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Site-Specific Conjugation of the Indolinobenzodiazepine DGN549 to Antibodies Affords Antibody–Drug Conjugates with an Improved Therapeutic Index as Compared with Lysine Conjugation
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Site-Specific Conjugation of the Indolinobenzodiazepine DGN549 to Antibodies Affords Antibody–Drug Conjugates with an Improved Therapeutic Index as Compared with Lysine Conjugation
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  • Chen Bai
    Chen Bai
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Emily E. Reid
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Alan Wilhelm
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Manami Shizuka
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Erin K. Maloney
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Dilrukshi Vitharana
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Yelena Kovtun
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    Michael L. Miller
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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    Orcidhttp://orcid.org/0000-0003-3762-3319
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    Ravi Chari
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    Nicholas C. Yoder
    Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
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Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2020, 31, 1, 93–103
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https://doi.org/10.1021/acs.bioconjchem.9b00777
Published November 20, 2019

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Abstract

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Antibody–drug conjugates have elicited great interest recently as targeted chemotherapies for cancer. Recent preclinical and clinical data have continued to raise questions about optimizing the design of these complex therapeutics. Biochemical methods for site-specific antibody conjugation have been a design feature of recent clinical ADCs, and preclinical reports suggest that site-specifically conjugated ADCs generically offer improved therapeutic indices (i.e., the fold difference between efficacious and maximum tolerated doses). Here we present the results of a systematic preclinical comparison of ADCs embodying the DNA-alkylating linker-payload DGN549 generated with both heterogeneous lysine-directed and site-specific cysteine-directed conjugation chemistries. Importantly, the catabolites generated by each ADC are the same regardless of the conjugation format. In two different model systems evaluated, the site-specific ADC showed a therapeutic index benefit. However, the therapeutic index benefit is different in each case: both show evidence of improved tolerability, though with different magnitudes, and in one case significant efficacy improvement is also observed. These results support our contention that conjugation chemistry of ADCs is best evaluated in the context of a particular antibody, target, and linker-payload, and ideally across multiple disease models.

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Introduction

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Antibody–drug conjugates (ADCs) are a class of biotherapeutics for targeted cancer chemotherapy that consist of a cytotoxic payload chemically conjugated to a monoclonal antibody via a linker. (1,2) Upon binding to receptors expressed on the surface of cancer cells, the ADC is internalized as a complex with the receptor and processed to release cytotoxic catabolites that induce cell death.
By combining the tumor specificity of the antibody with the potent cytotoxicity of the payload, ADCs offer the potential to selectively deliver cytotoxic drugs to cancer cells while minimizing systemic toxicity. To date, five have been approved: brentuximab vedotin (3) for Hodgkin’s lymphoma and some non-Hodgkin’s lymphomas; ado-trastuzumab emtansine (2) for metastatic HER2+ breast cancer; gemtuzumab ozogamicin (4) for acute myeloid leukemia; inotuzumab ozogamicin (5) for acute lymphoblastic leukemia; and, recently, polatuzumab vedotin (6) as part of triple combination therapy for diffuse large B-cell lymphoma. However, the promise of ADCs has yet to be fully realized, particularly in solid tumor indications. Inadequate therapeutic indices remain a barrier to the development of many ADCs. (7)
Recent advances in payload, antibody, and linker technologies have yielded ADCs with improved in vivo pharmacologic properties. (8) In addition to these innovations, there has been much interest in developing novel conjugation methods to produce more active and better tolerated ADCs. Traditionally, ADCs have been synthesized by linkage of the payload to cysteines derived from reduction of interchain disulfides, or to lysine residues. These processes yield heterogeneous conjugates that are mixtures of species with different numbers of payload molecules linked at different sites on the antibody surface. Newer methods involve conjugation of the payload at defined sites on the antibody to give homogeneous conjugates with uniform drug to antibody ratio (DAR). (9−11) In the past decade, numerous studies comparing ADCs prepared by heterogeneous chemistries to those synthesized by novel site-specific methods have concluded that the latter exhibit improved in vivo pharmacologic properties. (12−21)
We recently conducted a direct comparison of ADCs that were synthesized by conjugating the same maytansinoid effector to the same antibody via either heterogeneous lysine conjugation or site-specific, engineered cysteine conjugation. (22) These ADCs were similarly potent in vitro and were shown to release very similar catabolites. In contrast to previous reports, the lysine-linked ADC was more efficacious than the site-specific ADC at matched payload doses in a mouse model of cervical cancer. This result suggests that the benefits of site-specific conjugation may not be general to all combinations of antibody, linker, and payload.
In this study, we further evaluate the advantages and disadvantages of site-specific conjugation by comparing heterogeneous lysine- and site-specific engineered cysteine-conjugated (termed CYSMAB) ADCs of the DNA alkylator DGN549. (23) Anticipating, in light of the findings from our maytansinoid study, that the results of such a comparison may depend on the exact system being studied, we tested conjugates of two different antibodies that bind to different cell surface receptors. Our results show that, unlike the maytansinoid case study, site-specific conjugation can lead to improved efficacy or tolerability in the context of DGN549 ADCs. However, the advantages do not appear to translate across different antibodies. We conclude that the choice of conjugation chemistry defies general rulemaking and must be considered in every specific case.

Results and Discussion

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Chemistry

The structures of DGN549-L (1a) and DGN549-C (1b) are shown in Figure 1. (23) Compound 1a, bearing an N-hydroxysuccinimide ester, was utilized for antibody conjugation at lysine residues. Compound 1b, bearing a maleimide, was utilized for antibody conjugation at engineered cysteine residues, and was generated from 1a via acylation of aminoethylmaleimide hydrochloride (SI Figure 1). Additional chemical synthetic details can be found in the Supporting Information.

Figure 1

Figure 1. (A) Structures of DGN549-L (1a), used for lysine-conjugation; and DGN549-C (1b), used for cysteine conjugation. (B) Structural representations of lysine-linked and CYSMAB ADCs.

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Antibody Conjugation

Cysteine residues were introduced into the sequence of human IgG1s mAb1 and mAb2 via mutation as previously described. (22) Variants of FRα-targeting mAb1 with Cys mutations LC-V205C (14) and at HC-S442C (mAb1-C442) (24) were generated, as well as a double mutant containing both mutations (mAb1-C205-C442). Likewise, the CD123-targeting antibody mAb2 was used in a wild-type humanized IgG1 format (mAb2) or as an HC-S442C mutant (mAb2-C442). None of these mutants showed any reduction in antibody titer (compared to the wild-type antibodies) at the ∼10 mg scale.
Lysine conjugation of 1a to mAb1 and mAb2 was carried out as previously described to give ADCs 2a and 3a, respectively (Figure 1B). (23) ADCs site-specifically conjugated at HC-C442 are referred to as CYSMAB ADCs. In order to produce CYSMAB DGN549-C ADCs 2b and 3b (Figure 1C), global reduction and selective reoxidation of the Cys mutant antibodies were carried out as previously described. (22) The resulting partially reduced antibodies were conjugated with 1b in an aqueous buffer containing a small amount of dimethylacetamide (DMA), as well as a relatively large proportion of propylene glycol as nondenaturing cosolvents. Higher proportions of DMA supported conjugation with some antibodies, but other antibodies suffered from aggregate formation. As with lysine conjugation of 1a, free 1b could be readily purified away from the product ADC using disposable gel filtration columns. Preparation of a ∼4 DAR site-specific ADC via conjugation of mAb1-C205-C442 with 1b was carried out to give ADC 2c, using the same protocol used to produce ∼2 DAR ADCs 2b and 3b.

ADC Characterization

As previously reported, lysine conjugation of 1a could be titrated to give ADCs with DAR between 2.5 and 3.0, which was consistent across UV–vis, SEC, and LC-MS analyses. (23) Likewise, CYSMAB conjugation of 1b gave the expected DAR of approximately 2.0 by UV–vis spectroscopy. Analysis by LC-MS and HIC showed profiles consistent with conjugation largely at a single site, with some evidence for DAR 1 species and other ADC variants as reported by others. (12,14,25) Engineered cysteine conjugation to produce ADC 2c proceeded to an average DAR of 3.5. No attempt was made to optimize the conjugation reaction to increase the DAR. Unoptimized conjugation of 1b to a single mutant LC-V205C antibody achieved a DAR of 1.6, so we speculate that the cysteine in this position is simply less reactive to this linker-payload due to steric or electronic properties. (14,26)
Representative examples of lysine-directed and site-specific LC-MS profiles are shown in Figure 2, and a full data set, including the 3.5 DAR DGN549-C conjugate of mAb1-C205-C442 is shown in SI Figure 2. Both lysine-directed and site-specific conjugation gave the expected high monomer as measured by SEC (SI Table 1). A previous report showed site-specific conjugation to be an effective strategy for mitigating aggregation observed for heterogeneous pyrrolobenzodiazepine ADCs. (27) In some cases, site-specific conjugation may be useful to limit high DAR species which have undesirable biophysical or pharmacological properties. (28,29) In the present, and in some other (30) cases, there is little biophysical advantage to be gained from site-specific conjugation, since ADCs with favorable quality attributes can be produced by heterogeneous chemistries, at least at the DARs we report here. Sodium bisulfite was added to the formulation to reversibly sulfonate the imine functionality of DGN549 to improve its water solubility and thereby the biophysical properties of the ADCs, without compromising their potency. Preparation of ADC 2b without sodium bisulfite in the formulation was possible but required more extensive purification and the use of formulation buffer with high excipient concentrations (data not shown). The use of disposable gel filtration columns, sometimes with the additional step of dialysis, was enough to reduce free IGN impurities to the levels shown in SI Table 1 without the use of activated charcoal as has been reported for other hydrophobic ADC payloads. (27)

Figure 2

Figure 2. SEC-MS data for mAb1 ADCs: (A) ADC 2a and (B) ADC 2b.

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HIC

HIC has historically been a useful method for ADC characterization and measuring ADC hydrophobicity. (31,32) Some literature reports have claimed a correlation between hydrophobicity and the pharmacokinetic properties of ADCs, (18,33−36) with the hypothesis that the same properties that lead to binding to a HIC column can lead to nonspecific uptake in animal tissues. Therefore, a method for analyzing DGN549 ADCs using HIC was developed, which required the use of a relatively less hydrophobic stationary phase than that used for auristatin (37) or maytansinoid (38) ADCs. In this method, the lysine-linked DGN549 ADCs eluted as a broad unresolved peak over ∼45 min of the HIC gradient, as expected due to the heterogeneity in both DAR species as well as conjugation sites. (38) In contrast, the CYSMAB DGN549 ADCs appeared much less heterogeneous by HIC, eluting largely in a single primary peak with additional minor peaks for DAR 1 and other apparent minor impurities. Interestingly, the primary CYSMAB ADC peak eluted almost at the very end of the HIC gradient spanned by the lysine conjugate, suggesting that conjugation at this site results in relatively hydrophobic ADCs, at least as measured by this assay (Figure 3). Finally, we note that the HIC analyses are conducted after the formulation of ADCs with sodium bisulfite, but show little evidence of heterogeneity that might be expected from reversible sulfonation of DGN549. This suggests that the sulfonation equilibrium lies to one side under the conditions of the experiment.

Figure 3

Figure 3. (A) HIC chromatograms of ADCs 2a and 2b. (B) HIC chromatograms of ADCs 3a and 3b.

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In Vitro Catabolite Identification

Many previous studies comparing site-specific and heterogeneously conjugated ADCs have been confounded by differences in the expected catabolite profile of comparator ADCs. (13,15,16,39,40) In order to ensure that the catabolites generated by each method of DGN549 conjugation are similar, we subjected CYSMAB ADC 1b to the cell-based LC-MS catabolite identification assay previously described. (23) The only major catabolite species identified was the monoimine indolinobenzodiazepine aniline species 4 (Figure 4, SI Figure 3), which was previously identified as the catabolite of DGN549-L ADCs. (23)

Figure 4

Figure 4. Processing of ADCs 2a and 2b to release the same catabolite FGN849 (4).

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In Vitro Binding Cytotoxicity

Binding of mAb1 and mAb2 to FRα-positive and CD123-positive cell lines, respectively, was confirmed using a flow cytometry-based assay. For all ADCs, binding EC50’s were comparable to those of the parent antibodies (SI Table 2) indicating that conjugation did not affect antigen binding. Cytotoxicity comparison of lysine-linked and CYSMAB DGN549 ADCs was carried out with both mAb1 and mAb2-derived ADCs in FRα-positive and CD123-positive cell lines, respectively, and the cytotoxic IC50’s are summarized in Table 1 and Table 2. In all cases, potency was strongly specific with respect to antibody–antigen binding, as shown with either antigen blocking by excess unconjugated targeting antibody, or by comparison with a nontargeted ADC of the same format. Consistent with what we (22,41) and others (12,18,30,35) have generally observed, in vitro potency of ADCs derived from the same antibody and linker-payload generally correlates with the DAR. Thus, the cytotoxic potency derived on a molar DGN549 basis (Table 1, Table 2, right-hand columns) generally appears similar across each cell line, though in some cases the CYSMAB ADCs 2b and 3b were slightly more potent than the lysine-linked comparators 2a and 3a. Bystander activity (cytotoxic activity of an ADC toward antigen-negative cells in the presence of antigen-positive cells) of ADCs 2a and 2b was evaluated in a mixed population of FRα-negative and FRα-positive cells. Both ADCs effected killing of FRα-negative cells at subnanomolar concentrations (SI Figure 4). This observation is consistent with the release of matched cell-permeable catabolites from the lysine-linked and CYSMAB ADCs, independent of conjugation chemistry.
Table 1. Cytotoxic IC50 Values as a Function of [ADC] and [DGN549] for mAb1 ADCs against FRα-Expressing Cells in the Absence or Presence of Antigen Blocking by Excess Unconjugated Targeting Antibody
  IC50 (M ADC)IC50 (M DGN549)
Cell LineADC– Block+ Block– Block+ Block
KB2a5 × 10–129 × 10–101 × 10–112 × 10–9
2b5 × 10–122 × 10–97 × 10–125 × 10–9
2c2 × 10–121 × 10–95 × 10–125 × 10–9
T47D2a2 × 10–119 × 10–96 × 10–113 × 10–8
2b1 × 10–111 × 10–83 × 10–112 × 10–8
NCI-H21102a4 × 10–112 × 10–91 × 10–105 × 10–9
2b4 × 10–112 × 10–89 × 10–114 × 10–8
Table 2. Cytotoxic IC50 Values as a Function of [ADC] and [DGN549] for mAb2 ADCs and Nontargeting Controls against CD123-Expressing Cells
Cell LineADCIC50 (M ADC)IC50 (M DGN549)
EOL-13a2 × 10–126 × 10–12
3b2 × 10–124 × 10–12
Non-Targeting Lysine-Linked ADC8 × 10–102 × 10–9
Non-Targeting CYSMAB ADC2 × 10–94 × 10–9
HNT-343a8 × 10–132 × 10–12
3b1 × 10–122 × 10–12
Non-Targeting Lysine-Linked ADC7 × 10–102 × 10–9
Non-Targeting CYSMAB ADC1 × 10–92 × 10–9
MV4-113a5 × 10–132 × 10–12
3b8 × 10–132 × 10–12
Non-Targeting Lysine-Linked ADC3 × 10–116 × 10–11
Non-Targeting CYSMAB ADC2 × 10–114 × 10–11

In Vivo Efficacy

Efficacy comparison of lysine-linked and site-specific DGN549 ADCs of both mAb1 and mAb2 was carried out in xenograft models that express their respective antigens. Prior literature comparing site-specific ADCs with heterogeneous comparators has reported a variety of outcomes. Moreover, interpretation of many of the studies is confounded by differences in catabolism of the comparator ADCs. (13,15,16,39,40) For studies with similar catabolite profiles, most have reported similar or marginally better efficacy for site-specific ADCs, provided the stability of the conjugation chemistry is adequate to maintain PK exposure of the resulting ADC. (12,14,21) By contrast, our previous investigation of peptide-linked maytansinoid ADCs found superior efficacy of a heterogeneous lysine-linked conjugate compared to site-specific cysteine-linked counterparts. (22) Finally, a recent survey of the literature has claimed that site-specific ADCs trend toward improved efficacy. (42) While some of the underlying published reports utilize ADCs that generate different catabolites, this article provides a physiologically based mathematical model that suggests that the decreased DAR leads to increased antibody doses, which in turn results in improvements in tumor delivery that more than compensate for the decrease in payload delivered per binding event.
We evaluated anti-FRα ADCs 2a and 2b in the NCI-H2110 mouse xenograft model of NSCLC (Figure 5, Table 3). The ADCs were dosed at equivalent quantities of the payload DGN549. At 3 μg/kg, both ADCs were minimally active with similar percent tumor growth inhibition (%T/C), while at the higher dose of 9 μg/kg, both ADCs induced durable complete regressions.

Figure 5

Figure 5. Activity of mAb1 ADCs in SCID mice bearing NCI-H2110 non-small cell lung cancer xenografts expressing FRα.

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Table 3. Activity of mAb1 ADCs in SCID Mice Bearing NCI-H2110 Non-Small Cell Lung Cancer Xenografts Expressing FRα
 Dose Regressions
Treatmentμg DGN549/kgμg ADC/kgT/C Day 22 (%)PartialComplete
Vehicle---0/60/6
ADC 2a3160320/60/6
951016/66/6
ADC 2b3230390/60/6
968026/66/6
Anti-CD123 ADCs 3a and 3b were compared at equivalent payload doses in a disseminated model of AML derived from the cell line MOLM-13 (Figure 6, Table 4). In this survival model, both ADCs showed potent activity, with doses of 1 μg/kg of DGN549 or greater generating sustained lifespan increase in a majority of animals. Even at doses as low as 0.1 μg/kg, activity was observed, though it was discernibly improved for CYSMAB ADC 3b, showing a lifespan increase of 262% as compared with 59% for the equivalent dose of lysine-linked ADC 3a. Thus, in this case CYSMAB conjugation affords a significant increase in activity, while in the case of the FRα ADCs the two modes of conjugation give ADCs that appear similarly active.

Figure 6

Figure 6. Activity of mAb2 ADCs and nontargeting controls in SCID mice bearing Molm-13 acute myeloid leukemia xenografts expressing CD123.

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Table 4. Activity of mAb2 ADCs and Non-Targeting Controls in SCID Mice Bearing Molm-13 Acute Myeloid Leukemia Xenografts Expressing CD123
 Dose   
Treatmentμg DGN549/kgμg ADC/kgMedian Survival (Days)Tumor Growth DelayIncreased Life Span (%)
Vehicle- 28--
ADC 3a0.15.844.516.559
0.317.4>152>124>443
1.058>152>124>443
Non-Targeting Lysine-Linked ADC1.05431311
ADC 3b0.17.6101.573.5262.5
0.322.8>152>124>443
1.076>152>124>443
Non-Targeting CYSMAB ADC1.06.826.5--

Mouse Tolerability

Previous reports have claimed that site-specific conjugation can improve the tolerability of ADCs. (13,18,19,40) Although these data have primarily been established by comparison with auristatin ADCs conjugated via interchain disulfides, we reasoned that this benefit might be observed with other payload classes as well. Interestingly, for ADCs of mAb1, the CYSMAB ADC 2b was approximately 2-fold better tolerated in mouse on a DGN549 payload basis than the heterogeneous ADC 2a (Figure 7). In the case of mAb2, the MTD for both ADCs was ∼100 μg/kg (Figure 8). However, comparison of the 125 μg/kg doses shows that for site-specific ADC 3b, the majority of animals recovered and gained weight following the acute toxicity post-dose, while for 3a the majority of animals had to be euthanized. So, while the MTD was formally the same, the tolerability of 3b appears slightly improved over 3a. Two potential hypotheses for the improved tolerability of site-specific CYSMAB DGN549 ADCs 2b and 3b over their lysine-linked comparators are (1) the improved pharmacologic properties of the conjugate made only at HC-C442 relative to the amalgamated properties of all the lysine-linked species; and (2) the decreased DAR of the CYSMAB ADCs eliminating the population of higher DAR, potentially more toxic species within the heterogeneous ADCs. In order to examine these hypotheses further, we tested the MTD of the ∼4 DAR site-specific ADC 2c in mice. The MTD of 125 μg/kg (Figure 9) is intermediate between DAR 2 CYSMAB 2b and heterogeneous DAR 2.5 2a, suggesting that both the site-specificity and the lower DAR of 2b contribute to its greater tolerability in mouse. Neither mAb1 nor mAb2 shows significant cross-reactivity to their respective mouse antigens, indicating that antigen-mediated effects do not play a role in toxicity of the ADCs. Thus, the fact that site-specific ADC 3b shows a lower tolerability advantage over heterogeneous conjugate 3a than does site-specific 2b over 2a suggests that antibody-specific factors significantly influence the in vivo outcomes. While this result may appear surprising due to the conserved sequence and structural features of human IgG1s such as mAb1 and mAb2, it has been observed that many biophysical factors can lead to disparate PK properties and tolerability of monoclonal Abs. (43)

Figure 7

Figure 7. Tolerability of mAb1 ADCs in non-tumor-bearing CD-1 mice. (A) ADC 2a and (B) ADC 2b. 

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Figure 8

Figure 8. Tolerability of mAb2 ADCs in non tumor-bearing CD-1 mice: (A) ADC 3a and (B) ADC 3b.

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Figure 9

Figure 9. Tolerability of ADC 2c in non tumor-bearing CD-1 mice.

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Mouse Pharmacokinetics

Previous reports have claimed that site-specific ADCs can exhibit increased pharmacokinetic (PK) exposure because of reduced hydrophobicity. This has been linked to improved tolerability, with the rationale that increased ADC exposure reduces payload delivery to sensitive tissues. (12,18,21,33,34,36) For site-specific ADCs, site-specific conjugation near the C-terminus of the IgG1 heavy chain has been reported to be problematic with regard to both hydrophobicity and exposure. (33,35) ADC 2b therefore presented a bit of an enigma, being better tolerated in mouse but appearing more hydrophobic in toto than ADC 2a. We therefore assessed the PK of ADCs 2a and 2b in non-tumor-bearing CD1 mice (Figure 10, Table 5).

