Development and Properties of Valine-Alanine based Antibody-Drug Conjugates with Monomethyl Auristatin E as the Potent Payload

Antibody-drug conjugates (ADCs), designed to selectively deliver cytotoxic agents to antigen-bearing cells, are poised to become an important class of cancer therapeutics. Human epithelial growth factor receptor (HER2) is considered an effective target for cancer treatment, and a HER2-targeting ADC has shown promising results. Most ADCs undergoing clinical evaluation contain linkers that have a lysosomal protease-cleavable dipeptide, of which the most common is valine-citrulline (VC). However, valine-alanine (VA), another dipeptide comprising two human essential amino acids, has been used in next generation ADCs loading new toxins, but the druggable properties of ADCs loaded the most popular monomethyl auristatin E (MMAE) remain to be further explored. In this study, we generated VA-based ADCs that connected MMAE to an anti-HER2 antibody. We studied the differences in the preparation process, in vitro stability, cathepsin B activity and in vitro cytotoxicity of VA-based ADC compared to the ADC of VC. VA had comparable performance to VC, which preliminarily displays its practicability. Additional efficacy and safety studies in a xenograft model indicate this novel ADC exerted potent anti-tumor activity and negligible toxicity. The results of this study show the application potential of VA-based ADC with MMAE as the payload.


Introduction
Human epithelial growth factor receptor 2 (HER2/ErbB2) is a ligand-less tyrosine kinase receptor that acts as a pro-oncogene in different human cancers [1]. HER2 plays an important role in the development and prognosis of many cancer types such as metastatic breast, gastric, lung, colon, esophageal, and ovarian cancers [2,3]. For instance, it is overexpressed in approximately 20-25% of breast cancers, which were characterized by aggressive proliferation and poor prognosis before the introduction of targeted treatments [4,5]. HER2 is considered an effective target for antibody-based therapy in cancer treatment, especially breast cancer [6,7]. Trastuzumab (Herceptin ® ) and pertuzumab (Perjeta ® ) are two approved monoclonal antibody drugs that target the HER2 pathway, and their clinical application has greatly prolonged the survival of patients with metastatic breast cancer [8,9]. Despite these noteworthy advances in HER2-targeted therapy, patients eventually relapse, underscoring the need for alternative therapies [10].
Antibody-drug conjugates (ADCs), which combine the specificity, pharmacokinetics, and biodistribution of a monoclonal antibody (MAB) with the cytotoxic potency of a drug payload, is a promising new therapy for cancer [11]. Ado-trastuzumab emtansine (Kadcyla ® ), an anti-HER2 ADC that was designed with this new strategy, further prolonged the overall survival of trastuzumabresistant patients by more than six months [10,12]. The three components of ADCs are in the form of a targeted drug delivery system [13]. In addition to the development of antibodies and cytotoxic drugs, the design of the linker is of essential importance, as it impacts the efficacy and tolerability of the final product.
A suitable linker not only needs to provide sufficient stability during systemic circulation, but must also allow the rapid and efficient release of cytotoxic drug inside the tumor cells [14]. First-generation ADCs often contained unstable linkers, such as unhindered disulfides and hydrazones, which led to their failure [11,15]. On the other hand, next-generation ADCs contain linkers such as peptide linkers, glucuronides, and noncleavable linkers, which increase their stability [16,17]. Cleavage of the commonly used dipeptide linker depends on tumor-associated lysosomal cathepsin B and undergoes rapid hydrolysis, leading to release of the parent drug to kill the tumor [18,19]. The majority of auristatin-based ADCs use a dipeptide (valine-citrulline (VC)) linker conjugated to the auristatin analog monomethyl auristatin E (MMAE) [20,21]. In recent years, valine-alanine (VA), another dipeptide comprising two human essential amino acids, has been used in next generation ADCs loading new toxins such as pyrrolobenzodiazepine (PBD) [22,23]. In addition, the VA-based linker also been mentioned in patents for a novel internalizing ADC that connected MMAE to antibody via a dibromo-containing linker, with any relevant druggable properties were provided [24,25]. The effect of different cytotoxin on the ADC is very significant, in addition to PBD and MMAE being two completely different molecules with different drug release process ( Figure 1) [14,26]. The VA peptide has an advantage in the preparation process and cost of materials, and may have the potential to improve ADC stability and decrease product aggregation [27,28]. This has led to interest in exploring the physical and chemical properties, stability, enzymolysis kinetics, and efficacy of VA-based ADCs with MMAE as the potent payload.
In this study, we generated VA-based ADCs that connected MMAE to an anti-HER2 antibody. Compared to the VC-based ADC which payload been from a clinically approved ADC drug, we studied the preliminarily practicability of the VA-based ADCs in the preparation process, in vitro stability, cathepsin B activity and in vitro cytotoxicity. In addition, we further studied the anti-tumor activity and toxicity of VA-based ADC in a xenograft model. The results demonstrate that the VA-based ADC with MMAE as the payload also has application potential as an antitumor agent. that was designed with this new strategy, further prolonged the overall survival of trastuzumabresistant patients by more than six months [10,12]. The three components of ADCs are in the form of a targeted drug delivery system [13]. In addition to the development of antibodies and cytotoxic drugs, the design of the linker is of essential importance, as it impacts the efficacy and tolerability of the final product. A suitable linker not only needs to provide sufficient stability during systemic circulation, but must also allow the rapid and efficient release of cytotoxic drug inside the tumor cells [14]. Firstgeneration ADCs often contained unstable linkers, such as unhindered disulfides and hydrazones, which led to their failure [11,15]. On the other hand, next-generation ADCs contain linkers such as peptide linkers, glucuronides, and noncleavable linkers, which increase their stability [16,17]. Cleavage of the commonly used dipeptide linker depends on tumor-associated lysosomal cathepsin B and undergoes rapid hydrolysis, leading to release of the parent drug to kill the tumor [18,19]. The majority of auristatin-based ADCs use a dipeptide (valine-citrulline (VC)) linker conjugated to the auristatin analog monomethyl auristatin E (MMAE) [20,21]. In recent years, valine-alanine (VA), another dipeptide comprising two human essential amino acids, has been used in next generation ADCs loading new toxins such as pyrrolobenzodiazepine (PBD) [22,23]. In addition, the VA-based linker also been mentioned in patents for a novel internalizing ADC that connected MMAE to antibody via a dibromo-containing linker, with any relevant druggable properties were provided [24,25]. The effect of different cytotoxin on the ADC is very significant, in addition to PBD and MMAE being two completely different molecules with different drug release process ( Figure 1) [14,26]. The VA peptide has an advantage in the preparation process and cost of materials, and may have the potential to improve ADC stability and decrease product aggregation [27,28]. This has led to interest in exploring the physical and chemical properties, stability, enzymolysis kinetics, and efficacy of VAbased ADCs with MMAE as the potent payload.
In this study, we generated VA-based ADCs that connected MMAE to an anti-HER2 antibody. Compared to the VC-based ADC which payload been from a clinically approved ADC drug, we studied the preliminarily practicability of the VA-based ADCs in the preparation process, in vitro stability, cathepsin B activity and in vitro cytotoxicity. In addition, we further studied the anti-tumor activity and toxicity of VA-based ADC in a xenograft model. The results demonstrate that the VAbased ADC with MMAE as the payload also has application potential as an antitumor agent.

