BACKGROUND
sialoadhesin-expressing  macrophages have gained increased attention lately because of their unique distribution in lymphoid organs and their redistribution upon immune activation  <cit> .
situated in the spleen and other secondary lymphoid tissues, sn+ macrophages appear to be strategically placed for antigen acquisition and delivery to lymphocytes.
junt et al.
showed that in lymph nodes, sn+ macrophages capture viral particles  within minutes after subcutaneous injection, transport them across the subcapsular sinus floor and present them to migrating b cells in the underlying follicles  <cit> .
moreover, also particulate antigen and immune complexes have been shown to be captured and displayed by sn+ macrophages  <cit> .
besides, sn+ macrophages present lipid antigens in a cd1d dependent manner to inkt cells, leading to inkt cell activation and population expansion  <cit> .
also, sn+ macrophages have been implicated in the activation of cd8+ t cells by either directly presenting antigen to cd8+ t cells  <cit>  or by transferring the antigen to cd8+ dendritic cells in the spleen  <cit> .
ultimately, several independent research groups showed an enhanced cellular and/or humoral immune response upon sn-targeted antigen delivery  <cit> .
together, this suggests that sn+ macrophages may act as specialized antigen presenting cells involved in the antigen transport chain and contribute to the growing interest in sn+ macrophages for vaccination strategies, as recently reviewed by martinez-pomares and gordon  <cit> .
sn  is also present on inflammatory macrophages and activated monocytes  <cit> .
in affected tissue samples of rheumatoid arthritis patients for instance, high expression of sn was found on inflammatory macrophages  <cit> .
in addition, abundant sn expression on inflammatory monocytes/macrophages was shown to correlate with disease severity in pathological conditions like multiple sclerosis, atherosclerosis and breast cancer  <cit> .
these observations promote the idea of an sn-directed cell therapy aimed at elimination or immunomodulation of these cells.
recently, targeted delivery of an anti-tnf-α oligonucleotide to sn+ macrophages resulted in the relief of lupus-like symptoms in mice  <cit> , further showing the potential of sn+ macrophages as a target for immunomodulation.
sn seems thus a promising target for cell-directed therapy, a strategy that is further encouraged by the restricted expression pattern of sn limiting unwanted side effects.
furthermore, since sn was suggested to be an endocytic receptor  <cit> , the conjugation of antigens, toxins or drugs to an sn-specific antibody should allow intracellular delivery of these conjugates.
so far, three systems have been used to selectively target sn+ macrophages, namely antibodies, glycan-coated liposomes and cationic agarose hydrogels  <cit> .
while the latter was unintentionally identified as an sn-targeting system  <cit> , the glycans on the glycan-coated liposomes were specifically designed to be ligands with a high specificity and affinity for sn  <cit> .
previously, our research group has made use of an sn-specific monoclonal antibody  to generate functional sn-specific immunoconjugates via chemical coupling  <cit> .
although successful, this system implies, just like the two other described sn-targeting systems, that the cargo to be targeted is independently produced and purified, which translates into significant optimization for each separate conjugate.
also, chemical coupling to antibodies depends on the presence and distribution of reactive groups, e.g.
primary amines on lysine residues, that can be located in or near the antigen-binding region.
upon coupling, this might result in partial or complete loss of the antibody’s capacity to bind to the target antigen.
in addition, there is limited stoichiometric control because of the large number of reactive groups present in an antibody, leading to a heterogeneous mixture that makes batch to batch consistency hard to effectuate  <cit> .
to overcome these problems associated with chemical conjugation of a cargo to an sn-specific mab, we report here on the development of a versatile recombinant antibody vector that allows easy production and purification of defined genetic antibody fusion constructs.
the obtained vector will allow us to generate functional antibody-cargo constructs to evaluate sn-targeting strategies in a porcine animal model.