Figure 10

Figure 10. Pharmacokinetics of mAb1 ADCs 2a and 2b in non-tumor-bearing CD-1 mice, assayed by ELISA methods for total antibody and conjugated antibody.

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Table 5. Pharmacokinetic Parameters for mAb1 ADCs in Non-Tumor-Bearing CD-1 Mice
ADCCmax (μg/mL)AUC0-∞ (h·μg/mL)Cl (mL/h/kg)t1/2 (h/days)t1/2 (days)Vss (mL/kg)
2aTotal Antibody40.346550.542219.2163
2aConjugated Antibody45.355780.452329.7142
2bTotal Antibody52.1107950.2338315.6124
2bConjugated Antibody65.491590.2732213.4121
Despite its apparent hydrophobicity as read out by HIC, site-specific ADC 2b exhibits 1.6-fold greater exposure as well as 1.7-fold slower clearance in mice than heterogeneous ADC 2a. Because the clearance as read out by total antibody ELISA is slower for 2b, we speculate that there is some pharmacological advantage of ADC 2b, despite its higher apparent hydrophobicity (Figure 3, above). It is possible that DGN549 conjugation at the relatively solvent-exposed HC-C442 position may enable noncovalent binding of albumin to the conjugated DGN (44) effectively solubilizing the ADC. Alternatively, the HIC assay may not completely capture the ADC properties relevant to pharmacological disposition. Finally, it should be noted that several prior reports (21,33,35,36) evaluated PK in rat, rather than in mouse as presented here. One of the prior reports (33) observed significantly more rapid clearance in rat than in mouse for a site-specific ADC, so we cannot rule out the possibility of species-specific pharmacokinetic effects playing a role here.
Cysteine-maleimide-linked ADCs have been shown to undergo degradation in vivo via elimination of the maleimide which is subsequently scavenged by thiols present in the biological milieu. (45) Linkage at some cysteine mutation sites appear to be particularly prone to this “retro-Michael” mechanism of instability, leading to rapid formation of unconjugated antibody. (14,26,46) Although the ELISA method used to determine [ADC] is not DAR-sensitive, the accumulation of unconjugated antibody can be inferred from the difference between the total antibody and ADC ELISA readouts. By this metric, conjugation at HC-C442 appears quite stable chemically. In order to validate this further, we examined the intact LC-MS of ADC 2b affinity purified from mouse plasma 3 days after administration. The resulting LC-MS profile (SI Figure 5) showed only minor increase in the thiol adducts to the ADC, providing additional evidence for chemical stability of maleimide conjugation at HC-C442.

Conclusions

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Although some ADCs have had clinical success in treating cancer, development of these therapies has also been notoriously challenging. It is clear that the rules for generating successful therapies with this class of cancer therapeutic are not completely understood. (8,47) While publications in the field have emphasized the benefits of second-generation site-specific conjugation technologies versus the earlier heterogeneous conjugation chemistries used, both our prior report (22) and this work suggest that the benefits are highly context dependent.
In our previous report, site-specific 2 DAR conjugation reduced the efficacy of a maytansinoid ADC compared with a lysine-conjugated 3.5 DAR ADC with a similar catabolite profile. In the present case, site-specific conjugation of a highly potent, the peptide linked IGN payload gave an improvement in preclinical therapeutic index as compared with lysine conjugation. Both site-specific ADCs were better tolerated in mouse, and one antibody/antigen system also showed improved efficacy. The greater hydrophobicity of the IGN payload may contribute to the benefits observed with site-specific conjugation, although a uniform 4 DAR site-specific ADC was still somewhat better tolerated than the lysine linked with an average DAR of 2.5. Moreover, the ADC hydrophobicity as measured by HIC did not appear to correlate with either therapeutic index or PK exposure.
As long as site-specific conjugation does not decrease therapeutic index of a given ADC, it remains of interest for ADC design. Controlled, uniform DAR can enable higher antibody dosing, which may improve ADC targeting either by improved transport and delivery through tumor vasculature or by saturating a normal tissue antigen sink, (42,48,49) as demonstrated both preclinically and in a clinical program targeting MUC16. (47) While the present case clouds the relationship between PK exposure and ADC hydrophobicity, prior examples suggest that reduction in hydrophobicity via site-specific conjugation, or using other technologies, may help improve pharmacological properties. For example, while 8 DAR interchain cysteine-directed conjugates of the widely studied mc-Val-Cit-PAB-MMAE linker-payload show poor PK properties, (28) second-generation auristatin payloads can be used in this conjugation format. (34,36) Notably, camptothecin ADCs (50,51) can also be deployed as 8 DAR interchain cysteine conjugates, likely due to their modest hydrophobicity, and have seen encouraging clinical activity as such.
Our results reinforce our general conviction that site-specific conjugation is a useful tool to be applied in the development of new conjugate therapies. However, rather than providing a generic benefit, its use must be carefully considered in combination with each individual target, linker-payload, antibody, and disease in order to make an optimally safe and effective therapy.

Materials and Methods

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Preparation and Characterization of ADCs

Antibodies

Human IgG1 antibodies against FRα and CD123 were produced at ImmunoGen, Inc. IgG1 antibodies against Kunitz trypsin inhibitor (KTI), used as nontargeting controls that do not bind any human antigen, were also produced at ImmunoGen, Inc. Antibody mutants with engineered cysteines were produced by modifying the heavy chain gene sequence to give a serine to cysteine mutation at position 442 (EU numbering).

DGN549 Payloads

Synthesis of a lysine-reactive form of DGN549-L (1a) was previously described. (52) Synthesis of a cysteine-reactive form of DGN549-C (1b) from 1a is described in the Supporting Information.

Synthesis of Lysine-Linked ADCs

DGN549-L was reversibly sulfonated at the imine by treatment with a 5-fold molar excess of sodium bisulfite in a 9:1 mixture of N,N-dimethylacetamide (DMA) and 50 mM sodium succinate, pH 5.0. The sulfonated DGN549 was added in a 3–5-fold molar excess to a solution of antibody in 15 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) pH 8.5 and 10% DMA. The reaction was allowed to proceed for 3–4 h at 25 °C. The conjugate was purified into 10 mM succinate, 50 mM sodium chloride, 8.5% sucrose, 0.01% Tween-20, and 50 μM sodium bisulfite pH 6.2 formulation buffer using Sephadex G25 desalting columns.

Synthesis of CYSMAB ADCs

Antibodies bearing engineered cysteines at position 442 in the heavy chain in phosphate-buffered saline (PBS) pH 7.4 were treated with 50 mol equiv of tris(2-carboxyethyl)phosphine and incubated for 1 h at 37 °C to reduce the solvent-accessible cysteines. The reduced interchain disulfides were reoxidized by treating the crude reduced antibody with 100 mol equiv of dehydroascorbic acid for 90 min at 25 °C to afford an antibody intermediate with the two engineered cysteines in reduced state. This intermediate was purified into PBS pH 6.0 with 5 mM N,N,N′,N′-ethylenediaminetetraacetic acid (EDTA) by gel filtration using Sephadex G25 columns. Conjugation was then effected by adding propylene glycol and DGN549-C as a stock solution in DMA to give a reaction mixture with a final solvent composition of 2% v/v DMA and 38% v/v propylene glycol in PBS pH 6.0 containing 5 mM EDTA. The reaction was allowed to proceed overnight at 25 °C, then purified into 20 mM histidine, 50 mM sodium chloride, 8.5% sucrose, 0.01% Tween-20, and 50 μM sodium bisulfite pH 6.2 formulation buffer using Sephadex G25 desalting columns, and then dialyzed against the same buffer using a membrane with 10 kDa molecular weight cutoff to remove residual unconjugated DGN549 and any other side products.

Characterization of ADCs

Concentrations of antibody and DGN549, and DAR, were determined by UV–vis spectroscopy on an Agilent 8543 spectrophotometer. Percent monomeric ADC was determined by size exclusion chromatography (SEC) using a TSKGgel G3000SWXL column (Tosoh Bioscience #08541). ADCs were eluted isocratically with 400 mM sodium perchlorate, 50 mM sodium phosphate pH 7.0, 5% isopropanol mobile phase run at 0.5 mL/min for 30 min. Unconjugated DGN549 was assayed by tandem size exclusion and reversed phase chromatography using an Acquity UPLC (Waters), equipped with a Protein BEH SEC guard column (Waters Corporation #186005793) and an Acquity UPLC BEH C18 column (Waters Corporation #176000863) run in series. Mobile phases were 0.1% trifluoroacetic acid in water (mobile phase A) and 0.1% trifluoroacetic acid in acetonitrile (mobile phase B). The flow rate was 0.35 mL/min and the column temperature was set to 30 °C. The ADC was eluted from the SEC column isocratically over 2.5 min with 30% mobile phase B. Unconjugated DGN549 species retained in the SEC column were then separated on the C18 column on a linear gradient of 30% to 80% mobile phase B from 2.5 to 8.0 min and quantified based on a DGN549 standard curve. Intact ADCs were analyzed by SEC coupled to mass spectrometry as previously described. (53) Hydrophobic interaction chromatography (HIC) was performed on a Proteomix HIC Ethyl NP5 column (Sepax Technologies #432NP5-7810) using mobile phases consisting of 1.5 M ammonium sulfate, 50 mM potassium phosphate pH 5.8 (mobile phase A), and 50 mM potassium phosphate pH 6.6 with 25% isopropanol (mobile phase B). Analytes were eluted on a gradient of 0% to 100% B over 45 min at a flow rate of 1.0 mL/min.

In Vitro Experiments

Binding

Binding of mAb1 ADCs to FRα on T47D cells and of mAb2 ADCs to CD123 on HNT-34 cells wereconfirmed by flow cytometry as previously described. (29)

Cytotoxicity

Cytotoxic potencies were assayed by treating tumor cells in 96-well plates (1000 to 5000 cells per well, depending on the cell line) in appropriate culture media with ADC over a range concentrations for 5 days at 37 °C and determining cell viability using the WST-8 assay (Dojindo Molecular Technologies) as previously described. (54) Data were fit using Prism (GraphPad, San Diego CA), with independent IC50 values generated for ADC and DGN549 concentrations.

Bystander Cytotoxicity Assay

Bystander activity of mAb1 ADCs was determined by treating FRα-positive 300.19 cells (generated by transfection with cDNA encoding the human FRα gene), FRα-negative parental 300.19 cells, or a mixed population of transfected and parental cells with ADC over a range of concentrations such that the ADC was nontoxic to the antigen-negative cells, but cytotoxic to the antigen-positive cells. Cell viability after 4 days at 37 °C was determined by the CellTiter-Glo (Promega) luminescent assay. (55)

Identification of Catabolites from mAb1-DGN549 CYSMAB ADC

Catabolites from mAb1-DGN549 CYSMAB ADC were identified as previously described. (23) Briefly, KB cells expressing FRα were treated with a saturating concentration of ADC for 24 h at 37 °C. Catabolites in the media were concentrated by binding to an anti-IGN antibody (ImmuoGen, Inc.) immobilized on Protein A resin, extracted into acetone, and dried in vacuo. The residue was redissolved in 80:20 water:acetonitrile and analyzed using a Dionex UltiMate 3000 UPLC (Thermo Fisher, USA) fitted with a reverse phase column (BEH C8, 1.7 μm, 100 mm × 2.1 mm, Waters) coupled to a Q-Exactive high resolution mass spectrometer (Thermo Fisher, USA). A 30 min gradient elution method was applied with a flow rate of 0.35 mL/min (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile; gradient: 20% B for 1 min, 20–80% B in 19 min, 80–100% B in 0.4 min, 100% B for 4.6 min, 100–20% B in 2 min and 20% B for 3 min) and a column temperature of 30 °C. The MS method was set at positive ion mode with full MS scan. Extracted ion chromatograms were generated for a set of plausible catabolites, and signal above an established confidence threshold was considered positive for presence of a given mass. Positive hits were confirmed by comparison with a spiked synthetic standard.

In Vivo Studies

Efficacy of mAb1 ADCs in NCI-H2110 Xenografts

Female CB.17 SCID mice were inoculated with 1 × 107 NCI-H2110 cells in 0.1 mL serum free medium + Matrigel (1:1) by subcutaneous injection in the area on the right hind flank. The mice were randomized by tumor volume into groups of 6 and single doses of ADC were administered intravenously on day 8 post-inoculation when the mean tumor volume reached ∼100 mm3. Tumor size was measured two times per week in three dimensions using a caliper. The tumor volume was expressed in mm3 using the formula V = Length × Width × Height × 1/2. Percent tumor growth inhibition (% T/C), defined as T/C × 100 where T is the median tumor volume in treated mice and C is the median tumor volume in the control group, was calculated on day 22 post-inoculation when the median tumor volume in the control group reached 1000 mm3. A mouse was determined to have a partial regression (PR) when tumor volume was reduced by 50% or greater and complete tumor regression (CR) when no palpable tumor could be detected.

Efficacy of mAb2 ADCs in Molm-13 Xenografts

Female athymic nude mice were inoculated with 1 × 107 Molm-13 tumor cells suspended in 0.1 mL serum-free culture media by intravenous injection into the tail vein. The mice were randomized by body weight into groups of 10 and single doses of ADC were administered intravenously on day 7 post-inoculation. To block Fc receptors, single 400 mg/kg doses of KTI-targeting IgG1 antibody (this antibody binds human Fc receptor but does not bind any human antigen) were administered intraperitoneally 24 h before ADC dosing. To maintain Fc receptor blockade, single intraperitoneal injections of 100 mg/kg KTI-targeting antibody were then given on days 4 and 9 after ADC administration. Animals were monitored and sacrificed if (1) body weight dropped by more than 20% of initial body weight at any time during the study, (2) one or both hind legs were paralyzed, (3) tumor growth occurred anywhere on the animal, or (4) the animal was too sick to reach food and water. Mice found dead were recorded on the day of discovery. Tumor growth delay was defined as T – C, where T is median survival time in the treated group and C is the median survival time in the control group. Percent increased life span was defined as (T – C)/C × 100.

Tolerability

Non-tumor-bearing CD-1 mice were randomized by body weight into groups of 8, and single doses of ADC were administered intravenously. Mice were weighed once daily for the duration of the study. Toxicity was assessed based on body weight and observation. Results were reported as percent body weight change relative to the start of treatment. The maximum tolerated dose (MTD) was defined as the maximum dose at which no animals died or were euthanized due to >20% body weight loss.

Pharmacokinetics of mAb1 ADCs

Non-tumor-bearing CD-1 mice were randomized by body weight and single 2.5 mg/kg doses of ADC were administered intravenously. Plasma samples were collected at 2 and 30 min; 2, 4, and 8 h; and 1, 2, 3, 5, 7, 10, 14, 21, and 28 days post-administration. ADC concentrations in plasma were assayed by two ELISA methods, one specific for the antibody component and another specific for the DGN549 component. Costar high-binding plates (Corning) were coated with goat anti-human IgG1 antibody (Jackson ImmunoResearch) for capturing total antibody or murine anti-IGN antibody (ImmunoGen Inc.) for capturing DGN549-conjugated antibody. After blocking with casein, plasma samples were added and captured ADC was detected with anti-human IgG Fc antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch) using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Concentrations were determined from a standard curve generated using purified ADC and reported as the mean of samples from three individual mice. Pharmacokinetic parameters were calculated by noncompartmental analysis using Phoenix WinNonlin software (Certara).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.9b00777.

  • Detailed experimental procedures for synthesis of compound 1b, supplementary characterization data for ADCs, and supplementary methods (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Chen Bai - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Emily E. Reid - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Alan Wilhelm - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Manami Shizuka - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States;  Present Address: Pharmaron,404 Wyman Street, Waltham, MA 02451
    • Erin K. Maloney - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Rassol Laleau - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Lauren Harvey - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States;  Present Address: Kaleido Biosciences, 65 Hayden Avenue, Lexington, MA 02421
    • Katie E. Archer - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Dilrukshi Vitharana - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States;  Present Address: Anokion, 50 Hampshire Street, Cambridge, MA 02139
    • Sharlene Adams - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Yelena Kovtun - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Michael L. Miller - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States;  Orcidhttp://orcid.org/0000-0003-3762-3319
    • Ravi Chari - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
    • Thomas A. Keating - Science, Technology, and Translation, ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
  • Notes
    The authors declare no competing financial interest.