Linker Design
The design of the VA-based linker system was based on acetazolamide-drug conjugates [27]. Compared to counterparts with valine-lysine or valine-arginine linkers, small molecule-drug conjugates (SMDC) with VC and VA linkers exhibited greater stability and superior therapeutic activity. The SMDC containing the VA dipeptide linker not only showed the highest stability in vitro

Linker Design
The design of the VA-based linker system was based on acetazolamide-drug conjugates [27]. Compared to counterparts with valine-lysine or valine-arginine linkers, small molecule-drug conjugates (SMDC) with VC and VA linkers exhibited greater stability and superior therapeutic activity. The SMDC containing the VA dipeptide linker not only showed the highest stability in vitro but also the highest efficacy in vivo and in vitro. Applying the linker of this small molecule to ADCs may improve the performance of the final product. In addition, compared to the VC dipeptide, VA may have the potential to reduce ADC aggregation [28].
Both linkers with VA and VC were substrates for cathepsin B, and VA-based ADC could be easily cleaved by enzymes in an antibody-doxorubicin conjugate [28]. The VA dipeptide has also been used in clinical trials of ADCs containing PBD as the cytotoxic payload such as SGN-CD33A [22]. However, the effect of cytotoxin on the ADC is very significant, and the practicability of developing VA for MMAE-based ADC need to be further determined.
We generated VA-based internalizing ADCs with MMAE as the payload, as well as the corresponding VC-based ADC as a comparison. Theoretically, this type of ADC can also release the parent drug in a similar manner ( Figure 2A). We replaced the VC dipeptide with VA, with the goal of proving the feasibility of this design strategy. This study also explored the effects of increasing the polarity of the VA linker by shortening the carbon chain ( Figure 2B). These data can be used as evidence of the effects of carbon chains on drug release performance. but also the highest efficacy in vivo and in vitro. Applying the linker of this small molecule to ADCs may improve the performance of the final product. In addition, compared to the VC dipeptide, VA may have the potential to reduce ADC aggregation [28]. Both linkers with VA and VC were substrates for cathepsin B, and VA-based ADC could be easily cleaved by enzymes in an antibody-doxorubicin conjugate [28]. The VA dipeptide has also been used in clinical trials of ADCs containing PBD as the cytotoxic payload such as SGN-CD33A [22]. However, the effect of cytotoxin on the ADC is very significant, and the practicability of developing VA for MMAE-based ADC need to be further determined.
We generated VA-based internalizing ADCs with MMAE as the payload, as well as the corresponding VC-based ADC as a comparison. Theoretically, this type of ADC can also release the parent drug in a similar manner ( Figure 2A). We replaced the VC dipeptide with VA, with the goal of proving the feasibility of this design strategy. This study also explored the effects of increasing the polarity of the VA linker by shortening the carbon chain ( Figure 2B). These data can be used as evidence of the effects of carbon chains on drug release performance.

