Large Molecule Therapeutics

Milatuzumab–SN-38 Conjugates for the Treatment ofCD74þ Cancers

Serengulam V. Govindan1, Thomas M. Cardillo1, Robert M. Sharkey1, Fatma Tat1,David V. Gold2, and David M. Goldenberg1,2

AbstractCD74 is an attractive target for antibody–drug conjugates (ADC), because it internalizes and recycles

after antibody binding. CD74 mostly is associated with hematologic tumors but is expressed also in solid

cancers. Therefore, ADCs of the humanized anti-CD74 antibody, milatuzumab, were examined for the

therapy of CD74-expressing solid tumors. Milatuzumab–doxorubicin and two milatuzumab–SN-38 con-

jugates with cleavable linkers, differing in their stability in serum and how they release SN-38 in the

lysosome, were prepared. CD74 expression was determined by flow cytometry and immunohistology.

In vitro cytotoxicity and in vivo therapeutic studies were conducted in the human cancer cell lines A-375

(melanoma), HuH-7 and Hep-G2 (hepatoma), Capan-1 (pancreatic), NCI-N87 (gastric), and Raji Burkitt

lymphoma. The milatuzumab–SN-38 ADC was compared with SN-38 ADCs prepared with anti-Trop-2

and anti-CEACAM6 antibodies in xenografts expressing their target antigens. Milatuzumab–doxorubicin

was most effective in the lymphoma model, whereas in A-375 and Capan-1 solid tumors, only milatu-

zumab–SN-38 showed a therapeutic benefit. Despite much lower surface expression of CD74 than Trop-2

or CEACAM6, milatuzumab–SN-38 had similar efficacy in Capan-1 as anti-Trop-2–SN-38, but in NCI-N87,

anti-CEACAM6 and anti-Trop-2 conjugates were superior. Studies in two hepatoma lines at a single dose

level showed significant benefit over saline controls but not against an irrelevant immunoglobulin G

conjugate. CD74 is a suitable target for ADCs in some solid tumor xenografts, with efficacy largely

influenced by uniformity of CD74 expression and with SN-38 conjugates providing the best therapeutic

responses; SN-38 conjugates were preferable in solid cancers, whereas doxorubicin ADC was better in

lymphoma tested. Mol Cancer Ther; 12(6); 1–11. �2013 AACR.

IntroductionCD74, referred to as invariant chain or Ii, is a type II

transmembrane glycoprotein that associates with HLA-DR and inhibits the binding of antigenic peptides to theclass II antigen presentation structure (1–3). It serves as achaperone molecule, directing the invariant chain com-plexes to endosomes and lysosomes, an accessory mole-cule in the maturation of B cells, using a pathway medi-ated byNF-kB (4), and in T-cell responses via interactions

withCD44 (5). It is also a receptor for the proinflammatorycytokine, macrophage migration inhibitory factor (6),which is involved in activating cell proliferation andsurvival pathways.

In normal human tissues, CD74 is primarily expressedin B cells, monocytes, macrophages, dendritic cells, Lan-gerhans cells, subsets of activated T cells, and thymicepithelium (data on file; Immunomedics, Inc.), and it isexpressed in more than 90% of B-cell tumors (7, 8). EarlystudieshadconflictingdataonwhetherCD74 ispresent onthe membrane, in part because the antibodies to theinvariant chain were specific for the cytoplasmic portionof the molecule (9), but also because there are relativelyfew copies on the surface, and its half-life on the cellsurface is very short. Approximately, 80% of the CD74on the cell surface is associated with the MHC II antigen,HLA-DR (3). Using the murine anti-CD74 antibody, LL1,the Raji Burkitt lymphoma cell line was estimated to have4.8� 104 copies per cell, but because of rapid intracellulartransit, approximately 8 � 106 antibody molecules wereinternalized and catabolized per cell per day (10). Thus,CD74 internalization is highly dynamic,with the antibodybeing moved quickly from the surface and unloadedinside the cell, followed by CD74 reexpression on the

Authors' Affiliations: 1Immunomedics, Inc.; and 2Garden State CancerCenter at the Center for Molecular Medicine and Immunology, MorrisPlains, New Jersey

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Prior presentation: This work was presented in part at the 2012 ASCOAnnual Meeting [J Clin Oncol 30, 2012 (suppl; abstr 3091)].

