**7. Acknowledgement**

This work was supported by a grant from the Research and Technology Innovation Fund (KTIA-OTKA 80689) and the National Innovation Office (NIH)-ANR bilateral grant.

#### **8. References**

248 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

d. Human antibody production of transgenic mice expressing human immunoglobulin

attributes such as stability and aggregation besides molecular recognition.

genes

targeted antigen.

**6. Conclusions** 

medications.

**7. Acknowledgement** 

are still immunogenic (73). An explanation for this could be that *in vitro* affinity maturation lacks several control steps, as there is an additional *in vivo* selection for

Strategies to establish mouse strains with germ line modifications in their immunoglobulin genes usually aim for homologous recombination in mouse embryonic stem cells that disrupt endogenous Ig heavy and light chains, and introduce the human transgenes. In the past, different technologies were successfully applied to produce and deliver the human sequence transgenes: Lonberg et al. used pronuclear microinjection to introduce reconstructed minilocus transgenes (74), while Green et al. established transgenes with yeast artificial chromosome (YAC) (75). Initially, mouse heavy and light chain sequences were 'replaced' for several different VH, DH and JH regions with 1, or heavy chain constant region fragments and V, all five J and the C light chain genes. These transgenic animals were able to mount human antibodies in response to a

There have been many initiatives undertaken since then to broaden the size of the Vregion repertoire, as it has a strong influence on multiple checkpoints in B cell development and therefore the size of the mature B cell population (76). Following selection of the most efficient clones, for large-scale production usually a recombinant

In contrast to chimeric, humanized or *in vitro* generated therapeutic monoclonal antibodies, there are no reported cases of the generation of anti-human Ig responses towards transgenic therapeutic antibodies. Table 1 summarizes the currently available

Since the development of the hybridoma technique several monoclonal antibodies have been approved for the treatment of autoimmune diseases. Immunogenicity of murine sequences caused initial complications, which could be attenuated and finally overcome by the production of chimeric and humanized antibodies and with the generation of transgenic mouse strains for human Ig-sequences. One of the crucial steps by the design of a monoclonal antibody for therapeutic applications is the selection of the right target molecule. In autoimmune disorders several options exist: the blockade of the proinflammatory cytokines TNF, IL-1 or IL-6, the inhibition of T cell-B cell interactions, B cell depletion to reduce autoantibody production and the establishment of ectopic lymphoid structures or the blockade of B cell survival factors. Although we still need to face adverse events upon the application of these therapeutic antibodies, targeting specific molecules will help us to reduce the severity of occurring side effects and provide more efficient

This work was supported by a grant from the Research and Technology Innovation Fund

(KTIA-OTKA 80689) and the National Innovation Office (NIH)-ANR bilateral grant.

expression system is established to reduce costs (77).

monoclonal constructs and their origin.


Application of Monoclonal Antibody Therapies in Autoimmune Diseases 251

[32] Bingham, C. O. r., R. J. Looney, A. Deodhar, N. Halsey, M. Greenwald, C. Codding, B.

[33] Lund, F. E., and T. D. Randall. 2010. Effector and regulatory B cells: modulators of

[34] Chan, O. T., L. G. Hannum, A. M. Haberman, M. P. Madaio, and M. J. Shlomchik. 1999.

independent role for B cells in murine lupus. *J Exp Med* 189: 1639-1648. [35] Haas, K. M., R. Watanabe, T. Matsushita, H. Nakashima, N. Ishiura, H. Okochi, M.

systemic autoimmunity in NZB/W F1 mice. *J Immunol* 184: 4789-4800. [36] Carnahan, J., P. Wang, R. Kendall, C. Chen, S. Hu, T. Boone, T. Juan, J. Talvenheimo, S.

[37] Carnahan, J., R. Stein, Z. Qu, K. Hess, A. Cesano, H. J. Hansen, and D. M. Goldenberg.

[39] Bodmer, J. L., P. Schneider, and J. Tschopp. 2002. The molecular architecture of the TNF

[40] O'Connor, B. P., V. S. Raman, L. D. Erickson, W. J. Cook, L. K. Weaver, C. Ahonen, L. L.

