AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody–Drug Conjugate Production

The site-directed chemical conjugation of antibodies remains an area of great interest and active efforts within the antibody–drug conjugate (ADC) community. We previously reported a unique site modification using a class of immunoglobulin-G (IgG) Fc-affinity reagents to establish a versatile, streamlined, and site-selective conjugation of native antibodies to enhance the therapeutic index of the resultant ADCs. This methodology, termed “AJICAP”, successfully modified Lys248 of native antibodies to produce site-specific ADC with a wider therapeutic index than the Food and Drug Administration-approved ADC, Kadcyla. However, the long reaction sequences, including the reduction–oxidation (redox) treatment, increased the aggregation level. In this manuscript, we aimed to present an updated Fc-affinity-mediated site-specific conjugation technology named “AJICAP second generation” without redox treatment utilizing a “one-pot” antibody modification reaction. The stability of Fc affinity reagents was improved owing to structural optimization, enabling the production of various ADCs without aggregation. In addition to Lys248 conjugation, Lys288 conjugated ADCs with homogeneous drug-to-antibody ratio of 2 were produced using different Fc affinity peptide reagent possessing a proper spacer linkage. These two conjugation technologies were used to produce over 20 ADCs from several combinations of antibodies and drug linkers. The in vivo profile of Lys248 and Lys288 conjugated ADCs was also compared. Furthermore, nontraditional ADC production, such as antibody–protein conjugates and antibody–oligonucleotide conjugates, were achieved. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing site-specific antibody conjugates without antibody engineering.


Introduction
In the last decade, chemical conjugation to produce site-specific antibody-drug conjugates (ADCs) has received considerable attention in the oncology field. 1,2 he first example of sitespecific ADC approved by the Food and Drug Administration was relished by Daiichi-Sankyo's unique chemical conjugation approach using a high drug-to-antibody ratio (DAR) technology consisting of interchain disulfide bond reduction, followed by thiol-maleimide coupling with their original drug linker (deruxutecan). 3,4This elegant solution, even though the resultant ADCs contain a few lower DAR species, 5 enables the production of nearly homogeneous ADC with DAR=8 without aggregation and loss of antibody properties, such as antigen binding. 3,4However, this technology is limited by compatible drug linkers.For example, MC-VC-MMAE, a commonlyused ADC drug linker in the market, 6 cannot be applied to this high DAR technology because hydrophobicity causes aggregation, lowering the physical and biological profile of the ADCs. 7over, a high DAR ADC may not be an ideal molecular format for every ADC.Another ADC manufactured by Daiichi-Sankyo's conjugation technology using deruxutecan in the clinical stage has a DAR lower than 8, supporting that the appropriate DAR must be adjusted based on the antibody's characteristics. 8The Seattle Genetics (now Seagen) group published an in vivo pharmacokinetics (PK) and efficacy comparison study between chromatographically purified DAR = 2, DAR = 4, and DAR = 8 ADCs, suggesting that the lower DAR, especially DAR=2, had an ideal biological profile. 9g to the limitations in high DAR technology and the potential requirement of DAR=2 production, the Ajinomoto group commenced the development of site-specific conjugation technology utilizing Fc-affinity peptide reagents in 2019. 10The proof-of-concept study revealed that this affinity-guided approach enabled the modification of a specific lysine in the Fc region of various antibodies, including immunoglobulin-G (IgG)1, IgG2, and IgG4, to produce homogeneous DAR=2 ADCs.This site occupancy was proved via several analyses, including trypsin-digested peptide mapping, which revealed that the conjugation site of the resultant ADCs is solely Lys248. 11The Fc region is a constant domain of every antibody 12 ; therefore, this Fcaffinity conjugation technology, termed AJICAP, enables the application (theoretically) of all antibodies without complicated reaction optimization.Furthermore, biological evaluation of sitespecific AJICAP-ADC consisting of cytotoxic monomethyl auristatin E (MMAE) indicated that AJICAP first-generation technology enhanced therapeutic index compared with stochastic cysteine-based ADCs. 13This improvement in the in vivo profile was also observed in different payload (maytansinoid) cases.Site-specific AJICAP-ADC, consisting of maytansinoid, demonstrated higher in vivo efficacy and tolerability than Kadcyla, 14  Although AJICAP first-generation technology is a promising approach for manufacturing sitespecific ADCs, several challenges remain (Figure 1a).This previous approach requires tris-(2carboxyethyl)-phosphine hydrochloride (TCEP) reduction to cleave the linkage between antibodies and AJICAP peptide reagent to install thiol groups on specific lysine.However, this reduction also cleaves the disulfide bonds of interchain cysteines in antibodies.Therefore, the disulfides must be re-constructed following the re-oxidation step using dehydroascorbic acid.This redox treatment is also used in THIOMAB conjugation technology 15 ; however, it can lead to disulfide bond scrambling. 2 A more critical concern in this approach is the risk of aggregation.
Small amounts of aggregates (5-10%) were found in ADCs produced using the AJICAP first-generation technology. 13Additionally, a more streamlined manufacturing sequence consisting of fewer reaction steps is preferred from the chemical manufacturing control perspective.
Our research group performed optimization studies to streamline the conjugation reaction sequence to overcome these issues (Figure 1b).This improved technology, termed "AJICAP second generation," enables the production of site-specific ADC while preventing aggregation due to selective cleavage reaction utilizing thioester chemistry.The stability of the AJICAP peptide reagent in buffer solution was improved, resulting in higher selectivity to target lysine and improved DAR value of ADCs.This thioester-based strategy provides two different AJICAP peptide reagents to access two different conjugation sites (Lys248 and Lys288).In vivo biological studies of Lys248-and Lys288-based ADCs revealed comparable efficacy and tolerability.
Furthermore, the application of conjugation chemistry to non-traditional ADC production was investigated.

