**3. B cell depletion therapies mediated via CD20 and CD22**

While initially systemic autoimmunity was considered as a T cell-driven condition, multiple functions of B cells have been described in orchestrating autoimmune disorders, including self-reactive antibody production, auto-antigen presentation and co-stimulation of T lymphocytes, formation of ectopic lymphoid structures (neo-organogenesis) in the endtarget organs and pro-inflammatory cytokine production.

#### *CD20-mediated B cell depletion*

Due to their central role in the immune pathogenesis of systemic autoimmunity and the observation that patients treated with non-Hodgkin's lymphoma and coexisting RA showed

Application of Monoclonal Antibody Therapies in Autoimmune Diseases 243

number of resident CD20+ B cells in the damaged tissues, although with variable efficacy: 70% of cells residing in the spleen and lymph nodes are depleted after 24 hours, while access to peritoneal B cells is limited (30, 31). Success of the depletion also varies among B cell subpopulations: splenic marginal zone B cells, germinal center B cells and peritoneal B1 cells

The reemerging of the B cell population usually occurs in the majority of the patients after 4- 6 months and follows a definite pattern by immature CD5+ CD38high transitional B cells and

Monitoring of serum antibody levels in rituximab treated patients revealed that while titers of RF and anti-CCP antibodies significantly dropped, the humoral immune response towards most pathogens remained unaffected (e.g. pneumococcal capsular polysaccharide, tetanus toxoid) (32). Reduction in IgM-RF levels reflected to changes in total serum IgM levels, but the levels of IgA-RF, IgG-RF, and IgG anti-CCP antibodies decreased significantly more than those of their corresponding total serum immunoglobulin classes, which suggests that rituximab induces a selective reduction of short-lived autoantibody-secreting plasma cells. Two independent studies investigating changes in the synovial tissue composition of RA patients before and after rituximab treatment showed a significant decrease in B cell numbers in the synovium indicating that efficacy of the treatment lies in the disruption of

Data about the efficacy of B cell depletion in SLE are contradictory: several studies involving only a small group of patients reported a significant clinical improvement upon rituximab treatment, while phase II/III trials showed no difference in BILAG scores. In contrast, B cell deficient lupus-prone MRL/lpr mice do not develop nephritis due to the reduced activation of the T cell compartment while transgenic mice expressing only membrane bound BCR on the cell surface (mIgM.MRL/lpr) still develop the disease (34). Another study found that mouse strains prone to develop autoimmunity are a lot more resistant to B cell depletion. These data indicate a more complex role for B cells in SLE than the production of autoreactive antibodies. CD20-mediated B cell depletion at an early age in pristane-primed NZB/W F1 mice resulted in acceleration of the onset of disease, possibly due to the lack of IL-10 production by regulatory B cells. Treatment following the outbreak of symptoms on

Another B lymphocyte restricted target is the Ig-superfamily member CD22, a 135kDa glycoprotein that is first detected in the cytoplasm of pro- and pre-B cells, becoming present on the cell surface of mature peripheral B cells. It remains expressed on germinal center B cells but is absent on plasmablasts and terminally differentiated plasma cells. Known ligands of CD22 include the tyrosine phosphatase CD45 and the lectin CD33, both binding through 2,6-linked sialic acid motifs. While CD22 was reported to inhibit BCR-mediated cell activation *in vitro* via the recruitment of SHP-1 phosphatase to its cytoplasmic ITIM sequences upon phosphorylation by the tyrosine kinase Lyn (36), its *in vivo* role is less clarified. CD22-deficient mice develop hyper-proliferative B-lymphocytes and in

Epratuzumab, a humanized monoclonal IgG1 CD22-specific antibody recognizes a nonligand-binding site of the CD22 molecule. It is predicted to alter BCR-signaling by inducing disruption of cell surface signaling complexes and antibody-mediated depletion of B cells (37). Epratuzumab is shown to cause phosphorylation and internalization of CD22 on peripheral B cells *in vitro*. On a small cohort of SLE-patients, when administered four times

re-circulating plasmablasts appearing first and later circulating naïve B cells.

extrafollicular lymphoid structures and the inhibition of B-T cell interactions (33).

the other hand attenuated the intensity of the disease (35).

consequence increased levels of auto-antibodies.

*CD22-mediated B cell depletion* 

are significantly more protected.

improvements in symptoms of RA after anti-CD20 (Rituximab) treatment, several therapies target B cells.

