Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo–enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo–enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels–Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar KD values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts.


Introduction
Bispecific antibodies (bsAbs) form a heterogeneous family of biological therapeutics. This fast-growing class of therapeutics holds, among others, promise mainly for the treatment of cancer and inflammatory diseases [1][2][3]. In 2020, a total of three bispecific antibodies were approved and more than 100 are being studied in clinical trials alone for the treatment of cancer [4].
In contrast to conventional monoclonal antibodies (mAbs), bsAbs simultaneously bind to two different types of antigens or, in their biparatopic form, two different epitopes on the same antigen, e. g., a cell target with a receptor on an immune cell or several targets on a cell surface to increase the cytotoxic potential and achieve improved efficacy with lower target expression [2]. In addition to full-length bsAbs, small bsAbs are of similar therapeutic interest due to the absence of the Fc-region. Better tissue penetration, the lack of Fc-mediated antibody effector functions, and easier production are only a few of the advantages of using antibody fragments [1,3].
The development of bsAbs accelerated in the 1980s with the progress of the synthesis techniques for their production. In particular, quadroma technology should be mentioned, which led to non-uniform bsAbs due to incorrect chain pairings, but nevertheless made Tl. Enhanced Trypiligase (eTl), representing the most efficient next-generation Trypsiligase so far, reaches product yields close to the thermodynamic maximum of the reaction type catalyzed.
While enzyme catalysis guarantees a regioselective reaction process, click chemistry opens the possibility of a flexible and modular synthesis principle including Cto N-, Cto C-terminal, and Nto N-terminal linkages of antigen-binding domains as well. Like enzyme synthesis, the click reactions are also biorthogonal and can be conducted in aqueous systems [26]. SPAAC and the IEDDA have been proven to be particularly suitable for biological applications. Both click reactions are fast, proceed entirely without or with only a few side reactions, and require only small substrate excesses [27,28]. In some cases, hydrophobic, sometimes bulky product structures may lead to undesirable problems with hydrophobicity, yield, or functionality.
In the present study, a synthesis approach for bsAbs is presented that only requires a single enzyme and can optionally be combined with click chemistry. It enables simple and rapid post-translational domain shuffling of antigen-binding domains to assess the optimal architecture of the bsAb. The function of the approach was evaluated using the example of the synthesis of bsFabs consisting of the antigen-binding domains of the growth factors ErbB2 and ErbB3. Both domains were covalently linked in a flexible orientation, purely enzyme-catalyzed or chemo-enzymatically with the enzymatic linkage of click anchors and downstream click chemistry. All bsFabs bind with similar K D values as the individual Fabs. Tests in breast cancer cell lines demonstrated improved properties in the biological activity of the bsFabs compared to their single Fab counterparts.

Results and Discussion
The concept of posttranslational bsAbs assembling via eTl was investigated by fusing the antigen-binding moieties of two distinct Fabs, i.e., anti-ErbB2-and anti-ErB3-Fab. Both Fabs target epitopes on growth factor receptors of the EGFR family. ErbB2 is abundant in many tissue tumors, such as breast cancer [29,30]. At the same time, ErbB3 is the preferred interaction partner of ErbB2. Once they interact, they form the most robust signal within this family of receptors [31,32]. The ErbB2-specific Fab used is derived from the well-known antibody Trastuzumab, whereas the anti-ErbB3 counterpart is derived from CDX3379, a therapeutic antibody from Celldex therapeutics [33,34].