References

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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFWntrnN&md5=365b2432f4bc622eb92d01d5cb9c78a8
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    Behrens, C. R. and Liu, B. (2014) Methods for site-specific drug conjugation to antibodies. MAbs 6 (1), 46– 53,  DOI: 10.4161/mabs.26632
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    9
    Methods for site-specific drug conjugation to antibodies
    Behrens Christopher R; Liu Bin
    mAbs (2014), 6 (1), 46-53 ISSN:.
    Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over traditional chemotherapy. Drugs and linkers have been the current focus of ADC development, in addition to antibody and target selection. Recently, however,the importance of conjugate homogeneity has been realized. The current methods for drug attachment lead to a heterogeneous mixture, and some populations of that mixture have poor in vivo performance. New methods for site-specific drug attachment lead to more homogeneous conjugates and allow control of the site of drug attachment. These subtle improvements can have profound effects on in vivo efficacy and therapeutic index. This review examines current methods for site-specific drug conjugation to antibodies, and compares in vivo results with their non-specifically conjugated counterparts. The apparent improvement in pharmacokinetics and the reduced off target toxicity warrant further development of this site-specific modification approach for future ADC development.
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  10. 10
    Agarwal, P. and Bertozzi, C. R. (2015) Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 26 (2), 176– 192,  DOI: 10.1021/bc5004982
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    10
    Site-Specific Antibody-Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development
    Agarwal, Paresh; Bertozzi, Carolyn R.
    Bioconjugate Chemistry (2015), 26 (2), 176-192CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A review. Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small mols. to create targeted drugs. Despite the simplicity of this concept, generation of clin. successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepd. by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixts. that cannot be further purified. With advances in the fields of bioorthogonal chem. and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chem. and protein engineering.
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  11. 11
    Panowski, S., Bhakta, S., Raab, H., Polakis, P., and Junutula, J. R. (2014) Site-specific antibody drug conjugates for cancer therapy. MAbs 6 (1), 34– 45,  DOI: 10.4161/mabs.27022
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    11
    Site-specific antibody drug conjugates for cancer therapy
    Panowski Siler; Bhakta Sunil; Raab Helga; Polakis Paul; Junutula Jagath R
    mAbs (2014), 6 (1), 34-45 ISSN:.
    Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors--target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0-8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.
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  12. 12
    Junutula, J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D., Weir, S., Chen, Y., Simpson, M., Tsai, S. P., Dennis, M. S. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26 (8), 925– 932,  DOI: 10.1038/nbt.1480
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    12
    Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index
    Junutula, Jagath R.; Raab, Helga; Clark, Suzanna; Bhakta, Sunil; Leipold, Douglas D.; Weir, Sylvia; Chen, Yvonne; Simpson, Michelle; Tsai, Siao Ping; Dennis, Mark S.; Lu, Yanmei; Meng, Y. Gloria; Ng, Carl; Yang, Jihong; Lee, Chien C.; Duenas, Eileen; Gorrell, Jeffrey; Katta, Viswanatham; Kim, Amy; McDorman, Kevin; Flagella, Kelly; Venook, Rayna; Ross, Sarajane; Spencer, Susan D.; Wong, Wai Lee; Lowman, Henry B.; Vandlen, Richard; Sliwkowski, Mark X.; Scheller, Richard H.; Polakis, Paul; Mallet, William
    Nature Biotechnology (2008), 26 (8), 925-932CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogeneous conjugates with relatively narrow therapeutic index (max. tolerated dose/curative dose). Using leads from the authors' previously described phage display-based method to predict suitable conjugation sites, the authors engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb Ig folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepd. by conventional approaches. The favorable in vivo properties of the near-homogeneous compn. of this conjugate suggest that this strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
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  13. 13
    Junutula, J. R., Flagella, K. M., Graham, R. A., Parsons, K. L., Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, D. L., Li, G. (2010) Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer. Clin. Cancer Res. 16 (19), 4769– 4778,  DOI: 10.1158/1078-0432.CCR-10-0987
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    13
    Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer
    Junutula, Jagath R.; Flagella, Kelly M.; Graham, Richard A.; Parsons, Kathryn L.; Ha, Edward; Raab, Helga; Bhakta, Sunil; Nguyen, Trung; Dugger, Debra L.; Li, Guangmin; Mai, Elaine; Lewis Phillips, Gail D.; Hiraragi, Hajime; Fuji, Reina N.; Tibbitts, Jay; Vandlen, Richard; Spencer, Susan D.; Scheller, Richard H.; Polakis, Paul; Sliwkowski, Mark X.
    Clinical Cancer Research (2010), 16 (19), 4769-4778CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
    Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examd. the in vitro potency as well as in vivo preclin. efficacy and safety profiles of a heterogeneous prepn. of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate. To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) mol. was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 mols. per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti-cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equiv doses (μg/m2) and retained efficacy at equiv. antibody doses (mg/kg). An improved safety profile of >2-fold was obsd. in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6000 μg DM1/m2) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6000 μg DM1/m2). The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clin. development of HER2-pos. targeted breast cancer therapies.
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  14. 14
    Shen, B. Q., Xu, K., Liu, L., Raab, H., Bhakta, S., Kenrick, M., Parsons-Reponte, K. L., Tien, J., Yu, S. F., Mai, E. (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30 (2), 184– 189,  DOI: 10.1038/nbt.2108
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    14
    Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates
    Shen, Ben-Quan; Xu, Keyang; Liu, Luna; Raab, Helga; Bhakta, Sunil; Kenrick, Margaret; Parsons-Reponte, Kathryn L.; Tien, Janet; Yu, Shang-Fan; Mai, Elaine; Li, Dongwei; Tibbitts, Jay; Baudys, Jakub; Saad, Ola M.; Scales, Suzie J.; McDonald, Paul J.; Hass, Philip E.; Eigenbrot, Charles; Nguyen, Trung; Solis, Willy A.; Fuji, Reina N.; Flagella, Kelly M.; Patel, Darshana; Spencer, Susan D.; Khawli, Leslie A.; Ebens, Allen; Wong, Wai Lee; Vandlen, Richard; Kaur, Surinder; Sliwkowski, Mark X.; Scheller, Richard H.; Polakis, Paul; Junutula, Jagath R.
    Nature Biotechnology (2012), 30 (2), 184-189CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a pos. charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected pos. by succinimide ring hydrolysis and neg. by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chem. and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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  15. 15
    Pillow, T. H., Tien, J., Parsons-Reponte, K. L., Bhakta, S., Li, H., Staben, L. R., Li, G., Chuh, J., Fourie-O’Donohue, A., Darwish, M. (2014) Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering. J. Med. Chem. 57 (19), 7890– 7899,  DOI: 10.1021/jm500552c
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    15
    Site-Specific Trastuzumab Maytansinoid Antibody-Drug Conjugates with Improved Therapeutic Activity through Linker and Antibody Engineering
    Pillow, Thomas H.; Tien, Janet; Parsons-Reponte, Kathryn L.; Bhakta, Sunil; Li, Hao; Staben, Leanna R.; Li, Guangmin; Chuh, Josefa; Fourie-O'Donohue, Aimee; Darwish, Martine; Yip, Victor; Liu, Luna; Leipold, Douglas D.; Su, Dian; Wu, Elmer; Spencer, Susan D.; Shen, Ben-Quan; Xu, Keyang; Kozak, Katherine R.; Raab, Helga; Vandlen, Richard; Lewis Phillips, Gail D.; Scheller, Richard H.; Polakis, Paul; Sliwkowski, Mark X.; Flygare, John A.; Junutula, Jagath R.
    Journal of Medicinal Chemistry (2014), 57 (19), 7890-7899CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clin. activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clin. activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clin. activity was demonstrated. Here, we report that through chem. modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chem. synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clin. approved ADC, trastuzumab-MCC-DM1.
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    Tian, F., Lu, Y., Manibusan, A., Sellers, A., Tran, H., Sun, Y., Phuong, T., Barnett, R., Hehli, B., Song, F. (2014) A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. U. S. A. 111 (5), 1766– 1771,  DOI: 10.1073/pnas.1321237111
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    16
    A general approach to site-specific antibody drug conjugates
    Tian, Feng; Lu, Yingchun; Manibusan, Anthony; Sellers, Aaron; Tran, Hon; Sun, Ying; Phuong, Trung; Barnett, Richard; Hehli, Brad; Song, Frank; De Guzman, Michael J.; Ensari, Semsi; Pinkstaff, Jason K.; Sullivan, Lorraine M.; Biroc, Sandra L.; Cho, Ho; Schultz, Peter G.; Di Joseph, John; Dougher, Maureen; Ma, Dangshe; Dushin, Russell; Leal, Mauricio; Tchistiakova, Lioudmila; Feyfant, Eric; Gerber, Hans-Peter; Sapra, Puja
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (5), 1766-1771CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicol. study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These expts. support the notion that chem. defined antibody conjugates can be synthesized in com. relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chem. modification.
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    Jackson, D., Atkinson, J., Guevara, C. I., Zhang, C., Kery, V., Moon, S. J., Virata, C., Yang, P., Lowe, C., Pinkstaff, J. (2014) In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates. PLoS One 9 (1), e83865  DOI: 10.1371/journal.pone.0083865
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    17
    In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates
    Jackson, Dowdy; Atkinson, John; Guevara, Claudia I.; Zhang, Chunying; Kery, Vladimir; Moon, Sung-Ju; Virata, Cyrus; Yang, Peng; Lowe, Christine; Pinkstaff, Jason; Cho, Ho; Knudsen, Nick; Manibusan, Anthony; Tian, Feng; Sun, Ying; Lu, Yingchun; Sellers, Aaron; Jia, Xiao-Chi; Joseph, Ingrid; Anand, Banmeet; Morrison, Kendall; Pereira, Daniel S.; Stover, David
    PLoS One (2014), 9 (1), e83865/1-e83865/14, 14 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Antibody-drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are crit. to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous no. of drugs per antibody. The no. of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclin. comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclin. toxicol. profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.
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    Strop, P., Delaria, K., Foletti, D., Witt, J. M., Hasa-Moreno, A., Poulsen, K., Casas, M. G., Dorywalska, M., Farias, S., Pios, A. (2015) Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading. Nat. Biotechnol. 33 (7), 694– 696,  DOI: 10.1038/nbt.3274
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    18
    Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading
    Strop, Pavel; Delaria, Kathy; Foletti, Davide; Witt, Jody Melton; Hasa-Moreno, Adela; Poulsen, Kris; Casas, Meritxell Galindo; Dorywalska, Magdalena; Farias, Santiago; Pios, Ariel; Lui, Victor; Dushin, Russell; Zhou, Dahui; Navaratnam, Thayalan; Tran, Thomas-Toan; Sutton, Janette; Lindquist, Kevin C.; Han, Bora; Liu, Shu-Hui; Shelton, David L.; Pons, Jaume; Rajpal, Arvind
    Nature Biotechnology (2015), 33 (7), 694-696CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    There is no expanded citation for this reference.
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    Behrens, C. R., Ha, E. H., Chinn, L. L., Bowers, S., Probst, G., Fitch-Bruhns, M., Monteon, J., Valdiosera, A., Bermudez, A., Liao-Chan, S. (2015) Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs. Mol. Pharmaceutics 12 (11), 3986– 3998,  DOI: 10.1021/acs.molpharmaceut.5b00432
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    19
    Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs
    Behrens, Christopher R.; Ha, Edward H.; Chinn, Lawrence L.; Bowers, Simeon; Probst, Gary; Fitch-Bruhns, Maureen; Monteon, Jorge; Valdiosera, Amanda; Bermudez, Abel; Liao-Chan, Sindy; Wong, Tiffany; Melnick, Jonathan; Theunissen, Jan-Willem; Flory, Mark R.; Houser, Derrick; Venstrom, Kristy; Levashova, Zoia; Sauer, Paul; Migone, Thi-Sau; van der Horst, Edward H.; Halcomb, Randall L.; Jackson, David Y.
    Molecular Pharmaceutics (2015), 12 (11), 3986-3998CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    Conventional antibody-drug conjugates (ADCs) are heterogeneous mixts. of chem. distinct mols. that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs contg. four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacol. properties of ADCs in vivo is unknown. In order to det. the relative impact of interchain cysteine crosslinking on the therapeutic window and other properties of ADCs in vivo, we synthesized a deriv. of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF deriv. was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs contg. predominantly four drugs/antibody. The pharmacol. properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine crosslinking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.
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    Hamblett, K. J., Le, T., Rock, B. M., Rock, D. A., Siu, S., Huard, J. N., Conner, K. P., Milburn, R. R., O’Neill, J. W., Tometsko, M. E. (2016) Altering Antibody–Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability. Mol. Pharmaceutics 13 (7), 2387– 2396,  DOI: 10.1021/acs.molpharmaceut.6b00153
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    20
    Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability
    Hamblett, Kevin J.; Le, Tiep; Rock, Brooke M.; Rock, Dan A.; Siu, Sophia; Huard, Justin N.; Conner, Kip P.; Milburn, Robert R.; O'Neill, Jason W.; Tometsko, Mark E.; Fanslow, William C.
    Molecular Pharmaceutics (2016), 13 (7), 2387-2396CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two sep. maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the obsd. efficacy was similar. More severe thrombocytopenia was obsd. with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approx. 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance obsd. for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equiv. doses and exacerbation of clin. pathol. parameters (decreased tolerability) at equiv. doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.
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  21. 21
    Lhospice, F., Bregeon, D., Belmant, C., Dennler, P., Chiotellis, A., Fischer, E., Gauthier, L., Boedec, A., Rispaud, H., Savard-Chambard, S. (2015) Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models. Mol. Pharmaceutics 12 (6), 1863– 1871,  DOI: 10.1021/mp500666j
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    Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models
    Lhospice, F.; Bregeon, D.; Belmant, C.; Dennler, P.; Chiotellis, A.; Fischer, E.; Gauthier, L.; Boedec, A.; Rispaud, H.; Savard-Chambard, S.; Represa, A.; Schneider, N.; Paturel, C.; Sapet, M.; Delcambre, C.; Ingoure, S.; Viaud, N.; Bonnafous, C.; Schibli, R.; Romagne, F.
    Molecular Pharmaceutics (2015), 12 (6), 1863-1871CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    Antibody-drug conjugates (ADCs) have demonstrated clin. benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clin. use or development, including ADCETRIS, are produced by chem. conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, the authors describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivs. of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity expts. using two different CD30-pos. cell lines (Karpas 299 and Raji-CD30+) revealed comparable EC50 values for ADCETRIS (1.8±0.4 and 3.6±0.6 ng/mL, resp.) and the four cAC10-based ADCs (2.0±0.4 to 4.9±1.0 ng/mL). Quant. time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected 125I-radioiodinated cAC10 ADC and compared with that of 125I-ADCETRIS. The chemo-enzymically conjugated, radioiodinated ADC showed higher tumor uptake (17.84±2.2% ID/g 24 h p.i.) than 125I-ADCETRIS (10.5±1.8% ID/g 24 h p.i.). Moreover, 125I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the max. tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chem. modified ADCs with variable DARs.
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  22. 22
    Yoder, N. C., Bai, C., Tavares, D., Widdison, W. C., Whiteman, K. R., Wilhelm, A., Wilhelm, S. D., McShea, M. A., Maloney, E. K., Ab, O. (2019) A Case Study Comparing Heterogeneous Lysine- and Site-Specific Cysteine-Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates Benefits of Lysine Conjugation. Mol. Pharmaceutics 16 (9), 3926– 3937,  DOI: 10.1021/acs.molpharmaceut.9b00529
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    22
    A Case Study Comparing Heterogeneous Lysine- and Site-Specific Cysteine-Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates the Benefits of Lysine Conjugation
    Yoder, Nicholas C.; Bai, Chen; Tavares, Daniel; Widdison, Wayne C.; Whiteman, Kathleen R.; Wilhelm, Alan; Wilhelm, Sharon D.; McShea, Molly A.; Maloney, Erin K.; Ab, Olga; Wang, Lintao; Jin, Shan; Erickson, Hans K.; Keating, Thomas A.; Lambert, John M.
    Molecular Pharmaceutics (2019), 16 (9), 3926-3937CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small mol. effectors. First-generation mols. of this class often employed heterogeneous conjugation chem., but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chem., we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chem., target antigen, and other variables that det. efficacy in a given setting.
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  23. 23
    Miller, M. L., Shizuka, M., Wilhelm, A., Salomon, P., Reid, E. E., Lanieri, L., Sikka, S., Maloney, E. K., Harvey, L., Qiu, Q. (2018) A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs). Mol. Cancer Ther. 17 (3), 650– 660,  DOI: 10.1158/1535-7163.MCT-17-0940
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    23
    A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs)
    Miller, Michael L.; Shizuka, Manami; Wilhelm, Alan; Salomon, Paulin; Reid, Emily E.; Lanieri, Leanne; Sikka, Surina; Maloney, Erin K.; Harvey, Lauren; Qiu, Qifeng; Archer, Katie E.; Bai, Chen; Vitharana, Dilrukshi; Harris, Luke; Singh, Rajeeva; Ponte, Jose F.; Yoder, Nicholas C.; Kovtun, Yelena; Lai, Katharine C.; Ab, Olga; Pinkas, Jan; Keating, Thomas A.; Chari, Ravi V. J.
    Molecular Cancer Therapeutics (2018), 17 (3), 650-660CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
    Tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADCs) is now a clin. validated approach for cancer treatment. In an attempt to improve the clin. success rate of ADCs, emphasis has been recently placed on the use of DNA-crosslinking pyrrolobenzodiazepine compds. as the payload. Despite promising early clin. results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs contg. the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650-60. ©2018 AACR.
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  24. 24
    Stimmel, J. B., Merrill, B. M., Kuyper, L. F., Moxham, C. P., Hutchins, J. T., Fling, M. E., and Kull, F. C. (2000) Site-specific conjugation on serine -> cysteine variant monoclonal antibodies. J. Biol. Chem. 275 (39), 30445– 30450,  DOI: 10.1074/jbc.M001672200
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    24
    Site-specific conjugation on serine → cysteine variant monoclonal antibodies
    Stimmel, Julie B.; Merrill, Barbara M.; Kuyper, Lee F.; Moxham, Cary P.; Hutchins, Jeff T.; Fling, Mary E.; Kull, Frederick C., Jr.
    Journal of Biological Chemistry (2000), 275 (39), 30445-30450CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third const. domain (CH3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chem. conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following redn., reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence anal. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment contg. the engineered cysteine.
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  25. 25
    Cao, M., De Mel, N., Jiao, Y., Howard, J., Parthemore, C., Korman, S., Thompson, C., Wendeler, M., and Liu, D. (2019) Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis. MAbs 11 (6), 1064– 1076,  DOI: 10.1080/19420862.2019.1624127
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    25
    Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis
    Cao, Mingyan; De Mel, Niluka; Jiao, Yang; Howard, James; Parthemore, Conner; Korman, Samuel; Thompson, Christopher; Wendeler, Michaela; Liu, Dengfeng
    mAbs (2019), 11 (6), 1064-1076CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
    Site-specific ADCs are designed to overcome heterogeneity obsd. with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. An elevated level of size variants, such as heavy chain-light chain species, heavy chain-heavy chain-light chain species, and light chain species, is also obsd. with final site-specific ADC product. To understand root cause of heterogeneity generated during ADC conjugation process, we designed time-course studies for each conjugation step, including redn., oxidn., conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample anal. On basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidn. as a result of alternative disulfide bond arrangements. During oxidn., two hinge cysteines formed intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in unconjugated antibody. Time-course data also showed that elevated level of size variants, esp. heavy chain-heavy chain-light chain species and light chain species, resulted from quenching step, where quenching reagent engaged in disulfide bond exchange reaction with ADC and broke disulfide bonds connecting heavy chain and light chain. Underconjugated and overconjugated species arose from equil. established during conjugation reaction.
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  26. 26
    Vollmar, B. S., Wei, B., Ohri, R., Zhou, J., He, J., Yu, S. F., Leipold, D., Cosino, E., Yee, S., Fourie-O’Donohue, A. (2017) Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates. Bioconjugate Chem. 28 (10), 2538– 2548,  DOI: 10.1021/acs.bioconjchem.7b00365
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    26
    Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates
    Vollmar, Breanna S.; Wei, Binqing; Ohri, Rachana; Zhou, Jianhui; He, Jintang; Yu, Shang-Fan; Leipold, Douglas; Cosino, Ely; Yee, Sharon; Fourie-O'Donohue, Aimee; Li, Guangmin; Phillips, Gail L.; Kozak, Katherine R.; Kamath, Amrita; Xu, Keyang; Lee, Genee; Lazar, Greg A.; Erickson, Hans K.
    Bioconjugate Chemistry (2017), 28 (10), 2538-2548CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small mols. to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small mol. and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was obsd. between disulfide stability and efficacy for the conjugates. We hypothesized that the obsd. site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titrn. calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homol. modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.
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  27. 27
    Jeffrey, S. C., Burke, P. J., Lyon, R. P., Meyer, D. W., Sussman, D., Anderson, M., Hunter, J. H., Leiske, C. I., Miyamoto, J. B., Nicholas, N. D. (2013) A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. Bioconjugate Chem. 24 (7), 1256– 1263,  DOI: 10.1021/bc400217g
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    27
    A Potent Anti-CD70 Antibody-Drug Conjugate Combining a Dimeric Pyrrolobenzodiazepine Drug with Site-Specific Conjugation Technology
    Jeffrey, Scott C.; Burke, Patrick J.; Lyon, Robert P.; Meyer, David W.; Sussman, Django; Anderson, Martha; Hunter, Joshua H.; Leiske, Chris I.; Miyamoto, Jamie B.; Nicholas, Nicole D.; Okeley, Nicole M.; Sanderson, Russell J.; Stone, Ivan J.; Zeng, Weiping; Gregson, Stephen J.; Masterson, Luke; Tiberghien, Arnaud C.; Howard, Philip W.; Thurston, David E.; Law, Che-Leung; Senter, Peter D.
    Bioconjugate Chemistry (2013), 24 (7), 1256-1263CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A highly cytotoxic DNA crosslinking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity expts. demonstrated that the h1F6239C-PBD was potent and immunol. specific on CD70-pos. renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-pos. RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.
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    Hamblett, K. J., Senter, P. D., Chace, D. F., Sun, M. M. C., Lenox, J., Cerveny, C. G., Kissler, K. M., Bernhardt, S. X., Kopcha, A. K., Zabinski, R. F. (2004) Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate. Clin. Cancer Res. 10 (20), 7063– 7070,  DOI: 10.1158/1078-0432.CCR-04-0789
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    28
    Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate
    Hamblett, Kevin J.; Senter, Peter D.; Chace, Dana F.; Sun, Michael M. C.; Lenox, Joel; Cerveny, Charles G.; Kissler, Kim M.; Bernhardt, Starr X.; Kopcha, Anastasia K.; Zabinski, Roger F.; Meyer, Damon L.; Francisco, Joseph A.
    Clinical Cancer Research (2004), 10 (20), 7063-7070CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
    An antibody-drug conjugate consisting of monomethyl auristatin E (MMAE) conjugated to the anti-CD30 monoclonal antibody (mAb) cAC10, with eight drug moieties per mAb, was previously shown to have potent cytotoxic activity against CD30+ malignant cells. To det. the effect of drug loading on antibody-drug conjugate therapeutic potential, we assessed cAC10 antibody-drug conjugates contg. different drug-mAb ratios in vitro and in vivo. Coupling MMAE to the cysteines that comprise the interchain disulfides of cAC10 created an antibody-drug conjugate population, which was purified using hydrophobic interaction chromatog. to yield antibody-drug conjugates with two, four, and eight drugs per antibody (E2, E4, and E8, resp.). Antibody-drug conjugate potency was tested in vitro against CD30+ lines followed by in vivo xenograft models. The max.-tolerated dose and pharmacokinetic profiles of the antibody-drug conjugates were investigated in mice. Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC50 values E8<E4<E2), the in vivo antitumor activity of E4 was comparable with E8 at equal mAb doses, although the E4 contained half the amt. of MMAE per mAb. E2 was also an active antitumor agent but required higher doses. The max.-tolerated dose of E2 in mice was at least double that of E4, which in turn was twice that of E8. MMAE loading affected plasma clearance, as E8 cleared 3-fold faster than E4 and 5-fold faster than E2. By decreasing drug loading per antibody, the therapeutic index was increased demonstrating that drug loading is a key design parameter for antibody-drug conjugates.
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    Sun, X., Ponte, J. F., Yoder, N. C., Laleau, R., Coccia, J., Lanieri, L., Qiu, Q., Wu, R., Hong, E., Bogalhas, M. (2017) Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates. Bioconjugate Chem. 28 (5), 1371– 1381,  DOI: 10.1021/acs.bioconjchem.7b00062
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    29
    Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates
    Sun, Xiuxia; Ponte, Jose F.; Yoder, Nicholas C.; Laleau, Rassol; Coccia, Jennifer; Lanieri, Leanne; Qiu, Qifeng; Wu, Rui; Hong, Erica; Bogalhas, Megan; Wang, Lintao; Dong, Ling; Setiady, Yulius; Maloney, Erin K.; Ab, Olga; Zhang, Xiaoyan; Pinkas, Jan; Keating, Thomas A.; Chari, Ravi; Erickson, Hans K.; Lambert, John M.
    Bioconjugate Chemistry (2017), 28 (5), 1371-1381CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an av. drug to antibody ratio (DAR) of 3-4 (potentially ranging from 0-8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an av. DAR of 3.5. To understand the effect of DAR on the preclin. properties of ADCs using maytansinoid cytotoxic agents, we prepd. a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor alpha (FR)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR, and evaluated their biochem. characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (av. ∼2, range 0-4) to very high (av. 10, range 7-14) were prepd. in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR, at const. antibody concn. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic anal. showed that conjugates with an av. DAR below ∼6 had comparable clearance rates, but for those with an av. DAR around 9-10, rapid clearance was obsd. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with max. localization for this organ at 24-28% percentage injected dose per g (%ID/g) compared with 7-10% for lower DAR conjugates (all at 2-6 h post-injection). Our preclin. findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2-6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs, but suggest the exploration of lower or higher DAR may be warranted depending on the biol. of the target antigen.
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  30. 30
    Thompson, P., Fleming, R., Bezabeh, B., Huang, F., Mao, S., Chen, C., Harper, J., Zhong, H., Gao, X., Yu, X.-Q. (2016) Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics. J. Controlled Release 236, 100– 116,  DOI: 10.1016/j.jconrel.2016.06.025
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    Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics
    Thompson, Pamela; Fleming, Ryan; Bezabeh, Binyam; Huang, Fengying; Mao, Shenlan; Chen, Cui; Harper, Jay; Zhong, Haihong; Gao, Xizhe; Yu, Xiang-Qing; Hinrichs, Mary Jane; Reed, Molly; Kamal, Adeela; Strout, Patrick; Cho, Song; Woods, Rob; Hollingsworth, Robert E.; Dixit, Rakesh; Wu, Herren; Gao, Changshou; Dimasi, Nazzareno
    Journal of Controlled Release (2016), 236 (), 100-116CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepd. by chem. conjugation of small mol. cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the redn. of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophys. properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody mol. We demonstrate that the chem.-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addn., to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clin. development.
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    Ouyang, J. (2013) Drug-to-antibody ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography. Methods Mol. Biol. (N. Y., NY, U. S.) 1045, 275– 283,  DOI: 10.1007/978-1-62703-541-5_17
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    Drug-to-antibody ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography
    Ouyang Jun
    Methods in molecular biology (Clifton, N.J.) (2013), 1045 (), 275-83 ISSN:.
    Hydrophobic interaction chromatography (HIC) is the method of choice for determination of the drug-to-antibody ratio (DAR) and drug load distribution for cysteine (Cys)-linked antibody-drug conjugates (ADCs). The drug-loaded species are resolved based on the increasing hydrophobicity with the least hydrophobic, unconjugated form eluting first and the most hydrophobic, 8-drug form eluting last. The area percentage of a peak represents the relative distribution of the particular drug-loaded ADC species. The weighted average DAR is then calculated using the percentage peak area information and the drug load numbers. Reversed phase high-performance liquid chromatography (RP-HPLC) offers an orthogonal method to obtain DAR for Cys-linked ADCs. The method involves, first, a reduction reaction to completely dissociate the heavy and light chains of the ADC, then separation of the light and heavy chains and their corresponding drug-loaded forms on an RP column. The percentage peak area from integration of the light chain and heavy chain peaks, combined with the assigned drug load for each peak, is used to calculate the weighted average DAR.
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    Fekete, S., Veuthey, J. L., Beck, A., and Guillarme, D. (2016) Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products. J. Pharm. Biomed. Anal. 130, 3– 18,  DOI: 10.1016/j.jpba.2016.04.004
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    Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products
    Fekete, Szabolcs; Veuthey, Jean-Luc; Beck, Alain; Guillarme, Davy
    Journal of Pharmaceutical and Biomedical Analysis (2016), 130 (), 3-18CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
    A review. Hydrophobic interaction chromatog. (HIC) is a historical strategy used for the anal. purifn. and characterization of proteins. Similarly to what can be done in reversed-phase liq. chromatog. (RPLC), HIC is able to sep. protein species based on their hydrophobicity, but using different conditions. Compared to RPLC, the main benefit of HIC is its ability to perform sepns. under non denaturing conditions (i.e. physiol. pH conditions, ambient mobile phase temp. and no need for org. solvents) and so an orthogonal method. The goal of this review is to provide a general overview of theor. and practical aspects of modern HIC applied for the characterization of therapeutic protein biopharmaceuticals including monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) and bispecific antibodies (bsAbs). Therefore, method development approaches, state-of-the-art column technol., applications and future perspectives are described and critically discussed.
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  33. 33
    Strop, P., Liu, S. H., Dorywalska, M., Delaria, K., Dushin, R. G., Tran, T. T., Ho, W. H., Farias, S., Casas, M. G., Abdiche, Y. (2013) Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20 (2), 161– 167,  DOI: 10.1016/j.chembiol.2013.01.010
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    Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates
    Strop, Pavel; Liu, Shu-Hui; Dorywalska, Magdalena; Delaria, Kathy; Dushin, Russell G.; Tran, Thomas-Toan; Ho, Wei-Hsien; Farias, Santiago; Casas, Meritxell Galindo; Abdiche, Yasmina; Zhou, Dahui; Chandrasekaran, Ramalakshmi; Samain, Caroline; Loo, Carole; Rossi, Andrea; Rickert, Mathias; Krimm, Stellanie; Wong, Teresa; Chin, Sherman Michael; Yu, Jessica; Dilley, Jeanette; Chaparro-Riggers, Javier; Filzen, Gary F.; O'Donnell, Christopher J.; Wang, Fang; Myers, Jeremy S.; Pons, Jaume; Shelton, David L.; Rajpal, Arvind
    Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 161-167CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compds. at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chem. instability, as was obsd. with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.
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  34. 34
    Lyon, R. P., Bovee, T. D., Doronina, S. O., Burke, P. J., Hunter, J. H., Neff-LaFord, H. D., Jonas, M., Anderson, M. E., Setter, J. R., and Senter, P. D. (2015) Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nat. Biotechnol. 33 (7), 733– 735,  DOI: 10.1038/nbt.3212
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    Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index
    Lyon, Robert P.; Bovee, Tim D.; Doronina, Svetlana O.; Burke, Patrick J.; Hunter, Joshua H.; Neff-LaFord, Haley D.; Jonas, Mechthild; Anderson, Martha E.; Setter, Jocelyn R.; Senter, Peter D.
    Nature Biotechnology (2015), 33 (7), 733-735CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    The in vitro potency of antibody-drug conjugates (ADCs) increases with the drug-to-antibody ratio (DAR); however, ADC plasma clearance also increases with DAR, reducing exposure and in vivo efficacy. Here we show that accelerated clearance arises from ADC hydrophobicity, which can be modulated through drug-linker design. We exemplify this using hydrophilic auristatin drug linkers and PEGylated ADCs that yield uniform, high-DAR ADCs with superior in vivo performance.
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  35. 35
