Development of Novel Anti-Cd20 Monoclonal Antibodies and Modulation in Cd20 Levels on Cell Surface: Looking to Improve Immunotherapy Response

Rituximab has been revolutionized and validated CD20 targeting monoclonal antibody. Although, it is widely used for lymphoma therapy and many patients have been benefited. However significant numbers of patients are refractory or developed resistance to current therapies due to low level of CD20 expression and/or availability on cells surface. Thus development of novel anti-CD20 mAbs with great cell killing ability and enhance CD20 levels on cell surface can potentially exploit lymphoma therapy. In this scenario, we are summarizing the recently developed mAbs against CD20 and compounds that have ability to induce CD20 expression at significant level. We also are providing information regarding combination strategy for use of radiation and anti-CD20 mAbs in vitro. However, it will need to be determined by rigorous at pre-clinical and clinic testing. We hope this review will be beneficial for current research in the area of immunotherapy or radio-immunotherapy.


Introduction
Cancer remain is a global concern and great challenge to medical management. It has emerged as the second leading cause of death globally after cardiovascular diseases. The International Agency for Research on Cancer (IARC) recently estimated that 8.2 million deaths worldwide were due to cancer with 14.1 million new cases per year being reported worldwide [1]. In India, deaths from the disease have increased by 60% according to the 'Global Burden of Cancer-2013' report [2]. Among them non-Hodgkin lymphoma is the tenth most common type of cancer in the world. Approximately 71,850 new cases and 19,790 deaths were reported due to non-Hodgkin lymphoma in 2015 (Surveillance, Epidemiology and End Results Program 2015).
It is a type of blood cancer that occurs when lymphocytes begin behaving abnormally. Lymphocytes are white blood cells that protect the body from infection and disease. Abnormal lymphocytes may divide faster than normal cells or they may live longer than they are supposed to. Lymphoma may develop in many parts of the body such as the lymph nodes, spleen, bone marrow, blood or other organs of the human body.
There are two main types of lymphomas:

•
Hodgkin lymphoma (HL): There are 6 types of HL an uncommon form of lymphoma that involves the Reed-Sternberg cells.
• Non-Hodgkin lymphoma (NHL): There are more than 61 types of NHL some of which are more common than others. In other words any lymphoma that does not involve Reed-Sternberg cells is classified as non-Hodgkin lymphoma.
Classification of non-Hodgkin lymphoma (NHL) can be quite confusing (even for doctors) because there are so many types and several different organs are involved. The most recent WHO classification is based on microscopic observations, the chromosome features of the lymphoma cells and the presence of certain proteins on the surface of the cells • B-cell lymphomas: B-cell lymphomas make up most (about 85%) of non-Hodgkin lymphomas in the United States (http://www.cancer.org/cancer/nonhodgkinlymphoma).
• T-cell lymphomas: T-cell lymphomas make up less than 15% of non-Hodgkin lymphomas in the United States. There are many types of T-cell lymphoma but they are all fairly rare (http://www.cancer.org/cancer/non-hodgkinlymphoma).
Doctors put non-Hodgkin lymphomas into two groups depending on how quickly they are likely to grow and spread (Table 1).
alone may be used as curative treatment for stages I and II in patients with indolent NHL. For the more extensive and aggressive conditions RT is used in combination with chemotherapeutic substances. While indolent and aggressive NHLs are responsive to RT and chemotherapy 50%-70% of patients are relapsed [3,4]. Most side effects are associated with conventional therapies due to the non-specific nature of the treatments. Thus, there is a constant need for development of novel therapeutic strategies or approaches that may improve the outcome of NHL patients. Therefore, targeted immunotherapy is right option to improve clinical responses with decreasing toxicity. Targeted immunotherapy in cancer involves the administration of a substances which specifically interact with a molecules which may be directly or indirectly involved in oncogenesis [5]. These are tumor associated antigens which expressed on the cell surface, soluble factors, extracellular matrix proteins and proteins associated with vascularization of tumors. The expression of these antigens should ideally be limited to only cancerous cells to decrease any side effects which may results from targeting of normal cells.

