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Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells. Philip Brandish, Anthony Palmieri, Svetlana Antonenko, Maribel Beaumont, Lia Benso, Mark Cancilla, Mangeng Cheng, Laurence Fayadat-Dilman, Guo Feng, Isabel Figueroa, Juhi Firdos, Robert M. Garbaccio, Laura Garvin-Queen, Dennis Gately, Prasanthi Geda, Christopher Haines, SuChun Hseih, Douglas Hodges, Jeffrey C. Kern, Nick Knudsen, Kristen Kwasnjuk, Linda Liang, Huiping Ma, Anthony Manibusan, Paul Miller, Lily Moy, Yujie Qu, Sanjiv Shah, John Shin, Peter Stivers, Ying Sun, Daniela Tomazela, Hyun Chong Woo, Dennis Zaller, Shuli Zhang, Yiwei Zhang, and Mark Zielstorff Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00312 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Bioconjugate Chemistry

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Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells. Philip E. Brandish*†, Anthony Palmieri†, Svetlana Antonenko†, Maribel Beaumont†, Lia Benso†, Mark Cancilla†, Mangeng Cheng†, Laurence Fayadat-Dilman†, Guo Feng†, Isabel Figueroa†, Juhi Firdos‡, Robert Garbaccio†, Laura Garvin-Queen†, Dennis Gately‡, Prasanthi Geda†, Christopher Haines†, SuChun Hseih†, Douglas Hodges†, Jeffrey Kern†, Nickolas Knudsen‡, Kristen Kwasnjuk†, Linda Liang†, Huiping Ma†, Anthony Manibusan‡, Paul L. Miller†, Lily Y. Moy†, Yujie Qu†, Sanjiv Shah†, John S. Shin†, Peter Stivers†, Ying Sun‡, Daniela Tomazela†, Hyun Chong Woo†, Dennis Zaller†, Shuli Zhang†, Yiwei Zhang†, and Mark Zielstorff†

†

Merck & Co., Inc., 2000 Galloping Hill Road Kenilworth, NJ 07033 U.S.A.

‡

Ambrx, Inc., 10975 North Torrey Pines Road La Jolla, CA 92037

*Correspondence should be addressed to P.E.B. 33 Avenue Louis Pasteur Boston, MA 02115, ([email protected]), Phone, 617-992-3085

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Abstract Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody drug conjugates (ADCs) using antibodies containing sitespecific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

Introduction The value to patients of ADCs as a means to enable use of otherwise toxic agents has been established in the setting of oncology 1,2. These successes beg the question whether the approach can be applied in other areas of medicine where clinically valuable drugs are dose-limited by toxicity. We selected delivery of glucocorticoids as a case study outside of the field of oncology to test the feasibility of the general approach because they are highly effective anti-inflammatory drugs with broad utility, yet their use is limited by toxicities arising from systemic exposure and a role in homeostasis for the glucocorticoid receptor (GR) in most cells in the body 3,4. Using antibodies to target glucocorticoids has been tested with anti-E-selectin (activated endothelial cells) or anti-CD163 (macrophage subsets) coupled to dexamethasone, but these approaches have failed or have given equivocal results 5,6. We set out to test this concept using plasma-stable linkers to deliver selectivity and duration of action, and a defined attachment site using non-natural amino acids to afford homogeneity and the attendant improved pharmacokinetics 7,8. We selected CD74, often called MHC class II invariant chain, as the targeting antigen for two main reasons. First, it is present on myeloid antigen-presenting cells as well as B cells, both of which are relevant to systemic lupus erythematosus pathogenesis, a disease where physicians depend heavily on glucocorticoids and the unmet medical need is acute. Second, CD74 is known to internalize rapidly upon antibody ligation, and detailed work by Ong, Hansen and colleagues showed the capacity to be relatively large 9,10. In addition, human anti-



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human CD74 monoclonal antibodies that internalize well have been described, and a human CD74 transgenic mouse was available to facilitate testing in vivo 11. In previous works we described in detail our investigations of the structure-activity relationship and the discovery of the pyrophosphate linker that enabled this work 12,13. Here we describe the preparation, characterization and properties of anti-CD74-glucocorticoid conjugates and the progress toward our goal thus far.

Results and Discussion We incorporated the variable domains including complementarity-determining regions of the human anti-human CD74 antibody, originally described as clone 011, 14 into a human IgG4 antibody stabilized with the S228P mutation and expressed it transiently in CHO cells. When incubated with PBMCs, purified fluorescently labeled recombinant antibody bound to monocytes and B cells but not T cells as expected (Supplementary Fig. 1). Similarly, it bound to the T and B lymphoma cell lines HUT78 and SUDHL-6 which have been reported to express CD74 (Supplementary Fig. 1) 10,15. Hansen et al. found that HUT78 cells could internalize and catabolize relatively large amounts of anti-CD74 antibody prompting us to make use of the cell line for initial studies 10. Our anti-CD74 variant was time- and temperature-dependently internalized and efficiently catabolized by HUT78 cells (Supplementary Fig. 1). These data gave us confidence in the experimental system and the IgG4(S228P) clone 011 variant in particular. The antibody was stable in mouse and human plasma (Supplementary Table 1). Observed pharmacokinetic (PK) profiles in wild type mice were comparable to those typically seen for other human IgG4 antibodies in C57/b6 mice. In human CD74 transgenic mice, which are described in detail in the online methods section, concentration-time profiles show dose-dependent PK properties consistent with target-mediated clearance (Supplementary Fig. 2). Anti-CD74 clone 011 therefore formed the starting point for preparation of ADCs. In order to make site-specific homogeneous antibody drug conjugates and to control the drug-antibody ratio (DAR) we engineered into the IgG4 clone 011 variant an amber codon in place of alanine-114 in the heavy chain and used nonnatural amino acid incorporation technology to produce a second variant carrying a para-azido phenylalanine (pAF) residue instead of the native alanine

12,16

. Purification of the recombinant anti-CD74 clone 011-pAF antibody afforded highly pure

monomeric material which we then used to prepare ADCs (Supplementary Fig. 3). We took a decision early on to restrict our initial investigation to using known, well characterized glucocorticoids as payloads to limit the complexity of the work. Among payloads fitting this description, fluticasone propionate has a very high intrinsic potency at the glucocorticoid


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receptor that is driven by a very low off-rate 17. It has the added advantage of being subject to high clearance in vivo owing to rapid metabolism in the liver 18, i.e. we hypothesized that GC activation in target cells would persist while systemic exposure would be minimized. To attach the steroid payload to the antibody, we used a novel pyrophosphate acetal linker designed to be stable in plasma but labile in the lysosome and which would yield fluticasone propionate free of any linker moieties 12. We designated this linker-payload molecule flu449. It contains a cyclo-octyne ring distal to the fluticasone propionate payload which allows a copper-free Click coupling reaction to generate the conjugate shown in Fig. 1a under mild conditions. We also prepared an isotype-matched control ADC derived from palivizumab, a monoclonal antibody that binds to the F-protein of the two major strains of respiratory syncytial virus (RSV). This recombinant anti-RSV did not bind to human or mouse cells (not shown), and so any functional activity observed represents the sum of non-specific activity resulting from any Fcmediated or other uptake of antibody, any degradation in situ or in storage, and any payload carryover from ADC synthesis. Quality control testing of anti-human CD74(IgG4/S228P)-clone011-pAF-flu449 (anti-CD74-flu449) and the analogous antiRSV conjugate found that both materials were highly pure and lacked any significant amount of multimeric or aggregated material (Supplementary Fig. 3). More than one batch of antibody and ADC was used in the studies reported here. All batches of ADC were ≥ 96% monomer and DAR was ≥ 1.7 (Supplementary Fig. 3). Stability testing of anti-CD74-flu449 in mouse plasma at 37 °C for up to 14 days found no measurable decrease in DAR (Supplementary Fig. 4). These data were extremely encouraging, especially when coupled with the observed lability of the drug-linker molecule in lysosomal lysates reported by Garbaccio 12. Thus, all of the components for targeted delivery – specificity, purity, stability, lysosomal lability, antibody internalization and catabolism – were embodied in anti-CD74-flu449.