    Tumey, L. N., Li, F., Rago, B., Han, X., Loganzo, F., Musto, S., Graziani, E. I., Puthenveetil, S., Casavant, J., Marquette, K. (2017) Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates. AAPS J. 19, 1– 13,  DOI: 10.1208/s12248-017-0083-7
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    Burke, P. J., Hamilton, J. Z., Jeffrey, S. C., Hunter, J. H., Doronina, S. O., Okeley, N. M., Miyamoto, J. B., Anderson, M. E., Stone, I. J., Ulrich, M. L. (2017) Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates. Mol. Cancer Ther. 16 (1), 116– 123,  DOI: 10.1158/1535-7163.MCT-16-0343
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    36
    Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates
    Burke, Patrick J.; Hamilton, Joseph Z.; Jeffrey, Scott C.; Hunter, Joshua H.; Doronina, Svetlana O.; Okeley, Nicole M.; Miyamoto, Jamie B.; Anderson, Martha E.; Stone, Ivan J.; Ulrich, Michelle L.; Simmons, Jessica K.; McKinney, Erica E.; Senter, Peter D.; Lyon, Robert P.
    Molecular Cancer Therapeutics (2017), 16 (1), 116-123CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
    The emergence of antibody-drug conjugates (ADC), such as brentuximab vedotin and ado-trastuzumab emtansine, has led to increased efforts to identify new payloads and develop improved drug-linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC, resulting in accelerated plasma clearance and suboptimal in vivo activity, particularly for conjugates with high drug-to-antibody ratios (DAR). We recently reported on the incorporation of a discrete PEG24 polymer as a side chain in a β-glucuronidase-cleavable monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. In this work, we optimized the drug-linker by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo. Multiple PEG-glucuronide-MMAE linkers were prepd. with PEG size up to 24 ethylene oxide units, and homogeneous DAR 8 ADCs were evaluated. A clear relationship was obsd. between PEG length and conjugate pharmacol. when tested in vivo. Longer PEG chains resulted in slower clearance, with a threshold length of PEG8 beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize plasma clearance provided a wider therapeutic window relative to faster clearing conjugates bearing shorter PEGs. A lead PEGylated glucuronide-MMAE linker was identified incorporating a self-stabilizing maleimide and a PEG12 side chain emerged from these efforts, enabling highly potent, homogeneous DAR 8 conjugates and is under consideration for future ADC programs.
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    Lyon, R. P., Meyer, D. L., Setter, J. R., and Senter, P. D. (2012) Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine residues. Methods Enzymol. 502, 123– 138,  DOI: 10.1016/B978-0-12-416039-2.00006-9
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    Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine residues
    Lyon, Robert P.; Meyer, David L.; Setter, Jocelyn R.; Senter, Peter D.
    Methods in Enzymology (2012), 502 (Protein Engineering for Therapeutics, Part A), 123-138CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
    A review. Many methods have been described for the conjugation of drugs to monoclonal antibodies. The presence of a discrete no. of readily reducible disulfides in the common IgG subtypes presents a convenient opportunity for conjugation to cysteine residues with thiol-reactive drug-linkers. Such conjugates can be prepd. by a straightforward two-step reaction scheme involving the redn. of the antibody disulfides to the desired no. of av. thiols per antibody, followed by addn. of the drug-linker, ideally with a maleimido functionality for rapid, selective reaction. In a discovery setting, this basic method can be scaled down to produce microgram quantities of conjugate for early screening, and in a manufg. setting can be scaled up to produce grams or kilograms of conjugate for clin. trials and commercialization. The resulting conjugates are readily characterized using common HPLC methods.
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  38. 38
    Bhakta, S., Raab, H., and Junutula, J. R. (2013) Engineering THIOMABs for site-specific conjugation of thiol-reactive linkers. Methods Mol. Biol. 1045, 189– 203,  DOI: 10.1007/978-1-62703-541-5_11
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    Engineering THIOMABs for site-specific conjugation of thiol-reactive linkers
    Bhakta Sunil; Raab Helga; Junutula Jagath R
    Methods in molecular biology (Clifton, N.J.) (2013), 1045 (), 189-203 ISSN:.
    Antibody conjugates are used in many therapeutic and research applications and are generated by chemically linking a cysteine or lysine residue to potent chemotherapeutic drugs or other functional groups through a flexible linker. Recently, we have engineered THIOMABs (antibodies with engineered reactive cysteine residues) for site-specific conjugation and showed that these antibody conjugates display homogeneous labeling with optimal in vitro and in vivo characteristics. Here, we describe protocols for engineering, selection, and site-specific conjugation of THIOMABs with thiol-reactive linkers.
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  39. 39
    Garcia-Echeverria, C. (2014) Developing second generation antibody-drug conjugates: the quest for new technologies. J. Med. Chem. 57 (19), 7888– 7889,  DOI: 10.1021/jm501298k
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    Developing Second Generation Antibody-Drug Conjugates: The Quest for New Technologies
    Garcia-Echeverria, Carlos
    Journal of Medicinal Chemistry (2014), 57 (19), 7888-7889CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. The field of antibody-drug conjugates (ADCs) has gained significant momentum after the recent regulatory approval of two ADCs, and significant research efforts are directed to identify more effective payloads and simplify current manufg. challenges.
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  40. 40
    Drake, P. M., Albers, A. E., Baker, J., Banas, S., Barfield, R. M., Bhat, A. S., de Hart, G. W., Garofalo, A. W., Holder, P., Jones, L. C. (2014) Aldehyde Tag Coupled with HIPS Chemistry Enables the Production of ADCs Conjugated Site-Specifically to Different Antibody Regions with Distinct in Vivo Efficacy and PK Outcomes. Bioconjugate Chem. 25 (7), 1331– 1341,  DOI: 10.1021/bc500189z
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    40
    Aldehyde Tag Coupled with HIPS Chemistry Enables the Production of ADCs Conjugated Site-Specifically to Different Antibody Regions with Distinct in Vivo Efficacy and PK Outcomes
    Drake, Penelope M.; Albers, Aaron E.; Baker, Jeanne; Banas, Stefanie; Barfield, Robyn M.; Bhat, Abhijit S.; de Hart, Gregory W.; Garofalo, Albert W.; Holder, Patrick; Jones, Lesley C.; Kudirka, Romas; McFarland, Jesse; Zmolek, Wes; Rabuka, David
    Bioconjugate Chemistry (2014), 25 (7), 1331-1341CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addn. to the DAR and site of conjugation, linker compn. also plays an important role in the properties of an ADC. The authors have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chem. results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled the authors to undertake a systematic evaluation of the impact of conjugation site and linker compn. on ADC properties. Here, the authors describe the prodn. and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chem. The authors demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.
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    Catcott, K. C., McShea, M. A., Bialucha, C. U., Miller, K. L., Hicks, S. W., Saxena, P., Gesner, T. G., Woldegiorgis, M., Lewis, M. E., Bai, C. (2016) Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates. MAbs 8 (3), 513– 523,  DOI: 10.1080/19420862.2015.1134408
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    Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates
    Catcott, Kalli C.; McShea, Molly A.; Bialucha, Carl Uli; Miller, Kathy L.; Hicks, Stuart W.; Saxena, Parmita; Gesner, Thomas G.; Woldegiorgis, Mikias; Lewis, Megan E.; Bai, Chen; Fleming, Michael S.; Ettenberg, Seth A.; Erickson, Hans K.; Yoder, Nicholas C.
    mAbs (2016), 8 (3), 513-523CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
    Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Prepn. of ADCs for early stage screening is constrained by purifn. and biochem. anal. techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼100 μg of antibody per well and requires <5 μg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput prepn. and screening of different types of protein conjugates.
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    Cilliers, C., Guo, H., Liao, J., Christodolu, N., and Thurber, G. M. (2016) Multiscale Modeling of Antibody-Drug Conjugates: Connecting Tissue and Cellular Distribution to Whole Animal Pharmacokinetics and Potential Implications for Efficacy. AAPS J. 18 (5), 1117– 1130,  DOI: 10.1208/s12248-016-9940-z
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    Multiscale Modeling of Antibody-Drug Conjugates: Connecting Tissue and Cellular Distribution to Whole Animal Pharmacokinetics and Potential Implications for Efficacy
    Cilliers, Cornelius; Guo, Hans; Liao, Jianshan; Christodolu, Nikolas; Thurber, Greg M.
    AAPS Journal (2016), 18 (5), 1117-1130CODEN: AJAOB6; ISSN:1550-7416. (Springer)
    Antibody-drug conjugates exhibit complex pharmacokinetics due to their combination of macromol. and small mol. properties. These issues range from systemic concerns, such as deconjugation of the small mol. drug during the long antibody circulation time or rapid clearance from nonspecific interactions, to local tumor tissue heterogeneity, cell bystander effects, and endosomal escape. Math. models can be used to study the impact of these processes on overall distribution in an efficient manner, and several types of models have been used to analyze varying aspects of antibody distribution including physiol. based pharmacokinetic (PBPK) models and tissue-level simulations. However, these processes are quant. in nature and cannot be handled qual. in isolation. For example, free antibody from deconjugation of the small mol. will impact the distribution of conjugated antibodies within the tumor. To incorporate these effects into a unified framework, we have coupled the systemic and organ-level distribution of a PBPK model with the tissue-level detail of a distributed parameter tumor model. We used this math. model to analyze new exptl. results on the distribution of the clin. antibody-drug conjugate Kadcyla in HER2-pos. mouse xenografts. This model is able to capture the impact of the drug-antibody ratio (DAR) on tumor penetration, the net result of drug deconjugation, and the effect of using unconjugated antibody to drive ADC penetration deeper into the tumor tissue. This modeling approach will provide quant. and mechanistic support to exptl. studies trying to parse the impact of multiple mechanisms of action for these complex drugs.
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    Bumbaca, D., Boswell, C. A., Fielder, P. J., and Khawli, L. A. (2012) Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics. AAPS J. 14 (3), 554– 558,  DOI: 10.1208/s12248-012-9369-y
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    Physiochemical and Biochemical Factors Influencing the Pharmacokinetics of Antibody Therapeutics
    Bumbaca, Daniela; Boswell, C. Andrew; Fielder, Paul J.; Khawli, Leslie A.
    AAPS Journal (2012), 14 (3), 554-558CODEN: AJAOB6; ISSN:1550-7416. (Springer)
    A review. Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncol., immunol., neurol., and ophthalmol. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biol., IgG (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromol. therapeutics, which in turn could have ramifications on their efficacy and safety. This mini-review examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was obsd. that a net charge modification of at least a 1-unit shift in isoelec. point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to det. if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.
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    Bohnert, T. and Gan, L. S. (2013) Plasma protein binding: from discovery to development. J. Pharm. Sci. 102 (9), 2953– 2994,  DOI: 10.1002/jps.23614
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    Plasma protein binding: From discovery to development
    Bohnert, Tonika; Gan, Liang-Shang
    Journal of Pharmaceutical Sciences (2013), 102 (9), 2953-2994CODEN: JPMSAE; ISSN:0022-3549. (John Wiley & Sons, Inc.)
    A review. The importance of plasma protein binding (PPB) in modulating the effective drug concn. at pharmacol. target sites has been the topic of significant discussion and debate amongst drug development groups over the past few decades. Free drug theory, which states that in absence of energy-dependent processes, after steady state equil. has been attained, free drug concn. in plasma is equal to free drug concn. at the pharmacol. target receptor(s) in tissues, has been used to explain pharmacokinetics/pharmacodynamics relationships in a large no. of cases. Any sudden increase in free concn. of a drug could potentially cause toxicity and may need dose adjustment. Free drug concn. is also helpful to est. the effective concn. of drugs that potentially can ppt. metab. (or transporter)-related drug-drug interactions. Disease models are extensively validated in animals to progress a compd. into development. Unbound drug concn., and therefore PPB information across species is very informative in establishing safety margins and guiding selection of First in Human (FIH) dose and human efficacious dose. The scope of this review is to give an overview of reported role of PPB in several therapeutic areas, highlight cases where PPB changes are clin. relevant, and provide drug metab. and pharmacokinetics recommendations in discovery and development settings. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Assocn. J Pharm Sci.
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    Alley, S. C., Benjamin, D. R., Jeffrey, S. C., Okeley, N. M., Meyer, D. L., Sanderson, R. J., and Senter, P. D. (2008) Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjugate Chem. 19 (3), 759– 765,  DOI: 10.1021/bc7004329
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    Contribution of Linker Stability to the Activities of Anticancer Immunoconjugates
    Alley, Stephen C.; Benjamin, Dennis R.; Jeffrey, Scott C.; Okeley, Nicole M.; Meyer, Damon L.; Sanderson, Russell J.; Senter, Peter D.
    Bioconjugate Chemistry (2008), 19 (3), 759-765CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technol. advancement, since the linker half-lives in circulation (t1/2 ∼ 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepd. contg. a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 wk postadministration in mice. In order to assess the impact of improving linker stability beyond mc contg. ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac contg. ADCs, although the bac linker technol. led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide deriv. became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.
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  46. 46
    Ohri, R., Bhakta, S., Fourie-O’Donohue, A., Dela Cruz-Chuh, J., Tsai, S. P., Cook, R., Wei, B., Ng, C., Wong, A. W., Bos, A. B. (2018) High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers. Bioconjugate Chem. 29 (2), 473– 485,  DOI: 10.1021/acs.bioconjchem.7b00791
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    High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers
    Ohri, Rachana; Bhakta, Sunil; Fourie-O'Donohue, Aimee; dela Cruz-Chuh, Josefa; Tsai, Siao Ping; Cook, Ryan; Wei, Binqing; Ng, Carl; Wong, Athena W.; Bos, Aaron B.; Farahi, Farzam; Bhakta, Jiten; Pillow, Thomas H.; Raab, Helga; Vandlen, Richard; Polakis, Paul; Liu, Yichin; Erickson, Hans; Junutula, Jagath R.; Kozak, Katherine R.
    Bioconjugate Chemistry (2018), 29 (2), 473-485CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    THIOMAB antibody technol. utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technol. has enabled the exploration of different attachment sites on the antibody in combination with small mols., peptides, or proteins to yield antibody conjugates with unique properties. As reported previously, the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepd. by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purifn. method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivs. with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approx. 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivs. were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.
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    Drawing lessons from the clinical development of antibody-drug conjugates
    Lyon Robert
    Drug discovery today. Technologies (2018), 30 (), 105-109 ISSN:.
    The antibody-drug conjugate (ADC) field has seen a remarkable expansion in the number of entrants in clinical studies. Many of these agents employ newer conjugation technologies that have been developed over the last decade that confer various attributes to the ADCs prepared with them, including stability, potency, and homogeneity. In many cases, these new ADCs appear demonstrably superior to earlier technologies in preclinical models of activity and toxicology, but the degree to which these improvements will translate to the clinic is only starting to be seen. Many of these technologies are now competing head-to-head by targeting the same antigen in similar patient populations, allowing for a direct comparison of their clinical performance properties. As lessons from these experiences feed back into discovery research, future iterations of ADC design may be expected to bring improved therapeutics into the clinic.
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    Khera, E., Cilliers, C. M., Bhatnagar, S., and Thurber, G. M. (2018) Computation transport analysis of antibody-drug conjugate bystander effects and payload tumoral distribution: implications for therapy. Mol. Syst. Des. Eng. 3, 73– 88,  DOI: 10.1039/C7ME00093F
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    Computational transport analysis of antibody-drug conjugate bystander effects and payload tumoral distribution: implications for therapy
    Khera, Eshita; Cilliers, Cornelius; Bhatnagar, Sumit; Thurber, Greg M.
    Molecular Systems Design & Engineering (2018), 3 (1), 73-88CODEN: MSDEBG; ISSN:2058-9689. (Royal Society of Chemistry)
    Antibody drug conjugates (ADCs) have a proven clin. record with four FDA approved drugs and dozens more in clin. trials. However, a better understanding of the relationship between delivery and efficacy of ADCs is needed to improve the rate of successful clin. development. Recent evidence indicates that heterogeneous distribution can play a large role in the efficacy of these drugs. However, the impact of the drug payload, particularly the ability of the payload to diffuse outside of the original targeted cell into adjacent cells (the bystander effect), is not completely understood. Given the challenges in directly measuring the payload distribution within tumors, we developed a predictive computational model to study payload distribution as a function of antibody dose, payload dose, and payload properties. The computational results indicate that: 1. the heterogeneous tumoral distribution of ADCs impacts efficacy, and increasing the antibody dose improves penetration and efficacy. 2. The increased penetration of payloads with bystander effects can partially compensate for poor antibody penetration, but larger antibody doses still result in further improvement. This occurs because of the higher efficiency of direct cell killing than bystander killing. 3. Bystander effects are important for killing antigen neg. cells, and an optimum in physicochem. properties exists. Payloads with a balance in cellular uptake vs. tissue diffusion enter cells fast enough to avoid tumor washout but slow enough to reach distant cells. Therefore, optimizing the antibody dose, payload dose, and payload physicochem. properties results in ideal delivery to the site of action and max. efficacy.
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    Cilliers, C., Menezes, B., Nessler, I., Linderman, J., and Thurber, G. M. (2018) Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival. Cancer Res. 78 (3), 758– 768,  DOI: 10.1158/0008-5472.CAN-17-1638
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    Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival
    Cilliers, Cornelius; Menezes, Bruna; Nessler, Ian; Linderman, Jennifer; Thurber, Greg M.
    Cancer Research (2018), 78 (3), 758-768CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    Current ADC have made advances in engineering the antibody, linker, conjugation site, small-mol. payload, and drug-to-antibody ratio (DAR). However, the relationship between heterogeneous intratumoral distribution and efficacy of ADCs is poorly understood. Here, we compared trastuzumab and ado-trastuzumab emtansine (T-DM1) to study the impact of ADC tumor distribution on efficacy. In a mouse xenograft model insensitive to trastuzumab, coadministration of trastuzumab with a fixed dose of T-DM1 at 3:1 and 8:1 ratios dramatically improved ADC tumor penetration and resulted in twice the improvement in median survival compared with T-DM1 alone. In this setting, the effective DAR was lowered, decreasing the amt. of payload delivered to each targeted cell but increasing the no. of cells that received payload. This result is counterintuitive because trastuzumab acts as an antagonist in vitro and has no single-agent efficacy in vivo, yet improves the effectiveness of T-DM1 in vivo. Novel dual-channel fluorescence ratios quantified single-cell ADC uptake and metab. and confirmed that the in vivo cellular dose of T-DM1 alone exceeded the min. required for efficacy in this model. In addn., this technique characterized cellular pharmacokinetics with heterogeneous delivery after 1 day, degrdn. and payload release by 2 days, and in vitro cell killing and in vivo tumor shrinkage 2 to 3 days later. This work demonstrates that the intratumoral distribution of ADC, independent of payload dose or plasma clearance, plays a major role in ADC efficacy. This study shows how lowering the drug-to-antibody ratio during treatment can improve the intratumoral distribution of a antibody-drug conjugate, with implications for improving the efficacy of this class of cancer drugs.
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    Sahota, S. and Vahdat, L. T. (2017) Sacituzumab govitecan: an antibody-drug conjugate. Expert Opin. Biol. Ther. 17 (8), 1027– 1031,  DOI: 10.1080/14712598.2017.1331214
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    Sacituzumab govitecan: an antibody-drug conjugate
    Sahota, Sheena; Vahdat, Linda T.
    Expert Opinion on Biological Therapy (2017), 17 (8), 1027-1031CODEN: EOBTA2; ISSN:1471-2598. (Taylor & Francis Ltd.)
    : Despite advances in the diagnosis and treatment of patients with cancer, patients with metastatic cancer have limited therapeutic options after initial lines of therapy. Understanding tumor biol. has translated into the identification of actionable targets that resulted in therapeutics. Antibody-drug conjugates (ADC) are capitalizing on this explosion of scientific information. ADCs allow an antibody to a unique target to be conjugated via an innovative linker, to a highly toxic drug which is delivered to its target. Sacituzumab govitecan is an ADC that combines the active mol. in irinotecan, SN-38, to an antibody targeting trop2.: In this review, the authors introduce the reader to the ADC sacituzumab govitecan providing the reader with details about its pharmacokinetics, pharmacodynamics, efficacy and safety. The authors also give their expert anal. about its potential future use.: Sacituzumab govitecan is a novel and well-tolerated therapeutic showing promising results in difficult to treat cancers. Further studies are underway to optimize the group of patients that would benefit from it. Given its excellent performance, we are cautiously optimistic it will be approved by the FDA.
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    Nakada, T., Sugihara, K., Jikoh, T., Abe, Y., and Agatsuma, T. (2019) The Latest Research and Development into the Antibody-Drug Conjugate, [fam-] Trastuzumab Deruxtecan (DS-8201a), for HER2 Cancer Therapy. Chem. Pharm. Bull. 67 (3), 173– 185,  DOI: 10.1248/cpb.c18-00744
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    The latest research and development into the antibody-drug conjugate, [fam-] trastuzumab deruxtecan (DS-8201a), for HER2 cancer therapy
    Nakada, Takashi; Sugihara, Kiyoshi; Jikoh, Takahiro; Abe, Yuki; Agatsuma, Toshinori
    Chemical & Pharmaceutical Bulletin (2019), 67 (3), 173-185CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)
    A review. A major limitation of traditional chemotherapy for cancer is dose-limiting toxicity, caused by the exposure of non-tumor cells to cytotoxic agents. Use of mol. targeted drugs, such as specific kinase inhibitors and monoclonal antibodies, is a possible soln. to overcome this limitation and has achieved clin. success so far. Use of an antibody-drug conjugate (ADC) is a rational strategy for improving efficacy and reducing systemic adverse events. ADCs use antibodies selectively to deliver a potent cytotoxic agent to tumor cells, thus drastically improving the therapeutic index of chemotherapeutic agents. Lessons learned from clin. failure of early ADCs during the 1980s to 90s have recently led to improvements in ADC technol., and resulted in the approval of four novel ADCs. Nonetheless, further advances in ADC technol. are still required to streamline their clin. efficacy and reduce toxicity. [fam-] Trastuzumab deruxtecan (DS-8201a) is a next-generation ADC that satisfies these requirements based on currently available evidence. DS-8201a has several innovative features; a highly potent novel payload with a high drug-to-antibody ratio, good homogeneity, a tumor-selective cleavable linker, stable linker-payload in circulation, and a short systemic half-life cytotoxic agent in vivo; the released cytotoxic payload could exert a bystander effect. With respect to its preclin. profiles, DS-8201a could provide a valuable therapy with a great potential against HER2-expressing cancers in clin. settings. In a phase I trial, DS-8201a showed acceptable safety profiles with potential therapeutic efficacy, with the wide therapeutic index.
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    Reid, E. E., Archer, K. E., Shizuka, M., Wilhelm, A., Yoder, N. C., Bai, C., Fishkin, N. E., Harris, L., Maloney, E. K., E, H. (2019) Effect of linker stereochemistry on the antitumor activity of anti-body-drug conjugates (ADCs) containing indolinobenzodiazepine payloads. ACS Med. Chem. Lett. 10 (8), 1193– 1197,  DOI: 10.1021/acsmedchemlett.9b00240
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    Effect of Linker Stereochemistry on the Activity of Indolinobenzodiazepine Containing Antibody-Drug Conjugates (ADCs)
    Reid, Emily E.; Archer, Katie E.; Shizuka, Manami; Wilhelm, Alan; Yoder, Nicholas C.; Bai, Chen; Fishkin, Nathan E.; Harris, Luke; Maloney, Erin K.; Salomon, Paulin; Hong, Erica; Wu, Rui; Ab, Olga; Jin, Shan; Lai, Katharine C.; Sikka, Surina; Chari, Ravi V. J.; Miller, Michael L.
    ACS Medicinal Chemistry Letters (2019), 10 (8), 1193-1197CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
    Antibody-drug conjugates (ADCs) that incorporate potent indolinobenzodiazepine DNA alkylators as the payload component are currently undergoing clin. evaluation. In one ADC design, the payload mols. are linked to the antibody through a peptidase-labile L-Ala-L-Ala linker. In order to det. the role of amino acid stereochem. on antitumor activity and tolerability, we incorporated L- and D-alanyl groups in the dipeptide, synthesized all four diastereomers, and prepd. and tested the corresponding ADCs. Results of our preclin. evaluation showed that the L-Ala-L-Ala configuration provided the ADC with the highest therapeutic index (antitumor activity vs toxicity).
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    Lazar, A. C., Wang, L., Blättler, W. A., Amphlett, G., Lambert, J. M., and Zhang, W. (2005) Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry. Rapid Commun. Mass Spectrom. 19 (13), 1806– 1814,  DOI: 10.1002/rcm.1987
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    Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry
    Lazar, Alexandru C.; Wang, Lintao; Blaettler, Walter A.; Amphlett, Godfrey; Lambert, John M.; Zhang, Wei
    Rapid Communications in Mass Spectrometry (2005), 19 (13), 1806-1814CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody prepns. often contain several mol. variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepd. by covalently linking several drug mols. per antibody mol. As part of the product characterization, the mol. wts. of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the anal. of recombinant antibodies and immunoconjugates. Sample prepn. is an important element of ESI-MS anal., in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liq. chromatog. (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aq. acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid mols. generated charge envelope profiles that differ from those of the non-conjugated antibody. For the detn. of the distribution of the various conjugate species in an immunoconjugate sample prepd. by randomly linking in the av. 3.6 drug mols. per antibody mol., the exptl. conditions needed to be carefully selected to allow acquisition of the whole spectrum contg. the charge envelopes of all species.
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    Kovtun, Y. V., Audette, C. A., Mayo, M. F., Jones, G. E., Doherty, H., Maloney, E. K., Erickson, H. K., Sun, X., Wilhelm, S., Ab, O. (2010) Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Res. 70 (6), 2528– 2537,  DOI: 10.1158/0008-5472.CAN-09-3546
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    Antibody-Maytansinoid Conjugates Designed to Bypass Multidrug Resistance
    Kovtun, Yelena V.; Audette, Charlene A.; Mayo, Michele F.; Jones, Gregory E.; Doherty, Heather; Maloney, Erin K.; Erickson, Hans K.; Sun, Xiuxia; Wilhelm, Sharon; Ab, Olga; Lai, Katharine C.; Widdison, Wayne C.; Kellogg, Brenda; Johnson, Holly; Pinkas, Jan; Lutz, Robert J.; Singh, Rajeeva; Goldmacher, Victor S.; Chari, Ravi V. J.
    Cancer Research (2010), 70 (6), 2528-2537CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    Conjugation of cytotoxic compds. to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compds. used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG4Mal. Following uptake into target cells, conjugates made with the PEG4Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepd. with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG4Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addn., PEG4Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance. Cancer Res; 70(6); 2528-37.
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    Ab, O., Whiteman, K. R., Bartle, L. M., Sun, X., Singh, R., Tavares, D., LaBelle, A., Payne, G., Lutz, R. J., Pinkas, J. (2015) IMGN853, a Folate Receptor-α (FRα)-Targeting Antibody-Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors. Mol. Cancer Ther. 14 (7), 1605– 1613,  DOI: 10.1158/1535-7163.MCT-14-1095
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    IMGN853, a Folate Receptor-α (FRα)-Targeting Antibody-Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors
    Ab, Olga; Whiteman, Kathleen R.; Bartle, Laura M.; Sun, Xiuxia; Singh, Rajeeva; Tavares, Daniel; LaBelle, Alyssa; Payne, Gillian; Lutz, Robert J.; Pinkas, Jan; Goldmacher, Victor S.; Chittenden, Thomas; Lambert, John M.
    Molecular Cancer Therapeutics (2015), 14 (7), 1605-1613CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
    A majority of ovarian and non-small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody-drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-pos. cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non-small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochem. IMGN853 displayed cytotoxic activity against FRα-neg. cells situated near FRα-pos. cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clin. development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors. Mol Cancer Ther; 14(7); 1605-13. ©2015 AACR.
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  11. Rita Khoury, Khalil Saleh, Nadine Khalife, Mohamad Saleh, Claude Chahine, Rebecca Ibrahim, Axel Lecesne. Mechanisms of Resistance to Antibody-Drug Conjugates. International Journal of Molecular Sciences 2023, 24 (11) , 9674. https://doi.org/10.3390/ijms24119674
  12. Arnaud C. Tiberghien, Balakumar Vijayakrishnan, Arman Esfandiari, Mahammad Ahmed, Raul Pardo, John Bingham, Lauren Adams, Kathleen Santos, Gyoung-Dong Kang, Kathryn M. Pugh, Shameen Afif-Rider, Kapil Vashisht, Kemal Haque, Ravinder Tammali, Edward Rosfjord, Adriana Savoca, John A. Hartley, Philip W. Howard. Comparison of Pyrrolobenzodiazepine Dimer Bis-imine versus Mono-imine: DNA Interstrand Cross-linking, Cytotoxicity, Antibody–Drug Conjugate Efficacy and Toxicity. Molecular Cancer Therapeutics 2023, 22 (2) , 254-263. https://doi.org/10.1158/1535-7163.MCT-21-0693
  13. Juniper A. Scribner, Stuart W. Hicks, Kerstin W. Sinkevicius, Nicholas C. Yoder, Gundo Diedrich, Jennifer G. Brown, Jacquelynn Lucas, Megan E. Fuller, Thomas Son, Anahita Dastur, Jeff Hooley, Christopher Espelin, Marian Themeles, Francine Z. Chen, Ying Li, Michael Chiechi, Jenny Lee, Bhaswati Barat, Lusiana Widjaja, Sergey Gorlatov, James Tamura, Valentina Ciccarone, Olga Ab, Kerry A. McEachem, Scott Koenig, Eric H. Westin, Paul A. Moore, Thomas Chittenden, Richard J. Gregory, Ezio Bonvini, Deryk Loo. Preclinical Evaluation of IMGC936, a Next-Generation Maytansinoid-based Antibody–drug Conjugate Targeting ADAM9-expressing Tumors. Molecular Cancer Therapeutics 2022, 21 (7) , 1047-1059. https://doi.org/10.1158/1535-7163.MCT-21-0915
  14. Ning Zou, Amy Han. Application of Pyrrolobenzodiazepines in Antibody Drug Conjugates. 2022, 293-339. https://doi.org/10.1002/9781119627784.ch13
  15. Charlotte Sornay, Valentine Vaur, Alain Wagner, Guilhem Chaubet. An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins. Royal Society Open Science 2022, 9 (1) https://doi.org/10.1098/rsos.211563
  16. M. W. Giese, R. H. Woodman, G. T. Hermanson, P. D. Davis. The Use of Uniform PEG Compounds in the Design of ADCs. 2021, 286-376. https://doi.org/10.1039/9781839165153-00286
  17. Anna Kaempffe, Stephan Dickgiesser, Nicolas Rasche, Andrea Paoletti, Elisa Bertotti, Ilse De Salve, Federico Riccardi Sirtori, Roland Kellner, Doreen Könning, Stefan Hecht, Jan Anderl, Harald Kolmar, Christian Schröter. Effect of Conjugation Site and Technique on the Stability and Pharmacokinetics of Antibody-Drug Conjugates. Journal of Pharmaceutical Sciences 2021, 110 (12) , 3776-3785. https://doi.org/10.1016/j.xphs.2021.08.002
  18. Jeffrey Y.-K. Wong, Raja Mukherjee, Jiayuan Miao, Olena Bilyk, Vivian Triana, Mark Miskolzie, Antoine Henninot, John J. Dwyer, Serhii Kharchenko, Anna Iampolska, Dmitriy M. Volochnyuk, Yu-Shan Lin, Lynne-Marie Postovit, Ratmir Derda. Genetically-encoded discovery of proteolytically stable bicyclic inhibitors for morphogen NODAL. Chemical Science 2021, 12 (28) , 9694-9703. https://doi.org/10.1039/D1SC01916C
  19. Ming-Chen Yang, Chi-Sheng Shia, Wan-Fen Li, Chun-Chung Wang, I-Ju Chen, Teng-Yi Huang, Yu-Jung Chen, Hui-Wen Chang, Chi-Huan Lu, Yueh-Chin Wu, Nan-Hsuan Wang, Jiann-Shiun Lai, Cheng-Der Yu, Ming-Tain Lai. Preclinical Studies of OBI-999: A Novel Globo H–Targeting Antibody–Drug Conjugate. Molecular Cancer Therapeutics 2021, 20 (6) , 1121-1132. https://doi.org/10.1158/1535-7163.MCT-20-0763
  20. Jose F. Ponte, Leanne Lanieri, Eshita Khera, Rassol Laleau, Olga Ab, Christopher Espelin, Neeraj Kohli, Bahar Matin, Yulius Setiady, Michael L. Miller, Thomas A. Keating, Ravi Chari, Jan Pinkas, Richard Gregory, Greg M. Thurber. Antibody Co-Administration Can Improve Systemic and Local Distribution of Antibody–Drug Conjugates to Increase In Vivo Efficacy. Molecular Cancer Therapeutics 2021, 20 (1) , 203-212. https://doi.org/10.1158/1535-7163.MCT-20-0451
  21. Amissi Sadiki, Shefali R Vaidya, Mina Abdollahi, Gunjan Bhardwaj, Michael E Dolan, Harpreet Turna, Varnika Arora, Athul Sanjeev, Timothy D Robinson, Andrea Koid, Aashka Amin, Zhaohui Sunny Zhou. Site-specific conjugation of native antibody. Antibody Therapeutics 2020, 3 (4) , 271-284. https://doi.org/10.1093/abt/tbaa027
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Cite this: Bioconjugate Chem. 2020, 31, 1, 93–103
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  • Abstract

