**4. Inhibition of cell-cell contacts and survival factors**

Several other monoclonal antibodies currently under clinical trial target B cell survival factors and cytokines like B-cell activating factor (BAFF), a proliferation inducing ligand (APRIL), IL-6 or IL-10.

*Inhibition of the B cell survival factors BAFF and APRIL* 

BAFF and APRIL are members of the TNF superfamily that maintain peripheral B cell- and plasma cell homeostasis by supporting cell survival. Access to BAFF modifies the stringency of negative selection of naïve B cells, as auto-reactive B cells depend more on BAFF relative to naïve mature cells. BAFF is produced by neutrophil granulocytes, monocytes, macrophages, dendritic cells and T cells as a trans-membrane protein and cleaved from the cell surface by the protease furin (39). In the serum, BAFF and APRIL are found as both homo- and hetero-trimers. The expression of receptors for soluble BAFF (BR3/BAFFR, TACI and BCMA) varies depending on the B-cell developmental stage. Highest levels of BR3/BAFFR are observed on primary and activated follicular and marginal B cells, while expression is decreased but still detectable on germinal center B cells. BR3/BAFFR is reduced or absent on antibody producing plasma cells, whereas TACI and BCMA are abundantly expressed on these cells. Memory B cells express all three BAFF-receptors. Neutralizing antibodies against BAFF cause a loss of transitional-2, marginal zone and follicular B cells *in vivo*, but transitional-1, B1 B cells, and plasma cells are not affected because latter cells receive survival signals by TACI as well (40).


Table 1. Examples of therapeutic antibodies for the treatment of autoimmune disorders approved by the FDA

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 treatment of RA (43, 44, 45).

#### *Inhibition of B-T cell interactions*

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

every second week it was reported to improve clinical symptoms in all of the patients based

Several other monoclonal antibodies currently under clinical trial target B cell survival factors and cytokines like B-cell activating factor (BAFF), a proliferation inducing ligand

BAFF and APRIL are members of the TNF superfamily that maintain peripheral B cell- and plasma cell homeostasis by supporting cell survival. Access to BAFF modifies the stringency of negative selection of naïve B cells, as auto-reactive B cells depend more on BAFF relative to naïve mature cells. BAFF is produced by neutrophil granulocytes, monocytes, macrophages, dendritic cells and T cells as a trans-membrane protein and cleaved from the cell surface by the protease furin (39). In the serum, BAFF and APRIL are found as both homo- and hetero-trimers. The expression of receptors for soluble BAFF (BR3/BAFFR, TACI and BCMA) varies depending on the B-cell developmental stage. Highest levels of BR3/BAFFR are observed on primary and activated follicular and marginal B cells, while expression is decreased but still detectable on germinal center B cells. BR3/BAFFR is reduced or absent on antibody producing plasma cells, whereas TACI and BCMA are abundantly expressed on these cells. Memory B cells express all three BAFF-receptors. Neutralizing antibodies against BAFF cause a loss of transitional-2, marginal zone and follicular B cells *in vivo*, but transitional-1, B1 B cells, and plasma cells are not affected

Table 1. Examples of therapeutic antibodies for the treatment of autoimmune disorders

on the 6-, 10- or 18-week assessments (38).

(APRIL), IL-6 or IL-10.

approved by the FDA

**4. Inhibition of cell-cell contacts and survival factors** 

because latter cells receive survival signals by TACI as well (40).

*Inhibition of the B cell survival factors BAFF and APRIL* 

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

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 antibody producing plasma cells.

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 of infections and provides no additional benefits (11).

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 monoclonal antibody therapies**

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

Application of Monoclonal Antibody Therapies in Autoimmune Diseases 247

after digestion of the variable region with DNase I (62, 63).

frameworks, combined with synthetically created CDR cassettes.

genes need to be inserted into a human frame sequence.

(64, 65, 66).

affinity towards the antigen.

immunosorbent assay (ELISA).