RESULTS
development of a sialoadhesin-specific recombinant antibody, rec41d3
to allow production and purification of genetic fusion proteins of an sn-specific mab and other peptides/proteins, a recombinant sn-specific mab was made.
mab 41d <dig> was previously described to selectively bind porcine sn  and to be internalized into psn-expressing macrophages  <cit> .
the variable domains of mab 41d <dig> were sequenced and cloned in frame with a mouse igg <dig> backbone.
the igg <dig> backbone was modified so that the resulting plasmid p41d <dig> contains a c-terminal heavy chain multiple cloning site, allowing removal of the antibody’s heavy chain stop codon and insertion of protein encoding sequences .
transfection of hek293t cells with plasmid p41d <dig> led to the secretion of a fully assembled igg into the culture medium of the same size as mab 41d <dig>  as shown by western blot analysis of cell culture supernatants using polyclonal antibodies specific for mouse immunoglobulins .
recombinant antibodies were purified from cell culture medium using protein g chromatography and dialyzed to pbs.
to assess the purity of rec41d <dig> after purification, protein samples were resolved via sds-page under non-reducing and reducing conditions and coomassie blue staining was performed .
the presence of a single band under non-reducing conditions and the presence of two bands under reducing conditions consistent with the sizes of the heavy and light chains of an immunoglobulin showed that protein g purification yields pure rec41d <dig>  western blot analysis further confirmed that the bands present were antibody light and heavy chain fragments .
immunofluorescence stainings of cho cells expressing recombinant psn  were performed to assess the specificity of rec41d <dig> for psn .
with rec41d <dig>  a bright surface staining, similar to that of the native mab 41d <dig>  was seen only on cho-psn cells, but not on wild type cho cells, indicating specific psn recognition of rec41d <dig>  control stainings were performed with isotype matched irrelevant mab 13d <dig> , and its recombinant form rec13d <dig>  development, production and purification of rec13d <dig> was identical to the procedures used for rec41d <dig>
since the final goal is to use rec41d <dig> as a targeting molecule for psn, the affinity of rec41d <dig> for psn is highly important.
therefore, its affinity was determined using biacore.
as a target, a soluble form of the psn receptor, consisting of the first 4 n-terminal immunoglobulin-like domains of psn fused to a human igg fc , was coated on the affinity chips.
previously, mab 41d <dig> was shown to bind to this psn4d-fc  <cit> .
as seen in figure 1f, both mab 41d <dig> and rec41d <dig> bound with high affinity to psn4d-fc, their equilibrium dissociation constants  were determined to be  <dig> and  <dig> pm respectively.
rec41d <dig> induces psn endocytosis in primary macrophages
as our ultimate goal is to target psn+ macrophages in vivo, we studied the capacity of rec41d <dig> to bind psn and induce its internalization in in vitro cultivated primary cells.
primary porcine alveolar macrophages  were isolated and incubated with the recombinant antibody for different time periods, after which they were fixed and stained to visualize membrane-bound and internalized antibodies.
as for mab 41d <dig>  a clear membrane staining was observed at time zero, while with increasing time, psn-positive endocytic vesicles became readily apparent .
also, at early time points, endocytic vesicles of both antibodies were mainly present in the vicinity of the plasma membrane, while with increasing time endocytosed psn was also localized closer to the perinuclear region.
similar to mab 41d3-induced psn endocytosis, rec41d3-induced psn endocytosis is only partial, as confocal microscopical analysis showed that a clear membrane staining remains visible besides the endocytic vesicles.
as a control, pam were incubated with irrelevant, isotype matched mab 13d <dig> and rec13d <dig>  no cell staining was observed with these antibodies .
in a previous study, we have shown that mab 41d <dig> resides for prolonged times in early endosomes  <cit> .
to analyze the intracellular localization of internalized rec41d <dig> in comparison to mab 41d <dig>  double immunofluorescence stainings were performed with eea <dig>  ci-m6p or lamp <dig>  markers for early endosomes, late endosomes and lysosomes respectively.
for both antibodies, the majority of internalized antibody was localized to early endosomes , while the remainder was localized to late endosomes.
occasionally, a very limited number of internalized antibodies were localized in a lysosomal compartment.
these results show that rec41d <dig> follows an endocytic pathway similar to mab 41d <dig> and resides for prolonged times in early/late endosomes.
rec41d <dig> targets its cargo v <dig> as well as egfp towards psn+ cells
the previous results clearly show that rec41d <dig> can be used to target psn-expressing macrophages.
to be able to evaluate targeting of a cargo, we aimed at generating functional antibody fusion constructs in which a cargo is coupled to the c-terminus of the heavy chain of the antibody.
during the generation of the rec41d <dig> plasmid, a multiple cloning site was introduced at the c-terminus to facilitate this.
also, a flexible glycine-serine  linker  <cit>  was introduced between the c-terminal fc part and the cargo linked to rec41d <dig>  this minimizes the risk that unwanted interactions occur between rec41d <dig> and its cargo, which could result in non-functional antibody and/or cargo.