Evaluation of Antibody-Drug Conjugates (ADC) Preparation
The drug payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. The number of drug payloads per antibody, commonly referred to as the drug-toantibody ratio (DAR), can vary between 0 and 8 drugs per antibody. Reduction of inter-molecule chain disulfide bonds to produce free sulfhydryl groups allowed conjunction at specific residues

Evaluation of Antibody-Drug Conjugates (ADC) Preparation
The drug payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. The number of drug payloads per antibody, commonly referred to as the drug-to-antibody ratio (DAR), can vary between 0 and 8 drugs per antibody. Reduction of inter-molecule chain disulfide bonds to produce free sulfhydryl groups allowed conjunction at specific residues using maleimide-containing linkers, and generated conjugate mixtures at a limited number of defined positions. Hydrophobic interaction chromatography (HIC) analysis of the three ADCs (mil40-12a, mil40-12b, and mil40-12c; mil40 is a humanized monoclonal antibody that binds to HER2) allowed resolution of the conjugates into several major peaks corresponding to 0, 2, 4, 6, and 8 drug molecules per antibody ( Figure 3), with an average DAR of about 4.
Similar to other biologics, it is important to monitor high molecular weight species in ADC. Size exclusion chromatography (SEC) is a long-established method to measure size variants of proteins, especially high molecular weight species. To confirm the effects of dipeptide type on the degree of aggregation, we synthesized two types of ADCs using VA and VC as linkers. Then, we analyzed aggregation of the two prepared ADCs with an average DAR of about 4. The dimer of the aggregated antibody peak of VA-based ADC (mil40-12b) was 0.27% in the high-performance liquid chromatography (HPLC) spectrum (Agilent 1260; Wilmington, DE, USA), whereas that of VC-based ADC (mil40-12c) was 0.83%. To enlarge this effect, we synthesized the two ADCs with an average DAR of about 7. In these samples, VA-based ADC had no obvious increase in dimeric peak; however, the aggregation of VC-based ADC elevated to 1.80% with an increase in DAR from 4 to 7. In addition, the reaction system of VA-based conjugates had higher limpidity in the conjugate process, leading the recovery rate of the antibody to increase by 8.4%. Thus, VA was chosen over VC to reduce aggregation in ADCs with MMAE as the potent payload. Similar to other biologics, it is important to monitor high molecular weight species in ADC. Size exclusion chromatography (SEC) is a long-established method to measure size variants of proteins, especially high molecular weight species. To confirm the effects of dipeptide type on the degree of aggregation, we synthesized two types of ADCs using VA and VC as linkers. Then, we analyzed aggregation of the two prepared ADCs with an average DAR of about 4. The dimer of the aggregated antibody peak of VA-based ADC (mil40-12b) was 0.27% in the high-performance liquid chromatography (HPLC) spectrum (Agilent 1260; Wilmington, DE, USA), whereas that of VC-based ADC (mil40-12c) was 0.83%. To enlarge this effect, we synthesized the two ADCs with an average DAR of about 7. In these samples, VA-based ADC had no obvious increase in dimeric peak; however, the aggregation of VC-based ADC elevated to 1.80% with an increase in DAR from 4 to 7. In addition, the reaction system of VA-based conjugates had higher limpidity in the conjugate process, leading the recovery rate of the antibody to increase by 8.4%. Thus, VA was chosen over VC to reduce aggregation in ADCs with MMAE as the potent payload.