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Figure 1. Anti-CD74-flu449 causes up-regulation of GR target genes and inhibits B cell proliferation in vitro. (a), Fluticasone propionate was coupled to heavy chain residue 114, para-azidophenylalanine, via a pyrophosphate containing linker attached to the steroid 11-hydroxyl position using click chemistry as described in the Materials and Methods. This linker including an acetal spacer afforded efficient generation of parental fluticasone propionate compared to other linkers as reported in Garbaccio et al. (b, c) . Anti-CD74-flu449 regulates GILZ gene expression in CD74 positive cells (HUT78). The EC50 for anti-CD74-flu449 was 0.03 µg/ml (~ 0.4 nM fluticasone propionate equivalents) compared to 0.7 nM for fluticasone propionate. (d, e) Splenic B cells from hCD74 transgenic mice or wild type littermates were stimulated to proliferate with LPS in the presence of anti-CD74-flu449 or anti-RSV-flu449 and pulsed with 3H-thymidine after 43 hr. Data shown are means / s.d. of the aggregate of two independent studies with three technical replicates per study with radioactivity counts normalized to control. The IC50s for anti-CD74-flu449 and anti-RSV-flu449 in transgenic B cells were 0.08 and 5.8 µg/mL, respectively. (f, g) Purified human B cells were incubated with varying concentrations of anti-CD74-flu449 or fluticasone propionate for 24 hr at either 0.1 or 2 million cells per mL of culture medium, and ZBTB16 mRNA levels were measured by RT-QPCR. The inset table shows EC50 values calculated from curve fitting. The dotted line represents maximum gene response from free fluticasone propionate. Filled circles represent higher density and empty circles represent lower density


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culture conditions. The data represent mean / s.d. of two technical replicates. The data shown are one of two independent experiments with the same qualitative results. We were uncertain what to expect with respect to the kinetics of payload release in cells so we elected to use as simple a system as possible to test anti-CD74-flu449. GCs block immune signaling in many cell types with a variety of functional outputs, but the proximal activity of the GR is to regulate transcription. Most cell types in culture in vitro will upregulate a number of mRNA transcripts in response to GCs even in the absence of an immune or other stimulus. Well known examples are GILZ and FKBP5. We made use of this rapid response to test anti-CD74-flu449 in HUT78 cells, which express CD74, and 786-O cells, which do not express CD74, but which are both responsive to GR agonists. In HUT78 cells, treatment with anti-CD74-flu449 for 18 hours caused an increase in GILZ mRNA levels similar to that with fluticasone propionate (Fig. 1b). The control anti-RSV ADC was three orders of magnitude less potent than the anti-CD74 ADC in HUT78 cells and the activities of both ADCs were similarly weak in 786-O cells, all of which indicated CD74 dependent activity (Figs. 1b & 1c). A similar activity profile was found in the CD74-expressing B lymphoma cell line SUDHL-6 (Supplementary Fig. 5). Furthermore, the activity of anti-CD74-flu449 in HUT78 cells could be blocked with naked (nonconjugated) anti-CD74-pAF (Supplementary Fig. 5). Anti-CD74-flu449 also blocked LPS-induced proliferation of splenic B cells isolated from human CD74 transgenic mice but not from wild type littermates (Figs. 1d & 1e), as well as anti-IgMinduced proliferation of human B cells (data not shown). These experiments established that the depth of response to antiCD74-flu449 was comparable with that to fluticasone propionate and it was dependent on both the presence of human CD74 and the ADC specificity. Although encouraged by the results, we were cognizant that the permeable nature of fluticasone propionate means that released payload will diffuse out of the cell at some rate. Therefore we questioned whether the observed activity might be driven by a cell-intrinsic effect (payload acts in the cell in which it was liberated from the conjugate) or by payload accumulating in the medium. We investigated this question using two approaches. First, we looked for bystander activity in a mixed cell culture (human PBMCs in vitro) and found that gene expression was similarly regulated in both B cells (CD74 positive) and T cells, even though T cells express little or no CD74 (Supplementary Fig. 6). In the same experiment we found that fluticasone propionate accumulated in the medium above anti-CD74-flu449-treated cells which might account for the activity measured in the cells (Supplementary Fig. 6). We found similar levels of accumulation for four other cell-permeable payloads in various culture systems suggesting a common phenomenon (data not shown). Second, we examined the



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dependence of apparent potency of anti-CD74-flu449 on cell density, the rationale being that more cells per unit volume will generate a higher concentration of free payload in the culture medium which would manifest as an increase in apparent potency. Indeed, we found that the apparent potency of anti-CD74-flu449 on ZBTB16 mRNA up-regulation in isolated primary human B cells varied with the cell density in the experiment, whereas the potency of fluticasone propionate did not (Fig. 1f). We demonstrated the impact of this with a second, more defined bystander experiment. ZBTB16 gene expression was up-regulated by fluticasone propionate in human B and T cells to a similar degree whether cells were cultured separately or together. In contrast, anti-CD74-flu449 up-regulated ZBTB16 gene expression only in B cells when cells were cultured separately, but when B and T cells were cultured together ZBTB16 was up-regulated in both cell types exactly as if free fluticasone propionate had been added to the well (Supplementary Fig. 7). The permeability of fluticasone propionate and the findings from our mechanism of action studies were a concern, but we did not want to assume that anti-CD74-flu449 would be inactive in vivo. A study was therefore conducted in human CD74 transgenic mice to measure FKBP5 mRNA levels in splenic B cells (target CD74 positive cells) and splenic T cells (bystander CD74 negative cells). Additionally, plasma corticosterone was measured to assess systemic exposure to free payload. We found anti-CD74-flu449 was inactive on both endpoints even with plasma concentration of antibody ~20 ug/mL 24 hours after dosing, consistent with expectations based on a preceding PK study (Supplementary Fig. 8). We interpreted the negative in vivo result to mean that payload dissipated and was cleared before measurable GR activation could happen, i.e. the shortcoming of a permeable payload highlighted by our in vitro studies illustrated in Figure 1. Modification of payload permeability as a solution for ADCs has been investigated before: Doronina and colleagues showed that a cell-impermeant analog of monomethylauristatin, designated MMAF, could be delivered into cells using an anti-CD30 antibody with dramatic positive results in vitro 19. We found similar results with anti-CD74 clone 011 which supported trying a low permeability payload approach for GR agonism (data not shown). We synthesized a number of phosphonate analogs of fluticasone propionate designed to have low cellular permeability at neutral pH but moderate cellular permeability at pH 5-6 to enable escape from the lysosome. Compound A, whose structure is shown in Fig. 2, was selected for conjugation because its intrinsic binding affinity to GR was similar to fluticasone propionate, its measured permeability in a standard assay was low, and its apparent potency in cells was greatly reduced compared to fluticasone propionate while still functioning as an agonistic ligand (Fig. 2a-c). Conjugation of Compound A to anti-CD74 using a pyrophosphonate linkage yielded an ADC (anti-CD74-AXC496, Fig. 2a) that was active in human B cells (Fig. 2e-f), albeit less so than fluticasone


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propionate or anti-CD74-flu449. We knew from earlier work that a 24 hr incubation of cells with other anti-CD74 ADCs generated in the range of 1 – 10 nM free payload in the culture medium. Given that the measured EC50 of compound A in B cells was ~1.7 µM (Fig. 2b), payload accumulating in the medium to such levels should not produce measurable activity. Indeed, since 10 µg/ml ADC equates to approximately 130 nM payload, even complete conversion of ADC to free payload would be expected to produce only minimal activity if any. Therefore we measured free payload in the medium under identical conditions to the activity assay (by pooling samples from many replicate wells) and found 47 nM Compound A after 24 hr treatment with anti-CD74-AXC496 (Fig. 2g). Similar levels of Compound A were measured after treatment with anti-RSV-AXC496 (43 nM), where no activity on ZBTB16 mRNA was observed. The differential activity of the conjugates with comparable supernatant concentrations of Compound A suggests anti-CD74-AXC496 is acting via a cell intrinsic mechanism. The similar supernatant payload concentrations in control and active ADC experiments after 24 hr indicate a degree of instability or CD74-independent extracellular processing of conjugate to free payload that was not observed with the fluticasone propionate conjugates. Indeed, incubation of anti-CD74-AXC496 in cell culture medium for 24 hr (but not 4 hr) led to release of payload (Supplementary Table 2). We do not know the mechanism of degradation, but it is reasonable to suspect that it is due to the difference in the linker position and chemistry. As such, that is a potential root cause of the lower maximal activity of anti-CD74-AXC496 versus free Compound A or anti-CD74-flu449 (Figs. 2d and 2e).



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Figure 2. Identification of a low permeability payload with activity as an ADC in vitro. (a) Chemical structures of compound A and anti-CD74-AXC496. (b) Summary of the physical-chemical properties and bioactivity profile of compound A, compared to fluticasone propionate, that led to its selection for testing as antibody-drug conjugates. (c-e) Dose response curves for fluticasone propionate, compound A and the corresponding anti-RSV and anti-CD74 ADCs for regulation of ZBTB16 mRNA levels in primary human B cells in vitro. (f) Estimated potencies of free compounds, and ADCs calculated from the dose-response curves shown, with ADC concentrations expressed in terms of nanomolar equivalents of payload using predicted molecular weights and the drug-antibody ratios reported in the table in Supplementary Fig. 3. (g) Free payload concentrations measured in the supernatant above human B cells after 24 hr of the indicated treatments. BLQ, below limit of quantitation. Despite the evident reduced stability compared to earlier conjugates, we moved to test whether the apparent cellintrinsic activity would translate into cell-specific targeting, the critical and central goal of our work. We moved from purified human B cells back to a mixed cell culture system (human PBMCs). PBMCs were treated with anti-CD74-AXC496


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overnight, then B and T cells were separately recovered from the culture and activity of the conjugate was determined as ZBTB16 mRNA levels. Increased gene expression was measured in B cells treated with anti-CD74-AXC496, but not in T cells, whereas anti-CD74-flu449, free fluticasone propionate and free compound A all caused increased gene expression in B and T cells alike (Fig. 3). Levels of free payload quantified in the supernatant after 24hr were proportionally similar to levels in the B cell monoculture experiments (16.8 nM) and again cannot account for the measured activity. All these data are consistent with anti-CD74-AXC496 delivering payload and GR activation specifically to antigen-positive cells and not antigen-negative cells in a native co-culture of primary human immune cells. This was the objective of our collaboration and our research. While further optimization of conjugate stability is needed prior to in vivo testing (or indeed clinical development), our view is that the concept is proved.