two fusion constructs were generated: one construct containing a  <dig> linker fused to a v <dig> peptide tag , the other one containing a  <dig> linker fused to egfp .
the gs linker used to generate rec41d3-egfp is long, to ensure egfp has the opportunity to fold into a functional protein.
hek293t cell transfection followed by protein g purification of the supernatant clearly predominantly yields intact fusion proteins as shown by sds-page .
under non-reducing conditions a single band was visible for both fusion proteins, shifted in size in comparison to unmodified rec41d <dig>  under reducing conditions, it became clear that the antibody’s light chains remained unchanged, while the heavy chains were shifted in size, showing the acquisition of extra protein sequences.
the presence of v <dig> or egfp at the heavy chains of rec41d <dig> was further confirmed by western blot analysis .
these results indicate that rec41d3-v <dig> contains a v <dig> peptide tag, while rec41d3-egfp contains an egfp molecule.
to further analyze whether or not rec41d3-v <dig> and rec41d3-egfp carry their cargo towards psn+ cells and induce internalization into the cells, we incubated cho-psn and pam with the fusion constructs for one hour at 37°c.
as shown in figure 3c, both v <dig> and egfp were co-internalized together with rec41d <dig> in both cell types.
as a control, cho-psn and pam were incubated with irrelevant, isotype matched rec13d <dig> fusion constructs rec13d12-v <dig> and rec13d12-egfp.
no staining was observed with these constructs .
in conclusion, these data show that rec41d <dig> allows the targeting of peptides and/or proteins towards psn+ cells.
discussion
monoclonal antibodies and derivatives are currently the fastest growing class of therapeutic molecules  <cit> .
their inherent promise to minimize side effects by selectively targeting specific target cells has fuelled their development, leading to several fda-approved antibody therapeutics so far and many more in the pipeline.
although unmodified mabs proved their worth, the conjugation of effector molecules  to mabs broadened their therapeutic potential, especially in the domain of cancer therapeutics.
besides cancer, other diseases could also benefit from antibody-directed therapies, the only prerequisite being the identification of a receptor exclusively expressed on those immune cells involved in the induction of pathology.
in this respect, sialic acid-binding immunoglobulin-like lectins  are compelling candidates for therapy, as they display very restricted expression patterns on subsets of immune cells and may regulate immune cell functions.
furthermore, siglecs are endocytic receptors allowing therapeutic agents conjugated to a mab to be carried efficiently into the cell  <cit> .
sialoadhesin or siglec- <dig> is expressed on cells of the monocyte/macrophage lineage, notably on subsets of resident tissue macrophages and inflammatory monocytes/macrophages  <cit> .
a recent report describes the expression of sn on human mature dcs treated with lps in vitro <cit> , suggesting that sn may be present on mature dcs during inflammation in vivo as well.
not only have sn+ monocytes/macrophages been described in several diseases like inflammatory and autoimmune disorders as well as viral infections, they also appear to play a role in the initiation of an adaptive immune response as recently shown by different independent research groups and nicely reviewed by martinez-pomares and gordon  <cit> .
together, this makes these sn+ cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination.
in our previous study, we developed immunoconjugates by the chemical linkage of the model antigen hsa or a toxin to the psn-specific mab 41d <dig>  <cit> .
although these immunoconjugates proved efficient for boosting immune responses and killing psn-expressing cells respectively, the chemical linkage of the cargo to a targeting antibody has many disadvantages.
first of all, chemical coupling procedures rely on the presence and distribution of reactive groups, like e.g.
primary amines on lysine residues, that can be located in or near the antigen-binding region, which upon coupling might result in partial or complete loss of the antibody’s affinity for the target antigen.
secondly, because of the large number of reactive groups present in antibody molecules, a typical distribution can be observed of zero to eight molecules per antibody  <cit> , resulting in high variation of the final conjugate.
this variation is unwanted, as it may lead to a heterogeneous mixture of components with distinct affinities, stabilities, pharmacokinetics, efficacies, and safety profiles  <cit> .
moreover, chemical coupling implies that both antibody and cargo to be linked are independently produced and purified, which represents a significant challenge, especially when the cargo is also a biologic.
to circumvent these problems, we opted to generate a recombinant form of the psn-specific mab 41d <dig>  as shown in this study, this recombinant antibody displays a comparable affinity for psn compared to the native mab.
in addition, the recombinant mab also induces psn endocytosis in primary macrophages, a feature important to allow functionality of antibody-cargo constructs.