Stability Assays In Vitro
ADCs and their payloads were tested for stability in phosphate-buffer saline (PBS). To compare the linker stability of the two dipeptides, the reactive maleimide double bond of 12b and 12c were reduced with excess N-acetyl-L-cysteine (NAC) to afford NAC-12b and NAC-12c respectively [29]. These compounds were added to PBS buffer and incubated at 37 °C for seven days. Aliquots were taken at various time points and stored at −20 °C before HPLC analysis. Results were based on the area under the curve (AUC) of NAC-12b and NAC-12c at each time point as a percentage of the AUC at t = 0. After seven days, the AUCs for NAC-12b and NAC-12c were 97.92% and 97.05%, respectively, of the two samples taken immediately at t = 0 ( Figure 4A). In addition, it could be seen from the HPLC spectrum that the lost fraction was converted to the ring-opened succinimide hydrolysis adduct.
Next, we analyzed the in vitro stability of these two ADCs with an average DAR of about 4 and 7. Analogously, the two ADCs having 4 and 7 drugs per antibody, respectively, were diluted with PBS (1 mg/mL, pH 7.4) and incubated at 37 °C in a shaking incubator. All of the samples were analyzed by HIC HPLC. As shown in Figure 4B, none of the four ADCs had significant drug off-

Stability Assays In Vitro
ADCs and their payloads were tested for stability in phosphate-buffer saline (PBS). To compare the linker stability of the two dipeptides, the reactive maleimide double bond of 12b and 12c were reduced with excess N-acetyl-L-cysteine (NAC) to afford NAC-12b and NAC-12c respectively [29]. These compounds were added to PBS buffer and incubated at 37 • C for seven days. Aliquots were taken at various time points and stored at −20 • C before HPLC analysis. Results were based on the area under the curve (AUC) of NAC-12b and NAC-12c at each time point as a percentage of the AUC at t = 0. After seven days, the AUCs for NAC-12b and NAC-12c were 97.92% and 97.05%, respectively, of the two samples taken immediately at t = 0 ( Figure 4A). In addition, it could be seen from the HPLC spectrum that the lost fraction was converted to the ring-opened succinimide hydrolysis adduct.
Next, we analyzed the in vitro stability of these two ADCs with an average DAR of about 4 and 7. Analogously, the two ADCs having 4 and 7 drugs per antibody, respectively, were diluted with PBS (1 mg/mL, pH 7.4) and incubated at 37 • C in a shaking incubator. All of the samples were analyzed by HIC HPLC. As shown in Figure 4B, none of the four ADCs had significant drug off-targets in this test, and VA-and VC-based ADCs that having four payloads decreased stability by 1.58% and 1.34%, respectively, after seven days, whereas those that having seven payloads decreased stability by 2.52% and 3.88%, respectively, under the same test conditions. In short, ADCs with VA-and VC-based linker systems have substantially comparable in vitro stability in PBS.

Cathepsin B Reactivity
According to previous reports, drug linkers containing VA or VC dipeptides are substrates for cathepsin B [28]. Here, we confirmed the cleavable feasibility of VA-based conjugate under the reaction of cathepsin B. The susceptibility of the dipeptide linker conjugates (NAC-12b and NAC-12c) to enzymatic cleavage was determined by treatment of the acetyl-L-cysteine adduct with cathepsin B from human liver (EC 3.4.22.1, SIGMA-ALDRICH ® , St. Louis, MO, USA); NAC-12c was tested in parallel for comparison. The cleavage of dipeptide from both substrates resulted in 1,6elimination of MMAE, which was identified by liquid chromatography-mass spectrometry (LC-MS; Agilent 1100, Palo Alto, CA, USA). The amount of released MMAE over time is shown in Figure 5. These results demonstrated that the performance of VA-based conjugate is basically the same as VC, and is likely to be a suitable substrate for cathepsin B.

In Vitro Evaluation of Cytotoxic Agents and ADCs
To compare in vitro potency, the HER2-positive cell lines (BT-474, SK-BR-3, SK-OV-3, and NCI-N87), the weakly positive cell line MCF-7 (also known as a cell line expressing normal level of HER2) [30], and the HER2-negative cell line MDA-MB-468 were treated with various ADCs with an average

Cathepsin B Reactivity
According to previous reports, drug linkers containing VA or VC dipeptides are substrates for cathepsin B [28]. Here, we confirmed the cleavable feasibility of VA-based conjugate under the reaction of cathepsin B. The susceptibility of the dipeptide linker conjugates (NAC-12b and NAC-12c) to enzymatic cleavage was determined by treatment of the acetyl-L-cysteine adduct with cathepsin B from human liver (EC 3.4.22.1, SIGMA-ALDRICH ® , St. Louis, MO, USA); NAC-12c was tested in parallel for comparison. The cleavage of dipeptide from both substrates resulted in 1,6-elimination of MMAE, which was identified by liquid chromatography-mass spectrometry (LC-MS; Agilent 1100, Palo Alto, CA, USA). The amount of released MMAE over time is shown in Figure 5. These results demonstrated that the performance of VA-based conjugate is basically the same as VC, and is likely to be a suitable substrate for cathepsin B.