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    Figure 1

    Figure 1. (A) Structures of DGN549-L (1a), used for lysine-conjugation; and DGN549-C (1b), used for cysteine conjugation. (B) Structural representations of lysine-linked and CYSMAB ADCs.

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    Figure 2

    Figure 2. SEC-MS data for mAb1 ADCs: (A) ADC 2a and (B) ADC 2b.

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    Figure 3

    Figure 3. (A) HIC chromatograms of ADCs 2a and 2b. (B) HIC chromatograms of ADCs 3a and 3b.

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    Figure 4

    Figure 4. Processing of ADCs 2a and 2b to release the same catabolite FGN849 (4).

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    Figure 5

    Figure 5. Activity of mAb1 ADCs in SCID mice bearing NCI-H2110 non-small cell lung cancer xenografts expressing FRα.

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    Figure 6

    Figure 6. Activity of mAb2 ADCs and nontargeting controls in SCID mice bearing Molm-13 acute myeloid leukemia xenografts expressing CD123.

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    Figure 7

    Figure 7. Tolerability of mAb1 ADCs in non-tumor-bearing CD-1 mice. (A) ADC 2a and (B) ADC 2b. 

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    Figure 8

    Figure 8. Tolerability of mAb2 ADCs in non tumor-bearing CD-1 mice: (A) ADC 3a and (B) ADC 3b.

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    Figure 9

    Figure 9. Tolerability of ADC 2c in non tumor-bearing CD-1 mice.

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    Figure 10

    Figure 10. Pharmacokinetics of mAb1 ADCs 2a and 2b in non-tumor-bearing CD-1 mice, assayed by ELISA methods for total antibody and conjugated antibody.

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  • References

    This article references 55 other publications.