as protein sequences are attached to the c-terminus of the antibody’s heavy chain, they are less likely to hinder antigen binding by the variable immunoglobulin domains.
in addition, each heavy chain will contain only one cargo fused to the c-terminal end.
this will result in an antibody with  <dig> cargos in a defined position and a high intra and inter batch consistency.
furthermore, we could purify the antibody-cargo fusion proteins using standard protein g chromatography, which represents a major advantage compared to chemical coupling in which purification is needed for both cargo and antibody before, as well as after chemical conjugation.
in this study we managed to make genetic fusion constructs of a peptide or a protein linked to our recombinant mab.
obviously, the recombinant antibody vector does not allow to make genetic fusion constructs with chemical compounds.
for vaccination strategies however, this limitation is not expected to pose any problems, as most antigens used in vaccines are protein based.
one challenge however would be to ensure correct folding of the antigen upon genetic fusion to the antibody and to maintain this fold during purification procedures.
similarly, immunotoxins can be made using the sn targeting vector.
although the production of immunotoxins in eukaryotic cells has been limited due to potential toxicity to the producing cells, several independent research groups have reported on the successful production of immunotoxins in mammalian cell lines, including hek293t  <cit> .
in case a specific application would require the chemical linkage to an antibody, e.g.
when vaccines are based on glyco-epitopes, a recombinant mab has some major advantages.
it allows addition of specific amino acid modifications to the antibody, which will result in site-specific incorporation of drug molecules through chemical linkage yielding batch to batch consistency of antibody-drug conjugates.
examples of such already implemented modifications are the thiomabtm technology of genentech inc  <cit>  or the methodology of axup et al.
<cit> .
as our future plans include the use of the developed recombinant antibody to target antigens towards psn-expressing macrophages in vivo, one might be concerned about the immunogenicity of mouse antibodies in pigs.
poderoso et al.
previously used mouse mabs as surrogate antigens in pigs to evaluate the role of sn in the induction of humoral responses and noticed an enhanced anti-mouse antibody response in comparison with a non-targeting isotype control mab  <cit> .
the induction of anti-mouse antibodies was however low after primary injection of the mab, only after a booster vaccination antibody titres rose significantly.
previously, we have observed an enhanced anti-hsa antibody response after a single dose vaccination of hsa coupled to mab 41d <dig> without adverse clinical effects  <cit> .
therefore, in our future experiments, we will use a single dose of rec41d3-antigen to evaluate the protective efficacy of antigen targeting to psn.
if further experiments confirm the applicability of this targeting technology, ‘porcinization’ of the recombinant antibody will be examined to enable prime-booster vaccination schedules.
CONCLUSIONS
a recombinant antibody that targets sialoadhesin was developed.
in addition, we constructed a vector that allows the genetic linkage of a protein cargo in a defined position at the c-terminus of both heavy chains of a fully assembled antibody.
this vector was shown to be versatile, as both a peptide and a more complex, larger protein could be fused.
furthermore, production and purification of the antibody fusion constructs did not require major optimization.
in comparison to other sn targeting strategies , the one-step production of the final carrier-cargo product together with the high specificity and affinity of the recombinant antibody may be considered an advantage for drug development.
future research will mainly focus on the development of functional antibody-antigen fusions allowing the evaluation of sn-directed vaccination strategies.