Figure 3. Selective delivery of Compound A to B cells in mixed cell population in vitro. (a) ZBTB16 mRNA is induced in B cells isolated from PBMC treated with CD74-AXC496 after 24 hours at 37 °C, whereas ZBTB16 is not regulated in the isolated T cells (b) Quantification of payloads from PBMC supernatants.



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Conclusion To sum up, our goal was to explore the feasibility of targeting a non-cytotoxic payload, released from an antibody after entry into the cell, using glucocorticoid delivery as the test case. We employed novel fit-for-purpose linkers to study the requirements for payload permeability and we demonstrated the feasibility of the approach. Our work opens a path to development of a cell-targeted glucocorticoid therapy specifically, but also provides a framework and impetus for rescue of otherwise dose-limited potential medicines generally.

Materials and Methods Expression and purification of anti-CD74 antibody. CHO-S cells growing in suspension were transfected with human anti-CD74 antibody expression plasmid (1.3 µg DNA per million cells) via flow electroporation using a MaxCyte STX (Maxcyte corporation). The culture temperature was lowered to 32 °C for 24 hr following electroporation. Cultures were fed at day 1 and day 4 with a media optimized for antibody production and harvested at day 8 with 85-95% viability. Antibody was purified from clarified supernatant using Protein A chromatography (mAbSelect Sure LX, GE Healthcare) followed by anion exchange chromatography (Capto Q, GE Healthcare) and buffer exchange into the final formulation buffer, 20mM sodium acetate, 9% sucrose, pH 5.5. Purified antibody was checked for endotoxin levels (< 1 EU/mg) and aggregate content (< 1% high molecular weight species by SEC-UPLC) using, respectively, an Endosafe instrument (Charles River Laboratories) and a BEH200 UPLC-SEC analytical column (Waters corporation). Fluorescence labeling of antibody DyLight-650 labeling kits were used to conjugate an N-hydroxysuccinimide ester fluorescence dye (excitation/emission at 652 nm/672 nm) to the anti-CD74 antibody. Before labeling, the antibody preparation was buffer-exchanged to 50 mM sodium borate with 100 mM NaCl buffer at pH 8.5 using a 10 kDa molecular weight cut-off Slide-A-Lizer dialysis cassette. Each reaction mixture contained 1.2 mg of experimental antibody in a final volume 0.5 mL. The reaction was initiated by combining the antibody with the dye and continued for 1 hour at room temperature (RT) protected from light. Unconjugated dye was removed using a purification resin packed into spin columns. When necessary, samples were further concentrated


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by Amicon ultra centrifugation filter devices with a 10 kDa cut-off membrane. The labeled anti-CD74 mAb antibody was filtered using a 0.22 µm Durapore PVDF membrane. A NanoDrop apparatus (Thermo Scientific) was used to characterize the labeled reagents for determination of protein concentration and degree of labeling (DOL) as dye-to-protein, mole-to-mole ratio. The purity and integrity of the labeled antibody was also assessed by SEC-HPLC. Binding of anti-CD74 to human PBMCs and cell lines. Conjugated antibodies to CD3, CD4, CD19, and human IgG4/kappa (isotype control) were from BD Biosciences, anti-CD14 was from Beckman Coulter, and labeled anti-CD74 was conjugated in house (DyLight650). Frozen PBMCs (AccuCell #13075 from Precision Bioservice) were recovered for 1 hr at 37 °C in a 5% CO2 tissue culture incubator, (recovered PBMCs and the human cell lines (HUT78, SUDHL6, 786-O) incubated with Fc block (BioLegend #422301) for 20 min at room temperature, and then incubated with human IgG4/kappa or different concentrations of anti-CD74 and a cocktail of antibodies to CD3, CD4, CD14, and CD19 for 2 hr. Cells were washed and re-suspended in DPBS with 0.1% BSA plus sodium azide (0.09%). Flow cytometry was performed using an LSRII flow cytometer (BD Bioscience). Immunofluorescence sample preparation and laser scanning confocal microscopy (LSCM). HUT78 cells were incubated in RPMI media containing 10% FBS for up to 4 hr in the presence of 5 µg/mL of DyLight 650-labeled anti-CD74 antibody. Incubations at 37 °C in a 5% CO2 incubator and on ice were followed by several washes with ice-cold PBS containing 2% normal goat serum. Cells were then fixed with 2% paraformadehyde for 30 min. Cells were permeabilized with 1X saponin-based permeabilization buffer (eBioscience 00-8333-56) also containing 10% normal goat serum for 20 min followed by the addition of a mouse anti-human LAMP1 antibody (clone H4A3, Biolegend) at 5 µg/mL. After a 30 min incubation at room temperature, cells were washed and incubated for another 30 minutes with 2 µg/mL goat anti-mouse antibody conjugated to AlexaFluor 488 (Life Technologies, A11001). A final set of washes were performed and cells were centrifuged briefly in a 96-well glass-bottom plate with #1.5 cover glass (In Vitro Scientific, P96-1.5H-N). Imaging was performed with a Leica TCS SP5 MP confocal microscope outfitted with white light laser using 63x/NA1.3 glycerin objective configured for both fluorescent channels as well as DIC (differential interference contrast) to enhance cell morphology. Antibody catabolism using Capillary Fluorescent Electrophoresis (CAFÉ). Cell lysates were analyzed using a Caliper LifeSciences LabChip GX II microfluidic capillary electrophoresis apparatus (Perkin Elmer, Hopkinton, MA). Briefly, HUT78 cells were incubated in RPMI medium containing 10% fetal bovine serum at 100,000 per well in a 96-well flat



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bottom plate. DyLight 650- labeled antibody was added to each well to obtain a final concentration of 5 µg/mL. Cells were then incubated at 37 °C in a CO2 incubator for up to 24 hr. At the designated time, the cells were washed three times with icecold PBS containing 2% FBS in 96-well V-bottom plates. After the final wash and centrifugation, 50 µL of a diluted Caliper pico protein sample buffer (1:5 in water) was added to the cell pellet followed by vigorous pipetting to ensure a homogenous cell lysate. The samples were transferred to a 96-well PCR plate and heated for 10 min at 80 °C. The plate was then loaded onto the instrument and acquisition was performed using the pico protein 200 method with a Protein Express LabChip. Data was then processed using the PerkinElmer LabChip GX software, v4.2. Antibody stability and recovery in plasma. Fresh human and cynomolgus monkey plasma were obtained from Bioreclamation Inc. The plasma was pooled from whole blood from 3 individual donors. As a matrix control, bovine serum albumin (BSA) at a concentration level of 1 mg/mL in Dulbecco’s phosphate-buffered saline (DPBS), at pH 7.4 was used. DyLight-650-labeled antibody (at a final concentration of 80 µg/mL) was incubated with neat human or cynomolgus monkey plasma containing potassium EDTA at 37 °C. Aliquots of plasma (100 µL) were collected following 5 minutes and 3, 7, and 14 days of incubation. At each time point, 25 µL of plasma were applied onto a BioSep-SEC-S 3000 column equipped with a SecurityGuard cartridge (Phenomenex). They were separated using an Agilent 1200 HPLC system equipped with a DAD (Diode Array Detector), UV/Vis (Agilent Technologies Inc.), and fluorescent detector (Hamamatsu Photonics K.K). The size exclusion procedure was a 15 min isocratic run with DPBS as mobile phase at a flow rate of 1 mL/min at RT. The effluent was monitored optically by total fluorescent intensity at excitation/emission of 646/674 nm, respectively. Data collection and analysis were performed using the Agilent ChemStation software V2 (Agilent Technologies Inc.). Molecular weight markers were run before analysis of experimental samples for assessment of column performance. Stability was calculated as the percent of intact monomeric IgG peak area relative to the total fluorescent area in each chromatogram. The recovery was calculated as the percent of the intact monomeric IgG peak at each sequential time point relative to the initial incubation time point (5 min). Transgenic human CD74 mice. Honey et al. described transgenic expression of human CD74, including evidence that the recombinant gene product could rescue knock out of mouse CD74 indicating that the human transgene is functional11. A colony of the same C57b6 mice, wild type for mouse CD74, was re-established at Jackson Labs under contract. It was confirmed that the transgene was expressed in the immune system everywhere and only where mouse CD74 was expressed, and that whole body histology showed no extraneous expression that could or would confound in vivo studies with anti-