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      Nature Biotechnology (2012), 30 (7), 631-637CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      A review. Progress was made recently in developing antibody-drug conjugates (ADCs) that can selectively deliver cancer drugs to tumor cells. In principle, the idea is simple: by attaching drugs to tumor-seeking antibodies, target cells will be killed and nontarget cells will be spared. In practice, many parameters needed to be addressed to develop safe and effective ADCs, including the expression profiles of tumor vs. normal tissues, the potency of the drug, the linker attaching the drug and placement of the drug on the antibody, and the pharmacokinetic and stability profiles of the resulting ADC. All these issues had been taken into account in developing brentuximab vedotin (Adcetris), an ADC that recently received accelerated approval by the US Food and Drug Administration for the treatment of relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma (ALCL). Research is under way to extend the applications of brentuximab vedotin and to advance the field by developing other ADCs with new linker and conjugation strategies.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XpvFCqtbw%253D&md5=6940eb8178e8c8c150a7b21361e23234
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      Appelbaum, F. R. and Bernstein, I. D. (2017) Gemtuzumab ozogamicin for acute myeloid leukemia. Blood 130 (22), 2373– 2376,  DOI: 10.1182/blood-2017-09-797712
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      Gemtuzumab ozogamicin for acute myeloid leukemia
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      Blood (2017), 130 (22), 2373-2376CODEN: BLOOAW; ISSN:1528-0020. (American Society of Hematology)
      On 1 Sept. 2017, the US Food and Drug Administration (FDA) approved gemtuzumab ozogamicin (GO) for the treatment of adults with newly diagnosed CD33+ acute myeloid leukemia and for patients aged ≥2 years with CD33+ acute myeloid leukemia who have experienced a relapse or who have not responded to initial treatment. This signals a new chapter in the long and unusual story of GO, which was the first antibody-drug conjugate approved for human use by the FDA.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1erurfM&md5=970680b53cd9f2971e811d044ef9b6fd
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      Inotuzumab ozogamicin: a CD22 mAb-drug conjugate for adult relapsed or refractory B-cell precursor acute lymphoblastic leukemia
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      Drug Design, Development and Therapy (2018), 12 (), 2293-2300CODEN: DDDTAQ; ISSN:1177-8881. (Dove Medical Press Ltd.)
      Despite improved rates of remission and cure in newly diagnosed adult acute lymphoblastic leukemia (ALL), the prognosis for patients with relapsed or refractory disease remains poor and the 5-yr overall survival rate after relapse is under 10%. A recent paradigm shift has focused on the promise of targeted immunotherapy rather than std. chemotherapy, as ALL blast cells express a variety of antigens, and monoclonal antibodies may be developed to identify and destroy the leukemic cells. Inotuzumab ozogamicin is a CD22 monoclonal antibody conjugated to the cytotoxic antibiotic calicheamicin. CD22 expression is detected on leukemic blasts in over 90% of patients with ALL. Based on promising results from preclin. studies, inotuzumab ozogamicin was tested in Phase 1/2 and Phase 3 clin. trials and it demonstrated improved complete remission rates, progression-free survival and overall survival in relapsed or refractory adult ALL compared to std. therapy. Ongoing studies are evaluating the value of inotuzumab ozogamicin when given in combination with chemotherapy as part of upfront treatment. This review discusses the drug's biochem. properties and mechanism of action, preclin. research outcomes, clin. trial results, adverse events and toxicities, drug approval and ongoing investigations.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1OnsrrM&md5=de9ba79b0c4d5a13606416647011fe8c
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      Dornan, D., Bennett, F., Chen, Y., Dennis, M., Eaton, D., Elkins, K., French, D., Go, M. A., Jack, A., Junutula, J. R. (2009) Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 114 (13), 2721– 2729,  DOI: 10.1182/blood-2009-02-205500
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      Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma
      Dornan, David; Bennett, Fiona; Chen, Yvonne; Dennis, Mark; Eaton, Dan; Elkins, Kristi; French, Dorothy; Go, Mary Ann T.; Jack, Andrew; Junutula, Jagath R.; Koeppen, Hartmut; Lau, Jeffrey; McBride, Jacqueline; Rawstron, Andy; Shi, Xiaoyan; Yu, Nancy; Yu, Shang-Fan; Yue, Peng; Zheng, Bing; Ebens, Allen; Polson, Andrew G.
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      Here we describe the generation of an antibody-drug conjugate (ADC) consisting of a humanized anti-CD79b antibody that is conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABs) by a protease cleavable linker. By using flow cytometry, we detected the surface expression of CD79b in almost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia patients, suggesting that anti-CD79b-vcMMAE could be widely used in these malignancies. By using NHL cell lines to simulate a patient population we discovered that a minimal cell-surface expression level of CD79b was required for in vitro activity. Within the subpopulation of cell lines above this minimal threshold, we found that sensitivity to free MMAE, mutation of cancer genes, and cell doubling time were poorly correlated with in vitro activity; however, the expression level of BCL-XL was correlated with reduced sensitivity to anti-CD79b-vcMMAE. This observation was supported by in vivo data showing that a Bcl-2 family inhibitor, ABT-263, strikingly enhanced the activity of anti-CD79b-vcMMAE. Furthermore, anti-CD79b-vcMMAE was significantly more effective than a std.-of-care regimen, R-CHOP (ie, rituximab with a single i.v. injection of 30 mg/kg cyclophosphamide, 2.475 mg/kg doxorubicin, 0.375 mg/kg vincristine, and oral dosing of 0.15 mg/kg prednisone once a day for 5 days), in 3 xenograft models of NHL. Together, these data suggest that anti-CD79b-vcMMAE could be broadly efficacious for the treatment of NHL.
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      Donaghy, H. (2016) Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates. MAbs 8 (4), 659– 671,  DOI: 10.1080/19420862.2016.1156829
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      Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates
      Donaghy, Heather
      mAbs (2016), 8 (4), 659-671CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
      Antibody-drug conjugates (ADCs) represent a new class of cancer therapeutics. Their design involves a tumor-specific antibody, a linker and a cytotoxic payload. They were designed to allow specific targeting of highly potent cytotoxic agents to tumor cells while sparing normal cells. Frequent toxicities that may be driven by any of the components of an ADC have been reported. There are currently more than 50 ADCs in active clin. development, and a further ∼20 that have been discontinued. For this review, the reported toxicities of ADCs were analyzed, and the mechanisms for their effects are explored in detail. Methods to reduce toxicities, including dosing strategies and drug design, are discussed. The toxicities reported for active and discontinued drugs are important to drive the rational design and improve the therapeutic index of ADCs of the future.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtFant7k%253D&md5=b2ba3e43e8362d8a3f7c368a5af5f94e
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      Chari, R. V. J. (2016) Expanding the Reach of Antibody-Drug Conjugates. ACS Med. Chem. Lett. 7 (11), 974– 976,  DOI: 10.1021/acsmedchemlett.6b00312
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      Expanding the Reach of Antibody-Drug Conjugates
      Chari, Ravi V. J.
      ACS Medicinal Chemistry Letters (2016), 7 (11), 974-976CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      Antibody-drug conjugates (ADCs) represent an emerging new paradigm in cancer therapy. The approval of two ADCs has spurred considerable interest in this area of research, and over 55 ADCs are currently in clin. testing. In order to improve the clin. success rate of ADC therapy, all three components of the ADC: the antibody, linker, and payload have to be optimized. While considerable improvements have been made in antibody properties and target selection, medicinal chem. efforts have lagged behind, and there is a significant need for innovation in linker design and payloads.
      >> More from SciFinder ®
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      Behrens, C. R. and Liu, B. (2014) Methods for site-specific drug conjugation to antibodies. MAbs 6 (1), 46– 53,  DOI: 10.4161/mabs.26632
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      Methods for site-specific drug conjugation to antibodies
      Behrens Christopher R; Liu Bin
      mAbs (2014), 6 (1), 46-53 ISSN:.
      Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over traditional chemotherapy. Drugs and linkers have been the current focus of ADC development, in addition to antibody and target selection. Recently, however,the importance of conjugate homogeneity has been realized. The current methods for drug attachment lead to a heterogeneous mixture, and some populations of that mixture have poor in vivo performance. New methods for site-specific drug attachment lead to more homogeneous conjugates and allow control of the site of drug attachment. These subtle improvements can have profound effects on in vivo efficacy and therapeutic index. This review examines current methods for site-specific drug conjugation to antibodies, and compares in vivo results with their non-specifically conjugated counterparts. The apparent improvement in pharmacokinetics and the reduced off target toxicity warrant further development of this site-specific modification approach for future ADC development.
      >> More from SciFinder ®
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      Agarwal, P. and Bertozzi, C. R. (2015) Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 26 (2), 176– 192,  DOI: 10.1021/bc5004982
      10
      Site-Specific Antibody-Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development
      Agarwal, Paresh; Bertozzi, Carolyn R.
      Bioconjugate Chemistry (2015), 26 (2), 176-192CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A review. Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small mols. to create targeted drugs. Despite the simplicity of this concept, generation of clin. successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepd. by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixts. that cannot be further purified. With advances in the fields of bioorthogonal chem. and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chem. and protein engineering.
      >> More from SciFinder ®
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      Panowski, S., Bhakta, S., Raab, H., Polakis, P., and Junutula, J. R. (2014) Site-specific antibody drug conjugates for cancer therapy. MAbs 6 (1), 34– 45,  DOI: 10.4161/mabs.27022
      11
      Site-specific antibody drug conjugates for cancer therapy
      Panowski Siler; Bhakta Sunil; Raab Helga; Polakis Paul; Junutula Jagath R
      mAbs (2014), 6 (1), 34-45 ISSN:.
      Antibody therapeutics have revolutionized the treatment of cancer over the past two decades. Antibodies that specifically bind tumor surface antigens can be effective therapeutics; however, many unmodified antibodies lack therapeutic activity. These antibodies can instead be applied successfully as guided missiles to deliver potent cytotoxic drugs in the form of antibody drug conjugates (ADCs). The success of ADCs is dependent on four factors--target antigen, antibody, linker, and payload. The field has made great progress in these areas, marked by the recent approval by the US Food and Drug Administration of two ADCs, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). However, the therapeutic window for many ADCs that are currently in pre-clinical or clinical development remains narrow and further improvements may be required to enhance the therapeutic potential of these ADCs. Production of ADCs is an area where improvement is needed because current methods yield heterogeneous mixtures that may include 0-8 drug species per antibody molecule. Site-specific conjugation has been recently shown to eliminate heterogeneity, improve conjugate stability, and increase the therapeutic window. Here, we review and describe various site-specific conjugation strategies that are currently used for the production of ADCs, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases. In addition, we also summarize differences among these methods and highlight critical considerations when building next-generation ADC therapeutics.
      >> More from SciFinder ®
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      Junutula, J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D., Weir, S., Chen, Y., Simpson, M., Tsai, S. P., Dennis, M. S. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26 (8), 925– 932,  DOI: 10.1038/nbt.1480
      12
      Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index
      Junutula, Jagath R.; Raab, Helga; Clark, Suzanna; Bhakta, Sunil; Leipold, Douglas D.; Weir, Sylvia; Chen, Yvonne; Simpson, Michelle; Tsai, Siao Ping; Dennis, Mark S.; Lu, Yanmei; Meng, Y. Gloria; Ng, Carl; Yang, Jihong; Lee, Chien C.; Duenas, Eileen; Gorrell, Jeffrey; Katta, Viswanatham; Kim, Amy; McDorman, Kevin; Flagella, Kelly; Venook, Rayna; Ross, Sarajane; Spencer, Susan D.; Wong, Wai Lee; Lowman, Henry B.; Vandlen, Richard; Sliwkowski, Mark X.; Scheller, Richard H.; Polakis, Paul; Mallet, William
      Nature Biotechnology (2008), 26 (8), 925-932CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogeneous conjugates with relatively narrow therapeutic index (max. tolerated dose/curative dose). Using leads from the authors' previously described phage display-based method to predict suitable conjugation sites, the authors engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb Ig folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepd. by conventional approaches. The favorable in vivo properties of the near-homogeneous compn. of this conjugate suggest that this strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
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      Junutula, J. R., Flagella, K. M., Graham, R. A., Parsons, K. L., Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, D. L., Li, G. (2010) Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer. Clin. Cancer Res. 16 (19), 4769– 4778,  DOI: 10.1158/1078-0432.CCR-10-0987
      13
      Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer
      Junutula, Jagath R.; Flagella, Kelly M.; Graham, Richard A.; Parsons, Kathryn L.; Ha, Edward; Raab, Helga; Bhakta, Sunil; Nguyen, Trung; Dugger, Debra L.; Li, Guangmin; Mai, Elaine; Lewis Phillips, Gail D.; Hiraragi, Hajime; Fuji, Reina N.; Tibbitts, Jay; Vandlen, Richard; Spencer, Susan D.; Scheller, Richard H.; Polakis, Paul; Sliwkowski, Mark X.
      Clinical Cancer Research (2010), 16 (19), 4769-4778CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
      Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examd. the in vitro potency as well as in vivo preclin. efficacy and safety profiles of a heterogeneous prepn. of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate. To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) mol. was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 mols. per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti-cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equiv doses (μg/m2) and retained efficacy at equiv. antibody doses (mg/kg). An improved safety profile of >2-fold was obsd. in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6000 μg DM1/m2) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6000 μg DM1/m2). The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clin. development of HER2-pos. targeted breast cancer therapies.
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    14. 14
      Shen, B. Q., Xu, K., Liu, L., Raab, H., Bhakta, S., Kenrick, M., Parsons-Reponte, K. L., Tien, J., Yu, S. F., Mai, E. (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30 (2), 184– 189,  DOI: 10.1038/nbt.2108
      14
      Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates
      Shen, Ben-Quan; Xu, Keyang; Liu, Luna; Raab, Helga; Bhakta, Sunil; Kenrick, Margaret; Parsons-Reponte, Kathryn L.; Tien, Janet; Yu, Shang-Fan; Mai, Elaine; Li, Dongwei; Tibbitts, Jay; Baudys, Jakub; Saad, Ola M.; Scales, Suzie J.; McDonald, Paul J.; Hass, Philip E.; Eigenbrot, Charles; Nguyen, Trung; Solis, Willy A.; Fuji, Reina N.; Flagella, Kelly M.; Patel, Darshana; Spencer, Susan D.; Khawli, Leslie A.; Ebens, Allen; Wong, Wai Lee; Vandlen, Richard; Kaur, Surinder; Sliwkowski, Mark X.; Scheller, Richard H.; Polakis, Paul; Junutula, Jagath R.
      Nature Biotechnology (2012), 30 (2), 184-189CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a pos. charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected pos. by succinimide ring hydrolysis and neg. by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chem. and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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    15. 15
      Pillow, T. H., Tien, J., Parsons-Reponte, K. L., Bhakta, S., Li, H., Staben, L. R., Li, G., Chuh, J., Fourie-O’Donohue, A., Darwish, M. (2014) Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering. J. Med. Chem. 57 (19), 7890– 7899,  DOI: 10.1021/jm500552c
      15
      Site-Specific Trastuzumab Maytansinoid Antibody-Drug Conjugates with Improved Therapeutic Activity through Linker and Antibody Engineering
      Pillow, Thomas H.; Tien, Janet; Parsons-Reponte, Kathryn L.; Bhakta, Sunil; Li, Hao; Staben, Leanna R.; Li, Guangmin; Chuh, Josefa; Fourie-O'Donohue, Aimee; Darwish, Martine; Yip, Victor; Liu, Luna; Leipold, Douglas D.; Su, Dian; Wu, Elmer; Spencer, Susan D.; Shen, Ben-Quan; Xu, Keyang; Kozak, Katherine R.; Raab, Helga; Vandlen, Richard; Lewis Phillips, Gail D.; Scheller, Richard H.; Polakis, Paul; Sliwkowski, Mark X.; Flygare, John A.; Junutula, Jagath R.
      Journal of Medicinal Chemistry (2014), 57 (19), 7890-7899CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clin. activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clin. activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clin. activity was demonstrated. Here, we report that through chem. modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chem. synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clin. approved ADC, trastuzumab-MCC-DM1.
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    16. 16
      Tian, F., Lu, Y., Manibusan, A., Sellers, A., Tran, H., Sun, Y., Phuong, T., Barnett, R., Hehli, B., Song, F. (2014) A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. U. S. A. 111 (5), 1766– 1771,  DOI: 10.1073/pnas.1321237111
      16
      A general approach to site-specific antibody drug conjugates
      Tian, Feng; Lu, Yingchun; Manibusan, Anthony; Sellers, Aaron; Tran, Hon; Sun, Ying; Phuong, Trung; Barnett, Richard; Hehli, Brad; Song, Frank; De Guzman, Michael J.; Ensari, Semsi; Pinkstaff, Jason K.; Sullivan, Lorraine M.; Biroc, Sandra L.; Cho, Ho; Schultz, Peter G.; Di Joseph, John; Dougher, Maureen; Ma, Dangshe; Dushin, Russell; Leal, Mauricio; Tchistiakova, Lioudmila; Feyfant, Eric; Gerber, Hans-Peter; Sapra, Puja
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (5), 1766-1771CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicol. study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These expts. support the notion that chem. defined antibody conjugates can be synthesized in com. relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chem. modification.
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    17. 17
      Jackson, D., Atkinson, J., Guevara, C. I., Zhang, C., Kery, V., Moon, S. J., Virata, C., Yang, P., Lowe, C., Pinkstaff, J. (2014) In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates. PLoS One 9 (1), e83865  DOI: 10.1371/journal.pone.0083865
      17
      In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates
      Jackson, Dowdy; Atkinson, John; Guevara, Claudia I.; Zhang, Chunying; Kery, Vladimir; Moon, Sung-Ju; Virata, Cyrus; Yang, Peng; Lowe, Christine; Pinkstaff, Jason; Cho, Ho; Knudsen, Nick; Manibusan, Anthony; Tian, Feng; Sun, Ying; Lu, Yingchun; Sellers, Aaron; Jia, Xiao-Chi; Joseph, Ingrid; Anand, Banmeet; Morrison, Kendall; Pereira, Daniel S.; Stover, David
      PLoS One (2014), 9 (1), e83865/1-e83865/14, 14 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Antibody-drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are crit. to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous no. of drugs per antibody. The no. of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclin. comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclin. toxicol. profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.
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    18. 18
      Strop, P., Delaria, K., Foletti, D., Witt, J. M., Hasa-Moreno, A., Poulsen, K., Casas, M. G., Dorywalska, M., Farias, S., Pios, A. (2015) Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading. Nat. Biotechnol. 33 (7), 694– 696,  DOI: 10.1038/nbt.3274
      18
      Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading
      Strop, Pavel; Delaria, Kathy; Foletti, Davide; Witt, Jody Melton; Hasa-Moreno, Adela; Poulsen, Kris; Casas, Meritxell Galindo; Dorywalska, Magdalena; Farias, Santiago; Pios, Ariel; Lui, Victor; Dushin, Russell; Zhou, Dahui; Navaratnam, Thayalan; Tran, Thomas-Toan; Sutton, Janette; Lindquist, Kevin C.; Han, Bora; Liu, Shu-Hui; Shelton, David L.; Pons, Jaume; Rajpal, Arvind
      Nature Biotechnology (2015), 33 (7), 694-696CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      There is no expanded citation for this reference.
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    19. 19
      Behrens, C. R., Ha, E. H., Chinn, L. L., Bowers, S., Probst, G., Fitch-Bruhns, M., Monteon, J., Valdiosera, A., Bermudez, A., Liao-Chan, S. (2015) Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs. Mol. Pharmaceutics 12 (11), 3986– 3998,  DOI: 10.1021/acs.molpharmaceut.5b00432
      19
      Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs
      Behrens, Christopher R.; Ha, Edward H.; Chinn, Lawrence L.; Bowers, Simeon; Probst, Gary; Fitch-Bruhns, Maureen; Monteon, Jorge; Valdiosera, Amanda; Bermudez, Abel; Liao-Chan, Sindy; Wong, Tiffany; Melnick, Jonathan; Theunissen, Jan-Willem; Flory, Mark R.; Houser, Derrick; Venstrom, Kristy; Levashova, Zoia; Sauer, Paul; Migone, Thi-Sau; van der Horst, Edward H.; Halcomb, Randall L.; Jackson, David Y.
      Molecular Pharmaceutics (2015), 12 (11), 3986-3998CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      Conventional antibody-drug conjugates (ADCs) are heterogeneous mixts. of chem. distinct mols. that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs contg. four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacol. properties of ADCs in vivo is unknown. In order to det. the relative impact of interchain cysteine crosslinking on the therapeutic window and other properties of ADCs in vivo, we synthesized a deriv. of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF deriv. was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs contg. predominantly four drugs/antibody. The pharmacol. properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine crosslinking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.
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    20. 20
      Hamblett, K. J., Le, T., Rock, B. M., Rock, D. A., Siu, S., Huard, J. N., Conner, K. P., Milburn, R. R., O’Neill, J. W., Tometsko, M. E. (2016) Altering Antibody–Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability. Mol. Pharmaceutics 13 (7), 2387– 2396,  DOI: 10.1021/acs.molpharmaceut.6b00153
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      Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability
      Hamblett, Kevin J.; Le, Tiep; Rock, Brooke M.; Rock, Dan A.; Siu, Sophia; Huard, Justin N.; Conner, Kip P.; Milburn, Robert R.; O'Neill, Jason W.; Tometsko, Mark E.; Fanslow, William C.
      Molecular Pharmaceutics (2016), 13 (7), 2387-2396CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two sep. maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the obsd. efficacy was similar. More severe thrombocytopenia was obsd. with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approx. 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance obsd. for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equiv. doses and exacerbation of clin. pathol. parameters (decreased tolerability) at equiv. doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.
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    21. 21
      Lhospice, F., Bregeon, D., Belmant, C., Dennler, P., Chiotellis, A., Fischer, E., Gauthier, L., Boedec, A., Rispaud, H., Savard-Chambard, S. (2015) Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models. Mol. Pharmaceutics 12 (6), 1863– 1871,  DOI: 10.1021/mp500666j
      21
      Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models
      Lhospice, F.; Bregeon, D.; Belmant, C.; Dennler, P.; Chiotellis, A.; Fischer, E.; Gauthier, L.; Boedec, A.; Rispaud, H.; Savard-Chambard, S.; Represa, A.; Schneider, N.; Paturel, C.; Sapet, M.; Delcambre, C.; Ingoure, S.; Viaud, N.; Bonnafous, C.; Schibli, R.; Romagne, F.