The engrafting of the complementary determining regions (CDR) consists of different steps: the determination of the sequences in the nonhuman antibody that participate in the specific recognition of the antigen, the selection of the fitting human frame to engraft it in, and finally, the assembly of the nonhuman CDR and the human FR to a functional antibody via the insertion of back mutations. In order to minimize the presence of nonhuman sequences within the humanized antibody construct, several methods e.g. specificity determining residue (SDR) engrafting or superhumanization had been proposed. While the first is based on the computational analysis of the three-dimensional structure of the antigen-antibody complex, suggesting that only 20-33% of CDR residues are in contact with the antigen, the latter relies on the *in silico* selection of the best matching canonical structures of both the nonhuman and human sequences (59, 60). For the engraftment, most frequently human germline sequences are used to minimize potential immunogenicity (61). The objective of the back mutations by establishing the functional antibody is to maintain, or if possible, even improve the affinity of the antibody. To obtain this several methods exist, such as e.g. error-prone PCR with low fidelity polymerases under nonstandard conditions or pooling random DNA fragments

Despite the fact that humanized antibodies retain less than 5% of the murine sequences, a significant anti-drug response can still be observed in 0.1-9% of the treated patients

Besides attempts to avoid immunogenicity, antigen display also provides the best method to overcome limitations of the hybridoma technology concerning toxic or highly conserved antigen structures. Several display systems that apply insertion of variable region fragments to the phage genome have been developed for phage T7 (67), phage **(**68, 69, 70) and the Ff class (genus Inovirus) of the filamentous phage f1, fd, and M13 (71). The source for antibody fragments can either be a naive, a semi-synthetic or an entirely synthetic library. Naive libraries are generated by mRNA isolation and cDNA synthesis from B cells (naïve or antigen exposed cells), and the variable region genes are either expressed separately with a two or three step cloning strategy or fused as an scFv in a polymerase chain reaction (PCR). If the assembly PCR is involving randomization of the CDR3 region, namely the usage of oligonucleotide primers encoding various CDR3 and J gene segments (72), a semi-synthetic library is established. Entirely synthetic libraries use various different VH and VL germline master

The pIII minor coat protein of the filamentous phage M13 is widely used to fuse the antibody fragment of interest with, thus resulting in the expression of the antibody fragment on the surface of the phage. High affinity binding constructs can be then selected by panning, a method that consists of incubation cycles (2-5) with surfacebound antigen, followed by a restricting washing to remove non-specific clones. Specificity of selected constructs can be evaluated then using enzyme-linked

As a final step for the generation of therapeutic antibodies, the selected variable region

Clinical data show that despite the fully human sequence, many of the monoclonal antibodies produced by phage display technology e.g. the TNF-specific adalimumab

c. Selection of human antibody V regions using a phage library screen based on the

muronomab itself) had already shown us (49). The immune system may recognize these structures as foreign and develop an antibody response towards them, which may result in reduction of bioactivity. Factors like the sequence of the antibody, the secondary structure, the purity of the product, the dosage and frequency of administration, the diversity in MHC alleles within the population, the site of injection and the physical status of the individual all contribute to the outcome of the immunological response towards the protein-based medication (50, 51).

The clinical effects of the antibodies raised against the therapeutic protein depend on the epitope they recognize, their affinity and titer: some cause no reduction in bioactivity while others induce complications besides affecting the therapeutic benefits. These complications include increased risk of infusion reactions such as fever or rashes, pure red-cell aplasia or even cardiopulmonary and anaphylactic-like adverse events (52). In general, the emerging antibody response can inhibit the binding of the ligand to the receptor, or change the conformation and therefore the affinity and signaling properties of the soluble mediator (e.g. in case of IFNβ treatment). Immunological responses against monoclonal antibodies often enhance clearance by complex formation or block target recognition by binding to the variable region (idiotype). Rituximab, a mouse-human chimeric antibody induces the development of human anti-chimeric antibodies (HACA) in 1-5% of Non Hodgin's lymphoma and RA patients and in 65% of SLE patients, resulting in a reduction of the efficacy of B cell depletion and often in hypersensitivity reactions (53, 54).