AJICAP reagents design and antibody modification
There are two major challenges to improving AJICAP peptide reagents (Figure 1c): 1) replacing the unstable N-succinimide (NHS) group with an amine group of lysine in the antibody, and 2) replacing disulfide bonds in the AJICAP reagent requiring redox treatment in the ADC synthesis scheme.
Considering the shelf life and reactivity balance, the thiophenyl ester group was selected as a reactive group for antibody modification. 16The thiophenol group has higher stability and slightly lower reactivity than the NHS group.However, we expected that the proximity effect of the Fc affinity peptide would enhance reactivity for antibody modification.Additionally, the longer shelf life of thiophenyl ester in the reaction buffer solution compared to NHS ester improved the modification yield and site-specificity.An alkylthioester linkage was selected to replace the disulfide bond in the AJICAP first-generation reagent.Alkylthioesters are well-known protecting groups of thiols.N-succinimidyl S-acetylthioacetate is a widely used alkylthioester reagent for protein functionalization. 17Modification of lysine of protein using N-succinimidyl Sacetylthioacetate followed by specific deprotection using hydroxylamine can install sulfhydryl groups on the primary amine of lysine.An optimized AJICAP reagent (1b) was produced from these thioesters (alkyl and phenyl) (Figure 1d; Supporting Information (SI), Figure S1-S3).Furthermore, another AJICAP peptide reagent (6b) was designed to modify the Lys288 of antibodies (SI, Figure S4 and S5).In previous studies, the modification yield of Lys288 was lower than that of Lys248; therefore, we installed a rigid architecture 18 to allow the reactive thiophenyl ester group of the AJICAP reagent to be close to Lys288 in buffer conditions.

Table 1. Site-specific peptide conjugation
The conversion yield from antibodies to conjugates was calculated by measuring the peptide-toantibody ratio (PAR) using quadrupole time-of-flight mass spectrometry (Q-TOF MS) (SI, Figure S6-S10) 13 and hydrophobic interaction chromatography-HPLC (SI, Figure S11-S21). 19he previous Lys248 conjugation, AJICAP reagent (1a) provided higher PAR (entry 1) 10 ; however, AJICAP reagent (1b), consisting of a thiophenyl group, achieved further reactivity optimization to meet the targeted PAR and improved PAR=2 selectivity (entry 2).The conjugation efficiency of Lys288 conjugation was significantly improved.The AJICAP second-generation reagent (6b) provided PAR=2.0 conjugates, while the first-generation reagent (6a) produced limited conversion (entries 3 10 and 4).The predicted distance from the Z34C peptide, 20,21 the base peptide on reagents 6a and 6b, to Lys288 of the antibody is farther than that of the FcIII analog peptide 22 and Lys248 (SI, Figure S22).Simple NHS replacement with a thiophenylester was insufficient (SI, Figure S5, S8, and S14), 23 and linker tuning in the peptide reagent was required to overcome this gap.Several groups have reported unique affinity-guided protein modification technologies; in some cases, rigid architectures, such as piperazine 18 and proline 24 in the linker portion, are powerful tools for reaching the reactive group to target amino acids with improved conversion.These reports prompted us to screen linkers to improve the modification efficiency of Lys288, and the aromatic ring was found to be a suitable linkage.
In both reagents (1b and 6b), the following linker cleavage using hydroxylamine was completed (SI, Figure S15, S16, S20, and S21), indicating that the thiophenyl group reacted with lysine in the antibody, while no side reaction via alkylthioester occurred during peptide conjugation.