[42] Stohl, W., J. L. Scholz, and M. P. Cancro. 2011. Targeting BLyS in rheumatic disease: the sometimes-bumpy road from bench to bedside. *Curr Opin Rheumatol* 23: 305-310. [43] Seyler, T. M., Y. W. Park, S. Takemura, R. J. Bram, P. J. Kurtin, J. J. Goronzy, and C. M.

[44] Bracewell, C., J. D. Isaacs, P. Emery, and W. F. Ng. 2009. Atacicept, a novel B cell-

[45] Nestorov, I., A. Munafo, O. Papasouliotis, and J. Visich. 2008. Pharmacokinetics and

survival of long-lived bone marrow plasma cells. *J Exp Med* 199: 91-98. [41] Furie, R., W. Stohl, E. M. Ginzler, M. Becker, N. Mishra, W. Chatham, J. T. Merrill, A.

different mode of action from rituximab. *Mol Immunol* 44: 1331-1341. [38] Dorner, T., J. Kaufmann, W. A. Wegener, N. Teoh, D. M. Goldenberg, and G. R.

clinical trial. *Arthritis Rheum* 62: 64-74.

superfamily. *Trends Biochem Sci* 27: 19-26.

*Arthritis Res Ther* 10: R109.

*Biol Ther* 9: 909-919.

48: 406-417.

3990S.

R74.

CD4(+) T cell immunity. *Nat Rev Immunol* 10: 236-247.

Trzaskoma, F. Martin, S. Agarwal, and A. Kelman. 2010. Immunization responses in rheumatoid arthritis patients treated with rituximab: results from a controlled

A novel mouse with B cells but lacking serum antibody reveals an antibody-

Fujimoto, and T. F. Tedder. 2010. Protective and pathogenic roles for B cells during

Montestruque, J. Sun, G. Elliott, J. Thomas, J. Ferbas, B. Kern, R. Briddell, J. P. Leonard, and A. Cesano. 2003. Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties. *Clin Cancer Res* 9: 3982S-

2007. Epratuzumab, a CD22-targeting recombinant humanized antibody with a

Burmester. 2006. Initial clinical trial of epratuzumab (humanized anti-CD22 antibody) for immunotherapy of systemic lupus erythematosus. *Arthritis Res Ther* 8:

Lin, G. T. Mantchev, R. J. Bram, and R. J. Noelle. 2004. BCMA is essential for the

Weinstein, W. J. McCune, J. Zhong, W. Cai, and W. Freimuth. 2008. Biologic activity and safety of belimumab, a neutralizing anti-B-lymphocyte stimulator (BLyS) monoclonal antibody: a phase I trial in patients with systemic lupus erythematosus.

Weyand. 2005. BLyS and APRIL in rheumatoid arthritis. *J Clin Invest* 115: 3083-3092.

targeting biological therapy for the treatment of rheumatoid arthritis. *Expert Opin* 

biological activity of atacicept in patients with rheumatoid arthritis. *J Clin Pharmacol*


[17] Herber, D., T. P. Brown, S. Liang, D. A. Young, M. Collins, and K. Dunussi-

[19] Li, J., W. Shen, K. Kong, and Z. Liu. 2006. Interleukin-21 induces T-cell activation and

[20] Jungel, A., J. H. Distler, M. Kurowska-Stolarska, C. A. Seemayer, R. Seibl, A. Forster, B.

[22] von Boehmer, H., and F. Melchers. 2010. Checkpoints in lymphocyte development and

[23] Ruff, R. L., and V. A. Lennon. 2008. How myasthenia gravis alters the safety factor for

[24] Brown, R. S. 2009. Autoimmune thyroid disease: unlocking a complex puzzle. *Curr Opin* 

[25] Ngo, V. N., R. J. Cornall, and J. G. Cyster. 2001. Splenic T zone development is B cell

[26] Yanaba, K., J. D. Bouaziz, K. M. Haas, J. C. Poe, M. Fujimoto, and T. F. Tedder. 2008. A

[27] Weyand, C. M., Y. M. Kang, P. J. Kurtin, and J. J. Goronzy. 2003. The power of the third

[28] Tedder, T. F., and P. Engel. 1994. CD20: a regulator of cell-cycle progression of B

[29] Wilk, E., T. Witte, N. Marquardt, T. Horvath, K. Kalippke, K. Scholz, N. Wilke, R. E.