Initially, the Fabs were equipped with a nucleotide sequence encoding either the C-terminal YRH-or N-terminal RH-motif on the genomic level. Both enzyme recognition sequences were inserted into the respective termini of the heavy chain of the two Fabs. Biosynthesis and purification of the constructs were performed according to established protocols [23,33]. Final assembly of bsFabs was evaluated either by direct coupling of a C-terminally tagged anti-ErbB3-Fab-YRH with the N-terminally modified anti-ErbB2-RH-Fab via eTl (Scheme 1A) or indirectly via eTl-coupling of click anchors to the anti-ErbB2and anti-ErbB3-Fabs followed by click chemistry-mediated bsFab formation (Scheme 1B). Direct coupling via eTl according to Scheme 1A resulted in a Cto Nlinkage of anti-ErbB3and anti-ErbB2-Fab. On the contrary, Cto C-terminal linker structure was realized by click chemistry due to the eTl-coupling of the click anchors to the C-terminus of the two Fabs. The individual structure of the IEDDA and SPAAC based click linkers are shown in Figure 1. Previous studies showed that Tl also catalyzes the attachment of artificial functionalities to the N-terminus of proteins with high yields [24]. Finally, we tried to assemble a Cto C-linked anti-ErbB3-anti-ErbB2-bsFab by a purely enzymatic approach using the branched linker 5 (Figure 1) for Fab-coupling featuring two eTl-specific N-termini (Scheme 1C). Cto C-linkage was achieved by a two-step enzymatic reaction, ligating the first linker 5 to anti-ErbB2-Fab-YRH followed by the coupling of anti-ErbB3-Fab-YRH. The central lysine moiety in 5 allows the incorporation of a third functionality in addition to the two enzyme recognition sites. In our case, we used an azide to enable click chemistry with spectroscopic labels.

Enzymatic Synthesis of C-to N-Linked Anti-ErbB3-Anti-ErbB2-bsFab
The eTl-catalyzed direct coupling of anti-ErbB3-Fab-YRH and anti-ErbB2-RH-Fab, leading to the respective C-to N-linked bsFab conjugate, allows a simple one-step reaction regime, with a maximum yield of 60% of the bsFab product after reaction times of about 90 min at the reaction conditions used ( Figure 2). After isolation by size exclusion chromatography (SEC), a single homogeneous bsFab conjugate was verified by LC-MS ( Figure  2C), which was subsequently tested for biological functionality (Section 2.3). It should be mentioned that besides the main product and starting Fab substrates, only one further reaction product was found corresponding to the anti-ErbB3-Fab-Y-OH species in which the last two amino acid residues (RH) of the recognition sequence were missing. This indicated a certain enzymatic hydrolysis activity by eTl at the Tyr-Arg site, which is, however, usually negligible for this enzyme. The slightly reduced product yields indicated somewhat higher hydrolysis rates which may be due to limited accessibility of the enzyme recognition sequence at the anti-ErbB2-RH-Fab.

Enzymatic Synthesis of C-to N-Linked Anti-ErbB3-Anti-ErbB2-bsFab
The eTl-catalyzed direct coupling of anti-ErbB3-Fab-YRH and anti-ErbB2-RH-Fab, leading to the respective Cto N-linked bsFab conjugate, allows a simple one-step reaction regime, with a maximum yield of 60% of the bsFab product after reaction times of about 90 min at the reaction conditions used ( Figure 2). After isolation by size exclusion chromatography (SEC), a single homogeneous bsFab conjugate was verified by LC-MS ( Figure 2C), which was subsequently tested for biological functionality (Section 2.3). It should be mentioned that besides the main product and starting Fab substrates, only one further reaction product was found corresponding to the anti-ErbB3-Fab-Y-OH species in which the last two amino acid residues (RH) of the recognition sequence were missing. This indicated a certain enzymatic hydrolysis activity by eTl at the Tyr-Arg site, which is, however, usually negligible for this enzyme. The slightly reduced product yields indicated somewhat higher hydrolysis rates which may be due to limited accessibility of the enzyme recognition sequence at the anti-ErbB2-RH-Fab.

Generation of C-to C-Linked Anti-ErbB2-Anti-ErbB3-bsFab
Posttranslational synthesis of C-to C-linked anti-ErbB2-anti-ErbB3-bsFab was evaluated by chemo-enzymatic and purely enzymatic approaches as well. Both are two-step reaction processes in which the Fab substrates were first site-specifically modified and then converted into the final bsFabs.