Several different techniques exist to attenuate the break-through of tolerance against monoclonal antibodies, these include:

a. The replacement of murine constant regions with human sequences, where the specificity of the antibody remains intact (chimera design)

The hybridoma technology enabled us to produce monoclonal murine antibodies in large quantities (55). On the other hand, generation of human hybridomas is difficult because they produce only small amounts of IgM and are very unstable. Recombinant DNA techniques make it possible to change the constant regions of murine immunoglobulins to human domains (56). The heavy and light chain genes are clustered into exons that represent the domain structures, which facilitates domain exchanges in antibody molecules. The vector selection is a critical step in the *in vitro* production of these monoclonal antibody constructs, as glycosylation patterns highly vary between species. Mammalian non-immune cells (HeLa or CHO cells) or certain myeloma cell lines are frequently used for this purpose. (57)

The choice of the heavy chain isotype frequently defines the mechanism of action: IgG1 is binding with high affinity to FcRs, therefore, the therapeutic antibody of such isotype is more likely to cause additional cell depletion via antibody-dependent cellmediated cytotoxicity (ADCC). On the other hand IgG2 interacts only weakly with low affinity FcRs, so the therapeutic effects observed are mainly attributed to the capacity of the antibody for blocking or altering cellular signaling through the targeted receptor.

b. Humanization or reshaping of the variable region, thus partial exchange of framework residues to human sequences

The antigen specificity of the antibody is defined by only a few amino acids that are exposed to the surface of the antigen-binding pocket (paratope) and interact with the antigen. By transferring this set of residues from a nonhuman origin to a human frame (FR), the specificity of the antibody should remain (58).

muronomab itself) had already shown us (49). The immune system may recognize these structures as foreign and develop an antibody response towards them, which may result in reduction of bioactivity. Factors like the sequence of the antibody, the secondary structure, the purity of the product, the dosage and frequency of administration, the diversity in MHC alleles within the population, the site of injection and the physical status of the individual all contribute to the outcome of the immunological response towards the protein-based

The clinical effects of the antibodies raised against the therapeutic protein depend on the epitope they recognize, their affinity and titer: some cause no reduction in bioactivity while others induce complications besides affecting the therapeutic benefits. These complications include increased risk of infusion reactions such as fever or rashes, pure red-cell aplasia or even cardiopulmonary and anaphylactic-like adverse events (52). In general, the emerging antibody response can inhibit the binding of the ligand to the receptor, or change the conformation and therefore the affinity and signaling properties of the soluble mediator (e.g. in case of IFNβ treatment). Immunological responses against monoclonal antibodies often enhance clearance by complex formation or block target recognition by binding to the variable region (idiotype). Rituximab, a mouse-human chimeric antibody induces the development of human anti-chimeric antibodies (HACA) in 1-5% of Non Hodgin's lymphoma and RA patients and in 65% of SLE patients, resulting in a reduction of the

Several different techniques exist to attenuate the break-through of tolerance against

a. The replacement of murine constant regions with human sequences, where the

The hybridoma technology enabled us to produce monoclonal murine antibodies in large quantities (55). On the other hand, generation of human hybridomas is difficult because they produce only small amounts of IgM and are very unstable. Recombinant DNA techniques make it possible to change the constant regions of murine immunoglobulins to human domains (56). The heavy and light chain genes are clustered into exons that represent the domain structures, which facilitates domain exchanges in antibody molecules. The vector selection is a critical step in the *in vitro* production of these monoclonal antibody constructs, as glycosylation patterns highly vary between species. Mammalian non-immune cells (HeLa or CHO cells) or certain

The choice of the heavy chain isotype frequently defines the mechanism of action: IgG1 is binding with high affinity to FcRs, therefore, the therapeutic antibody of such isotype is more likely to cause additional cell depletion via antibody-dependent cellmediated cytotoxicity (ADCC). On the other hand IgG2 interacts only weakly with low affinity FcRs, so the therapeutic effects observed are mainly attributed to the capacity of the antibody for blocking or altering cellular signaling through the targeted receptor. b. Humanization or reshaping of the variable region, thus partial exchange of framework