[30] Hamaguchi, Y., J. Uchida, D. W. Cain, G. M. Venturi, J. C. Poe, K. M. Haas, and T. F.

[31] Uchida, J., Y. Hamaguchi, J. A. Oliver, J. V. Ravetch, J. C. Poe, K. M. Haas, and T. F.

regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-

dimension: tissue architecture and autoimmunity in rheumatoid arthritis. *Curr* 

Schmidt, and R. Jacobs. 2009. Depletion of functionally active CD20+ T cells by

Tedder. 2005. The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti-CD20 immunotherapy in mice. *J Immunol*

Tedder. 2004. The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody

peripheral B lymphocyte development. *Adv Immunol* 95: 1-50.

neuromuscular transmission. *J Neuroimmunol* 201-202: 13-20.

dependent inflammatory responses. *Immunity* 28: 639-650.

autoimmune disease. *Nat Immunol* 11: 14-20.

dependent. *J Exp Med* 194: 1649-1660.

lymphocytes. *Immunol Today* 15: 450-454.

immunotherapy. *J Exp Med* 199: 1659-1669.

rituximab treatment. *Arthritis Rheum* 60: 3563-3571.

*Opin Rheumatol* 15: 259-266.

174: 4389-4399.

1152-1163.

515-522.

*Pediatr* 21: 523-528.

Joannopoulos. 2007. IL-21 has a pathogenic role in a lupus-prone mouse model and its blockade with IL-21R.Fc reduces disease progression. *J Immunol* 178: 3822-3830. [18] Young, D. A., M. Hegen, H. L. Ma, M. J. Whitters, L. M. Albert, L. Lowe, M. Senices, P.

W. Wu, B. Sibley, Y. Leathurby, T. P. Brown, C. Nickerson-Nutter, J. C. J. Keith, and M. Collins. 2007. Blockade of the interleukin-21/interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis. *Arthritis Rheum* 56:

proinflammatory cytokine secretion in rheumatoid arthritis. *Scand J Immunol* 64:

A. Michel, R. E. Gay, F. Emmrich, S. Gay, and O. Distler. 2004. Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis. *Arthritis Rheum* 50: 1468-1476. [21] Monroe, J. G., and K. Dorshkind. 2007. Fate decisions regulating bone marrow and


Application of Monoclonal Antibody Therapies in Autoimmune Diseases 253

[61] Gonzales, N. R., E. A. Padlan, R. De Pascalis, P. Schuck, J. Schlom, and S. V. Kashmiri.

[62] Neylon, C. 2004. Chemical and biochemical strategies for the randomization of protein

[63] Stemmer, W. P. 1994. Rapid evolution of a protein in vitro by DNA shuffling. *Nature*

[64] Pendley, C., A. Schantz, and C. Wagner. 2003. Immunogenicity of therapeutic

[65] Hwang, W. Y., and J. Foote. 2005. Immunogenicity of engineered antibodies. *Methods* 36:

[66] Bartelds, G. M., C. L. Krieckaert, M. T. Nurmohamed, P. A. van Schouwenburg, W. F.

[67] Danner, S., and J. G. Belasco. 2001. T7 phage display: a novel genetic selection system

[68] Huse, W. D., L. Sastry, S. A. Iverson, A. S. Kang, M. Alting-Mees, D. R. Burton, S. J.

[71] McCafferty, J., A. D. Griffiths, G. Winter, and D. J. Chiswell. 1990. Phage antibodies: filamentous phage displaying antibody variable domains. *Nature* 348: 552-554. [72] Akamatsu, Y., M. S. Cole, J. Y. Tso, and N. Tsurushita. 1993. Construction of a human Ig

[73] Aarden, L., S. R. Ruuls, and G. Wolbink. 2008. Immunogenicity of anti-tumor necrosis

[74] Lonberg, N., L. D. Taylor, F. A. Harding, M. Trounstine, K. M. Higgins, S. R. Schramm,