2.2.1. Chemo-Enzymatic Synthesis of C-to C-Linked Anti-ErbB2-Anti-ErbB3-bsFab Chemo-enzymatic assembly of C-to C-linked anti-ErbB2-anti-ErbB3-bsFab begins with the site-specific coupling of click anchors to the starting Fabs by eTl-catalysis. For IEDDA click chemistry, the trans-cyclooctene (TCO) based click linker 1 was enzymatically coupled to anti-ErbB2-Fab-YRH, while the methyltetrazine (MeTz) anchor 2 was linked to anti-ErbB3-Fab-YRH. In the case of SPAAC chemistry ( Figure S1), pentanoic acid azide (PAA) (compound 3) instead of MeTz was used, and dibenzocyclooctyne (DBCO) (compound 4) instead of TCO. Regardless of the nature of the click reagent, similar reaction conditions were used for all couplings.
The typical course of the eTl-mediated coupling reactions is shown in Figure 3 as an example of the synthesis of anti-ErbB2-Fab-TCO and anti-ErbB3-Fab-MeTz. According to Figure 3A, product yields higher than 72% were reached after approximately 75 min of reaction time. The reactions proceeded cleanly and resulted in an equilibrium between the reaction product and starting substrate at the end of the reaction ( Figure 3B). Undesired by-products did not occur apart from traces of partially hydrolyzed anti-ErbB2-Y-OH ( Figure 3C). With respect to former studies, such reaction processes are rather typical for eTl-catalyzed transamidation reactions [25]. Comparable yields for the SPAAC-based products, i.e., PAA and DBCO, were found in reactions with linkers 3 and 4 ( Figure S1A). Following synthesis, the reaction products were isolated by affinity chromatography mainly to remove the excess click anchor substrates and eTl. The remaining quantities of Fab substrates, on the other hand, did not interfere with the further course of synthesis.

Generation of C-to C-Linked Anti-ErbB2-Anti-ErbB3-bsFab
Posttranslational synthesis of Cto C-linked anti-ErbB2-anti-ErbB3-bsFab was evaluated by chemo-enzymatic and purely enzymatic approaches as well. Both are two-step reaction processes in which the Fab substrates were first site-specifically modified and then converted into the final bsFabs.

Chemo-Enzymatic Synthesis of
Chemo-enzymatic assembly of Cto C-linked anti-ErbB2-anti-ErbB3-bsFab begins with the site-specific coupling of click anchors to the starting Fabs by eTl-catalysis. For IEDDA click chemistry, the trans-cyclooctene (TCO) based click linker 1 was enzymatically coupled to anti-ErbB2-Fab-YRH, while the methyltetrazine (MeTz) anchor 2 was linked to anti-ErbB3-Fab-YRH. In the case of SPAAC chemistry ( Figure S1), pentanoic acid azide (PAA) (compound 3) instead of MeTz was used, and dibenzocyclooctyne (DBCO) (compound 4) instead of TCO. Regardless of the nature of the click reagent, similar reaction conditions were used for all couplings.
The typical course of the eTl-mediated coupling reactions is shown in Figure 3 as an example of the synthesis of anti-ErbB2-Fab-TCO and anti-ErbB3-Fab-MeTz. According to Figure 3A, product yields higher than 72% were reached after approximately 75 min of reaction time. The reactions proceeded cleanly and resulted in an equilibrium between the reaction product and starting substrate at the end of the reaction ( Figure 3B). Undesired by-products did not occur apart from traces of partially hydrolyzed anti-ErbB2-Y-OH ( Figure 3C). With respect to former studies, such reaction processes are rather typical for eTl-catalyzed transamidation reactions [25]. Comparable yields for the SPAAC-based products, i.e., PAA and DBCO, were found in reactions with linkers 3 and 4 ( Figure S1A). Following synthesis, the reaction products were isolated by affinity chromatography mainly to remove the excess click anchor substrates and eTl. The remaining quantities of Fab substrates, on the other hand, did not interfere with the further course of synthesis. The subsequent SPAAC-and IEDDA-based click reactions were performed according to established protocols (Section 3.5). The course and analysis of the reactions are shown in Figure 4 using the IEDDA reaction as an example. As for the SPAAC reaction ( Figure S1), a quantitative product yield could also be obtained for the IEDDA coupling at a stoichiometry of 1:2 of the starting substrates. The only differences were in the reaction times, which ranged from several minutes to a few hours. In fact, the IEDDA-based click reaction was completed within about 30 min ( Figure 4A). The SPAAC reaction, on the other hand, took about 4 h to reach complete conversion ( Figure S1B,C). Regardless of the individual reaction time, in all cases, the formation of the desired conjugates ( Figures 4A,B and S1B,C) could be detected after only a few seconds ( Figures 4C and