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CD74 ADCs delivering glucocorticoids (data not shown). Therefore we had a high degree of confidence in the utility of these animals for our project. Pharmacokinetic studies in mice. To evaluate the pharmacokinetics of wild type anti-CD74 in absence of the antibody target, wild type C57b6 mice were randomized into four groups (n=18 each) and received single bolus intravenous doses of wild type (contains no non-natural amino acid substitutions) anti-CD74 of 30, 10, 3, 1 mg/kg, respectively. Blood for plasma was collected at 15 min, 1, 3, 6, and 16 hr, and 1, 2, 3, 5, and 7 days after dosing (Supplementary Fig. 2). Blood was collected from three mice at each time point: 0.4 mL for terminal bleeds (cardiac puncture) and 0.1 mL for non-terminal bleeds (tail vein). Individual mice were bled once or twice during the study, with at least one day between bleeds. A similar pharmacokinetic study was then carried out with wild-type anti-CD74, but now in hemizygous transgenic human CD74 C57b6 mice, but with n=7 mice per group, a modified range of doses, 30, 3, 1, 0.3 mg/kg , and a modified time course of 15 min, 1, 6, and 16 hr, 1, 3, 5 and 7 days (Supplementary Fig. 2). Mice were bled up to three times each. Likewise, to evaluate the pharmacokinetic properties of anti-CD74-flu449 in the presence of the human CD74 target, hemizygous transgenic human CD74 C57b6 mice were randomized into four groups (Supplementary Fig. 8). Group 1, 2 and 3 received 3, 10, or 30 mg/kg single bolus i.v. doses of anti-CD74-flu449, respectively. Animals in group 4 were dosed i.v. with 30 mg/kg of wild type anti-CD74. Plasma was collected at 5 and 15 min, 1, 3, and 6 hr, and 1, 2, 3 and 5 days after dosing and was analyzed for total anti-CD74 concentration by immunoassay and, to enable calculation of DAR, intact mass by mass spectrometry. Again, three mice replicates were collected at each time point. Additionally, for groups 1, 2, and 3 samples taken at 1 and 6 hr, and 1 , 2, and 5 days after test article administration were analyzed for free fluticasone propionate. Total anti-CD74 antibody concentrations (that is, anti-CD74 antibodies carrying zero, one or two payloads per molecule) were determined with an electrochemiluminescence-based immunoassay based on Meso Scale Discovery. The assay used recombinant human CD74 (R&D System cat# 3590-CD) as a capture reagent, and Sulfo-tagged mouse anti-human IgG (Southern Biotech cat# 9040-01) as a detection reagent. The LLOQ of the assay was determined to be 27.4 ng/mL. Immuno-affinity pull down of the anti-CD74-flu449 ADC from the thawed samples was carried out using biotinylated anti-human IgG (Southern Biotech, 2049-08) immobilized on streptavidin-coated magnetic beads (NanoLink Streptavidin Magnetic Beads, M-1002-010). Samples (30 µL) were incubated with 30 µL of beads carrying the anti-human polyclonal antibody for 1 hr at room temperature. Beads were settled in a magnetic rack, supernatant was discarded and beads were washed twice with 0.1% of RapiGest (Waters, 186001860) in 50 mM ammonium bicarbonate followed by three additional washes with 50 mM ammonium bicarbonate. The ADC was eluted with 40 µL of 0.25% acetic acid and 5 µL was injected in a POROS R2/10 2.1



Page 16 of 43

x 30 mm column (Life Technologies, 1-1112-12) with 100 µL/min flow rate. ADC was eluted from the column with a gradient of acetonitrile in water (30 - 58%). Data was acquired on a Waters Synapt G2-S mass spectrometer and deconvoluted to monoisotopic and singly-charged using the Waters MassEnt 1 software. Procedures involving the care and use of animals in the study were reviewed and approved by the Institutional Animal Care and Use Committee at Merck & Co., Inc., Palo Alto, CA, USA. During the study, the care and use of animals were conducted in accordance with the principles outlined in the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), the Animal Welfare Act, the American Veterinary Medical Association (AVMA) Euthanasia Panel on Euthanasia, and the Institute for Laboratory Animal Research (ILAR) Guide to the Care and Use of Laboratory Animals. Production and characterization of non-natural amino acid-containing antibodies and antibody-drug conjugates. The methods for expression, purification and characterization of non-natural amino acid-containing antibodies used in this work were essentially the same as for the antibodies in a companion paper and are described in detail there 12. Kern et al. also described the synthesis of the flu449 linker molecule, its conjugation to antibodies, and the characterization of purified conjugates. Again, essentially the same methods were used in the present work. ADCs were prepared at Ambrx and delivered to Merck sites for testing. The carrier solution for all ADCs and naked antibodies was 50 mM histidine pH 6.0, 100 mM NaCl and 5% trehelose. To assure consistency across experiments, ADCs were thawed on ice, dispensed to small aliquots, frozen to -80 °C, and those aliquots were then used for experiments with unused material discarded at the end of the day. For all in vitro studies in this report, none of the read outs were measurably different in the presence or absence of the carrier solution. Nevertheless, the final concentration of antibody carrier solution was maintained constant at 1 % v/v by serially diluting antibody solution prior to addition to the assay mixture and adding carrier solution to control wells. The same practice was observed for DMSO as the solvent for small molecules, with the final concentration being 0.1% v/v. In vitro stability of anti-CD74-flu449. Anti-CD74-flu449 ADC was spiked into C57b6 mice plasma at 0.1 µg/mL. Sample was split into 50 µL aliquots and tubes were capped under N2 and placed at 37 °C. Samples were frozen at -80 °C at 0, and 6 hr, and at 1, 3, 7 and 14 days and stored at -80 °C until analysis. Sample extraction and mass spectrometry methods for this experiment are the same as described for analysis of plasma samples in the mouse PK study described above. In vitro gene expression assays. HUT78 cells were cultured in IMEM plus 20% heat inactivated FBS and cell density was maintained between 0.1 to 1.2 million / mL. 786-O cells were cultured in RPMI plus 10% heat inactivated FBS. SUDHL6 cells were cultured in medium of RPMI-1640/10% FBS as suggested by ATCC. Actively growing cells were harvested and


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resuspended in HBSS with 2% FBS at 1.1 million cells per mL then dispensed to 384-well V-bottom plates at 45 µL per well. Serially diluted ADC solution was added to the cell plate (5 µL per well) and mixed for 2 min. Cells were then cultured at 37 °C, at 5% CO2 for a designated time before supernatant was removed. Cells were harvested in lysis buffer from the Cells-toCt kit (40 µL, Life Technologies, 4391851C) following the supplier’s protocol and mixed for 10 min followed by addition of 5 µL per well of stop solution from the kit. cDNA was synthesized with a reverse transcription kit (Life Technologies, 4391852C) followed by qPCR using the TaqMan gene expression master mix (Life Technologies, 4369016) with GILZ gene assay (Life Technologies, Hs00608272_m1) and GAPDH assay (Hs2758991_g1) in a duplex format with 3-4 technical replicates. The mRNA levels were normalized to GAPDH (internal control) using the formula ∆ threshold cycle (Ct) = Ct target – Ct reference. The differential expression signal were expressed as delta Ct (∆Ct) by subtracting the Ct values of the un-stimulated samples (containing only assay buffer or DMSO vehicle) from those of the stimulated samples and expressed as relative fold of change using the formula: 2-∆∆CT. The graphs were generated in GraphPad Prism and the EC50 values were calculated with non-linear regression curve fit of the data in GraphPad Prism. For the studies of ADCs bearing reduced permeability payloads frozen human CD19+ B cells purchased from Precision Biosciences (catalog # 84400, donor # 13108) were thawed and re-suspended in RPMI1640 plus 10% heat-inactivated FBS (cell culture medium) at 1 million cells/mL. ADC or fluticasone propionate was serially diluted to 100x the intended final concentrations. B cells were bulk treated in a 96-well block and subsequently dispensed to 96 well plates (100 µL and 1 million cells/mL) and incubated at 37 °C, 5% CO2 for 18 or 40 hr. Control wells containing equal percentages of DMSO (0.1%) and ADC buffer (1%) as sample wells were included. Cells were transferred to a 96 well v-bottom plate and spun at 500x g for 5 min. Medium was removed and cells were lysed in 150 µL RLT buffer containing beta-mercaptoethanol. RNA was isolated (RNeasy 96 kit, Qiagen, Cat # 74181) and cDNA was synthesized (iScript cDNA Synthesis Kit, BioRad, Cat # 170-8891) according to the supplier’s protocol. Quantitative PCR was performed on Applied Biosystem’s 7900 HT RealTime PCR System to measure expression of ZBTB16 (Life Technologies, Hs00957433_m1) and GAPDH (Life Technologies, Hs02758991_g1) in duplex format. The ∆∆Ct method was used to calculate fold change of expression of ZBTB16 relative to control sample, using 3 replicates per treatment condition. For free cell impermeable compounds, B cells were seeded at 100,000 cells/well in 90 µL in a 96-well flat bottom plate. Three fold serial dilutions were performed in DMSO, subsequent intermediate 10x stocks were made in tissue-culture medium and then added to the cell plate (10 µL/well). Cells were incubated at 37 °C, 5% CO2 for 18 hr, harvested and processed for PCR as described above. EC50 curves were calculated using Graph Pad Prism 6 software using nonlinear fit (agonist) vs. response – variable slope (four