 sialoadhesin-expressing macrophages have gained increased attention lately because of their unique distribution in lymphoid organs and their redistribution upon immune activation. situated in the spleen and other secondary lymphoid tissues, sn+ macrophages appear to be strategically placed for antigen acquisition and delivery to lymphocytes. junt et al. showed that in lymph nodes, sn+ macrophages capture viral particles within minutes after subcutaneous injection, transport them across the subcapsular sinus floor and present them to migrating b cells in the underlying follicles. moreover, also particulate antigen and immune complexes have been shown to be captured and displayed by sn+ macrophages. besides, sn+ macrophages present lipid antigens in a cd1d dependent manner to inkt cells, leading to inkt cell activation and population expansion. also, sn+ macrophages have been implicated in the activation of cd8+ t cells by either directly presenting antigen to cd8+ t cells or by transferring the antigen to cd8+ dendritic cells in the spleen. ultimately, several independent research groups showed an enhanced cellular and/or humoral immune response upon sn-targeted antigen delivery. together, this suggests that sn+ macrophages may act as specialized antigen presenting cells involved in the antigen transport chain and contribute to the growing interest in sn+ macrophages for vaccination strategies, as recently reviewed by martinez-pomares and gordon. sn is also present on inflammatory macrophages and activated monocytes. in affected tissue samples of rheumatoid arthritis patients for instance, high expression of sn was found on inflammatory macrophages. in addition, abundant sn expression on inflammatory monocytes/macrophages was shown to correlate with disease severity in pathological conditions like multiple sclerosis, atherosclerosis and breast cancer. these observations promote the idea of an sn-directed cell therapy aimed at elimination or immunomodulation of these cells. recently, targeted delivery of an anti-tnf-α oligonucleotide to sn+ macrophages resulted in the relief of lupus-like symptoms in mice, further showing the potential of sn+ macrophages as a target for immunomodulation. sn seems thus a promising target for cell-directed therapy, a strategy that is further encouraged by the restricted expression pattern of sn limiting unwanted side effects. furthermore, since sn was suggested to be an endocytic receptor, the conjugation of antigens, toxins or drugs to an sn-specific antibody should allow intracellular delivery of these conjugates. so far, three systems have been used to selectively target sn+ macrophages, namely antibodies, glycan-coated liposomes and cationic agarose hydrogels. while the latter was unintentionally identified as an sn-targeting system, the glycans on the glycan-coated liposomes were specifically designed to be ligands with a high specificity and affinity for sn. previously, our research group has made use of an sn-specific monoclonal antibody to generate functional sn-specific immunoconjugates via chemical coupling. although successful, this system implies, just like the two other described sn-targeting systems, that the cargo to be targeted is independently produced and purified, which translates into significant optimization for each separate conjugate. also, chemical coupling to antibodies depends on the presence and distribution of reactive groups, e.g. primary amines on lysine residues, that can be located in or near the antigen-binding region. upon coupling, this might result in partial or complete loss of the antibody’s capacity to bind to the target antigen. in addition, there is limited stoichiometric control because of the large number of reactive groups present in an antibody, leading to a heterogeneous mixture that makes batch to batch consistency hard to effectuate. to overcome these problems associated with chemical conjugation of a cargo to an sn-specific mab, we report here on the development of a versatile recombinant antibody vector that allows easy production and purification of defined genetic antibody fusion constructs. the obtained vector will allow us to generate functional antibody-cargo constructs to evaluate sn-targeting strategies in a porcine animal model.  development of a sialoadhesin-specific recombinant antibody, rec41d3 to allow production and purification of genetic fusion proteins of an sn-specific mab and other peptides/proteins, a recombinant sn-specific mab was made. mab 41d was previously described to selectively bind porcine sn and to be internalized into psn-expressing macrophages. the variable domains of mab 41d were sequenced and cloned in frame with a mouse igg backbone. the igg backbone was modified so that the resulting plasmid p41d contains a c-terminal heavy chain multiple cloning site, allowing removal of the antibody’s heavy chain stop codon and insertion of protein encoding sequences. transfection of hek293t cells with plasmid p41d led to the secretion of a fully assembled igg into the culture medium of the same size as mab 41d as shown by western blot analysis of cell culture supernatants using polyclonal antibodies specific for mouse immunoglobulins. recombinant antibodies were purified from cell culture medium using protein g chromatography and dialyzed to pbs. to assess the purity of rec41d after purification, protein samples were resolved via sds-page under non-reducing and reducing conditions and coomassie blue staining was performed. the presence of a single band under non-reducing conditions and the presence of two bands under reducing conditions consistent with the sizes of the heavy and light chains of an immunoglobulin showed that protein g purification yields pure rec41d western blot analysis further confirmed that the bands present were antibody light and heavy chain fragments. immunofluorescence stainings of cho cells expressing recombinant psn were performed to assess the specificity of rec41d for psn. with rec41d a bright surface staining, similar to that of the native mab 41d was seen only on cho-psn cells, but not on wild type cho cells, indicating specific psn recognition of rec41d control stainings were performed with isotype