S1D). The remaining bands after complete conversion at 40-55 kDa in the SDS-PAGE of Figure 4C (and Figure S1D, respectively) corresponded to unseparated, unmodified Fab species from the enzymatic reaction. These and the excess click component were finally separated by SEC in a one-step purification, yielding a final bispecific product of high purity and homogeneity ( Figures 4D and S1E). The subsequent SPAAC-and IEDDA-based click reactions were performed according to established protocols (Section 3.5). The course and analysis of the reactions are shown in Figure 4 using the IEDDA reaction as an example. As for the SPAAC reaction ( Figure S1), a quantitative product yield could also be obtained for the IEDDA coupling at a stoichiometry of 1:2 of the starting substrates. The only differences were in the reaction times, which ranged from several minutes to a few hours. In fact, the IEDDA-based click reaction was completed within about 30 min ( Figure 4A). The SPAAC reaction, on the other hand, took about 4 h to reach complete conversion ( Figure S1B,C). Regardless of the individual reaction time, in all cases, the formation of the desired conjugates ( Figures 4A,B and S1B,C) could be detected after only a few seconds (Figures 4C and S1D). The remaining bands after complete conversion at 40-55 kDa in the SDS-PAGE of Figure 4C (and Figure S1D, respectively) corresponded to unseparated, unmodified Fab species from the enzymatic reaction. These and the excess click component were finally separated by SEC in a one-step purification, yielding a final bispecific product of high purity and homogeneity ( Figures 4D and S1E).

Enzymatic Synthesis of C-to C-Linked Anti-ErbB2-Anti-ErbB3-bsFab
The eTl-catalyzed C-to C-linkage of two Fabs necessarily requires a special lin structure equipped with two nucleophilic recognition sequences for the biocatalyst ( motifs). Linker 5 obviously fulfilled this requirement. Furthermore, two Fab substr were required, each carrying the recognition sequence YRH for eTl at the C-terminus. reaction setting consisting of linker 5 and anti-ErbB2-Fab-YRH and anti-ErbB3-Fab-Y fulfilled both requirements but bore the general risk that, in addition to the bispe product, the respective homodimers are simultaneously formed from anti-ErbB2-Fa anti-ErbB3-Fab. A sequential reaction mode, starting first with the coupling of anti-Erb Fab-YRH with the linker and second, the coupling of anti-ErbB3-Fab-YRH to the resul intermediate could minimize this risk, especially if the nucleophilic component (linke was used in excess. Since the latter was the standard case in all previously performed reactions, the reaction conditions were maintained. The results of both reactions shown in Figure 5.

Enzymatic Synthesis of
The eTl-catalyzed Cto C-linkage of two Fabs necessarily requires a special linker structure equipped with two nucleophilic recognition sequences for the biocatalyst (RH-motifs). Linker 5 obviously fulfilled this requirement. Furthermore, two Fab substrates were required, each carrying the recognition sequence YRH for eTl at the C-terminus. The reaction setting consisting of linker 5 and anti-ErbB2-Fab-YRH and anti-ErbB3-Fab-YRH fulfilled both requirements but bore the general risk that, in addition to the bispecific product, the respective homodimers are simultaneously formed from anti-ErbB2-Fab or anti-ErbB3-Fab. A sequential reaction mode, starting first with the coupling of anti-ErbB2-Fab-YRH with the linker and second, the coupling of anti-ErbB3-Fab-YRH to the resulting intermediate could minimize this risk, especially if the nucleophilic component (linker 5) was used in excess. Since the latter was the standard case in all previously performed eTl reactions, the reaction conditions were maintained. The results of both reactions are shown in Figure 5. As it can be seen in Figure 5A, for the first reaction of the two-step procedure, the coupling of anti-ErbB2-Fab-YRH with linker 5, a product yield of higher 70% was obtained after approximately 75 min of reaction time. This corresponded to the results obtained with linkers 1 to 4. Interestingly, only about 3% of the homodimeric anti-ErbB2-linker 5anti-ErbB2-Fab was formed ( Figure 5A). Apparently, the 5-fold excess of the nucleophilic linker used was already sufficient to almost completely prevent unwanted homodimerization. After the monomeric anti-ErbB2-Fab-linker 5 product was purified by means of hydrophobic interaction chromatography (HIC) (Figure 5B), the second eTl-catalyzed coupling step was carried out by adding anti-ErbB3-Fab-YRH under again analogous reaction conditions. The result of this second enzymatic reaction is shown in Figure 5C,D.
