditional murine hybridoma technique starts by immunization of mice with desired antigens to trigger an immune response. Harvested splenocytes are fused with myeloma cells to produce hybridoma cells that persistently secrete the antibodies of interest. After the screening, selected leads are used to generate chimeric or humanized antibodies [9].

The main concern with this approach is the risk that might result in an immune response to the mouse antibody sequence. The consequence of this include allergic response and/or reduced bioavailability of mouse monoclonal antibodies. This immune response limited their clinical use [10].

Changes in the source of the molecule were determined as a solution to avoid the immune response. Introducing engineer changes, for example, recombinant DNA technologies, originated the chimeric, humanized, and human antibodies. Humanized mice allow for development of monoclonal antibodies with amino acid substitutions that lack mouse heavy chains and make them more similar to the human sequence system [2, 9].

The first chimeric antibody was approved in 1994 by the United States Food and Drug Administration (FDA) for inhibition of platelet aggregation in cardiovascular

diseases. The drug was developed by combining sequences of the murine variable domain with human constant region domain. In 1997, the first monoclonal antibody, rituximab – an immunoglobulin type 1 anti-CD20 -, was approved for non-Hodgkin's lymphoma by the FDA [9]. And the first humanized monoclonal antibody approved by the FDA also in 1997 was daclizumab, an anti-IL-2 receptor used for the prevention of transplant graft rejection [11].

Human monoclonal antibodies can either be obtained by phage display or transgenic animals [9]. Based on these techniques, the first fully human therapeutic antibody based on phage display was adalimumab, an anti-tumor necrosis factor α human antibody. It was approved in 2002 by the FDA for rheumatoid arthritis. Panitumumab, a monoclonal antibodies anti-epidermal growth factor receptor was the first human antibody generated in a transgenic mouse, approved by the FDA in 2006 and indicated for metastatic colorectal carcinoma, a type of cancer [11].

### **3. Biosimilar monoclonal antibodies in oncology**

As mentioned before, three biosimilar monoclonal antibodies are available in oncology: bevacizumab, rituximab, and trastuzumab. Cetuximab is in preliminary steps of developing a biosimilar.

Bevacizumab is a humanized inhibitor of vascular endothelial growth factor (VEGF) monoclonal antibody. It acts by selectively binding circulating VEGF, thereby inhibiting the binding of VEGF to its cell surface receptors, which results in a reduction of microvascular growth of tumor blood vessels, reducing the blood supply to tumor tissues. Other results observed are decrease interstitial pressure on tissues, increase vascular permeability, induction of apoptosis of tumor endothelial cells, and may increase delivery of chemotherapeutic agents [12].

Rituximab is a chimeric monoclonal antibody that has a high-affinity binding to B-lymphocyte antigen CD20 (CD20) on the surface of B cells. The death of B cells occurs by different ways, including antibody-dependent cellular cytotoxicity (ADCC) and apoptosis [13].

Trastuzumab is a recombinant humanized monoclonal antibody that binds to the domain of the extracellular segment of the human epidermal growth factor-2 receptor (HER2), and inhibits the proliferation and survival of HER2-dependent tumors [14]. When trastuzumab is biding to HER2 receptor might occur the degradation of the receptor, attraction of immune cells to tumor cells by ADCC and inhibition of some pathways involved in the suppression of cell growth and proliferation [15].

### **4. Assessment of biological activity of biosimilar monoclonal antibodies**

The biosimilar needs to demonstrate the proposed product is highly similar to the reference biological product and this is determined through a pathway that include comparative characterization made by evaluation of physicochemical, functional, and clinical characteristics of a biological product [16, 17].

The first step in biosimilar analytic characterization is identifying the characteristics associated with the quality, safety, and efficacy of reference biological product. These characteristics are known as critical quality attributes (CQAs) and represent physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality [18].

### *Monoclonal Antibodies for Cancer Treatment DOI: http://dx.doi.org/10.5772/intechopen.97915*

Analytic testing of CQAs is performed to detect differences in factors such as the expression system, the manufacturing process, physicochemical properties, functional activities, receptor binding, immunochemical properties, impurities, and clinical outcome of the biosimilar candidate [19, 20].

It may be useful to compare the quality attributes of the proposed biosimilar product with those of the reference product using a meaningful fingerprint-like analysis. It means the results obtained are extremely sensitive in identifying analytical differences and allow a very high level of confidence in the analytical similarity of the proposed biosimilar product [21].

Once the CQAs for the biosimilar candidate are identified, the next step is to categorize the relative importance or criticality of each attribute. In the case of monoclonal antibodies, that are more complex biological products, determining criticality may be more challenging due to the increased number of attributes to evaluate and the potential impact of each difference on the desired product [22].

Significant differences for a very important CQA of the biosimilar candidate, such as the primary amino acid structure, are enough to interrupt the biosimilarity pathway. The manufacturer will need change their process to reach the high level of similarity between this structure in the biosimilar compared with the reference product. In the other hand, differences detected among CQAs of very low importance, such as minor modifications in amino acid side chains, may be acceptable if they can be justified or understood as clinically irrelevant [22, 23].

Primary amino acid structure is the core DNA sequence, and it must be exactly the same for the biosimilar product and the reference product [22]. There are a range of methods commonly used for evaluating the primary structure, including the peptide mapping, characterization of disulfide linkages, and glycosylation [24]. If the amino acid sequence is not identical, it can happen unwanted amino acid interactions that will impact in the safety, efficacy, and immunogenicity of the product [22].

Antibody molecules are molecules consisting of three equalized portions, constructed in the same way from paired heavy and light polypeptide chains that consists of a series of similar, sequences, each about more than a hundred amino acids long [25].

Changes in the protein can occur during any step of the manufacturing process, for example, enzymatic modifications, aggregation, variable glycosylation, etc. These modifications are named as post translational modifications. They can influence the physicochemical and biological properties of a protein and affect immunogenicity, immune response, and clinical efficacy [26]. In general, proteins can differ in at least three ways: (i) primary amino acid sequence; (ii) modification of amino acids, such as glycosylation or other side chains; and (iii) higher order structure [23]. Glycosylation and phosphorylation can impact on the efficacy and safety of a protein, for this reason, during the development process, they are extensively tested [22].

When the primary amino acid structure and the three-dimensional structure are reached in the biosimilar product, the correct protein arrangement and structural integrity are obtained and then, the ability of the biological product to bind to the target receptor will result in pharmacologic action. For this reason, target binding is considered a very highly CQA [27].

Impurities can be product – or process-related, arising from cell substrates or cell culture component [28]. They have the potential to affect all aspects of the product's profile [22]. For this reason, the chosen analytical procedures should be adequate to detect, identify, and accurately quantify biologically significant levels of impurities [28].

Because the quality attributes of a biosimilar are not identical to those of the reference product, in addition to the analytic package, animal toxicology,

### *Biosimilars*

pharmacokinetic and pharmacodynamic testing, and immunogenicity studies are required by the regulatory agencies for demonstrating biosimilarity [29]. Then, to ensure that these differences do not lead to any clinically meaningful differences, comparative clinical studies are performed [30]. It is usually necessary to demonstrate comparable clinical efficacy of the biosimilar and the reference product in adequately powered, randomized, parallel group comparative clinical trial(s), preferably double-blinded and appropriate endpoints chosen [19].