      Molecular Pharmaceutics (2015), 12 (6), 1863-1871CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      Antibody-drug conjugates (ADCs) have demonstrated clin. benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clin. use or development, including ADCETRIS, are produced by chem. conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, the authors describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivs. of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity expts. using two different CD30-pos. cell lines (Karpas 299 and Raji-CD30+) revealed comparable EC50 values for ADCETRIS (1.8±0.4 and 3.6±0.6 ng/mL, resp.) and the four cAC10-based ADCs (2.0±0.4 to 4.9±1.0 ng/mL). Quant. time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected 125I-radioiodinated cAC10 ADC and compared with that of 125I-ADCETRIS. The chemo-enzymically conjugated, radioiodinated ADC showed higher tumor uptake (17.84±2.2% ID/g 24 h p.i.) than 125I-ADCETRIS (10.5±1.8% ID/g 24 h p.i.). Moreover, 125I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the max. tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chem. modified ADCs with variable DARs.
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    22. 22
      Yoder, N. C., Bai, C., Tavares, D., Widdison, W. C., Whiteman, K. R., Wilhelm, A., Wilhelm, S. D., McShea, M. A., Maloney, E. K., Ab, O. (2019) A Case Study Comparing Heterogeneous Lysine- and Site-Specific Cysteine-Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates Benefits of Lysine Conjugation. Mol. Pharmaceutics 16 (9), 3926– 3937,  DOI: 10.1021/acs.molpharmaceut.9b00529
      22
      A Case Study Comparing Heterogeneous Lysine- and Site-Specific Cysteine-Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates the Benefits of Lysine Conjugation
      Yoder, Nicholas C.; Bai, Chen; Tavares, Daniel; Widdison, Wayne C.; Whiteman, Kathleen R.; Wilhelm, Alan; Wilhelm, Sharon D.; McShea, Molly A.; Maloney, Erin K.; Ab, Olga; Wang, Lintao; Jin, Shan; Erickson, Hans K.; Keating, Thomas A.; Lambert, John M.
      Molecular Pharmaceutics (2019), 16 (9), 3926-3937CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small mol. effectors. First-generation mols. of this class often employed heterogeneous conjugation chem., but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chem., we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chem., target antigen, and other variables that det. efficacy in a given setting.
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    23. 23
      Miller, M. L., Shizuka, M., Wilhelm, A., Salomon, P., Reid, E. E., Lanieri, L., Sikka, S., Maloney, E. K., Harvey, L., Qiu, Q. (2018) A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs). Mol. Cancer Ther. 17 (3), 650– 660,  DOI: 10.1158/1535-7163.MCT-17-0940
      23
      A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs)
      Miller, Michael L.; Shizuka, Manami; Wilhelm, Alan; Salomon, Paulin; Reid, Emily E.; Lanieri, Leanne; Sikka, Surina; Maloney, Erin K.; Harvey, Lauren; Qiu, Qifeng; Archer, Katie E.; Bai, Chen; Vitharana, Dilrukshi; Harris, Luke; Singh, Rajeeva; Ponte, Jose F.; Yoder, Nicholas C.; Kovtun, Yelena; Lai, Katharine C.; Ab, Olga; Pinkas, Jan; Keating, Thomas A.; Chari, Ravi V. J.
      Molecular Cancer Therapeutics (2018), 17 (3), 650-660CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      Tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADCs) is now a clin. validated approach for cancer treatment. In an attempt to improve the clin. success rate of ADCs, emphasis has been recently placed on the use of DNA-crosslinking pyrrolobenzodiazepine compds. as the payload. Despite promising early clin. results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs contg. the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650-60. ©2018 AACR.
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    24. 24
      Stimmel, J. B., Merrill, B. M., Kuyper, L. F., Moxham, C. P., Hutchins, J. T., Fling, M. E., and Kull, F. C. (2000) Site-specific conjugation on serine -> cysteine variant monoclonal antibodies. J. Biol. Chem. 275 (39), 30445– 30450,  DOI: 10.1074/jbc.M001672200
      24
      Site-specific conjugation on serine → cysteine variant monoclonal antibodies
      Stimmel, Julie B.; Merrill, Barbara M.; Kuyper, Lee F.; Moxham, Cary P.; Hutchins, Jeff T.; Fling, Mary E.; Kull, Frederick C., Jr.
      Journal of Biological Chemistry (2000), 275 (39), 30445-30450CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third const. domain (CH3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chem. conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following redn., reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence anal. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment contg. the engineered cysteine.
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    25. 25
      Cao, M., De Mel, N., Jiao, Y., Howard, J., Parthemore, C., Korman, S., Thompson, C., Wendeler, M., and Liu, D. (2019) Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis. MAbs 11 (6), 1064– 1076,  DOI: 10.1080/19420862.2019.1624127
      25
      Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis
      Cao, Mingyan; De Mel, Niluka; Jiao, Yang; Howard, James; Parthemore, Conner; Korman, Samuel; Thompson, Christopher; Wendeler, Michaela; Liu, Dengfeng
      mAbs (2019), 11 (6), 1064-1076CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
      Site-specific ADCs are designed to overcome heterogeneity obsd. with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. An elevated level of size variants, such as heavy chain-light chain species, heavy chain-heavy chain-light chain species, and light chain species, is also obsd. with final site-specific ADC product. To understand root cause of heterogeneity generated during ADC conjugation process, we designed time-course studies for each conjugation step, including redn., oxidn., conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample anal. On basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidn. as a result of alternative disulfide bond arrangements. During oxidn., two hinge cysteines formed intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in unconjugated antibody. Time-course data also showed that elevated level of size variants, esp. heavy chain-heavy chain-light chain species and light chain species, resulted from quenching step, where quenching reagent engaged in disulfide bond exchange reaction with ADC and broke disulfide bonds connecting heavy chain and light chain. Underconjugated and overconjugated species arose from equil. established during conjugation reaction.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFOjsrrN&md5=565ad2567367d9fad04088d85523a876
    26. 26
      Vollmar, B. S., Wei, B., Ohri, R., Zhou, J., He, J., Yu, S. F., Leipold, D., Cosino, E., Yee, S., Fourie-O’Donohue, A. (2017) Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates. Bioconjugate Chem. 28 (10), 2538– 2548,  DOI: 10.1021/acs.bioconjchem.7b00365
      26
      Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates
      Vollmar, Breanna S.; Wei, Binqing; Ohri, Rachana; Zhou, Jianhui; He, Jintang; Yu, Shang-Fan; Leipold, Douglas; Cosino, Ely; Yee, Sharon; Fourie-O'Donohue, Aimee; Li, Guangmin; Phillips, Gail L.; Kozak, Katherine R.; Kamath, Amrita; Xu, Keyang; Lee, Genee; Lazar, Greg A.; Erickson, Hans K.
      Bioconjugate Chemistry (2017), 28 (10), 2538-2548CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small mols. to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small mol. and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was obsd. between disulfide stability and efficacy for the conjugates. We hypothesized that the obsd. site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titrn. calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homol. modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.
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    27. 27
      Jeffrey, S. C., Burke, P. J., Lyon, R. P., Meyer, D. W., Sussman, D., Anderson, M., Hunter, J. H., Leiske, C. I., Miyamoto, J. B., Nicholas, N. D. (2013) A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. Bioconjugate Chem. 24 (7), 1256– 1263,  DOI: 10.1021/bc400217g
      27
      A Potent Anti-CD70 Antibody-Drug Conjugate Combining a Dimeric Pyrrolobenzodiazepine Drug with Site-Specific Conjugation Technology
      Jeffrey, Scott C.; Burke, Patrick J.; Lyon, Robert P.; Meyer, David W.; Sussman, Django; Anderson, Martha; Hunter, Joshua H.; Leiske, Chris I.; Miyamoto, Jamie B.; Nicholas, Nicole D.; Okeley, Nicole M.; Sanderson, Russell J.; Stone, Ivan J.; Zeng, Weiping; Gregson, Stephen J.; Masterson, Luke; Tiberghien, Arnaud C.; Howard, Philip W.; Thurston, David E.; Law, Che-Leung; Senter, Peter D.
      Bioconjugate Chemistry (2013), 24 (7), 1256-1263CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A highly cytotoxic DNA crosslinking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity expts. demonstrated that the h1F6239C-PBD was potent and immunol. specific on CD70-pos. renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-pos. RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.
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    28. 28
      Hamblett, K. J., Senter, P. D., Chace, D. F., Sun, M. M. C., Lenox, J., Cerveny, C. G., Kissler, K. M., Bernhardt, S. X., Kopcha, A. K., Zabinski, R. F. (2004) Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate. Clin. Cancer Res. 10 (20), 7063– 7070,  DOI: 10.1158/1078-0432.CCR-04-0789
      28
      Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate
      Hamblett, Kevin J.; Senter, Peter D.; Chace, Dana F.; Sun, Michael M. C.; Lenox, Joel; Cerveny, Charles G.; Kissler, Kim M.; Bernhardt, Starr X.; Kopcha, Anastasia K.; Zabinski, Roger F.; Meyer, Damon L.; Francisco, Joseph A.
      Clinical Cancer Research (2004), 10 (20), 7063-7070CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
      An antibody-drug conjugate consisting of monomethyl auristatin E (MMAE) conjugated to the anti-CD30 monoclonal antibody (mAb) cAC10, with eight drug moieties per mAb, was previously shown to have potent cytotoxic activity against CD30+ malignant cells. To det. the effect of drug loading on antibody-drug conjugate therapeutic potential, we assessed cAC10 antibody-drug conjugates contg. different drug-mAb ratios in vitro and in vivo. Coupling MMAE to the cysteines that comprise the interchain disulfides of cAC10 created an antibody-drug conjugate population, which was purified using hydrophobic interaction chromatog. to yield antibody-drug conjugates with two, four, and eight drugs per antibody (E2, E4, and E8, resp.). Antibody-drug conjugate potency was tested in vitro against CD30+ lines followed by in vivo xenograft models. The max.-tolerated dose and pharmacokinetic profiles of the antibody-drug conjugates were investigated in mice. Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC50 values E8<E4<E2), the in vivo antitumor activity of E4 was comparable with E8 at equal mAb doses, although the E4 contained half the amt. of MMAE per mAb. E2 was also an active antitumor agent but required higher doses. The max.-tolerated dose of E2 in mice was at least double that of E4, which in turn was twice that of E8. MMAE loading affected plasma clearance, as E8 cleared 3-fold faster than E4 and 5-fold faster than E2. By decreasing drug loading per antibody, the therapeutic index was increased demonstrating that drug loading is a key design parameter for antibody-drug conjugates.
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    29. 29
      Sun, X., Ponte, J. F., Yoder, N. C., Laleau, R., Coccia, J., Lanieri, L., Qiu, Q., Wu, R., Hong, E., Bogalhas, M. (2017) Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates. Bioconjugate Chem. 28 (5), 1371– 1381,  DOI: 10.1021/acs.bioconjchem.7b00062
      29
      Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates
      Sun, Xiuxia; Ponte, Jose F.; Yoder, Nicholas C.; Laleau, Rassol; Coccia, Jennifer; Lanieri, Leanne; Qiu, Qifeng; Wu, Rui; Hong, Erica; Bogalhas, Megan; Wang, Lintao; Dong, Ling; Setiady, Yulius; Maloney, Erin K.; Ab, Olga; Zhang, Xiaoyan; Pinkas, Jan; Keating, Thomas A.; Chari, Ravi; Erickson, Hans K.; Lambert, John M.
      Bioconjugate Chemistry (2017), 28 (5), 1371-1381CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an av. drug to antibody ratio (DAR) of 3-4 (potentially ranging from 0-8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an av. DAR of 3.5. To understand the effect of DAR on the preclin. properties of ADCs using maytansinoid cytotoxic agents, we prepd. a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor alpha (FR)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR, and evaluated their biochem. characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (av. ∼2, range 0-4) to very high (av. 10, range 7-14) were prepd. in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR, at const. antibody concn. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic anal. showed that conjugates with an av. DAR below ∼6 had comparable clearance rates, but for those with an av. DAR around 9-10, rapid clearance was obsd. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with max. localization for this organ at 24-28% percentage injected dose per g (%ID/g) compared with 7-10% for lower DAR conjugates (all at 2-6 h post-injection). Our preclin. findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2-6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs, but suggest the exploration of lower or higher DAR may be warranted depending on the biol. of the target antigen.
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    30. 30
      Thompson, P., Fleming, R., Bezabeh, B., Huang, F., Mao, S., Chen, C., Harper, J., Zhong, H., Gao, X., Yu, X.-Q. (2016) Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics. J. Controlled Release 236, 100– 116,  DOI: 10.1016/j.jconrel.2016.06.025
      30
      Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics
      Thompson, Pamela; Fleming, Ryan; Bezabeh, Binyam; Huang, Fengying; Mao, Shenlan; Chen, Cui; Harper, Jay; Zhong, Haihong; Gao, Xizhe; Yu, Xiang-Qing; Hinrichs, Mary Jane; Reed, Molly; Kamal, Adeela; Strout, Patrick; Cho, Song; Woods, Rob; Hollingsworth, Robert E.; Dixit, Rakesh; Wu, Herren; Gao, Changshou; Dimasi, Nazzareno
      Journal of Controlled Release (2016), 236 (), 100-116CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepd. by chem. conjugation of small mol. cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the redn. of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophys. properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody mol. We demonstrate that the chem.-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addn., to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clin. development.
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    31. 31
      Ouyang, J. (2013) Drug-to-antibody ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography. Methods Mol. Biol. (N. Y., NY, U. S.) 1045, 275– 283,  DOI: 10.1007/978-1-62703-541-5_17
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      Drug-to-antibody ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography
      Ouyang Jun
      Methods in molecular biology (Clifton, N.J.) (2013), 1045 (), 275-83 ISSN:.
      Hydrophobic interaction chromatography (HIC) is the method of choice for determination of the drug-to-antibody ratio (DAR) and drug load distribution for cysteine (Cys)-linked antibody-drug conjugates (ADCs). The drug-loaded species are resolved based on the increasing hydrophobicity with the least hydrophobic, unconjugated form eluting first and the most hydrophobic, 8-drug form eluting last. The area percentage of a peak represents the relative distribution of the particular drug-loaded ADC species. The weighted average DAR is then calculated using the percentage peak area information and the drug load numbers. Reversed phase high-performance liquid chromatography (RP-HPLC) offers an orthogonal method to obtain DAR for Cys-linked ADCs. The method involves, first, a reduction reaction to completely dissociate the heavy and light chains of the ADC, then separation of the light and heavy chains and their corresponding drug-loaded forms on an RP column. The percentage peak area from integration of the light chain and heavy chain peaks, combined with the assigned drug load for each peak, is used to calculate the weighted average DAR.
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    32. 32
      Fekete, S., Veuthey, J. L., Beck, A., and Guillarme, D. (2016) Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products. J. Pharm. Biomed. Anal. 130, 3– 18,  DOI: 10.1016/j.jpba.2016.04.004
      32
      Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products
      Fekete, Szabolcs; Veuthey, Jean-Luc; Beck, Alain; Guillarme, Davy
      Journal of Pharmaceutical and Biomedical Analysis (2016), 130 (), 3-18CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
      A review. Hydrophobic interaction chromatog. (HIC) is a historical strategy used for the anal. purifn. and characterization of proteins. Similarly to what can be done in reversed-phase liq. chromatog. (RPLC), HIC is able to sep. protein species based on their hydrophobicity, but using different conditions. Compared to RPLC, the main benefit of HIC is its ability to perform sepns. under non denaturing conditions (i.e. physiol. pH conditions, ambient mobile phase temp. and no need for org. solvents) and so an orthogonal method. The goal of this review is to provide a general overview of theor. and practical aspects of modern HIC applied for the characterization of therapeutic protein biopharmaceuticals including monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) and bispecific antibodies (bsAbs). Therefore, method development approaches, state-of-the-art column technol., applications and future perspectives are described and critically discussed.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtVSgtb0%253D&md5=2e13c1de1d790e013013ee462660f69e
    33. 33
      Strop, P., Liu, S. H., Dorywalska, M., Delaria, K., Dushin, R. G., Tran, T. T., Ho, W. H., Farias, S., Casas, M. G., Abdiche, Y. (2013) Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20 (2), 161– 167,  DOI: 10.1016/j.chembiol.2013.01.010
      33
      Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates
      Strop, Pavel; Liu, Shu-Hui; Dorywalska, Magdalena; Delaria, Kathy; Dushin, Russell G.; Tran, Thomas-Toan; Ho, Wei-Hsien; Farias, Santiago; Casas, Meritxell Galindo; Abdiche, Yasmina; Zhou, Dahui; Chandrasekaran, Ramalakshmi; Samain, Caroline; Loo, Carole; Rossi, Andrea; Rickert, Mathias; Krimm, Stellanie; Wong, Teresa; Chin, Sherman Michael; Yu, Jessica; Dilley, Jeanette; Chaparro-Riggers, Javier; Filzen, Gary F.; O'Donnell, Christopher J.; Wang, Fang; Myers, Jeremy S.; Pons, Jaume; Shelton, David L.; Rajpal, Arvind
      Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 161-167CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compds. at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chem. instability, as was obsd. with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.
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    34. 34
      Lyon, R. P., Bovee, T. D., Doronina, S. O., Burke, P. J., Hunter, J. H., Neff-LaFord, H. D., Jonas, M., Anderson, M. E., Setter, J. R., and Senter, P. D. (2015) Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nat. Biotechnol. 33 (7), 733– 735,  DOI: 10.1038/nbt.3212
      34
      Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index
      Lyon, Robert P.; Bovee, Tim D.; Doronina, Svetlana O.; Burke, Patrick J.; Hunter, Joshua H.; Neff-LaFord, Haley D.; Jonas, Mechthild; Anderson, Martha E.; Setter, Jocelyn R.; Senter, Peter D.
      Nature Biotechnology (2015), 33 (7), 733-735CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      The in vitro potency of antibody-drug conjugates (ADCs) increases with the drug-to-antibody ratio (DAR); however, ADC plasma clearance also increases with DAR, reducing exposure and in vivo efficacy. Here we show that accelerated clearance arises from ADC hydrophobicity, which can be modulated through drug-linker design. We exemplify this using hydrophilic auristatin drug linkers and PEGylated ADCs that yield uniform, high-DAR ADCs with superior in vivo performance.
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    35. 35
      Tumey, L. N., Li, F., Rago, B., Han, X., Loganzo, F., Musto, S., Graziani, E. I., Puthenveetil, S., Casavant, J., Marquette, K. (2017) Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates. AAPS J. 19, 1– 13,  DOI: 10.1208/s12248-017-0083-7
      There is no corresponding record for this reference.
    36. 36
      Burke, P. J., Hamilton, J. Z., Jeffrey, S. C., Hunter, J. H., Doronina, S. O., Okeley, N. M., Miyamoto, J. B., Anderson, M. E., Stone, I. J., Ulrich, M. L. (2017) Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates. Mol. Cancer Ther. 16 (1), 116– 123,  DOI: 10.1158/1535-7163.MCT-16-0343
      36
      Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates
      Burke, Patrick J.; Hamilton, Joseph Z.; Jeffrey, Scott C.; Hunter, Joshua H.; Doronina, Svetlana O.; Okeley, Nicole M.; Miyamoto, Jamie B.; Anderson, Martha E.; Stone, Ivan J.; Ulrich, Michelle L.; Simmons, Jessica K.; McKinney, Erica E.; Senter, Peter D.; Lyon, Robert P.
      Molecular Cancer Therapeutics (2017), 16 (1), 116-123CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      The emergence of antibody-drug conjugates (ADC), such as brentuximab vedotin and ado-trastuzumab emtansine, has led to increased efforts to identify new payloads and develop improved drug-linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC, resulting in accelerated plasma clearance and suboptimal in vivo activity, particularly for conjugates with high drug-to-antibody ratios (DAR). We recently reported on the incorporation of a discrete PEG24 polymer as a side chain in a β-glucuronidase-cleavable monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. In this work, we optimized the drug-linker by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo. Multiple PEG-glucuronide-MMAE linkers were prepd. with PEG size up to 24 ethylene oxide units, and homogeneous DAR 8 ADCs were evaluated. A clear relationship was obsd. between PEG length and conjugate pharmacol. when tested in vivo. Longer PEG chains resulted in slower clearance, with a threshold length of PEG8 beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize plasma clearance provided a wider therapeutic window relative to faster clearing conjugates bearing shorter PEGs. A lead PEGylated glucuronide-MMAE linker was identified incorporating a self-stabilizing maleimide and a PEG12 side chain emerged from these efforts, enabling highly potent, homogeneous DAR 8 conjugates and is under consideration for future ADC programs.
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    37. 37
      Lyon, R. P., Meyer, D. L., Setter, J. R., and Senter, P. D. (2012) Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine residues. Methods Enzymol. 502, 123– 138,  DOI: 10.1016/B978-0-12-416039-2.00006-9
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      Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine residues
      Lyon, Robert P.; Meyer, David L.; Setter, Jocelyn R.; Senter, Peter D.
      Methods in Enzymology (2012), 502 (Protein Engineering for Therapeutics, Part A), 123-138CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
      A review. Many methods have been described for the conjugation of drugs to monoclonal antibodies. The presence of a discrete no. of readily reducible disulfides in the common IgG subtypes presents a convenient opportunity for conjugation to cysteine residues with thiol-reactive drug-linkers. Such conjugates can be prepd. by a straightforward two-step reaction scheme involving the redn. of the antibody disulfides to the desired no. of av. thiols per antibody, followed by addn. of the drug-linker, ideally with a maleimido functionality for rapid, selective reaction. In a discovery setting, this basic method can be scaled down to produce microgram quantities of conjugate for early screening, and in a manufg. setting can be scaled up to produce grams or kilograms of conjugate for clin. trials and commercialization. The resulting conjugates are readily characterized using common HPLC methods.
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    38. 38
      Bhakta, S., Raab, H., and Junutula, J. R. (2013) Engineering THIOMABs for site-specific conjugation of thiol-reactive linkers. Methods Mol. Biol. 1045, 189– 203,  DOI: 10.1007/978-1-62703-541-5_11
      38
      Engineering THIOMABs for site-specific conjugation of thiol-reactive linkers
      Bhakta Sunil; Raab Helga; Junutula Jagath R
      Methods in molecular biology (Clifton, N.J.) (2013), 1045 (), 189-203 ISSN:.
      Antibody conjugates are used in many therapeutic and research applications and are generated by chemically linking a cysteine or lysine residue to potent chemotherapeutic drugs or other functional groups through a flexible linker. Recently, we have engineered THIOMABs (antibodies with engineered reactive cysteine residues) for site-specific conjugation and showed that these antibody conjugates display homogeneous labeling with optimal in vitro and in vivo characteristics. Here, we describe protocols for engineering, selection, and site-specific conjugation of THIOMABs with thiol-reactive linkers.
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    39. 39
      Garcia-Echeverria, C. (2014) Developing second generation antibody-drug conjugates: the quest for new technologies. J. Med. Chem. 57 (19), 7888– 7889,  DOI: 10.1021/jm501298k
      39
      Developing Second Generation Antibody-Drug Conjugates: The Quest for New Technologies
      Garcia-Echeverria, Carlos
      Journal of Medicinal Chemistry (2014), 57 (19), 7888-7889CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. The field of antibody-drug conjugates (ADCs) has gained significant momentum after the recent regulatory approval of two ADCs, and significant research efforts are directed to identify more effective payloads and simplify current manufg. challenges.
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    40. 40
      Drake, P. M., Albers, A. E., Baker, J., Banas, S., Barfield, R. M., Bhat, A. S., de Hart, G. W., Garofalo, A. W., Holder, P., Jones, L. C. (2014) Aldehyde Tag Coupled with HIPS Chemistry Enables the Production of ADCs Conjugated Site-Specifically to Different Antibody Regions with Distinct in Vivo Efficacy and PK Outcomes. Bioconjugate Chem. 25 (7), 1331– 1341,  DOI: 10.1021/bc500189z
      40
      Aldehyde Tag Coupled with HIPS Chemistry Enables the Production of ADCs Conjugated Site-Specifically to Different Antibody Regions with Distinct in Vivo Efficacy and PK Outcomes