Immunotherapy/ Radio-immunotherapy
The concept of targeted immunotherapy was known almost a century before. Paul Ehrlich (1854-1915) the founder of immunology discovered a 'magic bullet' on the surface of an infected cell which able to selectively deliver a toxin to the bacterium inside the cell while sparing other tissues. This led to a discovery of therapy for syphilis in the pre-penicillin era for which Ehrlich received a Nobel Prize in 1908 [6]. The concept of the 'magic bullet' was successfully exploited by Milstein and Kohler in 1975 [7]. He successfully produced monoclonal antibodies using hybridoma technology and got Novel Prize for their intense scientific work. After two decades the concept of a 'therapeutic magic bullet' for cancer therapy was exist in 1997 with the approval of rituximab (anti-CD20 chimeric monoclonal antibody) by the US FDA for relapsed and refractory indolent lymphoma [8]. This was the first achievement of immunotherapy to kill B-lymphocytes by the use of anti-CD20 monoclonal antibody against the B-cell specific human CD20 cells surface molecules. The parallel successes of rituximab two other CD20 mAbs (Zevalin and Bexxar) were conjugated with radio-active materials to boost their therapeutic responses. Ibritumomab tiuxetan (Zevalin) is a CD20 mAb coupled with the radioactive isotope yttrium-90 or indium-111. Tositumumab (Bexxar) labeled with iodine-131. Both antibodies were approved by US FDA in 2002 and 2003 respectively. These are widely used for the treatment of follicular lymphoma (FL) patients and other NHLs as a part of radio-immunotherapy [9,10]. After that various mAbs have been raised against CD20 some of them have been approved for human use ( Figure 1).
The clinical success of CD20-targeted immunotherapy is limited expression of CD20 molecules. It is specifically expressed on tumor cells; it is not expressed in hematopoietic stem cell and differentiated B-cells. Therefore the B-cell hematopoiesis and other cell lineages are not in danger. CD20 is a non-glycosylated transmembrane phosphoprotein with four transmembrane domains. It has been a superb biomarker for immunotherapies targeting B-cell derived diseases defined by the monoclonal antibody tositumomab [11][12][13]. The success of rituximab prompted renewed interest in the study of a variety of clusters of differentiation (CD) molecules with the intent to use them as potential therapeutic targets.
The CD20 molecules play a crucial role in cell development and survival and when modulated by antibodies result in dysregulation of vital cell survival pathways. Furthermore, it exerts various effects upon ligation with anti-CD20 mAbs and can induced several cell death mechanism such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct induce programmed cell death (PCD). Recently, two newly characterized cell death pathways induced by anti-CD20 mAbs were reported such as lysosome mediated and reactive oxygen species mediated through NADPH [12,[14][15][16][17][18][19] (Figure 2).
Learning about the limitations of rituximab and other monoclonal antibodies lead to the development of new treatments strategies, appropriate modifications in the Fc region of mAbs or development of novel anti-CD20 mAbs as well as screening and identification of small molecules which have ability to increases levels of CD20 on surface of human tumor cells. The increases in CD20 levels on cell surface and developing these novel mAbs may be increase more CD20 and antibody associations, increases their binding affinity, reducing immunogenicity and improving ADCC, CDC and PCD. In an effort to increase their cytotoxic activity mAbs have also been conjugated to radioisotopes, chemo-toxins and made various modifications in Fc region. The purpose of this article is to update the scientific readers those are working in the area of recent advances in the biotechnology for the development of novel anti-CD20 mAbs and identification of CD20 modulators for the improvement of immunotherapeutic responses against lymphoproliferative disorders.

Development of novel anti-CD20 mAbs
The development of anti-CD20 mAbs against lymphoma diseases were started from concept of magic bullets in 1879. The main induction of monoclonal antibodies technology or generation was initiated after the Kohler and Milstein. His scientific work directed to a great expectation that mAbs would provide effective targeted therapy for cancer. Although the CD20 specific antibody B1 (renamed tositumomab) was first discovered in 1981. However rituximab became the first mAb approved by the U.S. Food and Drug Administration (FDA) for use in relapsed and indolent lymphoma [8,20]. It is a chimeric (human-mouse) mAb used to treatment of CD20 positive B-cell malignancies; eg. non-Hodgkin lymphoma and chronic lymphocytic leukemia (CLL) and for some autoimmune diseases including rheumatoid arthritis [8,21]. Rituximab is the first generation CD20 mAb. It can induce complementdependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and direct programmed cell death as well as showed cell growth inhibition [22,23]. It is widely used for lymphoma therapy alone or in combination regimens mainly for relapsed and refractory lymphomas [24,25]. R-CHOP chemotherapy remains the typical regimen for recently diagnosed DLBCL [26][27][28]. Ibritumomab tiuxetan (Zevalin) and tositumumab (Bexxar) both are murine based mAbs also used as radio-immunotherapeutic agents against indolent NHL and follicular lymphoma (FL) patients respectively [9,10,29]. However, the efficacy of rituximab is modest and often variable especially when used for CLL treatment with an objective response rates ranged between 25% and 35% [30,31]. Despite the unparalleled success of rituximab some patients still failed to respond or more commonly relapsed and become resistant after receiving rituximab administration.