Corresponding Author: David M. Goldenberg, Garden State CancerCenter at the Center for Molecular Medicine and Immunology, 300 TheAmerican Road, Morris Plains, NJ 07950. Phone: 973-605-8200, ext. 128;Fax: 973-605-8282; E-mail: dmg.gscancer@att.net

doi: 10.1158/1535-7163.MCT-12-1170

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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surface. Fab’ internalization occurs just as rapidly asimmunoglobulin G (IgG) binding, indicating that bivalentbinding is not required (3, 10). Later studies with a com-plementarity-determining region (CDR)-grafted versionof murine LL1, milatuzumab (hLL1), found that the anti-body could alter B-cell proliferation, migration, and adhe-sion molecule expression (8, 11, 12), but the exceptionalinternalization properties of the anti-CD74 antibody madeit an efficient carrier for the intracellular delivery of cancertherapeutics (13–16).On the basis ofpreclinical efficacyandtoxicology results, phase I clinical trials with milatuzumabin multiple myeloma (17), as well as milatuzumab-doxo-rubicin in multiple myeloma, non-Hodgkin lymphoma,and chronic lymphocytic leukemia, have been initiated.

Interestingly, CD74 also is expressed in nonhemato-poietic cancers, such as gastric, renal, urinary bladder,non–small cell lung cancers, certain sarcomas, and glio-blastoma (18–25), and therefore it may be a therapeutictarget for solid tumors expressing this antigen. Because amilatuzumab–doxorubicin conjugate was highly active inmodels of hematologic tumors (13, 14), it was a logicalchoice for this assessment. However, we developed pro-cedures for coupling the highly potent topoisomerase Iinhibitor, SN-38, to antibodies (26, 27). SN-38 is the activeform of irinotecan, whose pharmacology andmetabolismarewell known (28, 29). These conjugates have nanomolarpotency in solid tumor cell lines and were found to beactive with antibodies that were not actively internalized(26, 30). Prior studies indicated a preference for a linker(CL2A) that allowed SN-38 to dissociate from the conju-gate in serum with a half-life of approximately 1 day,rather than other linkers that were either more or lessstable in serum. However, given milatuzumab’s excep-tional internalization capability, a new linker that is highlystable in serum, but can release SN-38 when taken intothe lysosome, was developed (31).

The current investigation examines the prospects forusing these 3 milatuzumab anti-CD74 conjugates, 1 withdoxorubicin, and 2 as SN-38 conjugates, for effectivetherapy primarily against solid tumors.

Materials and MethodsHuman tumor cell lines

Raji Burkitt lymphoma, A-375 (melanoma), Capan-1(pancreatic adenocarcinoma), NCI-N87 (gastric carcino-ma),Hep-G2hepatoma, andMC/CARmyeloma cell lineswere purchased from American Type Culture Collection.HuH-7 hepatoma cell line was purchased from JapanHealth Science Research Resources Bank. All cell lineswere cultured in a humidified CO2 incubator (5%) at 37�Cin recommended media containing 10% to 20% fetal calfserum and supplements. Cells were passaged less than50 times and checked regularly for Mycoplasma.

Antibodies and conjugation methodsMilatuzumab (anti-CD74 MAb; refs. 8, 10, 11, 32), epra-

tuzumab (anti-CD22; ref. 33), veltuzumab (anti-CD20;

refs. 34, 35), labetuzumab (anti-CEACAM5; ref. 36),hMN15 anti-CEACAM6 (37), and hRS7 anti-Trop-2 (30)are humanized IgG1 monoclonal antibodies. CL2A andCL2E linkers and their SN-38 derivatives were preparedand conjugated to antibodies as reported earlier (30, 31), aswere the milatuzumab–doxorubicin conjugates (13, 14).All conjugates were prepared by disulfide reduction ofthe IgG, followed by reaction with the correspondingmaleimide derivatives of these linkers (Fig. 1). Spectro-photometric analyses estimated that the drug:IgGmolar substitution ratio was 5:7 (1.0 mg of the proteincontains �16 mg of SN-38 or 25 mg of doxorubicinequivalent).

In vitro cell binding and cytotoxicityAssays used to compare cell binding of the unconju-

gated and conjugatedmilatuzumabwith antigen-positivecells, and cytotoxicity testingusing theMTSdye reductionmethod (Promega), have been reported previously (27).