Corresponding to the course of the reaction shown in Figure 5C, a product yield of approx. 60% of the desired anti-ErbB2-Fab-linker 5-anti-ErbB3-bsFab could be obtained in a As it can be seen in Figure 5A, for the first reaction of the two-step procedure, the coupling of anti-ErbB2-Fab-YRH with linker 5, a product yield of higher 70% was obtained after approximately 75 min of reaction time. This corresponded to the results obtained with linkers 1 to 4. Interestingly, only about 3% of the homodimeric anti-ErbB2-linker 5-anti-ErbB2-Fab was formed ( Figure 5A). Apparently, the 5-fold excess of the nucleophilic linker used was already sufficient to almost completely prevent unwanted homodimerization. After the monomeric anti-ErbB2-Fab-linker 5 product was purified by means of hydrophobic interaction chromatography (HIC) (Figure 5B), the second eTl-catalyzed coupling step was carried out by adding anti-ErbB3-Fab-YRH under again analogous reaction conditions. The result of this second enzymatic reaction is shown in Figure 5C,D. Corresponding to the course of the reaction shown in Figure 5C, a product yield of approx. 60% of the desired anti-ErbB2-Fab-linker 5-anti-ErbB3-bsFab could be obtained in a reaction time of approx. 120 min. This yield corresponded to the Cto N-terminal coupling of the two Fabs by eTl (Section 2.1). Finally, the bispecific product was isolated and purified by SEC ( Figure 5D) and subsequently used for functional studies (Section 2.3). Noticeably, with this method, it was rather impossible to control which Fab was attached to which site of the linker, which is, however, without relevance if the linker structure is symmetric. In cases where control is essential, two orthogonal Trypsiligase variants could be used in a one-step procedure to address this problem [25]. The in vitro function of all synthesized and purified anti-ErbB2-anti-ErbB3-bsFabs was first analyzed regarding their epitope-binding behavior. For this purpose, the dissociation constants (K D ) were investigated using an SPR-based activity assay compared to the single ErbB2-and ErbB3-Fabs. The K D values obtained were determined sequentially by immobilizing the ectodomains of the receptors ErbB2 and ErbB3 on separate sensor chip surfaces and are summarized in Table 1. The complete SPR sensorgrams as well as the values for k on and k off are shown in Figure S6 and Figure S7. First, for the single anti-ErbB2-and anti-ErbB3-Fabs, it was noted that neither showed any cross-reactivity in their antigen-binding properties (Table 1). Second, it is clear from Table 1 that the additional N-terminal RH-motif in the anti-ErbB2-Fab did not affect the dissociation constant. As a consequence, the K D values were in a narrow range and correspond to those described in the literature [23,33]. Third, the results further showed that all bsFabs synthesized retained their binding functionality. The determined dissociation constants were comparable to those of the individual single Fabs. Fourth, even the binding behavior of the inner domain of the Cto N-terminal linked bsFab format (anti-ErbB2-domain) did not appear to be affected by the outer anti-ErbB3-domain. Thus, it can be concluded that the type of linkage in which the two individual ErbB2 and ErbB3 Fabs are fused (at least as it is in the synthesized formats), has no significant influence on the binding properties to the antigens. Table 1. Comparison of dissociation constants of single and bispecific ErbB2-and ErbB3-Fabs determined by SPR. Biotinylated ErbB2 or ErbB3 ectodomains were immobilized separately on streptavidin sensor chips; no binding was detected. SPAAC: strain-promoted alkyne-azide cycloaddition, IEDDA: inverse electron-demand Diels-Alder reaction.