[75] Green, L. L., M. C. Hardy, C. E. Maynard-Currie, H. Tsuda, D. M. Louie, M. J. Mendez,

immunoglobulin repertoire in phage lambda. *Science* 246: 1275-1281. [69] Mullinax, R. L., and J. A. Sorge. 2003. Preparing lambda libraries for expression of proteins in prokaryotes or eukaryotes. *Methods Mol Biol* 221: 271-287. [70] Kang, A. S., T. M. Jones, and D. R. Burton. 1991. Antibody redesign by chain shuffling

Lems, J. W. Twisk, B. A. Dijkmans, L. Aarden, and G. J. Wolbink. 2011. Development of antidrug antibodies against adalimumab and association with disease activity and treatment failure during long-term follow-up. *JAMA* 305: 1460-

for cloning RNA-binding proteins from cDNA libraries. *Proc Natl Acad Sci U S A* 98:

Benkovic, and R. A. Lerner. 1989. Generation of a large combinatorial library of the

from random combinatorial immunoglobulin libraries. *Proc Natl Acad Sci U S A* 88:

combinatorial library from genomic V segments and synthetic CDR3 fragments. *J* 

factor antibodies-toward improved methods of anti-antibody measurement. *Curr* 

C. C. Kuo, R. Mashayekh, K. Wymore, J. G. McCabe, and a. et. 1994. Antigenspecific human antibodies from mice comprising four distinct genetic

H. Abderrahim, M. Noguchi, D. H. Smith, Y. Zeng, and a. et. 1994. Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and

to minimize its immunogenicity. *Mol Immunol* 41: 863-872.

monoclonal antibodies. *Curr Opin Mol Ther* 5: 172-179.

*Nucleic Acids Res* 32: 1448-1459.

370: 389-391.

3-10.

1468.

12954-12959.

11120-11123.

*Immunol* 151: 4651-4659.

*Opin Immunol* 20: 431-435.

modifications. *Nature* 368: 856-859.

light chain YACs. *Nat Genet* 7: 13-21.

2004. SDR grafting of a murine antibody using multiple human germline templates

encoding DNA sequences: library construction methods for directed evolution.


[46] Nurieva, R. I., P. Treuting, J. Duong, R. A. Flavell, and C. Dong. 2003. Inducible costimulator is essential for collagen-induced arthritis. *J Clin Invest* 111: 701-706. [47] Galicia, G., A. Kasran, C. Uyttenhove, K. De Swert, J. Van Snick, and J. L. Ceuppens.

[48] Matsumoto, I., H. Zhang, T. Yasukochi, K. Iwanami, Y. Tanaka, A. Inoue, D. Goto, S. Ito,

[49] Schellekens, H., and N. Casadevall. 2004. Immunogenicity of recombinant human

[50] Hermeling, S., D. J. Crommelin, H. Schellekens, and W. Jiskoot. 2004. Structureimmunogenicity relationships of therapeutic proteins. *Pharm Res* 21: 897-903. [51] Cohen, B. A., J. Oger, A. Gagnon, and G. Giovannoni. 2008. The implications of

[52] Edwards, J. C., L. Szczepanski, J. Szechinski, A. Filipowicz-Sosnowska, P. Emery, D. R.

[55] Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody

[56] Jones, P. T., P. H. Dear, J. Foote, M. S. Neuberger, and G. Winter. 1986. Replacing the

[57] Whittle, N., J. Adair, C. Lloyd, L. Jenkins, J. Devine, J. Schlom, A. Raubitschek, D.

[58] Studnicka, G. M., S. Soares, M. Better, R. E. Williams, R. Nadell, and A. H. Horwitz.

[59] Gonzales, N. R., E. A. Padlan, R. De Pascalis, P. Schuck, J. Schlom, and S. V. Kashmiri.

autoimmune encephalomyelitis. *J Clin Immunol* 29: 426-433.

proteins: causes and consequences. *J Neurol* 251 Suppl 2: II4-9.

*Arthritis Res Ther* 10: R66.

erythematosus. *Drugs* 66: 1933-1948.

mouse. *Nature* 321: 522-525.

of predefined specificity. *Nature* 256: 495-497.

chimaeric B72.3 antibody. *Protein Eng* 1: 499-505.

application to an anti-CD28. *J Immunol* 169: 1119-1125.