Flow cytometry and immunohistologyFlow cytometry was carried out using the primary

humanized monoclonal antibodies listed earlier, whichwere then revealed with a goat anti-human IgG–FITCconjugate (see Supplementary Materials). Cells fromculture were processed in a manner that provided anassessment of only membrane-bound or membrane andcytoplasmic antigen (Supplementary Materials). Immu-nohistology was carried out on formalin-fixed, paraffin-embedded sections of subcutaneous tumor xenografts,staining without antigen retrieval methods, using theprimary humanized monoclonal antibodies listed ear-lier (except hRS7 anti-Trop-2), at 10 mg/mL, which wererevealed with an anti-human IgG conjugate (19). Trop-2expression was assessed using a goat polyclonal anti-body, as indicated in Supplementary Fig. S1.

SN-38 release from linker and in vitro serumstabilityof SN-38 conjugates

The stability of SN-38 conjugates under acidic condi-tions (pH 5.0) with or without cathepsin B, and in serum,are provided in the Supplementary Materials.

In vivo studiesAll studies were conducted in accordance with institu-

tional-approved animal welfare protocols. Female nudemice (4–8-week old) or female severe combined immu-nodeficient (SCID) mice (7-week old) were purchasedfrom Taconic and used after a 1-week quarantine. Ani-mals bearing solid tumor xenografts were treated intra-peritoneallywith test and control articles twiceweekly for4 weeks. Specific doses are given in Results. Toxicity wasassessed by weekly weight measurements.

For the Raji Burkitt lymphoma model, SCID micewere injected intravenously with 2.5 � 106 Raji cells in0.1 mL media. Five days later, animals received a singleintravenous injection (0.1 mL) of the conjugate or saline(N ¼ 10/group). Mice were observed daily for signs of

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distress and paralysis and were euthanized when eitherhind-limb paralysis developed more than 15% loss ofinitial weight or if otherwise moribund (surrogate sur-vival endpoints).Subcutaneous tumors were measured by caliper in

2 dimensions, and the tumor volume calculated as L �w2/2, where L is the longest diameter and w isthe shortest. Measurements were made at least onceweekly, with animals terminated when tumors grewto 1.0 cm3 (i.e., surrogate survival endpoint). The A-375melanoma cell line (6 � 106 cells in 0.2 mL) wasimplanted in nude mice and therapy was initiated whentumors averaged 0.23� 0.06 cm3 (N¼ 8/group). Capan-1 was implanted subcutaneously in nude mice using acombination of tumor suspension from serially pas-saged tumors (0.3 mL of a 15% w/v tumor suspension)combined with 8 � 106 cells from tissue culture. Treat-mentswere initiatedwhen tumor volume averaged 0.27�0.05 cm3 (N ¼ 10/group). NCI-N87 gastric tumor xeno-grafts were initiated by injecting 0.2 mL of a 1:1 (v/v)mixture ofMatrigel and 1� 107 cells from terminal culturesubcutaneously. Therapy was started when the tumorvolume averaged 0.249 � 0.045 cm3 (N ¼ 7/group). The

same procedure was followed for developing the Hep-G2and HuH-7 hepatoma xenografts in nude mice. Therapywas started when Hep-G2 averaged 0.364 � 0.062 cm3

(N ¼ 5/group) and HuH-7 averaged 0.298 � 0.055 cm3

(N ¼ 5/group).Efficacy is expressed in Kaplan–Meier survival curves,

using the surrogate endpointsmentioned earlier for deter-mining the median survival times. Analysis was con-ducted by a log-rank (Mantel–Cox) test using PrismGraphPad software with significance at P < 0.05.

ResultsCD74 expression in human tumor cell lines andxenografts

Surface CD74 expression in Raji was 19.6-fold higherthan background staining, whereas in most solid tumorthe mean fluorescent intensity (MFI) of membrane-onlyCD74 was often �2-fold higher than the backgroundMFI (Table 1). Therefore, identification of CD74-positivi-tity for solid tumorswasmadeprimarily in permeabilizedcells. In permeabilized cells, the MFI for 6 solid tumorcell lines was similar to Raji.

Figure 1. Schematic representationof the CL2A- and CL2E–SN-38linkers and the doxorubicin linkerused to prepare the antibodyconjugates.