The capability of AJICAP technology
The compatibility of this conjugation chemistry using several antibodies was evaluated via hydrophobic interaction chromatography-HPLC analysis (SI, Figure S23-S39).In the traditional cysteine-conjugation case, the antibody characteristics affected the reactivity and DAR of the resultant ADCs 25 ; therefore, a conjugation examination using several ADCs with different isoelectric points (PIs) 26 was conducted to understand the reaction tolerability of AJICAP secondgeneration technology.In addition to trastuzumab (PI=8.8,entries 1 and 2) and rituximab (PI=9.1,entries 3 and 4), infliximab (PI=7.6,entries 5 and 6), cetuximab (PI=8.7,entries 7 and 8), and pembrolizumab (PI=8.7,entries 9 and 10) were evaluated.In all cases, both AJICAP reagents (1b and 6b) reacted sufficiently with these mAbs to produce conjugates with PAR=2.0,indicating that this conjugation does not depend on the PI difference or IgG subtypes.These AJICAP reagents also modified the Fc protein (entries 11 and 12, confirmed using Q-TOF analysis (SI, Figure S40 and S41)), demonstrating the potential use of this Fc-modification technology for Fc-fusion protein production.Fc-Fusion is a commonly-used technique for prolonging the in vivo half-life of smalland medium-sized molecules. 27Furthermore, this powerful conjugation technology enabled the modification of polyclonal antibodies (whole IgGs purified from human serum) (entries 13 and 14).To the best of our knowledge, this is the first example of site-specific modification of polyclonal antibodies. 28Sequential linker cleavage using excess equivalents of hydroxylamine enables the release of an affinity peptide portion, providing site-specific thiol-incorporating antibodies (4).In all reaction cases, aggregation percentages analyzed using size exclusion chromatography (SEC) were retained post-conjugation or linker cleavage (SI, Figure S42-S55).

Table 2. Stepwise and one-pot conversion from native mAbs to antibody-thiol (4)
In addition, we succeeded in omitting the purification step after AJICAP peptide conjugation.
As described in table 2, the one-pot reaction of peptide conjugation followed by linker cleavage modified all antibodies, with the same efficiency and aggregation profile as the stepwise conversion.This streamlined process is applicable to ADC manufacturing.

ADC syntheses
Over 20 ADCs were synthesized to demonstrate the compatibility of antibody-thiols (4) produced by AJICAP second-generation technology (Table 3).

Table 3. ADC syntheses
There are various drug linkers in the current ADC field, each with unique hydrophobicity and reactivity. 29Therefore, we evaluated five drug linkers (MCC-maytansinoid, MC-VC-PAB-MMAE, MC-MMAF, MC-PEG8-VA-tesirine, and MC-GGFG-Dxd; figure S56 in SI presents their structures).All ADCs converted from trastuzumab-Lys248-thiol, trastuzumab-Lys288-thiol, rituximab-Lys248-thiol, and rituximab-Lys288-thiol had higher DAR values than previously reported. 10DAR was determined using two different analytical methods (Q-TOF MS and HIC-HPLC) (SI, Figure S57-S76 and S77-S96).The average DAR values derived from two different analyses were unidentical.The measured differences are attributed to method sensitivity, accuracy, or linearity. 30,31 he aggregation percentage of all ADCs was less than 3.0%, comparable with one of the naked antibodies (SI, Figure S97-S116).These results indicated that this improved technology could be a more practical approach for various ADCs production than previous AJICAP technology. 32