matched irrelevant mab 13d, and its recombinant form rec13d development, production and purification of rec13d was identical to the procedures used for rec41d since the final goal is to use rec41d as a targeting molecule for psn, the affinity of rec41d for psn is highly important. therefore, its affinity was determined using biacore. as a target, a soluble form of the psn receptor, consisting of the first 4 n-terminal immunoglobulin-like domains of psn fused to a human igg fc, was coated on the affinity chips. previously, mab 41d was shown to bind to this psn4d-fc. as seen in figure 1f, both mab 41d and rec41d bound with high affinity to psn4d-fc, their equilibrium dissociation constants were determined to be and pm respectively. rec41d induces psn endocytosis in primary macrophages as our ultimate goal is to target psn+ macrophages in vivo, we studied the capacity of rec41d to bind psn and induce its internalization in in vitro cultivated primary cells. primary porcine alveolar macrophages were isolated and incubated with the recombinant antibody for different time periods, after which they were fixed and stained to visualize membrane-bound and internalized antibodies. as for mab 41d a clear membrane staining was observed at time zero, while with increasing time, psn-positive endocytic vesicles became readily apparent. also, at early time points, endocytic vesicles of both antibodies were mainly present in the vicinity of the plasma membrane, while with increasing time endocytosed psn was also localized closer to the perinuclear region. similar to mab 41d3-induced psn endocytosis, rec41d3-induced psn endocytosis is only partial, as confocal microscopical analysis showed that a clear membrane staining remains visible besides the endocytic vesicles. as a control, pam were incubated with irrelevant, isotype matched mab 13d and rec13d no cell staining was observed with these antibodies. in a previous study, we have shown that mab 41d resides for prolonged times in early endosomes. to analyze the intracellular localization of internalized rec41d in comparison to mab 41d double immunofluorescence stainings were performed with eea ci-m6p or lamp markers for early endosomes, late endosomes and lysosomes respectively. for both antibodies, the majority of internalized antibody was localized to early endosomes, while the remainder was localized to late endosomes. occasionally, a very limited number of internalized antibodies were localized in a lysosomal compartment. these results show that rec41d follows an endocytic pathway similar to mab 41d and resides for prolonged times in early/late endosomes. rec41d targets its cargo v as well as egfp towards psn+ cells the previous results clearly show that rec41d can be used to target psn-expressing macrophages. to be able to evaluate targeting of a cargo, we aimed at generating functional antibody fusion constructs in which a cargo is coupled to the c-terminus of the heavy chain of the antibody. during the generation of the rec41d plasmid, a multiple cloning site was introduced at the c-terminus to facilitate this. also, a flexible glycine-serine linker was introduced between the c-terminal fc part and the cargo linked to rec41d this minimizes the risk that unwanted interactions occur between rec41d and its cargo, which could result in non-functional antibody and/or cargo. two fusion constructs were generated: one construct containing a linker fused to a v peptide tag, the other one containing a linker fused to egfp. the gs linker used to generate rec41d3-egfp is long, to ensure egfp has the opportunity to fold into a functional protein. hek293t cell transfection followed by protein g purification of the supernatant clearly predominantly yields intact fusion proteins as shown by sds-page. under non-reducing conditions a single band was visible for both fusion proteins, shifted in size in comparison to unmodified rec41d under reducing conditions, it became clear that the antibody’s light chains remained unchanged, while the heavy chains were shifted in size, showing the acquisition of extra protein sequences. the presence of v or egfp at the heavy chains of rec41d was further confirmed by western blot analysis. these results indicate that rec41d3-v contains a v peptide tag, while rec41d3-egfp contains an egfp molecule. to further analyze whether or not rec41d3-v and rec41d3-egfp carry their cargo towards psn+ cells and induce internalization into the cells, we incubated cho-psn and pam with the fusion constructs for one hour at 37°c. as shown in figure 3c, both v and egfp were co-internalized together with rec41d in both cell types. as a control, cho-psn and pam were incubated with irrelevant, isotype matched rec13d fusion constructs rec13d12-v and rec13d12-egfp. no staining was observed with these constructs. in conclusion, these data show that rec41d allows the targeting of peptides and/or proteins towards psn+ cells. discussion monoclonal antibodies and derivatives are currently the fastest growing class of therapeutic molecules. their inherent promise to minimize side effects by selectively targeting specific target cells has fuelled their development, leading to several fda-approved antibody therapeutics so far and many more in the pipeline. although unmodified mabs proved their worth, the conjugation of effector molecules to mabs broadened their therapeutic potential, especially in the domain of cancer therapeutics. besides cancer, other diseases could also benefit from antibody-directed therapies, the only prerequisite being the identification of a receptor exclusively expressed on those immune cells involved in the induction of pathology. in this respect, sialic acid-binding immunoglobulin-like lectins are compelling candidates for therapy, as they display very restricted expression patterns on subsets of immune cells and may regulate immune cell functions. furthermore, siglecs are endocytic receptors allowing therapeutic agents conjugated to a mab to be carried efficiently into the cell. sialoadhesin or siglec- is expressed on cells of the monocyte/macrophage lineage, notably on subsets of resident tissue macrophages and inflammatory monocytes/macrophages. a recent report describes the