Receptor Internalization Assay
The internalization studies were conducted with three breast cancer cell lines. These cell lines differ in the number of ErbB2 and ErbB3 receptors expressed on their cell surfaces. SKBR-3 cells show high levels of both receptors, while HCC-1954 cells are known for high ErbB2 and low ErbB3 receptor expression. In contrast, the reverse holds true for MCF-7 cells. They are characterized by low ErbB2 and high ErbB3 receptor levels [35]. It was expected that the bsFab formats, in addition to binding the respective specific antigens, would also exhibit synergistic binding in the presence of both antigens on the cell surface.
First, all single and bispecific Fabs to be measured were modified non-specifically with an AlexaFluor568 NHS ester for the internalization studies. The resulting dye loading varied from 2 to 11 depending on the protein used. SDS-PAGE and UV/Vis absorption spectroscopic analyses showed no evidence of aggregate formation, especially at higher dye loadings ( Figure S8). In addition, a lysosomal dye was used to follow the path of the proteins in the cells. The progression of internalization is shown for anti-ErbB3-IEDDAanti-ErbB2-bsFab and SKBR-3 cells as an example of all bsFabs in Figure 6. In addition, the course of internalization of every single anti-ErbB2-and anti-ErbB3-Fab is shown. The complete data sets for all bsFabs and cell lines can be found in Figures S2-S5 in the Supplementary Materials. In general, little fluorescence intensities for all Fabs/bsFabs were observed for the MCF-7 cell line ( Figure S3). In contrast, for SKBR-3 cells, significant fluorescence signals of all added proteins corresponding to that of the lysosomes were found, indicating internalization ( Figure 6). Remarkably, the fluorescence signals of the bsFabs in the case of the SKBR-3 cells were much stronger than that of the single Fabs alone. The fluorescence intensity of the single anti-ErbB3-Fab was worse compared to that of the single anti-ErbB2 counterpart. A more in-depth quantification of these tendencies based on the performed fluorescence microscopic analyses was, however, not possible with certainty. Additional flow cytometric studies are recommended for this purpose, but these were beyond the focus of this work. First, all single and bispecific Fabs to be measured were modified non-specifically with an AlexaFluor568 NHS ester for the internalization studies. The resulting dye loading varied from 2 to 11 depending on the protein used. SDS-PAGE and UV/Vis absorption spectroscopic analyses showed no evidence of aggregate formation, especially at higher dye loadings ( Figure S8). In addition, a lysosomal dye was used to follow the path of the proteins in the cells. The progression of internalization is shown for anti-ErbB3-IEDDAanti-ErbB2-bsFab and SKBR-3 cells as an example of all bsFabs in Figure 6. In addition, the course of internalization of every single anti-ErbB2-and anti-ErbB3-Fab is shown. The complete data sets for all bsFabs and cell lines can be found in Figures S2-S5 in the Supplementary Materials. In general, little fluorescence intensities for all Fabs/bsFabs were observed for the MCF-7 cell line ( Figure S3). In contrast, for SKBR-3 cells, significant fluorescence signals of all added proteins corresponding to that of the lysosomes were found, indicating internalization ( Figure 6). Remarkably, the fluorescence signals of the bsFabs in the case of the SKBR-3 cells were much stronger than that of the single Fabs alone. The fluorescence intensity of the single anti-ErbB3-Fab was worse compared to that of the single anti-ErbB2 counterpart. A more in-depth quantification of these tendencies based on the performed fluorescence microscopic analyses was, however, not possible with certainty. Additional flow cytometric studies are recommended for this purpose, but these were beyond the focus of this work. In the case of HCC-1954 cells, a similar trend to SKBR-3 cells was observed ( Figure  7). While the fluorescence spots for the single Fabs were still congruent with the lysosomes, this was only partially the case for the bsFabs. A plausible explanation could be that the bsFab-receptor complexes are translocated into the nucleus via the non-canonical pathway [36][37][38]. To test this hypothesis initially, we performed the same experiment with nuclear instead of lysosome staining (Figure 8). While the fluorescence signal of the single anti-ErbB2-Fab-YRH was mainly found distant from that of the nucleus, the signals of all In the case of HCC-1954 cells, a similar trend to SKBR-3 cells was observed (Figure 7). While the fluorescence spots for the single Fabs were still congruent with the lysosomes, this was only partially the case for the bsFabs. A plausible explanation could be that the bsFabreceptor complexes are translocated into the nucleus via the non-canonical pathway [36][37][38].