Conjugation site determination
The conjugation site of these trastuzumab-thiols (4a derived from 1b and 4b derived from 6b) was determined using peptide mapping analysis.In our previous peptide mapping analysis, the sequence coverage was approximately 80% because of a single enzyme (trypsin) digestion. 10To increase this coverage, double enzymatic digestion was performed using trypsin and Lys-C.In addition, we optimized HPLC condition including a long gradient and newly constructed HPLC system to increase peak resolution (see experimental section).Consequently, the sequence coverage was improved to more than 95%.The thiol adduct was compared to the results, and target-specific modification of AJICAP-antibody-thiols (Lys248 and Lys288) was indicated (Figure 3).Among detected lysines, the target site (Lys248 and Lys288, respectively) was specifically subject to 3-(2-amino-2-oxo-ethyl) sulfanylpropionate modification.In fact, for the AJICAP-Lys248-thiol sample, we detected a peptide fragment including two candidate residues Lys246 and Lys248 (THTCPPCPAPELLGGPSVFLFPPK 246 PK 248 DTLMISR) for modification.
In this analysis, Lys-C and trypsin for digestion were used, therefore lysines and arginines excepting followed by proline are recognized and cleaved.Lys246 is located just behind the proline and is inhibited from enzymatic cleavage, while Lys248 should be cleaved by the enzyme.
From these considerations, we concluded Lys248 is specifically modified.Similarly for AJICAP-Lys288-thiol, we identified FNWYVDGVEVHNAK288TK290PR, having two lysines Lys288 and Lys290.We concluded that AJICAP-Lys288-thiol achieved specific modification at Lys288

In vivo biological evaluation of AJICAP-ADCs
In previous studies, our research group revealed enhanced therapeutic index using AJICAP conjugation technology than stochastically conjugated ADCs. 13,14 ollowing these evaluations, we examined the biological evaluation of AJICAP-ADCs produced using second-generation methods (Figure 4).In addition, Lys288-conjugated ADC was evaluated to compare the in vivo efficacy of these two conjugation sites.Trastuzumab-Lys248-MMAE (5a) and trastuzumab-Lys288-MMAE (5b) were selected in these biological studies.4a).The Lys248 conjugated ADC (5a) at a dose of 5 mg/kg displayed significant tumor regression, comparable to previous ADC produced using the AJICAP first-generation technology. 13The other AJICAP-ADC (5b) (conjugation site: Lys288, payload: MMAE) also displayed significant efficacy at a 5 mg/kg dosage (Figure 4b).These results indicate that AJICAP conjugation technology can produce ADCs with comparable efficacy that are independent of conjugation site and generation.In previous studies, 13 the minimum effective dose of stochastic DAR=4 trastuzumab-MMAE ADC produced via native cysteine conjugation was 2.5 mg/kg.Despite the DAR number difference (stochastic ADC = 4, AJICAP-ADC = 2), the in vivo efficacy of AJICAP-ADCs was comparable to that of stochastic ADCs.AJICAP conjugation technology modified the antibody's Fc region; therefore, the resultant ADC was at risk of losing binding affinity to FcRn because of steric interference by the payload linker conjugated close to the FcRn binding site. 22However, in 2021, our research group discovered that AJICAP-ADC (conjugation site: Lys248, payload: MMAE) retained FcRn binding affinity postconjugation. 34These findings are reasonable considering the plasma stability of AJICAP-ADCs measured in a rat PK study. 13In this study, the FcRn binding affinity and PK profile of two different AJICAP-ADCs (5a and 5b) (conjugation site: Lys248 or Lys288, payload: MMAE) produced using second-generation technology were confirmed.Biolayer interferometry analysis 35 of these ADCs, as in a previous study, 34 revealed that both ADCs had comparable binding strength for immobilized FcRn with naked trastuzumab (Figure 4c).These affinity results of two different ADCs provided via biolayer interferometry can be rationalized from their molecular structures (SI, figure S119).Lys248 and Lys288 are acceptable sites for FcRn binding that avoids steric hindrance due to conjugated payload linkers.Rat PK studies supported this FcRn binding result (Figures 4d     and 4e).Total antibody levels from two AJICAP-conjugated ADCs (5a and 5b) (conjugation site: Lys248 or Lys288, payload: MMAE) indicated a half-life similar to that of an unconjugated antibody.Total ADC analysis in rat PK studies indicated that these AJICAP-ADCs demonstrated sufficient stability in blood circulation.In contrast, stochastic ADC produced via cysteine conjugation technology showed insufficient stability in rats. 13Furthermore, we conducted an initial toxicology study of AJICAP-ADC (5b) (conjugation site: Lys288, payload: MMAE), which showed that this ADC did not negatively impact body weight changes in rats at the 80 mg/kg dosage (SI, Figure S120).The other ADC (5a) (conjugation site: Lys248, payload: MMAE) has already been evaluated in a rat toxicology study, which indicated that the maximum tolerated dose was > 80 mg/kg. 13In previous studies, 13 the estimated maximum tolerated dose value of trastuzumab-stochastic MMAE was approximately 10 mg/kg.These results indicate that the two AJICAP-ADCs have significantly wider tolerability than traditional stochastic ADCs.Further toxicological studies to determine the maximum tolerated dose of Lys288 conjugated ADC are underway.
Based on these in vivo efficacy, PK, and initial safety studies, we concluded that AJICAP secondgeneration technology enables the production of site-specific ADCs with higher therapeutic indexes than traditional stochastic ADCs.
In these in vivo datasets, no significant differences were observed in Lys248-ADCs and Lys288-ADCs.In the antibody development, some point mutations in the Fc region of an antibody to enhance FcRn binding were reported. 36Some of their mutated antibodies might lose their affinity for the AJICAP-Lys248 peptide reagent.The discovery of a novel AJICAP-ADC conjugated to Lys288 described herein may provide another alternative to using Fc region-mutated antibodies for AJICAP technology.