Page 18 of 43

parameters) calculation. ADCs were also tested for activity on mRNA levels in isolated mouse splenocytes: Spleens were harvested from C57b6 human CD74 hemizygous transgenic and wild type littermate mice. A single cell suspension in RPMI 1640 media with 5% FBS was made and cells were collected by centrifugation. Red blood cells were lysed using 3 mL per spleen of RBC lysis buffer (BD Pharm Lyse Buffer, BD 555899) for 3 min at room temperature. Cells were washed in RPMI 1640 with 5% FBS, passed through a 70 µM filter, and diluted to 3 million cells/mL. Cells were plated at 1.5 million cells/mL in deep well plates and incubated at 37 °C for 30 min before addition of compound or vehicle (5 µL in 2% DMSO) and ADCs or vehicle (5 µL). After the indicated times, cells were pelleted, resuspended in 0.35 mL Qiagen buffer RLT and stored at -80 °C. FKBP5 mRNA levels (Mm00487401_m1) were determined and normalized to GAPDH mRNA levels (Mm99999915_g1) as described for human B cells immediately above. In vitro mouse B cell proliferation assay. Spleens were harvested from C57b6 human CD74 hemizygous transgenic and wild type littermate mice. A single cell suspension in RPMI 1640 media was made and cells were collected by centrifugation. Red blood cells were lysed using 2 mL per spleen of RBC lysis buffer (Sigma #R7757) for 5 min at room temperature. Cells were washed in RPMI 1640 and passed through a 70 µM filter. B cells were isolated using a MACS negative selection purification kit (Miltenyi, 130-090-862). Purity was assessed by flow cytometry using anti-CD19-FITC (BD), and B cell purity was greater than 78%. Cells were resuspended in growth medium: RPMI (Cellgro), 10% heatinactivated FCS (Hyclone), 1X HEPES (Gibco), 1X penicillin, streptomycin, L-glutamine (Biowhittaker), 1X betamercaptoethanol (Gibco), sodium pyruvate (CellGro). They were then plated at 100,000 per well in a 96-well U bottom plate. Cells were incubated with either fluticasone propionate diluted in DMSO (0.1% final concentration) or ADCs diluted in carrier solution (1% final concentration) for 1 hr prior to the addition of 1.25 µg/mL LPS (Salmonella typhosa, Sigma L2387). Cells were incubated for 43 hr at 37 °C and 5% CO2 before pulsing for 6 hr with 0.1 µCi per well of 3H-thymidine, followed by harvesting and counting by standard methods. Measurement of released payload in tissue culture supernatants. Instrument and Analytical Conditions: LC-APCI(-)MS/MS was used to analyze fluticasone propionate from HBSS with 2% FBS. LC-APCI(-)-MS/MS was carried out in a Waters Acquity (Milford, MA) – Thermo TSQ Vantage (San Jose, CA) system. Liquid chromatography separation was performed on an Ascentis C8 (5 cm x 2.1 mm; 3µm particle size) column at 40 °C. A 3 min binary gradient system from 15 to 90% buffer B (acetonitrile:water (80:20), 0.1% formic acid) from buffer A (water:acetonitrile (95:5)) was used at 0.4


Page 19 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mL/min flow rate. Negative APCI was performed with 4 kV for the corona discharge, 400 oC for the vaporizer temperature, 20 psi for sheath gas and 5 psi for auxiliary gas pressure. The mass spectrometer was set in SRM mode, with Q1 and Q3 resolution of 0.7 Da. The transitions selected for detection of fluticasone propionate were: 545.2 > 329.1, 423.2, 433.2 and 479.2; and the transition selected for the detection of dexamethasone, used an internal standard, was 437.12 > 361.26. The calibration curve was prepared by spiking fluticasone propionate in HBSS, 2% FBS buffer from a primary stock solution of 2 mg/mL of fluticasone propionate in methanol. An intermediary calibration curve from 19.6 to 10,000 ng/mL was prepared followed by a 100x single-step dilution of all calibration points (from 0.196 to 100 ng/mL). Dexamethasone stock solution of 2 mg/mL in methanol was prepared and a working solution of 1000 ng/mL was prepared by diluting the stock solution with methanol. Ten microliters of dexamethasone was added to one 1 mL of each sample before extraction. Samples were prepared by solid phase extraction using the Waters Oasis HLB µElution plate (WAT058951 - Waters, Milford - MA) conditioned according to supplier instructions. One milliliter of each sample was added to individual wells, washed with aqueous-organic solution (water:acetonitrile, 5:95) and eluted with 100% acetonitrile. The eluate was dried in a speed-vac, reconstituted with methanol:water (1:1) and 5 uL was injected in the LC-APCI(-)-MS/MS. The lower limit of quantification (LLOQ) for the method was 0.39 nM in HBSS + 2% FBS Buffer. Thermo Quanlynx software was used for the data analysis. Quantification of Compound A in cell culture medium. Sample Preparation: The standard solutions and quality control samples were dispensed directly into a 96 well plate by the HP D300 into the wells assigned. Different standard levels from 0.0001 to 10 µM were prepared by dispensing the appropriate volume of stock working solutions. Six quality control (QC) levels (0.0005, 0.0025, 0.05, 1.0, 3.75 and 7.5 µM), were prepared by dispensing the appropriate volume of the stock working solutions. After preparation, an aliquot of 50 µL of drug free sample matrix (RPMI1640 media + 10% FBS) was transferred into each of the wells. Three replicates for each QC concentration were prepared. Unknown samples from the B cell and PBMC experiments were added for analysis in a 96-well plate format. An aliquot (50 µL) of individual subject samples was disposed into the well assigned. A single step protein precipitation technique was used for sample preparation. A 200 µL aliquot of acetonitrile containing the following IS mix (labetalol, imipramine and diclofenac) was added to each sample (standard, QCs and biological samples) in order to precipitate the proteins. Plates were mixed by vortex for homogeneity, and then subjected to centrifugation at 3000 rpm for 5 minutes. The supernatant (200 µL) was transferred into a clean 96 deep well plates and injected into the LCMS/MS system.



Page 20 of 43

LC-MS/MS analysis: The levels of compound A was measured by LC-MS/MS method. LC analysis was carried out on an LX-2 Thermo Cohesive systems equipped with ThermoFisher Allegros 1250 Pumps and a HTS PAL CTC autosampler refrigerated at 10C during analysis. Chromatography was performed on a Waters Acquity HSS T3 (2.1 mm x 50mm, 1.8 µm) column at room temperature with an injection volume of 5 µL. The mobile phase consisting of a solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) was delivered at a flow rate of 750 µL/min. The LC gradient started from 95/5 (A/B) and changed to 5/95 (A/B) from 0.25 to 1.75 min (ramp) and remaining constant to this ratio for 0.42 min (step). The gradient decreased to 95/5 (A/B) at 2.17 min (step) remaining constant to this ratio for 1.0 min. Detection was carried out using a triple quadrupole tandem mass spectrometer (API5000, Applied Biosystems) equipped with an electrospray interface (ESI). Ions were created in the positive ion mode setting the sprayer voltage at 5.0 kV and the ion source temperature at 500°C. The common parameters and the nitrogen flow values for nebulizer gas (Gas 1), auxiliary gas (Gas 2), curtain gas and the gas for collision-activated dissociation (CAD) were set at 60, 60, 35, and 5, respectively. The Analyst 1.6.2 software (Applied Biosystems) was used to control the MS-MS system and MultiQuant 3.0.1 for data analyses. Detection of Compound A was performed in the multiple reaction-monitoring (MRM) mode and the following the sum of precursor to product ion pairs m/z 689.000 → 293.3 (DP/CE/CXP/EP: 30/45/15/10); 690.000 → 294.3 (DP/CE/CXP/EP: 30/45/15/10); 689.000 → 275.3 (DP/CE: 30/45/15/10); ); 690.000 → 276.3 (DP/CE: 30/45/15/10) were used for quantitation. The following MRM transitions were monitored for the IS, 329.200 → 162.100 (DP/CE: 76/37/16/10); 296.000 → 214.000 (DP/CE: 76/49/15/10) and 281.3 → 193.1 (DP/CE: 70/50/15/10). The dwell time was set as 15 msec for each compound. Chromagraphic data were collected and integrated by MultiQuant 3.0.1 data analysis program. Peak area ratios of the analyte to IS were utilized for construction of the calibration curve. A weighting of 1/x2 (least-squares linear regression analysis, where x is the concentration of a given standard) was used for curve fit. Concentrations in unknown samples were calculated from the best-fit equation (y = mx+b) where y is the peak area ratio. The regression equation for the calibration curve was also used to back-calculate the measured concentration at each quality control level, and the results were compared with the theoretical concentration to obtain the accuracy expressed as a percentage of the theoretical value. Accuracy was defined as the degree of deviation of the determined value from the nominal value: [(measured value − nominal value) / nominal value] × 100.