Physiological autoimmunity, thus the production of auto-antibodies in healthy individuals emerges upon infection and facilitates the clearing of apoptotic cells at the site of inflammation. Defects in down-regulation of this response can lead to the development of pathologic conditions. One of the several criteria by the diagnosis of autoimmunity is the presence of self-reactive antibodies in the circulation that are often present decades before the onset of clinical symptoms. During the course of B cell development, many checkpoints exist to prevent the escape of self-reactive B cells to the periphery these include receptor revision, clonal deletion and anergy (21, 22). Once an auto-reactive B cell is activated though by a self-structure first extra-follicular short-lived plasma cells are formed that produce lowaffinity antibodies. Some of these auto-reactive cells also enter the germinal centers where they undergo affinity maturation and class switch, and develop into long-lived auto-reactive memory cells.

Antibodies can contribute to disease pathogenesis in two different ways: direct action by binding to its target e.g. in myasthenia gravis where anti-acetylcholine receptor antibodies bind post-synaptic receptors and compromise motor functions in neurons (23), or in Graves' disease, where the anti-thyroid stimulating hormone (TSH) receptor auto-antibodies can act as receptor agonists (24). The indirect contribution of auto-antibodies to autoimmunity consists of the formation of immune complexes inducing Fc-receptor mediated phagocytosis and/or activation of the complement system and production of pro-inflammatory cytokines, thus leading to tissue damage.

In addition to antibody production B cells also have an important role as antigen presenting cells. B cell deficiency in mice results in a disrupted lymphoid structure in the spleen, lack of follicular dendritic cell network and absence of Peyer's patches (25). B cell depletion studies in mice showed a defect in CD4+ T cell priming in the absence of B cell co-stimulation, especially when the antigen is available only at low concentrations. B cells not only provide support to T cells via direct cell-cell contact, but also shape the immune response by producing either pro-inflammatory cytokines including IL-6, IFN and LT. Certain subsets are also able to produce IL-10 that has a regulatory function and contributes to the attenuation of the disease (26).

Tertiary ectopic lymphoid structures have been described at the end-organ in several autoimmune disorders: in the synovium of RA patients organized zones of B, T and follicular dendritic cells can be found in more than 50% of the cases, while kidneys of SLE patients also often contain such organized structures. B cell depletion or the blockade of B cell-T cell contacts has been proved to disrupt these ectopic lymphoid follicles and attenuate disease severity in several animal models of autoimmunity (27).

One of the most effective disease modifying anti-rheumatic drugs (DMARDs) is rituximab, a human CD20-specific chimeric monoclonal antibody. CD20 is a 35-37kDa tetra-spanning integral membrane protein first expressed on late pre-B cells in the bone marrow, present on naïve and mature B cells, down-regulated on antibody-secreting plasmablasts and extinguishing on plasma cells. It has been shown to regulate early steps of cell cycle progression, B cell proliferation and apoptosis. Upon cell activation, it gets trans-located to membrane lipid rafts, where it can act as a Ca2+-channel (28). CD20 is also expressed on a small subset of basally activated IL1- and TNF producing T cells (0.1-6.8% in healthy individuals) that showed enhanced susceptibility to apoptosis (29). Within an hour Rituximab treatment induces a ~90% reduction of the pre-treatment state in circulating CD20+ B cell numbers that lasts for at least 3 months and also mediates a decrease in the

improvements in symptoms of RA after anti-CD20 (Rituximab) treatment, several therapies

Physiological autoimmunity, thus the production of auto-antibodies in healthy individuals emerges upon infection and facilitates the clearing of apoptotic cells at the site of inflammation. Defects in down-regulation of this response can lead to the development of pathologic conditions. One of the several criteria by the diagnosis of autoimmunity is the presence of self-reactive antibodies in the circulation that are often present decades before the onset of clinical symptoms. During the course of B cell development, many checkpoints exist to prevent the escape of self-reactive B cells to the periphery these include receptor revision, clonal deletion and anergy (21, 22). Once an auto-reactive B cell is activated though by a self-structure first extra-follicular short-lived plasma cells are formed that produce lowaffinity antibodies. Some of these auto-reactive cells also enter the germinal centers where they undergo affinity maturation and class switch, and develop into long-lived auto-reactive

Antibodies can contribute to disease pathogenesis in two different ways: direct action by binding to its target e.g. in myasthenia gravis where anti-acetylcholine receptor antibodies bind post-synaptic receptors and compromise motor functions in neurons (23), or in Graves' disease, where the anti-thyroid stimulating hormone (TSH) receptor auto-antibodies can act as receptor agonists (24). The indirect contribution of auto-antibodies to autoimmunity consists of the formation of immune complexes inducing Fc-receptor mediated phagocytosis and/or activation of the complement system and production of pro-inflammatory cytokines,