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Immunohistology showed Raji subcutaneous xeno-grafts had a largely uniform and intense staining, withprominent cell-surface labeling (Fig. 2A). The Hep-G2hepatoma cell line had the most uniform uptake of thesolid tumors, with moderately strong, but predominantlycytoplasmic, staining (Fig. 2D), followed by the A-375melanoma cell line that had somewhat less uniform stain-ingwithmore intense, yetmostly cytoplasmic, expression(Fig. 2B). The Capan-1 pancreatic (Fig. 2C) and NCI-N87(Fig. 2F) gastric carcinoma cell lines had moderate

(Capan-1) to intense (NCI-N87) CD74 staining, but it wasnot uniformly distributed. The HuH-7 hepatoma cell line(Fig. 2E) had the least uniform and the weakest staining.

Immunoreactivity of the conjugatesKd values for unconjugated milatuzumab, milatuzu-

mab-CL2A-, and CL2E–SN-38 conjugates were not sig-nificantly different, averaging 0.77, 0.59, and 0.80 nmol/L,respectively. Kd values for the unconjugated and doxo-rubicin-conjugated milatuzumab measured in the MC/CAR multiple myeloma cell line were 0.5 � 0.02 nmol/Land 0.8 � 0.2 nmol/L, respectively (14).

In vitro drug release and serum stability ofconjugates

Figure 3A illustrates the release mechanisms of SN-38from the mercaptoethanol-capped CL2A and CL2E lin-kers in an environment, partially simulating lysosomalconditions; namely, low pH (pH 5.0) and in the presenceor absence of cathepsin B. The CL2E–SN-38 substratewas inert at pH 5 in the absence of the enzyme (Sup-plementary Table S1), but in the presence of cathepsin B,cleavage at the Phe-Lys site proceeded quickly with ahalf-life of 34 minutes (Fig. 3A, left). The formation ofactive SN-38 requires intramolecular cyclization at thecarbamate bond at the tenth position of SN-38, whichoccurred more slowly with a half-life of 10.7 hours(Fig. 3A, left; additional data in Supplementary TableS1 and Supplementary Fig. S2).

As expected, cathepsin B had no effect on the releaseof active SN-38 in the CL2A linker. However, CL2A hasa cleavable benzyl carbonate bond, releasing activeSN-38 at a rate similar to the CL2E linker at pH 5.0,with a half-life of approximately 10.2 hours (Fig. 3B,right; additional data Supplementary Table S2). Themilatuzumab–doxorubicin conjugate, which has apH-sensitive acylhydrazone bond, had a half-life of7 to 8 hours at pH 5.0 (Supplementary Fig. S3A andSupplementary Table S3).

While all of these linkers release the drug at relativelysimilar rates under lysosomally relevant conditions, they

Table 1. CD74 expression by flow cytometry expressed as MFI of milatuzumab-positive gated cells

Surface Surface and cytoplasmic

Cell linehLL1

(bkgd)aMFI ratiohLL1:bkgd

hLL1(bkgd)

MFI ratiohLL1:bkgd

Panc CA Capan-1 22 (12) 1.8 248 (5) 49.6Gastric Hs746T 17 (8) 2.1 144 (5) 28.8

NCI-N87 5 (4) 1.3 220 (6) 36.7Melanoma A-375 16 (3) 5.3 185 (6) 30.8Hepatoma Hep-G2 9 (6) 1.5 156 (5) 31.2

HuH-7 8 (5) 1.6 114 (4) 28.5Lymphoma Raji 59 (3) 19.6 143 (5) 28.6

aBackground MFI of cells incubated with GAH-FITC only.

Figure 2. CD74 expression in specimens of several human cancers grownas subcutaneous xenografts in athymic of SCID nudemice: A, Raji Burkittlymphoma; B, A-375 melanoma; C, Capan1 pancreatic carcinoma; D,Hep-G2 hepatoma; E, HuH-7 hepatoma; and F, NCI-N87 gastriccarcinoma. Scale bar in A corresponds to 200 mm (50 mm the highermagnification insets). A nonbinding, isotype-matched antibody showedno evidence of staining (shown only for the Raji xenograft, top right insetin A; scale, 200 mm).

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have very different stability in serum. Milatuzumab–CL2A–SN-38 released 50% of free SN-38 in 21.55 � 0.17hours (Fig. 3C), consistent with other CL2A–SN-38 con-jugates (30, 31). The CL2E–SN-38 conjugate, however,was highly inert, with a half-life extrapolated to approx-imately 87.5 days. The milatuzumab–doxorubicin conju-gate released 50% of the doxorubicin in 98 hours, whichwas similar to 2 other antibody–doxorubicin conjugates(Supplementary Fig. S3B and Supplementary Table S3).