While successes, limitations and elucidations of the mechanism of action of rituximab have increased our understanding or knowledge and helped in our goal to improve the efficacy and decreasing the associated adverse effects as well as providing effective therapies for those patients who have developed resistance to rituximab. The interest in development of anti-CD20 mAbs continues to provide a major focus for scientific and clinical investigators alike and it seems to be highly probable that this research interest will continue to grow as the new generation of anti-CD20 mAbs have developed and tested clinically. Currently, there are several new generation anti-CD20 mAbs have been engineered and/or modified to improve antitumor activity and Fc binding affinity and provide advantages over rituximab that are currently undergoing clinical investigation. They may be grouped in two categories: second or third generation anti-CD20 mAb.
Second generation mAbs designed as humanized or fully human with unmodified Fc domain, the purpose of reducing immunogenicity compared to chimeric mAb rituximab. Second generation mAbs include Ocrelizumab, veltuzumab and ofatumumab. Ocrelizumab and veltuzumab are humanized while ofatumumab is fully human antibody. Ocrelizumab (PRO70769, 2H7) is a humanized type I anti-CD20 IgG1 mAb. It has differences in several amino acid positions within the CDRs variable regions of the light and heavy chain as compared to rituximab. Thus it demonstrated superior binding affinity for the low-affinity variants of the FcγRIIIa receptor (CD16). Moreover it showed higher ADCC and lower CDC activity as compared to rituximab toward lymphoid malignancies. Currently, this mAb has been evaluated through a phase I/II study in patients with relapsed/ refractory follicular lymphoma (FL) after rituximab failed therapy and showed superior efficacy and safety [32,33]. Veltuzumab is another humanized type I anti-CD20 IgG1 mAb identical to rituximab with single amino acid substitution (Asp101 instead of Asn101) within the CDR3 of the variable heavy chain resulting showed reduced off-rate [13,34]. It also showed antiproliferative, apoptotic and ADCC effects in vitro similar to rituximab but this modification results more potent binding avidities and a stronger effects on CDC as compared to rituximab [32,35]. In addition, the administration of very low doses either intravenous or subcutaneous routes it showed a potent anti B-cell lymphoma activity in cynomolgus monkeys (Macaca fascicularis) and reduced tumor growth in mice bearing human B-cell lymphomas [34]. Moreover, it achieves efficient delivery into the blood and pharmacologically active when administration subcutaneously compared to other routes. Of these novel mAbs, ofatumumab is at the most advance stage of clinical development with slow off-rate and high CDC activity. Ofatumumab (OFA) is a fully human type I anti-CD20 IgG1κ mAb. It recognizes an overlapping epitope on the small and big extracellular loop of CD20. Moreover, it showed better complement activation as compared to rituximab against both rituximab sensitive and resistant non-Hodgkin lymphomas cell lines expressing high levels of complement defense proteins and low levels of CD20 antigen which failed to undergo CDC with rituximab [36-42]. In addition, it showed higher potential activity than rituximab because of the high capacity for C1q activation. It also showed better response against relapsed/ refractory FL and successively in a phase I/II dose escalation study with an overall response rate (ORR) of 43% [43]. Importantly, in a phase I/II studies on lymphoma and leukemia (specially on CLL) also showed increased complement activity without further increase in toxicity [40,43]. Possibly, the ofatumumab may be give rises to superior efficacy in combination with chemotherapy for tumor clearance and this is being investigated in ongoing trials in both FL and DLBCL.
Likewise, the third generation humanized mAbs are modified mAbs in the Fc region. The Fc domain was engineered with the glycol or protein. The main goal of this modification is to improving the therapeutic efficacy in all patients; particularly patients in which expression with low affinity version of the Fc receptor are found on their tumor cells.