In addition to antibody production B cells also have an important role as antigen presenting cells. B cell deficiency in mice results in a disrupted lymphoid structure in the spleen, lack of follicular dendritic cell network and absence of Peyer's patches (25). B cell depletion studies in mice showed a defect in CD4+ T cell priming in the absence of B cell co-stimulation, especially when the antigen is available only at low concentrations. B cells not only provide support to T cells via direct cell-cell contact, but also shape the immune response by producing either pro-inflammatory cytokines including IL-6, IFN and LT. Certain subsets are also able to produce IL-10 that has a regulatory function and contributes to the

Tertiary ectopic lymphoid structures have been described at the end-organ in several autoimmune disorders: in the synovium of RA patients organized zones of B, T and follicular dendritic cells can be found in more than 50% of the cases, while kidneys of SLE patients also often contain such organized structures. B cell depletion or the blockade of B cell-T cell contacts has been proved to disrupt these ectopic lymphoid follicles and attenuate

One of the most effective disease modifying anti-rheumatic drugs (DMARDs) is rituximab, a human CD20-specific chimeric monoclonal antibody. CD20 is a 35-37kDa tetra-spanning integral membrane protein first expressed on late pre-B cells in the bone marrow, present on naïve and mature B cells, down-regulated on antibody-secreting plasmablasts and extinguishing on plasma cells. It has been shown to regulate early steps of cell cycle progression, B cell proliferation and apoptosis. Upon cell activation, it gets trans-located to membrane lipid rafts, where it can act as a Ca2+-channel (28). CD20 is also expressed on a small subset of basally activated IL1- and TNF producing T cells (0.1-6.8% in healthy individuals) that showed enhanced susceptibility to apoptosis (29). Within an hour Rituximab treatment induces a ~90% reduction of the pre-treatment state in circulating CD20+ B cell numbers that lasts for at least 3 months and also mediates a decrease in the

disease severity in several animal models of autoimmunity (27).

target B cells.

memory cells.

thus leading to tissue damage.

attenuation of the disease (26).

number of resident CD20+ B cells in the damaged tissues, although with variable efficacy: 70% of cells residing in the spleen and lymph nodes are depleted after 24 hours, while access to peritoneal B cells is limited (30, 31). Success of the depletion also varies among B cell subpopulations: splenic marginal zone B cells, germinal center B cells and peritoneal B1 cells are significantly more protected.

The reemerging of the B cell population usually occurs in the majority of the patients after 4- 6 months and follows a definite pattern by immature CD5+ CD38high transitional B cells and re-circulating plasmablasts appearing first and later circulating naïve B cells.

Monitoring of serum antibody levels in rituximab treated patients revealed that while titers of RF and anti-CCP antibodies significantly dropped, the humoral immune response towards most pathogens remained unaffected (e.g. pneumococcal capsular polysaccharide, tetanus toxoid) (32). Reduction in IgM-RF levels reflected to changes in total serum IgM levels, but the levels of IgA-RF, IgG-RF, and IgG anti-CCP antibodies decreased significantly more than those of their corresponding total serum immunoglobulin classes, which suggests that rituximab induces a selective reduction of short-lived autoantibody-secreting plasma cells. Two independent studies investigating changes in the synovial tissue composition of RA patients before and after rituximab treatment showed a significant decrease in B cell numbers in the synovium indicating that efficacy of the treatment lies in the disruption of extrafollicular lymphoid structures and the inhibition of B-T cell interactions (33).

Data about the efficacy of B cell depletion in SLE are contradictory: several studies involving only a small group of patients reported a significant clinical improvement upon rituximab treatment, while phase II/III trials showed no difference in BILAG scores. In contrast, B cell deficient lupus-prone MRL/lpr mice do not develop nephritis due to the reduced activation of the T cell compartment while transgenic mice expressing only membrane bound BCR on the cell surface (mIgM.MRL/lpr) still develop the disease (34). Another study found that mouse strains prone to develop autoimmunity are a lot more resistant to B cell depletion. These data indicate a more complex role for B cells in SLE than the production of autoreactive antibodies. CD20-mediated B cell depletion at an early age in pristane-primed NZB/W F1 mice resulted in acceleration of the onset of disease, possibly due to the lack of IL-10 production by regulatory B cells. Treatment following the outbreak of symptoms on the other hand attenuated the intensity of the disease (35).