CytotoxicityA significant issue related to the evaluation of these

conjugates was the relative potency of free doxorubicinand SN-38 in hematopoietic and solid tumor cell lines.Our group reported previously that SN-38 was active inseveral B-cell lymphoma and acute leukemia cell lines,with potencies ranging from 0.13 to 2.28 nmol/L (31).SN-38 potency in 4 of the solid tumor cell lines that

were later used for in vivo therapy studies ranged from2.0 to 6 nmol/L (Table 2). Doxorubicin had a mixedresponse, with 3 to 4 nmol/L potency in the Raji lym-phoma and the A-375 melanoma cell lines, but it wasnearly 10 times less potent than SN-38 against Capan-1,NCI-N87, and Hep-G2 cell lines. Other studies compar-ing the potency of SN-38 with doxorubicin found 2additional cell lines with similar potency for both drugs:LS174T colon cancer, 18 versus 18 (nmol/L potency ofSN-38 vs. doxorubicin, respectively) and MDA-MB-231breast cancer, 2 versus 2 nmol/L. In 4 other cell lines,SN-38 was 5- to 20-fold more potent than doxorubicin:SK-OV-4 ovarian cancer: 18 versus 90 nmol/L; Calu-3lung adenocarcinoma, 32 versus 582 nmol/L; Capan-2pancreatic cancer, 37 versus 221 nmol/L; and NCI-H466small cell lung cancer, 0.1 versus 2 nmol/L. Collectively,these data suggest that doxorubicin may be less effec-tive against solid tumors than SN-38, whereas SN-38

Figure 3. Cleavage of ME-cappedCL2A–SN-38 and CL2E–SN-38derivatives with or withoutcathepsin-B at pH 5 and conjugatesin human serum in vitro. A, theliberation of SN-38 from CL2E linkeris a 2-step process, whereas being a1-step process for CL2A linker. B,drug release kinetics for ME-cappedCL2E–SN-38 (left) and CL2A–SN-38(right). C, in vitro stability of antibodyconjugates in human serum at 37�C.

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seems to be equally effective in solid and hematopoietictumors.

As expected, the 3 conjugate forms were often someorder of magnitude less potent than the free drug in vitro,as both drugs are expected to be transported readily intothe cells, whereas drug conjugates require antibody bind-ing to transport drug inside the cell, and with the solidtumor cell lines having such low surface expression, thiswas expected (Table 2). TheCL2A-linked SN-38 conjugateis an exception, as more than 90% of the SN-38 is releasedfrom the conjugate into the media over the 4-day assayperiod (30, 31). Thus, even if this conjugate was internal-ized rapidly, it would be difficult to discern differencesbetween the free drug and the CL2A-linked drug.

The stable CL2E-linked SN-38 conducted well in theRaji cell line, compared with free SN-38, but it had sub-stantially (7- to 16-fold) lower potency in the 4 solid tumorcell lines, suggesting the relatively low surface expressionof CD74 may be playing a role in minimizing drug trans-port in these solid tumors. Themilatuzumab–doxorubicinconjugate had substantial differences in its potency whencomparedwith the free doxorubicin in all cell lines, whichwas of similar magnitude as the CL2E–SN-38 conjugatesto free SN-38 in the solid tumor cell lines.

In the 6 additional cell lines mentioned earlier, themilatuzumab–CL2A–SN-38 conjugate was 9- to 60-timesmore potent than themilatuzumab–doxorubicin conjugate(not shown), but again, this resultwas influenced largelybythe fact that the CL2A-linked conjugate releases most of itsSN-38 into the media over the 4-day incubation period,whereas the doxorubicin conjugate would at most release50% of its drug over this same time. The CL2E-linkedmilatuzumab was not examined in these other cell lines.

In vivo therapy for human tumor xenograftsPrevious in vivo studies with the milatuzumab–doxo-

rubicin or -SN-38 conjugates prepared with various anti-bodies had indicated they were efficacious at dosesfar lower than their maximum-tolerated dose (13, 14,26, 30, 31), and thus in vivo testing focused on comparingsimilar, but fixed, amounts of each conjugate at levels thatwere tolerated. For example, weight loss never exceeded

10% of the starting weight, except in the intravenousRaji model, where weight loss was often indicative ofprogressive disease, not toxicity. It also is important tonote that milatuzumab does not bind to murine bloodcells (i.e., murine CD74).