The antigen specificity of the antibody is defined by only a few amino acids that are exposed to the surface of the antigen-binding pocket (paratope) and interact with the antigen. By transferring this set of residues from a nonhuman origin to a human frame

efficacy of B cell depletion and often in hypersensitivity reactions (53, 54).

specificity of the antibody remains intact (chimera design)

myeloma cell lines are frequently used for this purpose. (57)

(FR), the specificity of the antibody should remain (58).

medication (50, 51).

monoclonal antibodies, these include:

residues to human sequences

The engrafting of the complementary determining regions (CDR) consists of different steps: the determination of the sequences in the nonhuman antibody that participate in the specific recognition of the antigen, the selection of the fitting human frame to engraft it in, and finally, the assembly of the nonhuman CDR and the human FR to a functional antibody via the insertion of back mutations. In order to minimize the presence of nonhuman sequences within the humanized antibody construct, several methods e.g. specificity determining residue (SDR) engrafting or superhumanization had been proposed. While the first is based on the computational analysis of the three-dimensional structure of the antigen-antibody complex, suggesting that only 20-33% of CDR residues are in contact with the antigen, the latter relies on the *in silico* selection of the best matching canonical structures of both the nonhuman and human sequences (59, 60).

For the engraftment, most frequently human germline sequences are used to minimize potential immunogenicity (61). The objective of the back mutations by establishing the functional antibody is to maintain, or if possible, even improve the affinity of the antibody. To obtain this several methods exist, such as e.g. error-prone PCR with low fidelity polymerases under nonstandard conditions or pooling random DNA fragments after digestion of the variable region with DNase I (62, 63).

Despite the fact that humanized antibodies retain less than 5% of the murine sequences, a significant anti-drug response can still be observed in 0.1-9% of the treated patients (64, 65, 66).

c. Selection of human antibody V regions using a phage library screen based on the affinity towards the antigen.

Besides attempts to avoid immunogenicity, antigen display also provides the best method to overcome limitations of the hybridoma technology concerning toxic or highly conserved antigen structures. Several display systems that apply insertion of variable region fragments to the phage genome have been developed for phage T7 (67), phage **(**68, 69, 70) and the Ff class (genus Inovirus) of the filamentous phage f1, fd, and M13 (71). The source for antibody fragments can either be a naive, a semi-synthetic or an entirely synthetic library. Naive libraries are generated by mRNA isolation and cDNA synthesis from B cells (naïve or antigen exposed cells), and the variable region genes are either expressed separately with a two or three step cloning strategy or fused as an scFv in a polymerase chain reaction (PCR). If the assembly PCR is involving randomization of the CDR3 region, namely the usage of oligonucleotide primers encoding various CDR3 and J gene segments (72), a semi-synthetic library is established. Entirely synthetic libraries use various different VH and VL germline master frameworks, combined with synthetically created CDR cassettes.

The pIII minor coat protein of the filamentous phage M13 is widely used to fuse the antibody fragment of interest with, thus resulting in the expression of the antibody fragment on the surface of the phage. High affinity binding constructs can be then selected by panning, a method that consists of incubation cycles (2-5) with surfacebound antigen, followed by a restricting washing to remove non-specific clones. Specificity of selected constructs can be evaluated then using enzyme-linked immunosorbent assay (ELISA).

As a final step for the generation of therapeutic antibodies, the selected variable region genes need to be inserted into a human frame sequence.

Clinical data show that despite the fully human sequence, many of the monoclonal antibodies produced by phage display technology e.g. the TNF-specific adalimumab 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 attributes such as stability and aggregation besides molecular recognition.

Application of Monoclonal Antibody Therapies in Autoimmune Diseases 249

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d. Human antibody production of transgenic mice expressing human immunoglobulin genes

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 targeted antigen.

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 expression system is established to reduce costs (77).

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 monoclonal constructs and their origin.