Third generation anti-CD20 mAbs include AME133v, Pro131921 (v114), GA101, R603/ EMAB-6 and TRU-015. They are ongoing in early phases of clinical development. AME-133v (LY2469298, ocaratuzumab) is an Fc protein engineered humanized type I IgG1 mAb which currently being evaluated in a Phase I/II dose escalation study in patients with relapsed/refractory follicular B-cell NHL [44]. In vitro study suggested that it has 13 to 20 fold more binding affinity with CD20 and 5-7 fold higher avidity to the low affinity (F/F and F/V) variants of FcγRIIIa receptor thereby improving killing of B-cell NHL ~10 fold as compared to rituximab [44][45][46]. Although, the clinical trial with AME-133v are currently ongoing and it will need to be compared to rituximab in randomized clinical trials to substantiate its potential clinical advantages. Pro131921 (v114)

Modulation in CD20 Surface Levels
A number of CD20 mAbs are now used in clinical practice or are in different stages of development ( Table 3). Most of them such as rituximab, 90Y-Ibritumomab, tositumomab, ofatumumab and Obinutuzumab (GA 101) have been FDA approved for use in NHLs and RA. All anti-CD20 mAbs are biochemically and functionally divided into two distinct subtypes such as rituximab-like type I and tositumomab-like type II as shown in Table 2 [75,76].
In clinical applications, the efficacy of anti-CD20 mAbs seems to be decline after a period of months of treatments due to therapeutic resistance. Actually the explanation for this therapeutic resistance is not clear. The possible mechanisms of this resistance of B-cell NHLs against anti-CD20 mAbs therapy may be include three patterns: (I) Protection of the tumor cells from mAbs mediated depletion of B-cell lymphoma by ADCC, CDC and apoptotic stimulation (II) Inadequate binding of mAbs to the CD20 molecule and (III) Low levels of CD20 antigens on cells surface or reduce CD20 surface levels on cells.
Although, some investigators provide information that decreased levels of CD20 expression and/ or harbor low levels of CD20 on surface of malignant B-cells may be one of the major contributing factors for antibody response [103,104]. However, there is general agreement that diseases such as chronic lymphocytic leukemia display the CD20 cell surface molecules in fairly low levels and respond proportionally less as compared to others low grade B-cell malignancies [30, [104][105][106]. Some studies are strongly suggested that cytokines, some inhibitors and radiation exposure showed strong ability to significantly induced expression of CD20, HER2 and EFGR at both total protein levels as well as availability on cell surface specifically in malignant cells, not on normal cell lineages. In relation to CD20 expression some reports provide strong evident that bryostatin-1, interleukin-4, granulocyte macrophage colony stimulating factor, tumor necrosis factor-α, interferon-α and γ radiation have strong ability to induce changes in CD20 expression at transcription, translation and epigenetically as well as their associated transcription factors as showing in Table 4 [107][108][109][110][111][112][113].
The bryostatin-1 induced increases in CD20 expression were found at the transcriptional level. The effects of bryostatin-1 on CD20 expression in NHL derived cells was apparently mediated through the MAPK/ERK signal transduction pathway and involved protein kinase C [111]. An increase in CD20 transcription was also shown to be triggered by CpG independently of PU.1 transcription factor in CLL cells [128]. Recently, it was also showed that L-744,832 induced inhibition of farnesyltransferase activity leads to up-regulation of CD20 levels and to improved human tumor cell killing activity followed by anti-CD20 mAbs. Moreover, the inhibition of farnesyltransferase activity was found to be associated with increased binding affinity of PU.1 and Oct-2 to the CD20 promoter sequences [117]. Bortezomib a proteasome inhibitor have potential to induced expression of COOH-terminal region of the internal domain of CD20 but not the whole CD20 molecule [118]. Recent study addressed the potential activity of bortezomib in more detail that the unexpected negative influence of proteasome inhibitors on the CD20 levels as well as rituximab mediated CDC and ADCC toward CD20 positive B-cell malignancies [119]. The CD20 expression is also regulated by epigenetic mechanisms. For example 5-azacytidine (inhibitor of DNA methyltransferase activity) can significantly increases the CD20 expression in Bcell lymphoma [120] and trichostatin-A (a modulator of histone-acetylation status) also have ability to increases CD20 mRNA and protein levels in RRBL1 cells, a B-cell lymphoma cell line [121,122]. Two other HDAC inhibitors such as valproic acid (VPA) and romidepsin both have ability to increased CD20 expression at protein and mRNA levels in B-cell lymphoma cell lines. The VPA-mediated increase in CD20 expression is clinically achievable