Application to novel format antibody conjugates
Recently, innovations have enlightened the oncology or bioconjugation community and broadened the research and development area to include ADC alternative antibody-oligonucleotide conjugates 37 and antibody-protein conjugates. 38Feasibility studies were performed using model conjugates to meet the potential requirements for producing these novel format antibody conjugates (Figure 5).As a model siRNA compound, we used a siRNA compound whose sequence targets peptidylprolyl isomerase B (PPIB, cyclophilin B) reported by the Genentech group. 39Following their report, model siRNA was converted to a maleimide conjugate (7), which afforded the antibody-siRNA conjugate (8) by thiol-maleimide coupling with antibody-Lys248-thiol (4a).The resulting conjugate (8) was purified using preparative SEC-HPLC to remove excess unreacted siRNA from the antibody-conjugate composition.SDS-PAGE analysis of this conjugate revealed a considerable conversion yield (SI, Figure S121).Lysozyme was selected as the model compound for protein conjugate production.Random lysine conjugation using an azide-NHS reagent produced the azide-incorporated lysozyme (11).Antibody-DBCO (10) transformed from antibody-Lys248-thiol (4a) and reacted with this azide-lysozyme (11) to form an antibody-protein conjugate (12).A Q-TOF MS analysis of (12) revealed that this conjugation proceeded smoothly (SI, Figure S122-124).These results indicate that the AJICAP conjugation strategy can be applied to produce ADCs with small cytotoxic molecules and ADC alternatives whose payloads are medium-sized molecules.Further investigations, including DAR determination, process development, and in vitro and in vivo evaluation, are currently ongoing.

Conclusion
Our research group has completed proof-of-concept studies of AJICAP second-generation and improved chemical conjugation technology using Fc-affinity reagents.This novel methodology is compatible with various antibody formats, including antibody fragments and polyclonal antibodies.The linker structural tuning enabled site-specific modification of native IgGs at the novel conjugation site (Lys288) with a high conversion yield.This conjugation technology can easily introduce two payload linkers per native antibody, producing over 20 site-specific ADCs without aggregation during the reaction.In vivo biological studies, including mouse efficacy, rat PK, and toxicology studies, of ADCs produced by this chemical site-specific conjugation technology indicate a widened therapeutic index.Furthermore, preliminary feasibility studies for application to non-traditional ADCs, including antibody-oligonucleotide conjugates and antibody-protein conjugates, were completed.This technology enables rapid, versatile, streamlined, site-specific conjugation to various native antibodies with several linker payloads, including medium-sized molecules, to produce nextgeneration antibody conjugates.
Biolayer interferometry assay to analyze FcRn binding was performed as previously reported. 35

Synthetic protocols to produce AJICAP reagents
Supporting Information contains detailed synthetic protocols for producing AJICAP reagents.