#### *CD22-mediated B cell depletion*

Another B lymphocyte restricted target is the Ig-superfamily member CD22, a 135kDa glycoprotein that is first detected in the cytoplasm of pro- and pre-B cells, becoming present on the cell surface of mature peripheral B cells. It remains expressed on germinal center B cells but is absent on plasmablasts and terminally differentiated plasma cells. Known ligands of CD22 include the tyrosine phosphatase CD45 and the lectin CD33, both binding through 2,6-linked sialic acid motifs. While CD22 was reported to inhibit BCR-mediated cell activation *in vitro* via the recruitment of SHP-1 phosphatase to its cytoplasmic ITIM sequences upon phosphorylation by the tyrosine kinase Lyn (36), its *in vivo* role is less clarified. CD22-deficient mice develop hyper-proliferative B-lymphocytes and in consequence increased levels of auto-antibodies.

Epratuzumab, a humanized monoclonal IgG1 CD22-specific antibody recognizes a nonligand-binding site of the CD22 molecule. It is predicted to alter BCR-signaling by inducing disruption of cell surface signaling complexes and antibody-mediated depletion of B cells (37). Epratuzumab is shown to cause phosphorylation and internalization of CD22 on peripheral B cells *in vitro*. On a small cohort of SLE-patients, when administered four times

Application of Monoclonal Antibody Therapies in Autoimmune Diseases 245

In the serum of patients with active SLE and Sjogren's syndrome, the expression levels of soluble BAFF have been found elevated. Therefore, it is a potential target molecule in autoimmune disorders. Belimumab a humanized IgG1 monoclonal antibody blocks BAFFbinding to its receptors, thereby inhibiting the persistence of antibody producing B cells by mediating apoptotic cell death of early plasmablasts, naïve B cells and activated B cells (41) (42). Restriction of BAFF levels might facilitate the function of regulatory B cell populations. Atacicept is a fully human chimeric molecule consisting of the TACI ligand-binding extracellular domain fused to the Fc-portion of a human IgG1. It blocks both TACI- and BAFF-binding to their receptors and resulted to be successful in phase I clinical trials for the

For RA patients who give an inadequate response to anti-TNF therapy, there is an increasing number of DMARDs that offer improvement of clinical symptoms by the

Activation of T cells by antigens not only requires TCR-binding to the specific peptide-MHC complex on the APC, but also the ligation of co-stimulatory molecules like CD40, inducible T cell co-stimulator (ICOS) and CD28. Therefore, biologics that block these interactions may interfere with sufficient helper T cell activation and inhibit B cell differentiation into

Abatacept is a soluble, fully human fusion protein of the extracellular domain of CTLA-4 and the hinge region, the CH2 and CH3 domains of a human IgG1 molecule. It recognizes B7 (CD80/86) with a high affinity and blocks its interaction with CD28, this way selectively inhibiting T cell activation. Abatacept is approved for the treatment of RA and juvenile idiopathic arthritis, and in a phase I trial it was shown to improve the clinical symptoms of psoriasis patients via the reduction of the size of the intralesional T cell population. Combination of abatacept with TNF-blockers is not advised as it increases the occurrence

Although the role of ICOS in T cell signaling has not been completely resolved yet, experimental data demonstrate that ICOS/ICOSL is an important regulator of T cell activation. It is expressed on resting T cells only at low amounts, while it gets strongly upregulated upon activation. Highest level of expression is observed on T follicular helper cells. Blockade of ICOS/ICOSL interactions with monoclonal antibodies has been reported to improve collagen-induced arthritis and murine models of lupus (46). At the early stage of experimental autoimmune encephalomyelitis (EAE), a mouse model of sclerosis multiplex, inhibition of the ICOS/ICOSL interaction seemed to aggravate the disease, while it induced an improvement when administered at later phases (47). In glucose-6-phosphate isomerase (G6PI)-induced arthritis, early ICOSL-specific monoclonal antibody treatment resulted in significant loss of disease severity, but treatment at later stages of arthritis reduced symptoms only marginally. The number of G6PI-specific T helper cells decreased, but there was no difference in the antigen-specific antibody levels in the sera of the animals (48).

**5. Biotechnical approaches for the reduction of immunogenicity in** 

By the application of recombinant proteins in human medicine immunogenicity is one of the major concerns as several examples (insulin-, growth hormone-, factor VIII treatment or

inhibition of T cell-antigen presenting cell (APC) interactions.

of infections and provides no additional benefits (11).

treatment of RA (43, 44, 45). *Inhibition of B-T cell interactions* 

antibody producing plasma cells.

**monoclonal antibody therapies** 

every second week it was reported to improve clinical symptoms in all of the patients based on the 6-, 10- or 18-week assessments (38).