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Bystander and in vitro mechanism of action studies. Frozen human CD19+ B cells were purchased from Precision Biosciences. B cells were thawed, spun at 300 g for 8 min and resuspended at 4 million or 200,000 cells/mL in RPMI (Invitrogen) with 10% heat inactivated (HI)-FBS (GIBCO) at 37 °C and 5% CO2. B cells were incubated with an 8 point halflog titration of flucticasone proprionate (starting at 3 nM) or ADC (starting at 3 µg/mL) for 24 hr at 37 °C in a 96-well plate. DMSO (0.1%, Sigma) and ADC carrier solution (1%) were used as a control. Final cell concentrations were 2 million cells/mL or 100,000 cells/mL. The cells were transferred to a V-bottom 96-well plate and spun at 500 g for 5 min. Cells were then washed with PBS (Invitrogen) + 0.2% BSA (Sigma) twice at 500 g for 5 min. Cells were lysed on ice using buffer RLT and RNA was isolated using an RNeasy kit provided by Qiagen. Reverse transcription of the RNA was performed using iScript (Bio-Rad). Expression of ZBTB16 was determined by performing RT PCR in the 7900HT Fast Real-Time PCR system. Data was analyzed in Graphpad Prism. Data is represented as a fold change relative to the GAPDH housekeeping gene. For the B cell and T cell co-culture experiment, PBMCs were isolated from fresh, heparinized human whole blood using a FICOLL-Plaque Plus (GE Healthcare) gradient. Written informed consent was obtained from the donors under a study protocol reviewed and approved by Western Institutional Review Board. Cells were washed and cultured in RPMI with 10% HI-FBS at 37 °C and 5% CO2. CD19+ B cells were positively selected using the EasySep Human CD19 Positive Selection Kit (Stemcell Technologies). CD3+ T cells were selected from the first pour-off of the B cell isolation using the EasySep Human T Cell Enrichment Kit (Stemcell Technologies). Cells were diluted in RPMI + GlutaMAX/10% FBS to a final concentration of 2 million cells/mL. In a 12-well plate, 1 million cells per well were treated with 0.03 µg/mL ADC, 3 nM fluticasone propionate, or control solution, DMSO and ADC carrier buffer. All wells contained 0.1% DMSO and 1% ADC carrier buffer. Cells were incubated for 24 hr at 37 °C. CD19+ B cells and CD3+ T cells were purified using the same selection kits as above. Isolated cells were spun for 5 min at 500 g. Following the removal of the supernatant, cells were placed on ice and lysed by adding RLT buffer. RNA was isolated using an RNeasy kit provided by Qiagen. Reverse transcription of the RNA was performed using iScript (Bio-Rad). Expression of ZBTB16 was determined by performing RT PCR in the 7900HT Fast Real-Time PCR system. Data was analyzed in Graphpad Prism. Data is represented as a fold change relative to the GAPDH housekeeping gene. For PBMC studies using ADCs bearing reduced permeability payloads, cells were thawed in RPMI1640+10%FBS and rested for 1 hour at 37°C. Cells were diluted to 1x10^6 cells/mL and 5 mLs of cells were seeded into 6 well plates. CD74-AXC496 or Compound A was added to reach concentrations of 3 ug/mL or 10 µM and CD74-flu449 or fluticasone propionate was added to reach 1 µg/mL or 10 nM, respectively. Cells were incubated for 24 hours at 37°C. B cells were purified using Stem Cell CD19+ selection kit and T cells were purified using Stem Cell



Page 22 of 43

CD3+ selection kit. Cells were lysed in RLT buffer and processed for Real Time PCR as described above. For cell free ADC stability experiments, CD74-AXC496 or RSV-AXC496 was diluted to 10 ug/mL in either ADC buffer or RPMI1640+10%FBS media (no cells). 4 °C samples were kept on ice, immediately collected and stored at -80°C. 37 °C samples were incubated for either 4 or 24 hours, collected and stored -80 °C. Samples were submitted for analysis by mass spec to measure concentration of Compound A. An additional set of samples of Compound A (10 µM) was submitted as spike in controls. In vivo pharmacodynamic assays. Inbred male C57b6 hemizygous human CD74 transgenic or wild type littermates were purchased from Jackson Laboratories (Sacramento, CA) at 8-10 weeks of age, and given a minimum of 7 days acclimation time. Animals were housed in pathogen-free conditions in the animal research facility at MRL Boston in individually ventilated Tecniplast 1291 cages with autoclaved Maple Sani Chips bedding (Teklad 7090M). Cages were ventilated at 70 air changes per hour under negative pressure relative to the room. Mice were fed Teklad Global 18% Protein Rodent Diet (Teklad 2018SX) ad libitum. Autoclaved water bottles were checked daily and changed at least weekly. Animal housing rooms were maintained at 70 °F ± 1° and 30-70% relative humidity under a 12 hr light/12 hr dark schedule. All animals were treated according to the guidelines of the MSD Institutional Animal Care and Use Committee. For the pharmacodynamic in vivo experiments, matched cohorts of transgenic and wild type for each condition were dosed intravenously through the lateral vein of the tail with a solution of either vehicle 1 (70:10:20 PEG400:EtOH:Water), fluticasone propionate solution prepared in vehicle 1, vehicle 2 (50 mM histidine, 100 mM NaCl, 2.5% trehalose, pH 6.0), or anti-RSV-flu449 or anti-CD74flu449 solution prepared in vehicle 2. Animals dosed with vehicle 2, anti-RSV-flu449, and anti-CD74-flu449 were dosed on day 1 at 24 hr prior to sacrificed at a volume of 5 mL/kg. Animals dosed with vehicle 1 and fluticasone propionate were dosed on day 2 at 2 hr prior to sacrifice at a volume of 1 mL/kg. On day 2 at 30 min prior to sacrifice all dosed animals received a change of caging to induce stress hormone release. At the time of sacrifice 5 naïve animals of each genotype were euthanized via cervical dislocation to establish basal stress hormone levels. The remaining dosed animals were euthanized via carbon dioxide inhalation, an additional stressor. Post sacrifice, blood (via intracardiac puncture) and spleen samples were collected from all animals. Syringes used for blood collection were pre-loaded with 10 µL 14% ethylenediaminetetraacetic acid (Boston BioProducts, Ashland, MA) to prevent clotting during collection. Blood samples were deposited into untreated 2 mL microcentrifuge tubes (Eppendorf, Hamburg, Germany) and placed on a rocker plate (ThermoFisher Scientific, Waltham, MA). Spleens were collected into microcentrifuge tubes containing 1 mL Gibco RPMI 1640 (ThermoFisher Scientific) and placed on wet ice. Post collection, whole blood samples were centrifuged at 10,000 g for 2.5 min to obtain


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plasma. Plasma aliquots were plated and frozen at -80 °C until the time of analysis. Spleens isolated from wild type or transgenic mice were processed into splenocytes using the Miltenyi gentleMacs Octo Cell Dissociator. Spleens were placed in C tubes containing PBS + 2% serum + 1 mM EDTA and ran on mouse spleen program 1. Homogenate was poured over a 70 µm strainer and washed. Cells were pelleted, a red blood cell lysis was performed and cells counted. Two million splenocytes were collected in a 96-well block and the remaining cells were used as input into B and T cell negative selection isolation kits from Stem Cell technologies (Cat #s 19854 and 19851) using the EasyEights EasySep Magnets (Cat# 18103). Negatively selected B and T cells were collected in 96-well deep well blocks on ice. Cells were pelleted and lysed in 350 µL RLT buffer and RNA isolated using Qiagen’s RNeasy kits (Cat # 74104). For the fluticasone propionate only pharmacodynamic study, splenocytes were not further separated into B and T cells. cDNA was synthesized (iScript cDNA Synthesis Kit, BioRad, Cat # 170-8891) according to the supplier’s protocol and qPCR was performed on Applied Biosystem’s 7900 HT Real-Time PCR System to measure expression of GAPDH (Life Technologies, Mm99999915_g1) and FKBP5 (Life Technologies Mm00487401_m1). The delta delta Ct method was used to measure fold change of expression of FKBP5 relative to control sample for each cell population. Plasma fluticasone propionate levels in the fluticasone propionateonly PD study were measured as follows using LC-MS/MS following protein precipitation. Briefly, 50 µL aliquots were treated with 200 µL acetonitrile containing internal standard, vortexed and centrifuged. The supernatants were mixed with an equal volume of water and injected into the LC-MS/MS system which consisted of an API 4000 mass spectrometer, PE 200 Series Micro Pumps and a Leap CTC PAL autosampler. Chromatographic separation was achieved on a Waters Atlantis T3 column eluted with a linear gradient from 20% A (0.1 % formic acid in water) to 80% B (0.1 % formic acid in acetonitrile). Detection of the analyte was performed in the multiple reaction-monitoring (MRM) mode. Quantification was accomplished using standard curves prepared in the appropriate matrix and was based on the peak area ratios of the analyte to the internal standard. Pharmacokinetic parameters were calculated using established non-compartmental methods. The area under the plasma concentration versus time curve was determined using the Watson software (version 7.3), with linear trapezoidal interpolation in the ascending slope and logarithmic trapezoidal interpolation in the descending slope. The plasma concentrations of adrenocorticotropic hormone and corticosterone were determined by ELISA (Alpco, Salem, NH, and Abcam, Cambridge, UK, respectively) under contract at Charles River Laboratories (Wilmington, MA).



Page 24 of 43

Synthesis of Compound A and antibody-AXC496 conjugates.