Lys248 conjugation using AJICAP reagent (1b) followed by linker cleavage (one-pot)
We added 2.5 equivalent of AJICAP reagent (1b) (20 mM in DMF) to each mAb (10 mg/mL, 20 mM acetate buffer, pH 5.5), and the mixture was incubated for 1 h at 20 °C.After 1 h, excess NH2OH HCl was added, and the mixture was stirred for an additional 1 h.This reaction mixture was purified using a NAP-25 desalting column and eluted with 20 mM acetate buffer at pH 5.5.

Lys288 conjugation using AJICAP reagent (6b) followed by linker cleavage (one-pot)
Six equivalents of AJICAP reagent (6b) (20 mM in DMF) were added to each mAb (10 mg/mL, 20 mM borate buffer, pH 8.2), and the mixture was incubated for 1 h at 20 °C.After 1 h, excess NH2OH HCl and 1 M acetate buffer (pH 4.7) for adjusting the pH of the reaction mixture (less than pH 6.0) were added and stirred for an additional 1 h.Subsequently, the reaction mixture was purified using a Centripure P50 desalting column and eluted with 20 mM acetate buffer (pH 5.5).

ADC synthesis
Payload conjugation with antibody-thiol (4) was achieved using a previously established procedure. 13The SI shows the payload linkers used in this study.

Peptide mapping for conjugation site determination
For peptide mapping, 20 µg of each sample (trastuzumab, trastuzumab-Lys248-thiol, and trastuzumab-Lys288-thiol) was diluted to 110 µL with 0.25 M Tris-HCl (pH 7.5)/0.75M GnHCl buffer.Reductive alkylation was achieved by adding dithiothreitol (DTT) and iodoacetamide (IAM) successively.The sample was then buffer exchanged to 100 mM Tris-HCl (pH 7.5) by Zeba Spin Desalting columns.For enzymatic digestion, we added 3:1 mixture of trypsin and Lys-C, then incubated at 37 °C for 18 h.Digestion was quenched by adding formic acid and acetonitrile.
The resulting peptide mixture was analyzed on an Orbitrap Fusion Tribrid (Thermo Fischer Scientific) interfaced with a Vanquish Duo (Thermo Fischer Scientific).We used an ACQUITY UPLC CSH C18 Column (1.7 µm, 2.1x150 mm, Cat # 186005298, Waters) with a column temperature of 50 °C.The chromatographic method consisted of a 2 min hold at 2% solvent B (0.1% formic acid in acetonitrile) and a 55 min linear gradient from 2 to 42% solvent B. After a linear rise to 90% solvent B in 5 min, a wash step was performed with a 5 min hold at 90% solvent B. Subsequently, solvent B was reduced to 2% in 1 min and maintained at 2% for 12 min.The flow rate was set to 250 µL/min, and solvent A contained 0.1% formic acid.Mass spectrometry analysis was conducted in data-dependent acquisition mode with full scans (350-2,000 m/z) acquired at a mass resolution of 120,000.Tandem mass spectra were produced using the higher energy collision-induced dissociation method.

Biological evaluation procedure
Detailed biological evaluation procedure can be found in the SI.
a clinical ADC approved by the Food and Drug Administration.These results indicate that AJICAP technology has great potential for producing next-generation ADCs.

Figure 1 .
Figure 1.Comparison of AJICAP first and second technology: a) AJICAP first-generation for the same reason.Detailed extracted ion chromatogram (XIC) and MS/MS results supporting the site-specificity of two antibody-thiols (Lys248 and Lys288) are shown in SI, figure S117 and S118.

Figure 3 .
Figure 3. Summary of lysine residue search by BioPharma Finder: a) analysis of trastuzumab-

Figure 5 .
Figure 5. Synthesis of novel format antibody conjugates.The production of an antibody-siRNA