expression of sn on human mature dcs treated with lps in vitro, suggesting that sn may be present on mature dcs during inflammation in vivo as well. not only have sn+ monocytes/macrophages been described in several diseases like inflammatory and autoimmune disorders as well as viral infections, they also appear to play a role in the initiation of an adaptive immune response as recently shown by different independent research groups and nicely reviewed by martinez-pomares and gordon. together, this makes these sn+ cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. in our previous study, we developed immunoconjugates by the chemical linkage of the model antigen hsa or a toxin to the psn-specific mab 41d. although these immunoconjugates proved efficient for boosting immune responses and killing psn-expressing cells respectively, the chemical linkage of the cargo to a targeting antibody has many disadvantages. first of all, chemical coupling procedures rely on the presence and distribution of reactive groups, like e.g. primary amines on lysine residues, that can be located in or near the antigen-binding region, which upon coupling might result in partial or complete loss of the antibody’s affinity for the target antigen. secondly, because of the large number of reactive groups present in antibody molecules, a typical distribution can be observed of zero to eight molecules per antibody, resulting in high variation of the final conjugate. this variation is unwanted, as it may lead to a heterogeneous mixture of components with distinct affinities, stabilities, pharmacokinetics, efficacies, and safety profiles. moreover, chemical coupling implies that both antibody and cargo to be linked are independently produced and purified, which represents a significant challenge, especially when the cargo is also a biologic. to circumvent these problems, we opted to generate a recombinant form of the psn-specific mab 41d as shown in this study, this recombinant antibody displays a comparable affinity for psn compared to the native mab. in addition, the recombinant mab also induces psn endocytosis in primary macrophages, a feature important to allow functionality of antibody-cargo constructs. as protein sequences are attached to the c-terminus of the antibody’s heavy chain, they are less likely to hinder antigen binding by the variable immunoglobulin domains. in addition, each heavy chain will contain only one cargo fused to the c-terminal end. this will result in an antibody with cargos in a defined position and a high intra and inter batch consistency. furthermore, we could purify the antibody-cargo fusion proteins using standard protein g chromatography, which represents a major advantage compared to chemical coupling in which purification is needed for both cargo and antibody before, as well as after chemical conjugation. in this study we managed to make genetic fusion constructs of a peptide or a protein linked to our recombinant mab. obviously, the recombinant antibody vector does not allow to make genetic fusion constructs with chemical compounds. for vaccination strategies however, this limitation is not expected to pose any problems, as most antigens used in vaccines are protein based. one challenge however would be to ensure correct folding of the antigen upon genetic fusion to the antibody and to maintain this fold during purification procedures. similarly, immunotoxins can be made using the sn targeting vector. although the production of immunotoxins in eukaryotic cells has been limited due to potential toxicity to the producing cells, several independent research groups have reported on the successful production of immunotoxins in mammalian cell lines, including hek293t. in case a specific application would require the chemical linkage to an antibody, e.g. when vaccines are based on glyco-epitopes, a recombinant mab has some major advantages. it allows addition of specific amino acid modifications to the antibody, which will result in site-specific incorporation of drug molecules through chemical linkage yielding batch to batch consistency of antibody-drug conjugates. examples of such already implemented modifications are the thiomabtm technology of genentech inc or the methodology of axup et al.. as our future plans include the use of the developed recombinant antibody to target antigens towards psn-expressing macrophages in vivo, one might be concerned about the immunogenicity of mouse antibodies in pigs. poderoso et al. previously used mouse mabs as surrogate antigens in pigs to evaluate the role of sn in the induction of humoral responses and noticed an enhanced anti-mouse antibody response in comparison with a non-targeting isotype control mab. the induction of anti-mouse antibodies was however low after primary injection of the mab, only after a booster vaccination antibody titres rose significantly. previously, we have observed an enhanced anti-hsa antibody response after a single dose vaccination of hsa coupled to mab 41d without adverse clinical effects. therefore, in our future experiments, we will use a single dose of rec41d3-antigen to evaluate the protective efficacy of antigen targeting to psn. if further experiments confirm the applicability of this targeting technology, ‘porcinization’ of the recombinant antibody will be examined to enable prime-booster vaccination schedules.  a recombinant antibody that targets sialoadhesin was developed. in addition, we constructed a vector that allows the genetic linkage of a protein cargo in a defined position at the c-terminus of both heavy chains of a fully assembled antibody. this vector was shown to be versatile, as both a peptide and a more complex, larger protein could be fused. furthermore, production and purification of the antibody fusion constructs did not require major optimization. in comparison to other sn targeting strategies, the one-step production of the final carrier-cargo product together with the high specificity and affinity of the recombinant antibody may be considered an advantage for drug development. future research will mainly focus on the development of functional antibody-antigen fusions allowing the evaluation of sn-directed vaccination strategies. 