HO Me

O Me H

SH O O Me

+

O HO P HO Br

H

F

a

O 1

F

HO Me

2

O HO P HO O S O Me O H

F

+

Fmoc

Me

O

N H

b

O OH P OH

H

O 3

F

R

4

(Compound A)

O O O P P HO HO O S O Me O O

N H HO Me

H

F O F

HO Me F

O O

d

OH

+

Me H 5, R = Fmoc 6, R = H

c

O Me H

S O O

7

O P

O P

O O HO HO

H N

O O

Me H

O F

(a) DIPEA, dioxane, 25 °C, 63%; (b) CDI, Et3N, DMF 25°C then 4, ZnCl2, 25 °C, 32% (c) DBU, CH2Cl2, 25 °C, 39%; (d) HATU, Et3N, DMF, 25 °C, 55%

Step A: Preparation of (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17carbonyl)thio)decyl)phosphonic acid (3, Compound A). In a dry round bottom flask equipped with a stir bar under nitrogen, (6S,8S,9R,10S,11S, 13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyl-oxy)6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H cyclopenta[a] phenanthrene-17-carbothioic S-acid (1, 200mg, 0.427 mmol) and (10-bromodecyl)phosphonic acid (2, 141 mg, 0.470 mmol, 1.10 eq.) were dissolved in anhydrous DMF (2.1mL, 0.2M). After 10 minutes of stirring, diisopropylethylamine (176 L, 1.07 mmol, 2.50 eq.) was added and the reaction stirred overnight at ambient temperature. Upon completion as determine by LCMS, the reaction was concentrated, diluted in DMSO


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and directly injected on a reverse phase acidic prep HPLC (Sunfire C18 30x150) with 5 to 95 gradient of organic (0.1% TFA/acetonitrile)/aqueous (0.1% TFA/water). The isolated fractions containing product were evaporated using a Genevac and isolated (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17carbonyl)thio)decyl)phosphonic acid (3, 185mg, 63% yield) as a white, foamy solid. LRMS (ES) (M+H)+ : observed = 689.6, calculated = 689.8. 1H NMR (DMSO-d6, 500 MHz): δH 7.26 (1H, d, J = 10.2 Hz), 6.29 (1H, d, J = 10.2 Hz), 6.11 (1H, s), 5.63 (1H, ddd, J = 48.7, 11.1, 6.7 Hz), 4.20 (1H, d, J = 9.3 Hz), 2.86 (2H, dddd, J = 20.9, 18.9, 13.5, 7.4 Hz), 2.31 (2H, q, J = 7.7 Hz), 2.24 (1H, br s), 2.05-2.11 (2H, m), 1.80-1.87 (2H, m), 1.47 (8H, d, J = 12.3 Hz), 1.31 (5H, br s), 1.25 (9H, s), 0.981.02 (6H, m), 0.89 (3H, d, J = 7.1 Hz). Step B: Preparation of ((9H-fluoren-9-yl)methyl-carbamoyl)-2-aminoethyl phosphoric) (10(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17-carbonyl)thio) decyl) phosphonic anhydride (5). In a dry glass vial equipped with a stir bar under nitrogen, (9H-fluoren-9-yl)methyl (2(phosphonooxy)ethyl)carbamate (4, 95mg, 0.261 mmol, 1 eq.) and carbonyldiimidazole (50.8mg, 0.314 mmol, 1.2 eq.) were dissolved in anhydrous DMF (0.50 mL) and treated with triethylamine (36.4 L, 0.261 mmol, 1 eq.). The reaction was stirred 20min and found to be complete as measured by LCMS. In a separate dry glass vial (10(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16 -trimethyl-3-oxo-17-(propionyloxy)6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17-carbonyl)thio)decyl)phosphonic acid (3, 180mg, 0.261 mmol, 1eq.) and zinc (II) chloride (214 mg, 1.57mmol, 6 eq.) were combined and dissolved in anhydrous DMF (0.80 mL, 0.2M final concentration of reaction). To this latter solution was added the solution of activated 4 and the resulting mixture was stirred overnight at ambient temperature. The reaction was judged to be complete by LCMS (basic conditions) after 12h and diluted with 1N HCl (5 mL). The mixture was extracted with DCM (5 x 5mL) and the combined extracts were concentrated. The resulting crude was dissolved in 1mL of methanol and injected onto a on a reverse phase basic prep HPLC (Phenomenex Gemini –NX C18 OBD 5 uM 30 x 100mm; 30-90%MeCN/water w/ 0.1% NH4OH modifier over 20 min). The isolated fractions containing product were evaporated using a lyophilizer and isolated ((9H-fluoren-9-yl)methyl-carbamoyl)2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S, 14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl)



Page 26 of 43

phosphonic anhydride (5, 88.0mg, 32% yield) as a white solid. LRMS (ES) (M+H)+ : observed = 1034.6, calculated = 1034.0. Step C: Preparation of (2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S, 16R,17R)-6,9-difluoro-11-hydroxy10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9, 10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene17-carbonyl)thio) decyl) phosphonic anhydride (6). In a dry round bottom flask equipped with a stir bar under nitrogen, ((9H-fluoren-9-yl)methyl-carbamoyl)-2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S, 14S,16R,17R)-6,9-difluoro11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a]phenanthrene-17-carbonyl)thio)decyl) phosphonic anhydride (5, 88.0mg, 0.085 mmol) was dissolved in anhydrous dichloromethane (0.42mL, 0.2M) and treated with DBU (65mL, 0.425 mmol, 5 eq). The resulting solution was stirred at ambient temperature for 1h and determined to be complete as judged by LCMS. The reaction was concentrated and taken up in methanol and injected onto a on a reverse phase basic prep HPLC (Phenomenex Gemini –NX C18 OBD 5 uM 30 x 100mm; 10-70%MeCN/water w/ 0.1% NH4OH modifier over 20 min, 235nM wavelength). The isolated fractions containing product were evaporated using a lyophilizer and isolated (2-aminoethyl phosphoric) (10(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio) decyl)phosphonic anhydride (6, 27.0mg, 39% yield) as a white solid. LRMS (ES) (M+H)+ : observed = 812.4, calculated = 812.8. Step D: Preparation (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3Hcyclopenta[a] phenanthrene-17-carbonyl)thio)decyl)phosphonic anhydride. In a dry round bottom flask equipped with a stir bar under nitrogen, (2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S, 16R,17R)-6,9-difluoro-11-hydroxy10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodeca-hydro-3H-cyclopenta[a]phenanthrene17-carbonyl)thio)decyl)phosphonic anhydride (6, 9.0mg, 11mmol, 1.0 eq) and 2-(cyclooct-2-yn-1-yloxy)acetic acid (7, 3.0 mg, 17 mmol, 1.5eq) were dissolved in anhydrous DMF (0.37mL, 0.03M). To the resulting solution was added HATU (6.3mg, 17mmol, 1.5eq) and triethylamine (6.2 L, 44 mmol, 4 eq.). The reaction became yellow immediately and stirred 20 min before completion as judged by basic LCMS. The reaction was injected onto a on a reverse phase basic prep HPLC (Phenomenex Gemini –NX C18 OBD 5 uM 30 x 100mm; 10-70%MeCN/water w/ 0.1% NH4OH modifier over 20 min, 235 nM wavelength). The isolated fractions containing product were evaporated using a lyophilizer and isolated (2-(2-(cyclooct-


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2-yn-1-yloxy)acetamido)ethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17carbonyl)thio)decyl) phos-phonic anhydride (Compound A, 6.0mg, 55% yield) as a white solid. 1H NMR (DMSO-d6, 500 MHz, presat to suppress water): δH 8.33 (1H, s), 7.35 (1H, d, J = 10.1 Hz), 6.27 (1H, dd, J = 10.0, 2.0 Hz), 6.10 (1H, d, J = 2.1 Hz), 5.63 (1H, ddd, J = 48.7, 10.7, 6.4 Hz), 4.29 (1H, t, J = 5.2 Hz), 4.20 (1H, d, J = 9.0 Hz), 3.89 (1H, d, J = 14.6 Hz), 3.73-3.76 (3H, m), 3.22-3.27 (3H, m), 2.86 (2H, dddd, J = 19.8, 13.9, 12.7, 7.0 Hz), 2.31 (2H, ddd, J = 8.4, 8.2, 6.5 Hz), 2.21-2.25 (3H, m), 2.15 (1H, t, J = 6.8 Hz), 2.05-2.11 (4H, m), 1.70-1.93 (7H, m), 1.45-1.58 (10H, m), 1.24-1.31 (15H, m), 0.98-1.01 (6H, m), 0.86-0.90 (4H, m). HRMS calcd for C46H69F2NO13P2S (M+H)+ 976.4011, found 976.4024.