Cathepsin B Reactivity
According to previous reports, drug linkers containing VA or VC dipeptides are substrates for cathepsin B [28]. Here, we confirmed the cleavable feasibility of VA-based conjugate under the reaction of cathepsin B. The susceptibility of the dipeptide linker conjugates (NAC-12b and NAC-12c) to enzymatic cleavage was determined by treatment of the acetyl-L-cysteine adduct with cathepsin B from human liver (EC 3.4.22.1, SIGMA-ALDRICH ® , St. Louis, MO, USA); NAC-12c was tested in parallel for comparison. The cleavage of dipeptide from both substrates resulted in 1,6elimination of MMAE, which was identified by liquid chromatography-mass spectrometry (LC-MS; Agilent 1100, Palo Alto, CA, USA). The amount of released MMAE over time is shown in Figure 5. These results demonstrated that the performance of VA-based conjugate is basically the same as VC, and is likely to be a suitable substrate for cathepsin B.

In Vitro Evaluation of Cytotoxic Agents and ADCs
To compare in vitro potency, the HER2-positive cell lines (BT-474, SK-BR-3, SK-OV-3, and NCI-N87), the weakly positive cell line MCF-7 (also known as a cell line expressing normal level of HER2) [30], and the HER2-negative cell line MDA-MB-468 were treated with various ADCs with an average

In Vitro Evaluation of Cytotoxic Agents and ADCs
To compare in vitro potency, the HER2-positive cell lines (BT-474, SK-BR-3, SK-OV-3, and NCI-N87), the weakly positive cell line MCF-7 (also known as a cell line expressing normal level of HER2) [30], and the HER2-negative cell line MDA-MB-468 were treated with various ADCs with an average DAR of 4. Both the naked antibodies (mil40 and trastuzumab) had relatively lower inhibitory effects on all HER2-positive cells, and both of them have almost the same activity in all of the tested cell lines. Meanwhile, the three ADCs (mil40-12a, mil40-12b, and mil40-12c) exhibited more potent activity in the HER2-positive cell line, both IC 50 and inhibition, while very weak or no inhibitory effects in the HER2-negative cell line, just as antibody drugs, indicating its significant antigen selectivity. As a comparison, MMAE had potent anti-tumor activity in both HER2-positive and HER2-negative cell lines ( Figure 6).
The above-mentioned three ADCs had almost identical cytotoxicity in vitro, indicating that the type of dipeptide and length of the carbon chain did not have significant effects on cytotoxicity and drug release in cells. Overall, VA-based ADCs with MMAE exhibited similar efficacy and selectivity to the VC-based ADC, and the selectivity between HER2-positive and HER2-negative cells increased by about 2400-26,000 fold. In addition, both antibodies exhibited very weak cell inhibitory activity in the HER2-positive ovarian cancer cell line SK-OV-3; however, the associated ADCs exhibited similar cytotoxicity to the other cell lines, with an increase in anti-tumor activity of tens of thousands (Table 1).  (Table  1). Each group was established three holes and results are shown as mean ± SD. IC50 is presented on Table 1.   Table 1.

In Vivo Evaluation
Carrying more payloads usually speeds up the elimination of ADC in vivo [2,31]. Here, the anti-HER2 antibody of mil40 conjugated with an average of four VA-MMAE drug payloads was selected for further in vivo characterization and evaluation. The ADC (mil40-12b) was evaluated in the BT-474 (HTB-20™; ATCC, Maryland, USA) breast tumor model in female non-obese diabetes / severe combined immunodeficiency (NOD/SCID) mice, with naked antibody and unconjugated MMAE as the control group. Compared with vehicle, the control group only caused a partial delay in tumor growth, whereas the ADC treatment group with the same therapeutic dose exhibited very obvious tumor inhibition ( Figure 7A). In the ADC medium dose treatment group (2.5 mg/kg), tumors in half of the tumor-bearing animals disappeared (3/6 cures), while all the tumors can still be observed after treatment in the vehicle or mixture treatment group. Furthermore, ADC treatment groups showed significant dose-efficacy dependence, and tumor in all tested animals at 5 mg/kg dose were all disappeared after treatment and no recurrence occurred after one month ( Figure 7B). In addition, there was no weight loss observed in all groups treated with ADCs, indicating that the treatments were preliminarily well tolerated ( Figure 7C). These results indicate that this VA-based ADC has potent anti-tumor activity and basic safety. Furthermore, the tumor suppression data of ADCs with VA and VC dipeptides showed that VA-based ADC had substantially comparable activity to the ADC of VC ( Figure 7D), also without obviously weight loss in all treatment groups (data not shown).