17.

814.

2009. ICOS deficiency results in exacerbated IL-17 mediated experimental

A. Tsutsumi, and T. Sumida. 2008. Therapeutic effects of antibodies to tumor necrosis factor-alpha, interleukin-6 and cytotoxic T-lymphocyte antigen 4 immunoglobulin in mice with glucose-6-phosphate isomerase induced arthritis.

immunogenicity for protein-based multiple sclerosis therapies. *J Neurol Sci* 275: 7-

Close, R. M. Stevens, and T. Shaw. 2004. Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. *N Engl J Med* 350: 2572-2581. [53] Saito, K., M. Nawata, S. Iwata, M. Tokunaga, and Y. Tanaka. 2005. Extremely high titer

of anti-human chimeric antibody following re-treatment with rituximab in a patient with active systemic lupus erythematosus. *Rheumatology (Oxford)* 44: 1462-1464. [54] Sabahi, R., and J. H. Anolik. 2006. B-cell-targeted therapy for systemic lupus

complementarity-determining regions in a human antibody with those from a

Colcher, and M. Bodmer. 1987. Expression in COS cells of a mouse-human

1994. Human-engineered monoclonal antibodies retain full specific binding activity by preserving non-CDR complementarity-modulating residues. *Protein Eng* 7: 805-

2003. Minimizing immunogenicity of the SDR-grafted humanized antibody CC49 by genetic manipulation of the framework residues. *Mol Immunol* 40: 337-349. [60] Tan, P., D. A. Mitchell, T. N. Buss, M. A. Holmes, C. Anasetti, and J. Foote. 2002.

"Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences:


[76] Green, L. L., and A. Jakobovits. 1998. Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. *J Exp Med* 188: 483-495.

**13** 

*Poland* 

**The Emerging Role of Monoclonal** 

**Lupus Erythematosus** 

Ewa Robak and Tadeusz Robak

*Medical University of Lodz* 

**Antibodies in the Treatmentof Systemic** 

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell hyperactivity and defective T-cell function, with production of high titer autoantibodies and clinical involvement in multiple organ systems. Patients with mild SLE can generally be maintained on a combination of non-steroidal anti-inflammatory drugs and antimalarials. Corticosteroids, azathioprine and cyclophosphamide remain important for long term management of most patients with active disease and even those in clinical remission. However, these agents have considerable side effects and are not effective in all patients with SLE. Novel immunological therapies include both B and T cell directed treatments, anticytokine and complement directed therapies. These modalities enable more specific immunosuppression, and include cyclosporin, high-dose intravenous immunoglobulin, mycophenolate mofetil, tacrolimus and new purine nucleoside analogs (Schröder and

In recent years, clinical studies have been undertaken with selected monoclonal antibodies (mAbs) in the treatment of several hematological diseases, especially in malignant disorders. However, some clinical observations indicate that mAbs may be an important alternative for

B-lymphocytes are an essential component of the acquired immune response (La Cava 2010). They randomly express cell-surface receptors which are often autoreactive and must be controlled by the process of B-cell tolerance. In SLE, the number of B-cells in the peripheral blood is often decreased, and those that are present have abnormal phenotypes indicative of activation. The important role of B cells in the pathogenesis of SLE has provided a strong rationale to target B cells in SLE. Selective therapeutic depletion of B-cells

The CD20 (B1) antigen is a 33–35 kDa integral membrane protein expressed on the surfaces of non-malignant and most malignant B cells (Cragg, Walshe et al. 2005). The CD20 protein consists of cytoplasmic N- and C-termini and four hydrophobic regions for anchoring the molecule in the membrane (Robak 2008). The characteristics that make CD20 a good target

the conventional therapy of some autoimmune disorders (Robak 2004).

became possible with the availability of the anti-CD20 antibody rituximab.

**2. Anti-CD20 monoclonal antibodies** 

**1. Introduction** 

Zeunerorts 2009).

[77] Lonberg, N. 2005. Human antibodies from transgenic animals. *Nat Biotechnol* 23: 1117- 1125.