Presaturation at 3.45 ppm exp1

presat

SAMPLE Date 09-Oct-2015 solvent DMSO source garbacci sam_id 0366571-0067 operator robot storfile wl124158 ACQUISITION sfrq 500.084 tn H1 at 2.289 np 32054 sw 7001.0 fb 4000 bs 16 ss 2 tpwr 55 pw 1.7 d1 0 tof 500.1 nt 128 ct 128 alock y gain 48 FLAGS il n in n dp y hs nn DISPLAY sp -501.9 wp 7000.7 vs 1696 sc 0 wc 250 hzmm 28.00 is 2223.89 rfl 502.1 rfp 0 th 2 ins 160.000 ai ph

12

SATURATION sspul n composit n satpwr -14 satfrq -774.567 satdly 1.500 satmode ynn DEC. & VT dfrq 500.083 dn H1 dpwr 10 dof -774.6 dm nnn dmm c dmf 200 dseq dres 1.0 homo n temp 25.0 PROCESSING lb 0.35 wtfile proc ft fn 65536 math f werr wexp wbs wnt

11

autorunwerr autorunwexp

10

9

8

7

6

5

4

3

2

1

-0

ppm

|sid|garbacci|H1|inova wp500l|0366571-0067|

Determination of apparent permeability. MDCKII cells (kindly provided by the Netherlands Cancer Institute, under a licensing agreement) were seeded on to 96-well transwell culture plates (Millipore Corp, Billerica, MA) and used in experiments after five days in culture. Test compound (1 µM) was prepared in Hank’s Balanced Salt Solution (HBSS), 10 mM (4-(2-hydroxyethyl)-1-piperrazineethanesulfonic acid) (HEPES, pH 7.4), with 10 µM cyclosporine A (to inhibit



Page 28 of 43

endogenous transport) and 1.2 µM dextran Texas red (to confirm monolayer integrity). Substrate solution (150 µL) was added to either the apical (A) or the basolateral (B) compartment of the culture plate, and buffer (150 µL; HBSS, 10 mM HEPES, pH 7.4) with 10 µM cyclosporine A was added to the compartment opposite to that containing the substrate. At t = 3 hr, 50 µL samples were removed from both sides of monolayers dosed with test compound and placed in 96 well plates, 50 µL internal standard (1 µM labetolol) and 100 µL HBSS was added to the samples. Samples were analyzed by LC/MS/MS using an Applied Biosystems SCIEX API 5000 triple quadruple mass spectrometer (Concord, ON, Canada) with a TurboIonSpray ion source in the positive ion mode. A Thermo Scientific Transcend LX-2 system (Franklin, MA.) was coupled to the API 5000 with a flow rate of 800 L/min to direct sample into the mass spectrometer. The apparent permeability (Papp) was calculated by the following formula for samples taken at t=3 hr:

=

ℎ ( ) ∆ () ( ) () ∆ ()

Where: Volume of Receiver Chamber is 0.15 mL; Area of membrane is 0.11 cm2; the initial concentration is the sum of the concentration measured in the donor plus concentration measured in receiver compartments at t=3 hr; ∆ in concentration is concentration in the receiver compartment at 3 hr; and ∆ in Time is the incubation time (3 x 60 x 60 = 10800 s). Papp was expressed as 10-6 cm/s. The Papp reported is the average of the A to B and B to A Papp values control MDCKII cells at t = 3 hr:

Papp =

Papp ( A → B ) + Papp ( B → A)

2

The B-A/A-B ratio was calculated by dividing the Papp from B to A by the Papp from A to B at t = 3 hr. Acknowledgements None Author contributions P.E.B. designed and interpreted experiments, led the project and wrote the paper. A.P. designed, conducted and interpreted experiments and wrote the paper. S.A., M.B., L.B., M.C., M.C., L.F-D., G.F., I.F., J.F., R.C., L.G.Q., D.G., P.G., C.H., D.H., J.K., N.K., K.K., L.L., H.M., A.M., P.M., L.M., Y.Q., S.S., J.S., P.S., Y.S., D.T., H.C.W., D.Z., S.Z., Y.Z., M.Z. designed, conducted and interpreted experiments.


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Competing financial interest At the time when the work was conducted, all authors were employees of †Merck & Co., Inc., Kenilworth, NJ, USA. or ‡

Ambrx, Inc. and some own stock or stock options.

Funding Sources None to report Supporting Information Additional in vitro cell line and primary human cell data, antibody-drug conjugate quality control data, and in vivo pharmacology results. Reference List 1. Beck, A., Goetsch, L., Dumontet, C., and Corvaia, N. (2017) Strategies and challenges for the next generation of antibodydrug conjugates. Nat. Rev. Drug Discov. 16, 315-337. 2. Lambert, J. M., and Berkenblit, A. (2018) Antibody-Drug Conjugates for Cancer Treatment. Annu. Rev. Med. 69, 191-207. 3. Van der Goes, M. C., Jacobs, J. W., and Bijlsma, J. W. (2014) The value of glucocorticoid co-therapy in different rheumatic diseases--positive and adverse effects. Arthritis Res. Ther. 16, 1-13. 4. Schacke, H., Docke, W. D., and Asadullah, K. (2002) Mechanisms involved in the side effects of glucocorticoids. Pharmacol. Ther. 96, 23-43. 5. Everts, M., Kok, R. J., Asgeirsdottir, S. A., Melgert, B. N., Moolenaar, T. J., Koning, G. A., van Luyn, M. J., Meijer, D. K., and Molema, G. (2002) Selective intracellular delivery of dexamethasone into activated endothelial cells using an Eselectin-directed immunoconjugate. J. Immunol. 168, 883-889. 6. Graversen, J. H., Svendsen, P., Dagnaes-Hansen, F., Dal, J., Anton, G., Etzerodt, A., Petersen, M. D., Christensen, P. A., Moller, H. J., and Moestrup, S. K. (2012) Targeting the hemoglobin scavenger receptor CD163 in macrophages highly increases the anti-inflammatory potency of dexamethasone. Mol. Ther. 20, 1550-1558. 7. Axup, J. Y., Bajjuri, K. M., Ritland, M., Hutchins, B. M., Kim, C. H., Kazane, S. A., Halder, R., Forsyth, J. S., Santidrian, A. F., Stafin, K., et al. (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. U. S. A. 109, 16101-16106. 8. Agarwal, P., and Bertozzi, C. R. (2015) Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjug. Chem. 26, 176-192. 9. Ong, G. L., Goldenberg, D. M., Hansen, H. J., and Mattes, M. J. (1999) Cell surface expression and metabolism of major histocompatibility complex class II invariant chain (CD74) by diverse cell lines. Immunology. 98, 296-302.



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10. Hansen, H. J., Ong, G. L., Diril, H., Valdez, A., Roche, P. A., Griffiths, G. L., Goldenberg, D. M., and Mattes, M. J. (1996) Internalization and catabolism of radiolabelled antibodies to the MHC class-II invariant chain by B-cell lymphomas. Biochem. J. 320, 293-300. 11. Honey, K., Forbush, K., Jensen, P. E., and Rudensky, A. Y. (2004) Effect of decreasing the affinity of the class IIassociated invariant chain peptide on the MHC class II peptide repertoire in the presence or absence of H-2M. J. Immunol. 172, 4142-4150. 12. Garbaccio, R. M., Kern, J., Brandish, P. E., Shah, S., Liang, L., Sun, Y., Wang, J., Knudsen, N., Beck, A., Manibusan, A., et al. Phosphate based linkers for intracellular delivery of drug conjugates. WIPO Pat. Appl. WO2015153401A1, October 8, 2015. 13. Kern, J. C., Dooney, D., Zhang, R., Liang, L., Brandish, P. E., Cheng, M., Feng, G., Beck, A., Bresson, D., Firdos, J., et al. (2016) Novel Phosphate Modified Cathepsin B Linkers: Improving Aqueous Solubility and Enhancing Payload Scope of ADCs. Bioconjug. Chem. 27, 2081-2088. 14. Verploegen, S., Overdijk, M., Van Dijkhuizen, R., Bleeker, W.K., Van Berkel, P., Parren, P., Lisby, S. Human antibodies and antibody-drug conjugates against cd74. WIPO Pat. Appl. WO2012104344A1, August 9, 2012. 15. Stein, R., Qu, Z., Cardillo, T. M., Chen, S., Rosario, A., Horak, I. D., Hansen, H. J., and Goldenberg, D. M. (2004) Antiproliferative activity of a humanized anti-CD74 monoclonal antibody, hLL1, on B-cell malignancies. Blood. 104, 37053711. 16. Tian, F., Lu, Y., Manibusan, A., Sellers, A., Tran, H., Sun, Y., Phuong, T., Barnett, R., Hehli, B., Song, F., et al. (2014) A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. U. S. A. 111, 1766-1771. 17. Hogger, P., and Rohdewald, P. (1994) Binding kinetics of fluticasone propionate to the human glucocorticoid receptor. Steroids. 59, 597-602. 18. Phillipps, G. H. (1990) Structure-activity relationships of topically active steroids: the selection of fluticasone propionate. Respir. Med. 84 Suppl A., 19-23. 19. Doronina, S. O., Mendelsohn, B. A., Bovee, T. D., Cerveny, C. G., Alley, S. C., Meyer, D. L., Oflazoglu, E., Toki, B. E., Sanderson, R. J., Zabinski, et al. (2006) Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug. Chem. 17, 114-124.

TOC Graphic


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Figure 1 Please see manuscript for Figure Legend 116x75mm (300 x 300 DPI)





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Supplementary Figure 1 Please see Supporting Information File for Figure Legend 97x55mm (300 x 300 DPI)


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Supplementary Table 1 Please see Supporting Information File for Figure Legend 38x9mm (300 x 300 DPI)





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Supplementary Table 2 Please see Supporting Information File for Figure Legend 26x5mm (300 x 300 DPI)



TOC Graphic 46x23mm (300 x 300 DPI)


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