In Vivo Evaluation
Carrying more payloads usually speeds up the elimination of ADC in vivo [2,31]. Here, the anti-HER2 antibody of mil40 conjugated with an average of four VA-MMAE drug payloads was selected for further in vivo characterization and evaluation. The ADC (mil40-12b) was evaluated in the BT-474 (HTB-20™; ATCC, Maryland, USA) breast tumor model in female non-obese diabetes / severe combined immunodeficiency (NOD/SCID) mice, with naked antibody and unconjugated MMAE as the control group. Compared with vehicle, the control group only caused a partial delay in tumor growth, whereas the ADC treatment group with the same therapeutic dose exhibited very obvious tumor inhibition ( Figure 7A). In the ADC medium dose treatment group (2.5 mg/kg), tumors in half of the tumor-bearing animals disappeared (3/6 cures), while all the tumors can still be observed after treatment in the vehicle or mixture treatment group. Furthermore, ADC treatment groups showed significant dose-efficacy dependence, and tumor in all tested animals at 5 mg/kg dose were all disappeared after treatment and no recurrence occurred after one month ( Figure 7B). In addition, there was no weight loss observed in all groups treated with ADCs, indicating that the treatments were preliminarily well tolerated ( Figure 7C). These results indicate that this VA-based ADC has potent anti-tumor activity and basic safety. Furthermore, the tumor suppression data of ADCs with VA and VC dipeptides showed that VA-based ADC had substantially comparable activity to the ADC of VC ( Figure 7D), also without obviously weight loss in all treatment groups (data not shown).

Hematological Analysis
To further verify the safety of the ADC (mil40-12b), changes in hematology between vehicle and treatment groups were compared at the end of administration and the observation period. The main test indicators included levels of white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), neutrophils (Neuts), and lymphocytes (Lymph). In contrast to the vehicle group, all of the above hematological parameters from the three ADC treatment groups were similar on Days 28 and 58

Hematological Analysis
To further verify the safety of the ADC (mil40-12b), changes in hematology between vehicle and treatment groups were compared at the end of administration and the observation period. The main test indicators included levels of white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), neutrophils (Neuts), and lymphocytes (Lymph). In contrast to the vehicle group, all of the above hematological parameters from the three ADC treatment groups were similar on Days 28 and 58 (Figure 8).
Analysis of variance (ANOVA) analysis was conducted on the hematology parameters of the treatment groups on Days 28 and 58, and at the end of drug administration. Unpaired two-tailed t-test for multiple comparisons was used. The level of confidence intervals was set at 95%, and the significance was set at p < 0.05. There was no significant difference between vehicle and ADC treatment groups (1, 2.5, and 5 mg/kg) regarding all parameters from both batches of blood samples (Table 2). Thus, this ADC had low blood toxicity and off-target bone marrow toxicity, which may be the reason it was well tolerated. significance was set at p < 0.05. There was no significant difference between vehicle and ADC treatment groups (1, 2.5, and 5 mg/kg) regarding all parameters from both batches of blood samples (Table 2). Thus, this ADC had low blood toxicity and off-target bone marrow toxicity, which may be the reason it was well tolerated.

Histopathological Studies
At the end of the xenograft model test, animals in specific groups were used for histopathological studies. Hematoxylin and Eosin (H&E) staining (heart, liver, and lung) and bone marrow smears showed no significant differences in histopathology evaluation, revealing no significant tissue damage produced in animals that received ADC relative to vehicle (Figure 9). This further confirms that this type of VA-based ADC has low systemic toxicity.

Histopathological Studies
At the end of the xenograft model test, animals in specific groups were used for histopathological studies. Hematoxylin and Eosin (H&E) staining (heart, liver, and lung) and bone marrow smears showed no significant differences in histopathology evaluation, revealing no significant tissue damage produced in animals that received ADC relative to vehicle (Figure 9). This further confirms that this type of VA-based ADC has low systemic toxicity.  Figure 9. Histopathological studies of VA-based ADC (mil40-12b). At the end of the xenograft test, animals (n = 6/group) in the vehicle and ADC treatment group (2.5 mg/kg) were sacrificed, and the tissue and bone marrow were used for histopathological studies. Scale bar: 46:1; the actual size of each picture is 425 × 320 μm.