      Drake, Penelope M.; Albers, Aaron E.; Baker, Jeanne; Banas, Stefanie; Barfield, Robyn M.; Bhat, Abhijit S.; de Hart, Gregory W.; Garofalo, Albert W.; Holder, Patrick; Jones, Lesley C.; Kudirka, Romas; McFarland, Jesse; Zmolek, Wes; Rabuka, David
      Bioconjugate Chemistry (2014), 25 (7), 1331-1341CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addn. to the DAR and site of conjugation, linker compn. also plays an important role in the properties of an ADC. The authors have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chem. results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled the authors to undertake a systematic evaluation of the impact of conjugation site and linker compn. on ADC properties. Here, the authors describe the prodn. and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chem. The authors demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.
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    41. 41
      Catcott, K. C., McShea, M. A., Bialucha, C. U., Miller, K. L., Hicks, S. W., Saxena, P., Gesner, T. G., Woldegiorgis, M., Lewis, M. E., Bai, C. (2016) Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates. MAbs 8 (3), 513– 523,  DOI: 10.1080/19420862.2015.1134408
      41
      Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates
      Catcott, Kalli C.; McShea, Molly A.; Bialucha, Carl Uli; Miller, Kathy L.; Hicks, Stuart W.; Saxena, Parmita; Gesner, Thomas G.; Woldegiorgis, Mikias; Lewis, Megan E.; Bai, Chen; Fleming, Michael S.; Ettenberg, Seth A.; Erickson, Hans K.; Yoder, Nicholas C.
      mAbs (2016), 8 (3), 513-523CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
      Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Prepn. of ADCs for early stage screening is constrained by purifn. and biochem. anal. techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼100 μg of antibody per well and requires <5 μg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput prepn. and screening of different types of protein conjugates.
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    42. 42
      Cilliers, C., Guo, H., Liao, J., Christodolu, N., and Thurber, G. M. (2016) Multiscale Modeling of Antibody-Drug Conjugates: Connecting Tissue and Cellular Distribution to Whole Animal Pharmacokinetics and Potential Implications for Efficacy. AAPS J. 18 (5), 1117– 1130,  DOI: 10.1208/s12248-016-9940-z
      42
      Multiscale Modeling of Antibody-Drug Conjugates: Connecting Tissue and Cellular Distribution to Whole Animal Pharmacokinetics and Potential Implications for Efficacy
      Cilliers, Cornelius; Guo, Hans; Liao, Jianshan; Christodolu, Nikolas; Thurber, Greg M.
      AAPS Journal (2016), 18 (5), 1117-1130CODEN: AJAOB6; ISSN:1550-7416. (Springer)
      Antibody-drug conjugates exhibit complex pharmacokinetics due to their combination of macromol. and small mol. properties. These issues range from systemic concerns, such as deconjugation of the small mol. drug during the long antibody circulation time or rapid clearance from nonspecific interactions, to local tumor tissue heterogeneity, cell bystander effects, and endosomal escape. Math. models can be used to study the impact of these processes on overall distribution in an efficient manner, and several types of models have been used to analyze varying aspects of antibody distribution including physiol. based pharmacokinetic (PBPK) models and tissue-level simulations. However, these processes are quant. in nature and cannot be handled qual. in isolation. For example, free antibody from deconjugation of the small mol. will impact the distribution of conjugated antibodies within the tumor. To incorporate these effects into a unified framework, we have coupled the systemic and organ-level distribution of a PBPK model with the tissue-level detail of a distributed parameter tumor model. We used this math. model to analyze new exptl. results on the distribution of the clin. antibody-drug conjugate Kadcyla in HER2-pos. mouse xenografts. This model is able to capture the impact of the drug-antibody ratio (DAR) on tumor penetration, the net result of drug deconjugation, and the effect of using unconjugated antibody to drive ADC penetration deeper into the tumor tissue. This modeling approach will provide quant. and mechanistic support to exptl. studies trying to parse the impact of multiple mechanisms of action for these complex drugs.
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    43. 43
      Bumbaca, D., Boswell, C. A., Fielder, P. J., and Khawli, L. A. (2012) Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics. AAPS J. 14 (3), 554– 558,  DOI: 10.1208/s12248-012-9369-y
      43
      Physiochemical and Biochemical Factors Influencing the Pharmacokinetics of Antibody Therapeutics
      Bumbaca, Daniela; Boswell, C. Andrew; Fielder, Paul J.; Khawli, Leslie A.
      AAPS Journal (2012), 14 (3), 554-558CODEN: AJAOB6; ISSN:1550-7416. (Springer)
      A review. Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncol., immunol., neurol., and ophthalmol. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biol., IgG (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromol. therapeutics, which in turn could have ramifications on their efficacy and safety. This mini-review examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was obsd. that a net charge modification of at least a 1-unit shift in isoelec. point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to det. if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.
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    44. 44
      Bohnert, T. and Gan, L. S. (2013) Plasma protein binding: from discovery to development. J. Pharm. Sci. 102 (9), 2953– 2994,  DOI: 10.1002/jps.23614
      44
      Plasma protein binding: From discovery to development
      Bohnert, Tonika; Gan, Liang-Shang
      Journal of Pharmaceutical Sciences (2013), 102 (9), 2953-2994CODEN: JPMSAE; ISSN:0022-3549. (John Wiley & Sons, Inc.)
      A review. The importance of plasma protein binding (PPB) in modulating the effective drug concn. at pharmacol. target sites has been the topic of significant discussion and debate amongst drug development groups over the past few decades. Free drug theory, which states that in absence of energy-dependent processes, after steady state equil. has been attained, free drug concn. in plasma is equal to free drug concn. at the pharmacol. target receptor(s) in tissues, has been used to explain pharmacokinetics/pharmacodynamics relationships in a large no. of cases. Any sudden increase in free concn. of a drug could potentially cause toxicity and may need dose adjustment. Free drug concn. is also helpful to est. the effective concn. of drugs that potentially can ppt. metab. (or transporter)-related drug-drug interactions. Disease models are extensively validated in animals to progress a compd. into development. Unbound drug concn., and therefore PPB information across species is very informative in establishing safety margins and guiding selection of First in Human (FIH) dose and human efficacious dose. The scope of this review is to give an overview of reported role of PPB in several therapeutic areas, highlight cases where PPB changes are clin. relevant, and provide drug metab. and pharmacokinetics recommendations in discovery and development settings. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Assocn. J Pharm Sci.
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    45. 45
      Alley, S. C., Benjamin, D. R., Jeffrey, S. C., Okeley, N. M., Meyer, D. L., Sanderson, R. J., and Senter, P. D. (2008) Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjugate Chem. 19 (3), 759– 765,  DOI: 10.1021/bc7004329
      45
      Contribution of Linker Stability to the Activities of Anticancer Immunoconjugates
      Alley, Stephen C.; Benjamin, Dennis R.; Jeffrey, Scott C.; Okeley, Nicole M.; Meyer, Damon L.; Sanderson, Russell J.; Senter, Peter D.
      Bioconjugate Chemistry (2008), 19 (3), 759-765CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technol. advancement, since the linker half-lives in circulation (t1/2 ∼ 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepd. contg. a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 wk postadministration in mice. In order to assess the impact of improving linker stability beyond mc contg. ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac contg. ADCs, although the bac linker technol. led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide deriv. became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.
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    46. 46
      Ohri, R., Bhakta, S., Fourie-O’Donohue, A., Dela Cruz-Chuh, J., Tsai, S. P., Cook, R., Wei, B., Ng, C., Wong, A. W., Bos, A. B. (2018) High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers. Bioconjugate Chem. 29 (2), 473– 485,  DOI: 10.1021/acs.bioconjchem.7b00791
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      High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers
      Ohri, Rachana; Bhakta, Sunil; Fourie-O'Donohue, Aimee; dela Cruz-Chuh, Josefa; Tsai, Siao Ping; Cook, Ryan; Wei, Binqing; Ng, Carl; Wong, Athena W.; Bos, Aaron B.; Farahi, Farzam; Bhakta, Jiten; Pillow, Thomas H.; Raab, Helga; Vandlen, Richard; Polakis, Paul; Liu, Yichin; Erickson, Hans; Junutula, Jagath R.; Kozak, Katherine R.
      Bioconjugate Chemistry (2018), 29 (2), 473-485CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      THIOMAB antibody technol. utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technol. has enabled the exploration of different attachment sites on the antibody in combination with small mols., peptides, or proteins to yield antibody conjugates with unique properties. As reported previously, the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepd. by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purifn. method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivs. with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approx. 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivs. were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.
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    47. 47
      Lyon, R. (2018) Drawing lessons from the clinical development of antibody-drug conjugates. Drug Discovery Today: Technol. 30, 105– 109,  DOI: 10.1016/j.ddtec.2018.10.001
      47
      Drawing lessons from the clinical development of antibody-drug conjugates
      Lyon Robert
      Drug discovery today. Technologies (2018), 30 (), 105-109 ISSN:.
      The antibody-drug conjugate (ADC) field has seen a remarkable expansion in the number of entrants in clinical studies. Many of these agents employ newer conjugation technologies that have been developed over the last decade that confer various attributes to the ADCs prepared with them, including stability, potency, and homogeneity. In many cases, these new ADCs appear demonstrably superior to earlier technologies in preclinical models of activity and toxicology, but the degree to which these improvements will translate to the clinic is only starting to be seen. Many of these technologies are now competing head-to-head by targeting the same antigen in similar patient populations, allowing for a direct comparison of their clinical performance properties. As lessons from these experiences feed back into discovery research, future iterations of ADC design may be expected to bring improved therapeutics into the clinic.
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    48. 48
      Khera, E., Cilliers, C. M., Bhatnagar, S., and Thurber, G. M. (2018) Computation transport analysis of antibody-drug conjugate bystander effects and payload tumoral distribution: implications for therapy. Mol. Syst. Des. Eng. 3, 73– 88,  DOI: 10.1039/C7ME00093F
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      Computational transport analysis of antibody-drug conjugate bystander effects and payload tumoral distribution: implications for therapy
      Khera, Eshita; Cilliers, Cornelius; Bhatnagar, Sumit; Thurber, Greg M.
      Molecular Systems Design & Engineering (2018), 3 (1), 73-88CODEN: MSDEBG; ISSN:2058-9689. (Royal Society of Chemistry)
      Antibody drug conjugates (ADCs) have a proven clin. record with four FDA approved drugs and dozens more in clin. trials. However, a better understanding of the relationship between delivery and efficacy of ADCs is needed to improve the rate of successful clin. development. Recent evidence indicates that heterogeneous distribution can play a large role in the efficacy of these drugs. However, the impact of the drug payload, particularly the ability of the payload to diffuse outside of the original targeted cell into adjacent cells (the bystander effect), is not completely understood. Given the challenges in directly measuring the payload distribution within tumors, we developed a predictive computational model to study payload distribution as a function of antibody dose, payload dose, and payload properties. The computational results indicate that: 1. the heterogeneous tumoral distribution of ADCs impacts efficacy, and increasing the antibody dose improves penetration and efficacy. 2. The increased penetration of payloads with bystander effects can partially compensate for poor antibody penetration, but larger antibody doses still result in further improvement. This occurs because of the higher efficiency of direct cell killing than bystander killing. 3. Bystander effects are important for killing antigen neg. cells, and an optimum in physicochem. properties exists. Payloads with a balance in cellular uptake vs. tissue diffusion enter cells fast enough to avoid tumor washout but slow enough to reach distant cells. Therefore, optimizing the antibody dose, payload dose, and payload physicochem. properties results in ideal delivery to the site of action and max. efficacy.
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    49. 49
      Cilliers, C., Menezes, B., Nessler, I., Linderman, J., and Thurber, G. M. (2018) Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival. Cancer Res. 78 (3), 758– 768,  DOI: 10.1158/0008-5472.CAN-17-1638
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      Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival
      Cilliers, Cornelius; Menezes, Bruna; Nessler, Ian; Linderman, Jennifer; Thurber, Greg M.
      Cancer Research (2018), 78 (3), 758-768CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      Current ADC have made advances in engineering the antibody, linker, conjugation site, small-mol. payload, and drug-to-antibody ratio (DAR). However, the relationship between heterogeneous intratumoral distribution and efficacy of ADCs is poorly understood. Here, we compared trastuzumab and ado-trastuzumab emtansine (T-DM1) to study the impact of ADC tumor distribution on efficacy. In a mouse xenograft model insensitive to trastuzumab, coadministration of trastuzumab with a fixed dose of T-DM1 at 3:1 and 8:1 ratios dramatically improved ADC tumor penetration and resulted in twice the improvement in median survival compared with T-DM1 alone. In this setting, the effective DAR was lowered, decreasing the amt. of payload delivered to each targeted cell but increasing the no. of cells that received payload. This result is counterintuitive because trastuzumab acts as an antagonist in vitro and has no single-agent efficacy in vivo, yet improves the effectiveness of T-DM1 in vivo. Novel dual-channel fluorescence ratios quantified single-cell ADC uptake and metab. and confirmed that the in vivo cellular dose of T-DM1 alone exceeded the min. required for efficacy in this model. In addn., this technique characterized cellular pharmacokinetics with heterogeneous delivery after 1 day, degrdn. and payload release by 2 days, and in vitro cell killing and in vivo tumor shrinkage 2 to 3 days later. This work demonstrates that the intratumoral distribution of ADC, independent of payload dose or plasma clearance, plays a major role in ADC efficacy. This study shows how lowering the drug-to-antibody ratio during treatment can improve the intratumoral distribution of a antibody-drug conjugate, with implications for improving the efficacy of this class of cancer drugs.
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    50. 50
      Sahota, S. and Vahdat, L. T. (2017) Sacituzumab govitecan: an antibody-drug conjugate. Expert Opin. Biol. Ther. 17 (8), 1027– 1031,  DOI: 10.1080/14712598.2017.1331214
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      Sacituzumab govitecan: an antibody-drug conjugate
      Sahota, Sheena; Vahdat, Linda T.
      Expert Opinion on Biological Therapy (2017), 17 (8), 1027-1031CODEN: EOBTA2; ISSN:1471-2598. (Taylor & Francis Ltd.)
      : Despite advances in the diagnosis and treatment of patients with cancer, patients with metastatic cancer have limited therapeutic options after initial lines of therapy. Understanding tumor biol. has translated into the identification of actionable targets that resulted in therapeutics. Antibody-drug conjugates (ADC) are capitalizing on this explosion of scientific information. ADCs allow an antibody to a unique target to be conjugated via an innovative linker, to a highly toxic drug which is delivered to its target. Sacituzumab govitecan is an ADC that combines the active mol. in irinotecan, SN-38, to an antibody targeting trop2.: In this review, the authors introduce the reader to the ADC sacituzumab govitecan providing the reader with details about its pharmacokinetics, pharmacodynamics, efficacy and safety. The authors also give their expert anal. about its potential future use.: Sacituzumab govitecan is a novel and well-tolerated therapeutic showing promising results in difficult to treat cancers. Further studies are underway to optimize the group of patients that would benefit from it. Given its excellent performance, we are cautiously optimistic it will be approved by the FDA.
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    51. 51
      Nakada, T., Sugihara, K., Jikoh, T., Abe, Y., and Agatsuma, T. (2019) The Latest Research and Development into the Antibody-Drug Conjugate, [fam-] Trastuzumab Deruxtecan (DS-8201a), for HER2 Cancer Therapy. Chem. Pharm. Bull. 67 (3), 173– 185,  DOI: 10.1248/cpb.c18-00744
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      The latest research and development into the antibody-drug conjugate, [fam-] trastuzumab deruxtecan (DS-8201a), for HER2 cancer therapy
      Nakada, Takashi; Sugihara, Kiyoshi; Jikoh, Takahiro; Abe, Yuki; Agatsuma, Toshinori
      Chemical & Pharmaceutical Bulletin (2019), 67 (3), 173-185CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)
      A review. A major limitation of traditional chemotherapy for cancer is dose-limiting toxicity, caused by the exposure of non-tumor cells to cytotoxic agents. Use of mol. targeted drugs, such as specific kinase inhibitors and monoclonal antibodies, is a possible soln. to overcome this limitation and has achieved clin. success so far. Use of an antibody-drug conjugate (ADC) is a rational strategy for improving efficacy and reducing systemic adverse events. ADCs use antibodies selectively to deliver a potent cytotoxic agent to tumor cells, thus drastically improving the therapeutic index of chemotherapeutic agents. Lessons learned from clin. failure of early ADCs during the 1980s to 90s have recently led to improvements in ADC technol., and resulted in the approval of four novel ADCs. Nonetheless, further advances in ADC technol. are still required to streamline their clin. efficacy and reduce toxicity. [fam-] Trastuzumab deruxtecan (DS-8201a) is a next-generation ADC that satisfies these requirements based on currently available evidence. DS-8201a has several innovative features; a highly potent novel payload with a high drug-to-antibody ratio, good homogeneity, a tumor-selective cleavable linker, stable linker-payload in circulation, and a short systemic half-life cytotoxic agent in vivo; the released cytotoxic payload could exert a bystander effect. With respect to its preclin. profiles, DS-8201a could provide a valuable therapy with a great potential against HER2-expressing cancers in clin. settings. In a phase I trial, DS-8201a showed acceptable safety profiles with potential therapeutic efficacy, with the wide therapeutic index.
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    52. 52
      Reid, E. E., Archer, K. E., Shizuka, M., Wilhelm, A., Yoder, N. C., Bai, C., Fishkin, N. E., Harris, L., Maloney, E. K., E, H. (2019) Effect of linker stereochemistry on the antitumor activity of anti-body-drug conjugates (ADCs) containing indolinobenzodiazepine payloads. ACS Med. Chem. Lett. 10 (8), 1193– 1197,  DOI: 10.1021/acsmedchemlett.9b00240
      52
      Effect of Linker Stereochemistry on the Activity of Indolinobenzodiazepine Containing Antibody-Drug Conjugates (ADCs)
      Reid, Emily E.; Archer, Katie E.; Shizuka, Manami; Wilhelm, Alan; Yoder, Nicholas C.; Bai, Chen; Fishkin, Nathan E.; Harris, Luke; Maloney, Erin K.; Salomon, Paulin; Hong, Erica; Wu, Rui; Ab, Olga; Jin, Shan; Lai, Katharine C.; Sikka, Surina; Chari, Ravi V. J.; Miller, Michael L.
      ACS Medicinal Chemistry Letters (2019), 10 (8), 1193-1197CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      Antibody-drug conjugates (ADCs) that incorporate potent indolinobenzodiazepine DNA alkylators as the payload component are currently undergoing clin. evaluation. In one ADC design, the payload mols. are linked to the antibody through a peptidase-labile L-Ala-L-Ala linker. In order to det. the role of amino acid stereochem. on antitumor activity and tolerability, we incorporated L- and D-alanyl groups in the dipeptide, synthesized all four diastereomers, and prepd. and tested the corresponding ADCs. Results of our preclin. evaluation showed that the L-Ala-L-Ala configuration provided the ADC with the highest therapeutic index (antitumor activity vs toxicity).
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    53. 53
      Lazar, A. C., Wang, L., Blättler, W. A., Amphlett, G., Lambert, J. M., and Zhang, W. (2005) Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry. Rapid Commun. Mass Spectrom. 19 (13), 1806– 1814,  DOI: 10.1002/rcm.1987
      53
      Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry
      Lazar, Alexandru C.; Wang, Lintao; Blaettler, Walter A.; Amphlett, Godfrey; Lambert, John M.; Zhang, Wei
      Rapid Communications in Mass Spectrometry (2005), 19 (13), 1806-1814CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody prepns. often contain several mol. variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepd. by covalently linking several drug mols. per antibody mol. As part of the product characterization, the mol. wts. of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the anal. of recombinant antibodies and immunoconjugates. Sample prepn. is an important element of ESI-MS anal., in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liq. chromatog. (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aq. acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid mols. generated charge envelope profiles that differ from those of the non-conjugated antibody. For the detn. of the distribution of the various conjugate species in an immunoconjugate sample prepd. by randomly linking in the av. 3.6 drug mols. per antibody mol., the exptl. conditions needed to be carefully selected to allow acquisition of the whole spectrum contg. the charge envelopes of all species.
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    54. 54
      Kovtun, Y. V., Audette, C. A., Mayo, M. F., Jones, G. E., Doherty, H., Maloney, E. K., Erickson, H. K., Sun, X., Wilhelm, S., Ab, O. (2010) Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Res. 70 (6), 2528– 2537,  DOI: 10.1158/0008-5472.CAN-09-3546
      54
      Antibody-Maytansinoid Conjugates Designed to Bypass Multidrug Resistance
      Kovtun, Yelena V.; Audette, Charlene A.; Mayo, Michele F.; Jones, Gregory E.; Doherty, Heather; Maloney, Erin K.; Erickson, Hans K.; Sun, Xiuxia; Wilhelm, Sharon; Ab, Olga; Lai, Katharine C.; Widdison, Wayne C.; Kellogg, Brenda; Johnson, Holly; Pinkas, Jan; Lutz, Robert J.; Singh, Rajeeva; Goldmacher, Victor S.; Chari, Ravi V. J.
      Cancer Research (2010), 70 (6), 2528-2537CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      Conjugation of cytotoxic compds. to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compds. used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG4Mal. Following uptake into target cells, conjugates made with the PEG4Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepd. with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG4Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addn., PEG4Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance. Cancer Res; 70(6); 2528-37.
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    55. 55
      Ab, O., Whiteman, K. R., Bartle, L. M., Sun, X., Singh, R., Tavares, D., LaBelle, A., Payne, G., Lutz, R. J., Pinkas, J. (2015) IMGN853, a Folate Receptor-α (FRα)-Targeting Antibody-Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors. Mol. Cancer Ther. 14 (7), 1605– 1613,  DOI: 10.1158/1535-7163.MCT-14-1095
      55
      IMGN853, a Folate Receptor-α (FRα)-Targeting Antibody-Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors
      Ab, Olga; Whiteman, Kathleen R.; Bartle, Laura M.; Sun, Xiuxia; Singh, Rajeeva; Tavares, Daniel; LaBelle, Alyssa; Payne, Gillian; Lutz, Robert J.; Pinkas, Jan; Goldmacher, Victor S.; Chittenden, Thomas; Lambert, John M.
      Molecular Cancer Therapeutics (2015), 14 (7), 1605-1613CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      A majority of ovarian and non-small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody-drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-pos. cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non-small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochem. IMGN853 displayed cytotoxic activity against FRα-neg. cells situated near FRα-pos. cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clin. development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors. Mol Cancer Ther; 14(7); 1605-13. ©2015 AACR.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.9b00777.

    • Detailed experimental procedures for synthesis of compound 1b, supplementary characterization data for ADCs, and supplementary methods (PDF)


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