and safe, but insufficient for inducing cell death. Moreover, it is also revealed that HDAC inhibitors trans-activated the CD20 gene promoter through hyper-acetylation and Sp1 recruitment [123]. Whereas, other reports are exploited that CD20 antigens is down-regulated by anti-CD20 mAb rituximab treatment. It is a well-recognized phenomenon in patients with non-Hodgkin's lymphomas particular in chronic lymphocytic leukemia (CLL). In CLL, rituximab mediated down modulation of CD20 is associated with reduced levels of CD20 mRNA at in vitro and in vivo indicating regulation of CD20 expression at the level of transcription [129,130]. Recently it is also reported that initially CD20 antigens disappeared in patients with CLL treated with rituximab containing salvage regimens occurred in 4 out of 8 (50%) tested patients after some time CD20 levels returned at progression or recovered. Half of whom developed Richter's syndrome [131]. One more report indicated that lenalidomide or CD40 ligation in normal B-cells down regulates CD20 levels [132,133]. Radiation induced changes in CD20 expression on B-cell lymphoma were identified first time in 1997 by Philippe et al. [131]. Later on, Kunala et al. was also suggested that exposure of ionizing radiation (10Gy) can significantly increases CD20 surface expression in a dose and time dependent manner in IM9, IM9/Bcl-2 and Ramos neoplastic B-cell lines. In contrast, he was also investigated that CD20 expression was not induced in CD20negative Molt-4 cell line whereas it was increases only about 25% in the GM1310B normal B-cell line. Moreover, the overexpression of Bcl-2 protein does not inhibited radiation induced CD20 expression. In addition, the treatment of cells with actinomycin-D is known to inhibit RNA synthesis followed by 10Gy γ-radiation. This suggests a transcriptional regulation of CD20 expression rather than a simple alteration in cell surface morphology or surface level of CD20 on the targets cells [126,127]. Gupta et al. strongly suggested that the significant increases in cell surface expression of CD20 were transient and cell type dependent manner in logarithmically growing Daudi and Raji cells followed by 0.5 and/or 1.5Gy radiation exposure. The enhanced expression of CD20 antigen was associated with transcriptional up-regulation of CD20 mRNA and CD20 regulatory transcription factors. Moreover, the changes in CD20 surface levels were found to be correlated with overall changes in oxidative stress and mitochondrial membrane potential [112]. Recently, Singh et al. demonstrated that sub-lethal dose (0.5Gy) of γ-radiation can induce ~3 fold CD20 levels on Burkitt's lymphoma cell line 'Daudi' and it was also associated with changes in oxidative condition in intracellular milieu [124,125]. Moreover, cytokines which involved in CD20 expression also cause robust intracellular oxidative bursts. Accumulating evidence indicates that CD20 expression in malignant cells can be modulated at transcriptional, transcriptional, posttranscriptional and even posttranslational levels and their occurrence and significance may be vary depending on the type of malignancies. However, the precise mechanisms of changes in CD20 expression still unclear and further need to be investigation.

Conclusion
In conclusion over the last 10 years rituximab is used against the treatment of all common Bcell malignancies. Based on this success, limitations and elucidation of the mechanism various novel anti-CD20 mAbs has been developed to improved clinical outcomes with outstanding performance in ADCC, CDC and PCD and reduced immunogenicity. Although, the mechanisms of action of each anti-CD20 mAbs has been well studied in preclinical settings. However, the variability seen in clinical responses of these mAbs may be depend on level of CD20 expression, levels of circulating soluble CD20, presence of effector cells, CD20 binding epitope and kinetics, binding with Fc receptors, tissue distribution and tumor burden. Singh et al. recently published a report provide information that sub-lethal dose of radiation can induced CD20 surface levels on cells determined efficacy of both type I (rituximab) and type II (tositumomab) anti-CD20 mAbs in vitro. However, more preclinical and clinical investigations need to be confirmed. Therefore, the ability to selectively control CD20 expression and appropriate modifications in Fc domain of mAbs may be great importance in enhancing therapeutic values and in optimizing anti-CD20 immunotherapy and radio-immunotherapy. The modulation in CD20 expression may provide more binding sites for anti-CD20 mAbs and may play a major role in therapeutic response. Based on this information and previous data we suggested that use of external beam radiotherapy (in a site selective manner) just prior to immunotherapy may be beneficial for tumor clearance and maximum clinical outcomes.      Table 3 List of anti-CD20 monoclonal antibodies.  High ADCC Low CDC