Chemistry
Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All of the other reagents and solvents were purchased as the highest grade available and used without further purification.
Thin layer chromatography (TLC) was performed using pre-coated silica gel plates (Yantai dexin Bio-Technology Co., Ltd., Yantai, China). Column chromatography was performed using silica gel  (mil40-12b). At the end of the xenograft test, animals (n = 6/group) in the vehicle and ADC treatment group (2.5 mg/kg) were sacrificed, and the tissue and bone marrow were used for histopathological studies. Scale bar: 46:1; the actual size of each picture is 425 × 320 µm.

Chemistry
Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All of the other reagents and solvents were purchased as the highest grade available and used without further purification.
Thin layer chromatography (TLC) was performed using pre-coated silica gel plates (Yantai dexin Bio-Technology Co., Ltd., Yantai, China). Column chromatography was performed using silica gel (200-300 mesh; Yantai Chemical Industry Research Institute, Yantai, China). NMR spectra were recorded on a JNM-ECA-400 400 MHz spectrometer (JEOL Ltd., Tokyo, Japan) using CDCl3 and DMSO-d6 as solvent. Chemical shifts are expressed in (ppm), with tetramethylsilane (TMS) functioning as the internal reference. MS was performed on an API 3000 triple-quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada) equipped with a Turbo Ion Spray electrospray ionization source (AB Sciex, Concord, ON, Canada) that was used for mass analysis and detection. Analyst 1.4 software (AB Sciex, Concord, ON, Canada) was used for data acquisition.

Synthesis of the Linkers and ADC Payloads
Synthetic routes and analytical data are provided in the experimental protocols.

Bioconjugation and Purification
Humanized anti-HER2 IgG1 antibodies mil40 (10 mg/mL) in L-Histidine buffer (20 mM), pH 7.5, were treated with tris(2-carboxyethyl)phosphine hydrochloride (TCEP; 2.3 or 4.0 equivalents) at 25 • C for 90 min. To the reduced mAb was added the maleimide drug derivatives (1.2 equivalents /SH group) in ice-cold dimethylacetamide (DMAC) (5% v/v). After 40 min, the reactions were quenched with excess NAC (8 equivalents). The mixture was placed on ice for 30 min before buffer exchange by elution through Sephadex G25, and concentrated by centrifugal ultrafiltration. The conjugates was sterile filtered through a 0.2 µm filter under sterile conditions and stored at −80 • C before use for analysis and testing.

HPLC Analysis
HIC HPLC was used to determine levels of the molar ratio of drug substitution: 1260 HPLC (Agilent; Wilmington, DE, USA); Butyl-NPR column (2.5 µm, 4.6 × 35 mm, #14947, TOSOH Bioscience; Tokyo, Japan). Here, HIC buffer A was 50 mM potassium phosphate, pH 7.0, and 1.5 M ammonium sulfate; and HIC buffer B was 50 mM potassium phosphate, pH 7.0, 20% isopropanol. The gradient was 100% to 100% B over 15 min; flow rate was 1 mL/min; and UV detection wavelength was 280 nm. The DAR was determined by peak area integration according to the reported method [32].
SEC HPLC was used to determine levels of aggregation within each ADC: 1260 HPLC (Agilent; Wilmington, DE, USA); G3000SWXL analytical column (7.8 mm × 30 cm, #08541, TOSOH Bioscience; Tokyo, Japan). The SEC buffer contained 40 mM sodium phosphate and 150 mM sodium chloride (pH 7.0, 1 mL/min flow rate); and the UV detection wavelength was 280 nm. We performed a needle wash after each injection and include blank runs between each analyte. The aggregation was determined by peak area integration according to the reported method [33].

Linker Stability Assays In Vitro
The acetyl-L-cysteine (NAC) solution (0.41 mg/mL, PBS, pH 7.4) was added to 12b and 12c (500 mg/mL, DMSO). The reaction mixture was incubated at room temperature for 10 min, after which HPLC analysis revealed complete conversion to NAC-12b and NAC-12c as previously reported [34]. mixtures were incubated at 37 • C in a shaking incubator. Aliquots (200 µL) were collected at subsequent time points (0, 1,2,4,8,12,24,48, 72 h, and 7 days) and frozen at −20 • C. After sampling, all samples were melted at room temperature and analyzed by HPLC. Results were based on the AUC of remaining NAC-12b and NAC-12c at each time point as a percentage of the AUC at t = 0.
Analogously, ADCs that contained different dipeptide linkers were incubated in PBS (pH 7.4; 1 mg/mL) at 37 • C in a shaking incubator. Aliquots (200 µL) were collected at different time points (0, 4, 8, 12, 24, 48, 96, and 168 h) and then stored at −20 • C. All samples were thawed at room temperature before HIC analysis. The DAR of the samples at different time points were calculated as previously described [32].