sialoadhesin -expressing monocytes/macrophages have been associated with several diseases like inflammatory and autoimmune disorders as well as viral infections, and they also appear to play a role in the initiation of an adaptive immune response. production and purification of antibody fusion constructs was possible without major optimization and with batch to batch consistency, confirming the development of a versatile antibody vector to evaluate sn-directed targeting strategies in a porcine animal model. transfection of hek293t cells with the resulting plasmid led to the secretion of fully assembled igg into the culture medium. in addition, rec41d also induced sn endocytosis in primary macrophages and resided for prolonged times in early/late endosomes. this recombinant antibody rec41d was shown to specifically bind to porcine sn with a comparable affinity as the native monoclonal antibody. both constructs were shown to be efficiently produced in hek293t cells and easily purified using standard protein g chromatography. previously, we developed functional sn-specific immunoconjugates that were generated via chemical coupling. to generate a more flexible and controlled system, we developed a recombinant antibody vector allowing the creation of genetic antibody fusion constructs. to allow the generation of antibody fusion constructs, a multiple cloning site was introduced at the c-terminus of the heavy chain. two fusion constructs were generated, one containing a v peptide tag and one containing an egfp molecule. this paper reports on the characterization of the recombinant antibody and the evaluation of its use for sn-directed targeting. a recombinant antibody allowing targeted delivery of peptides and proteins to sn-expressing macrophages was developed. this makes sn-expressing cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. the variable domains of the porcine sn-specific monoclonal antibody 41d were sequenced and cloned in frame with a mouse igg backbone. furthermore, since sn was shown to be an endocytic receptor, the conjugation of effector molecules to an sn-specific ligand should allow intracellular delivery of these conjugates. in addition, both v and egfp were shown to be co-internalized together with rec41d into sn-expressing primary macrophages. although successful, the system requires significant optimization for each immunoconjugate to be made.
 sialoadhesin -expressing monocytes/macrophages have been associated with several diseases like inflammatory and autoimmune disorders as well as viral infections, and they also appear to play a role in the initiation of an adaptive immune response. this makes sn-expressing cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. furthermore, since sn was shown to be an endocytic receptor, the conjugation of effector molecules to an sn-specific ligand should allow intracellular delivery of these conjugates. previously, we developed functional sn-specific immunoconjugates that were generated via chemical coupling. although successful, the system requires significant optimization for each immunoconjugate to be made. to generate a more flexible and controlled system, we developed a recombinant antibody vector allowing the creation of genetic antibody fusion constructs. this paper reports on the characterization of the recombinant antibody and the evaluation of its use for sn-directed targeting.  the variable domains of the porcine sn-specific monoclonal antibody 41d were sequenced and cloned in frame with a mouse igg backbone. transfection of hek293t cells with the resulting plasmid led to the secretion of fully assembled igg into the culture medium. this recombinant antibody rec41d was shown to specifically bind to porcine sn with a comparable affinity as the native monoclonal antibody. in addition, rec41d also induced sn endocytosis in primary macrophages and resided for prolonged times in early/late endosomes. to allow the generation of antibody fusion constructs, a multiple cloning site was introduced at the c-terminus of the heavy chain. two fusion constructs were generated, one containing a v peptide tag and one containing an egfp molecule. both constructs were shown to be efficiently produced in hek293t cells and easily purified using standard protein g chromatography. in addition, both v and egfp were shown to be co-internalized together with rec41d into sn-expressing primary macrophages.  a recombinant antibody allowing targeted delivery of peptides and proteins to sn-expressing macrophages was developed. production and purification of antibody fusion constructs was possible without major optimization and with batch to batch consistency, confirming the development of a versatile antibody vector to evaluate sn-directed targeting strategies in a porcine animal model. macrophagesialoadhesincd169siglec-1recombinant antibodytargetingcell-directed therapy 