Initial studiesfirst examined thedoxorubicin andSN-38conjugates in a disseminated Raji model of lymphomato gauge how the milatuzumab–doxorubicin conjugatecompared with the 2 SN-38 conjugates (Fig. 4A). Allspecific conjugates were significantly better than nontar-geting labetuzumab–SN-38 conjugate or saline-treatedanimals, which had a median survival of only 20 days(P < 0.0001). Despite in vitro studies indicating as much asan 8-fold advantage for the SN-38 conjugates in Raji, thebest survival was seen with the milatuzumab–doxorubi-cin conjugate,where all animals given a single 17.5mg/kg(350 mg) dose and 7 of 10 animals given 2.0 mg/kg (40 mg)were alive at the conclusion of the study (i.e., 17.5 mg/kgdose milatuzumab–doxorubicin versus milatuzumab–CL2A–SN-38 with median survival >112 days versus78.5 days, respectively; P ¼ 0.0012). Survival was signi-ficantly lower for the more stable CL2E–SN-38 conjugatethan with the CL2A–SN-38 conjugate (P < 0.0001 andP ¼ 0.0197, 17.5 and 2.0 mg/kg doses, respectively, forthe CL2A vs. CL2E), even though in vitro studies sug-gested that both conjugates would release active SN-38at similar rates when internalized.

Five solid tumor cell lines were examined, startingwiththe A-375 melanoma cell line, as it had the best in vitroresponse to both doxorubicin and SN-38. A-375 xeno-grafts grew rapidly (tumor sizes in individual animalsshown in Fig. 4D), with saline-treated control animalshaving a median survival of only 10.5 days (Fig. 4B). A12.5 mg/kg (0.25 mg per animal) twice weekly dose ofthe milatuzumab–CL2A–SN-38 conjugate extended sur-vival to 28 days (P ¼ 0.0006), which was significantlybetter than the control epratuzumab–CL2A–SN-38 con-jugate having a median survival of 17.5 days (P¼ 0.0089),with the latter not being significantly different from thesaline-treated animals (P ¼ 0.1967). The milatuzumab–CL2A conjugate provided significantly longer survivalthan the milatuzumab–CL2E–SN-38 conjugate (P ¼

Table 2. In vitro cytotoxicity in human cancer cell lines

IC50, nmol/L

Drug or conjugateMelanoma(A-375)

Pancreatic(Capan-1)

Gastric(NCI-N87)

Hepatic(Hep-G2)

NHL(Raji)

SN-38 2 6 6 3 2Milatuzumab–SN-38CL2A linkera 5 13 15 8 2CL2E linkera 34 210 130 78 4

Doxorubicin 3 43 29 46 4Milatuzumab–doxorubicina 29 540 280 628 32

aDrug/IgG mole ratio: CL2A, 6.5; CL2E, 6.6; doxorubicin, 7.3.

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0.0014), which had the samemedian survival of 14 days asits control epratuzumab–CL2E–SN-38 conjugate. Despitegiving a 2-fold higher dose of the milatuzumab–doxoru-bicin than the SN-38 conjugates, the median survival wasno better than the saline-treated animals (10.5 days).As with the A-375 melanoma model, only the CL2A-

linked SN-38 conjugate was effective in Capan-1, with amedian survival of 35 days, significantly different fromuntreated animals (P < 0.036; Fig. 4C), even at a lower dose(5 mg/kg; 100 mg per animal; P < 0.02). Neither themilatuzumab–CL2E nor the nontargeting epratuzu-mab–CL2A–SN-38 conjugates, or a 2-fold higher dose ofthe milatuzumab–doxorubicin conjugate, provided anysurvival advantage (P ¼ 0.44 vs. saline). It is noteworthythat in the same studywith animals given the samedose ofthe internalizing anti-Trop-2 CL2A–SN-38 conjugate(hRS7–SN-38; IMMU-132), themedian survivalwas equalto milatuzumab–CL2A–SN-38 (Fig. 5A). The hRS7–CL2A–SN-38 conjugate had been identified previously asan antibody–drug conjugate (ADC) of interest for treatinga variety of solid tumors (30). TheMFI for surface-bindinghRS7 on Capan-1 was 237, compared with 22 for milatu-zumab (see Table 1). Thus, despite having a substantiallylower surface antigen expression, the milatuzumab–CL2A–SN-38 conjugate did as well as the hRS7–CL2A–SN-38 conjugate in this model.With the milatuzumab–doxorubicin conjugate having

inferior therapeutic results in 2 of the solid tumor xeno-grafts, the focus shifted to comparing the milatuzumab–SN-38 conjugates with SN-38 conjugates prepared withother humanized antibodies against Trop-2 (hRS7) or

CEACAM6 (hMN-15), which are more highly expressedon the surface of many solid tumors (38, 39). Threeadditional xenograft models were examined.