To test this hypothesis initially, we performed the same experiment with nuclear instead of lysosome staining (Figure 8). While the fluorescence signal of the single anti-ErbB2-Fab-YRH was mainly found distant from that of the nucleus, the signals of all bsFabs could be found both distant and superimposed on the nucleus. This finding may indicate a different distribution of mono-and bispecifics in HCC-1954 cells. Whether the bsFabs can actually be found on or even in the cell nucleus cannot be concluded with certainty from this finding. bsFabs could be found both distant and superimposed on the nucleus. This finding may indicate a different distribution of mono-and bispecifics in HCC-1954 cells. Whether the bsFabs can actually be found on or even in the cell nucleus cannot be concluded with certainty from this finding.

SPR-Based Activity Assay
SPR spectroscopy was performed on a BIAcore X instrument (BIAcore (Uppsala, Sweden)). The biotinylated ErbB2 or ErbB3 ectodomains (BioCat, (Heidelberg, Germany)) were immobilized, each on a separate SAHC 200 M sensor chip (XanTec bioanalytics (Düsseldorf, Germany)), resulting in a surface density of approximately 1600 RU. The Fabs or bsFabs were applied in a dilution series using PBS as running buffer. Complex formation was observed at a continuous flow rate of 30 µL/min for 120 s. Kinetic parameters were determined by fitting the data to the 1:1 Langmuir binding model with the BIAevaluation software (BIAcore (Uppsala, Sweden)). After each injection, the surface was regenerated by injecting 10 µL 10 mM NaOH, 1 M NaCl.

Conclusions
Currently, more than one hundred formats of bsAbs have been described in the scientific literature. Not infrequently, the format determines the therapeutic efficacy. In this study, we presented an approach that can generate numerous amounts of such formats with only a single enzyme. By combining this with click chemistry techniques, a modular synthesis kit was created that allowed rapid and flexible shuffling of the individual antigenbinding domains in different but well-defined arrangements. The formats generated in this way can then be used to screen potentially suitable candidates for the respective application. The heterologous expression of each individual format or the use of different coupling procedures with individual reaction conditions, including the need for two distinct enzymes, as is currently the case, is not necessary. We were able to show that both the purely enzymatic synthesis and the chemo-enzymatic reactions enable high product yields with only short reaction times. The products were homogeneous and showed a uniform architecture. The formation of multimeric synthesis products, as found with the use of transglutaminase [18], could not be observed with eTl. We were also able to initially demonstrate the biological function of all bispecific constructs. As expected, we could show that the synthesized and fluorescence-labeled anti-ErbB2-anti-ErbB3-bsFabs exhibited improved fluorescence intensities in mammalian breast cancer cell lines compared with the single Fabs alone. Our results suggest that the bispecific formats produced might follow a partially different endocytotic pathway after internalization in certain cell lines than the individual Fabs from which they were constructed. In particular, the findings with labeled bsFabs, which in the case of HCC-1954 cells lead to additional fluorescence signals overlapping with those of the nuclei, give rise to further studies. The background for this is the clinical finding that the diagnosis of EGF receptors localized in the cell nucleus is associated with poor patient prognosis in the case of severe cancer progression [43,44]. On the basis of these findings, it would be very promising to investigate whether the transport of DNA-damaging toxins in the direction of the cell nucleus mediated by the bsFabs may lead to additional effects on the cancer cell compared to the monospecific Fabs. With the trifunctional linker already used in this study, this would be possible without any problems from a synthetic point of view and with high flexibility in terms of the active substance. Studies in this direction are presently in progress.