Cathepsin B Reactivity
NAC-12b and NAC-12c (500 µg/mL) were prepared according to the above method and the enzymatic reaction temperature was set at 37 • C to mimic physiological conditions. The buffer mixture contained 25 mM sodium acetate, and 1 mM EDTA, at pH 5.5. To 880 µL of the buffer was added the NAC-conjugates solution respectively (100 µL) followed by a solution of Cathepsin B (Sigma: EC 3.4.22.1; #C8571; 20 µL of buffered aqueous solution), and the reaction mixture was incubated at 37 • C [29,35]. Aliquots (100 µL) were taken at subsequent time points (t = 0, 5, 10, 20, 40, 80, 160, and 320 min), and analyzed by LC-MS. Results of the released MMAE was quantified with the standard curve method.

In Vitro Cytotoxicity
The HER2-positive breast cancer cell line BT-474, SK-BR-3, gastric cancer cell line NCI-N87, ovarian cancer cell line SK-OV-3, weakly positive breast cancer cell line MCF-7 and HER2-negative breast cancer cell line MDA-MB-468 were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium (Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and Glutamax (Invitrogen). Cells (3.3 × 10 4 cells/mL) were added to each well of a 384-well plate after which 10 µL compound was also added to the assay plate. The plate was incubated for 4 days at 37 • C, 5% CO 2 , 95% humidity. Then the plates were incubated at room temperature for about 10 min, 40 µL CTG reagent was added to each well, and the plates were incubated for 30 min at room temperature. Luminescence was detected using the EnSpire Plate Reader, and Prism5 for Windows (Graphpad software, Inc., La Jolla, CA, USA) was used for data analysis, including IC 50 calculations.

Xenograft Studies
Female NOD/SCID mice 6-8 weeks of age, with an average weight of approximately 22 g, were approved by Anikeeper Inc. Female NOD/SCID mice were inoculated subcutaneously with 1 × 10 7 BT-474 breast cancer cells in 0.2 mL DMEM-Matrigel mixture (1:1 ratio) for tumor development. The treatment started when the mean tumor size reached approximately 150 mm 3 . The animals were given 1, 2.5, and 5 mg/kg ADC (mil40-12b), antibody + free MMAE, and vehicle alone on Days 0, 7, 14, and 21 or given 3 mg/kg ADCs (mil40-12b and mil40-12c) and vehicle on Days 0, 7, and 14. The animals were monitored twice weekly for body weight and tumor size. Tumor volume was calculated using the formula: TV = a × b 2 /2, where "a" and "b" are long and short diameters of a tumor, respectively. All procedures related to animal handling, care, and the treatment in this study were performed according to guidelines approved by the Institutional Animal Care and Use Committee of Pharmaron following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care under the licence number ON-CELL-XEM-06012016 on 1 June 2016.

Hematology
The animals in the xenograft model were subjected to whole-blood hematological analysis at the end of drug administration (28 days) and after 1 month of recovery (58 days). Then, 100 µL whole blood from each animal was drawn. Some samples were diluted 3 times for a final CBC count because of the volume limitation. ADC treatment-related changes in hematology parameters included RBC, HGB, WBC, PLT, Neut, and Lymph compared to the control, and results are expressed as mean ± SD. The data were analyzed base on the number with corrected dilution factor.
For comparison of hematological indicators, unpaired two-tailed t-test or two-way ANOVA for multiple comparisons was used. The level of significance was set at p < 0.05. Statistical analyses were performed using Prism5 for Windows (Graphpad software, Inc.).

Histopathology
At the end of the xenograft model test, animals in specific groups were used for histopathological studies. The organs mainly included the heart, liver, and lung. Tissue samples were fixed in 10% buffered formalin for 24 h and transferred into 70% ethanol. Dehydration through 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol × 2, 100% ethanol × 2 followed by 3 changes of xylene and then paraffin. Finally, the tissues were paraffin block embedded and sectioned. Then, H&E staining, mounting with permount, observation, and photographing under a microscope were performed in addition to bone marrow smears.

Synthetic Routes
The synthetic routes for the linker-MMAE conjugates 12a-12c are shown in Scheme 1.