In the gastric tumor model, NCI-N87, animals given17.5 mg/kg/dose (350 mg) of milatuzumab–CL2A–SN-38provided some improvement in survival, but it failed tomeet statistical significance compared with the saline-treated animals (31 vs. 14 days; P ¼ 0.0760) or to thenonbinding veltuzumab anti-CD20–CL2A–SN39 conju-gate (21 days; P ¼ 0.3128; Fig. 5B). However, the hRS7-and hMN-15–CL2A conjugates significantly improvedthe median survival to 66 and 63 days, respectively (P¼ 0.0001). The MFI for surface-expressed Trop-2 andCEACAM6were 795 and 1,123, respectively,much higherthan CD74 that was just 5 (see Table 1). Immunohistologyshowed a relatively intense cytoplasmic expression ofCD74 in the xenograft of this cell line, but importantly itwas scattered, appearing only in defined pockets withinthe tumor (Fig. 2F). CEACAM6 and Trop-2 were moreuniformly expressed than CD74 (Supplementary Fig. S1Dand S1E, respectively), with CEACAM6 being moreintensely present both cytoplasmically and on the mem-brane, and Trop-2 primarily found on the membrane.Thus, the improved survival with the anti-CEACAM6and anti-Trop-2 conjugates most likely reflects higherantigen density and more uniform expression in NCI-N87.

In the Hep-G2 hepatoma cell line, immunohistologyshowed a very uniform expression with moderate cyto-plasmic staining of CD74 (Fig. 2D), and flow cytometryindicated a relatively low surface expression (MFI ¼ 9).

Figure 4. Comparing milatuzumab–SN-38 and doxorubicin ADCs in 3 human tumor xenograft models. A, disseminated model of lymphoma (Raji; 10/group)was given a single intravenous dose of the agents listed 5 days after intravenous injection of tumor cells. B, subcutaneous A-375 melanoma xenografts(8/group) and Capan-1 pancreatic adenocarcinoma xenografts (10/group; C) using milatuzumab (Mmab) conjugates given intraperitoneally on the daysindicated. D, individual animal data from the A375 study in B, showing tumor size progression (dashed lines) plotted with the average � SEM (solidline). Dotted horizontal line at 1.0 cm3marks the timewhenanimalswere removed fromstudy due to tumor progression (i.e., survival time). Lmab, labetuzumabhumanized anti-CEACAM5 IgG; Emab, epratuzumab humanized anti-CD22 IgG.

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The MFI with hMN-15 was 175 and immunohistologyshowed a fairly uniform membrane and cytoplasmicexpression of CEACAM6, with isolated pockets of veryintense membrane staining (Supplementary Fig. S1B). Astudy in animals bearing Hep-G2 xenografts found themilatuzumab–CL2A–SN-38 extended survival to 45 dayscompared with 21 days in the saline-treated group (P ¼0.0048), whereas the hMN-15–CL2A–SN-38 conjugateimproved survival to 35 days (Fig. 5C). There was a trendfavoring the milatuzumab conjugate over hMN-15–CL2A–SN-38, but it did not achieve statistical significance(46 vs. 35 days; P ¼ 0.0802). However, the nonbindingveltuzumab–CL2A–SN-38 conjugate provided a similarsurvival advantage as the milatuzumab conjugate. Wepreviously observed that therapeutic results with non-binding conjugates could be similar to the specific CL2A-linked conjugate, particularly at higher protein doses(30), but titration of the specific and control conjugatesusually revealed selectivity. Thus, neither of the specificconjugates provided a selective therapeutic advantage atthese doses in this cell line.

Another study using the HuH-7 hepatoma cell line,which had similar surface expression, but slightly lowercytoplasmic levels as Hep-G2 (see Table 1), found thehMN-15–SN-38 conjugate providing a longer (35 vs. 18days), albeit not significantly different, survival advan-tage than the milatuzumab–CL2A conjugate (P ¼0.2944; Fig. 5D).While both the hMN-15 andmilatuzumabconjugates were significantly better than the saline-trea-ted animals (P ¼ 0.008 and 0.009, respectively), againneither conjugate was significantly different from thenontargeted veltuzumab–SN-38 conjugate at this dose

level (P ¼ 0.4602 and 0.9033, respectively). CEACAM6surface expression was relatively low in this cell line(MFI ¼ 81), and immunohistology showed that bothCD74 (Fig. 2E) and CEACAM6 (Supplementary Fig.S1C) were very faint and highly scattered.

DiscussionADCs have been of considerable research interest

for many years, but only recently peaked, primarily dueto the clinical success of 2 conjugates prepared with so-called "supertoxic" agents that have subnanomolarpotency, which replaced many of the earlier ADCsprepared using chemotherapeutic agents that had poten-cies in the nanomolar levels (40–46). However, drugpotency or even its specific mechanism of action is notthe only defining property that affords optimal perfor-mance of an ADC.

CD74 is expressed at relatively low levels on the cellsurface (2, 3, 10), but its unique internalization and sur-face reexpression allows milatuzumab anti-CD74 ADCsto be effective in hematopoietic cancer xenograft models,even with a moderately toxic drug, such as doxorubi-cin (13, 14). This conjugate is currently being studiedclinically in patients with hematopoietic cancers(NCT01101594 andNCT01585688), butwith evidence thatCD74 is expressed on several types of solid tumors,additional preclinical studies were initiated to assess itspotential use in these cancers. In addition, as SN-38 andother camptothecins are used to treat solid tumors, the useof milatuzumab–SN-38 conjugates was assessed as well.Promising efficacy has been seen with SN-38 conjugates

Figure 5. Therapeutic efficacy of antibody–SN-38 conjugates in various tumor models. A, hRS7 anti-Trop-2 conjugates given intraperitoneally twiceweekly for 4 weeks in nude mice bearing subcutaneous Capan-1 human pancreatic cancer xenografts (N ¼ 10/group). B, animal bearingsubcutaneous NCI-N87 gastric carcinoma xenografts (7/group) treated with CL2A–SN-38 conjugates prepared with milatuzumab, hRS7, or hMN15anti-CEACAM6 IgG. C and D, Hep-G2 (C) and HuH-7 (D) human hepatoma xenografts (5/group) treated with milatuzumab or hMN15-CL2A–SN-38conjugates. Vmab, veltuzumab humanized anti-CD20 IgG.

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prepared with several antibodies against other antigensexpressed in solid and hematologic tumor models(13, 14, 30, 31, 47), and this has led to the developmentof 2 new SN-38 conjugates being pursued in phase Iclinical trials of colorectal and diverse epithelial cancers(NCT01270698 and NCT01631552).In vitro studies revealed unconjugated doxorubicin

and SN-38 had similar potency in the Raji lymphomacell line, but SN-38 was more potent in several of thesolid tumor cell lines, suggesting SN-38 was potentiallypreferred for solid tumors. Despite the similarities inpotency of the free drugs against Raji in vitro, themilatuzumab–doxorubicin conjugate provided a signif-icantly better response in mice bearing Raji xenograftsthan the milatuzumab–SN-38 conjugates. In contrast,even though in vitro testing had indicated that A-375melanoma was equally sensitive to free doxorubicinand free SN-38 when tested in vivo, milatuzumab–doxo-rubicin was less effective than the CL2A-linked SN-38milatuzumab conjugate in A-375 as well as in xenograftsof Capan-1 human pancreatic cancer. These results andthe in vitro studies showing unconjugated SN-38 had a5- to 20-fold higher potency than doxorubicin in moresolid tumor cell lines led to our decision to abandonfurther evaluation of the doxorubicin conjugate forsolid tumor therapy. However, to gauge the use of themilatuzumab–SN-38 conjugates, we conducted addi-tional comparative assessments to antibody–SN-38 con-jugates against other antigens present in a variety ofsolid tumors.The internalizing hRS7 anti-Trop-2 CL2A-linked SN-38

conjugate was evaluated previously in the Capan-1 cellline (30), and therefore the efficacy of milatuzumab andhRS7 SN-38 conjugates was examined. Milatuzumab andhRS7 CL2A-linked SN-38 conjugates had similar mediansurvivals that were significantly higher than with controlconjugates, and better than their respective CL2E-linkedconjugates. Flow cytometry had indicated Trop-2 expres-sion was approximately 10-fold higher than CD74 inCapan-1, which suggested that the transport capabilitiesof CD74, which were known to be exceptional (10), weremore efficient than Trop-2. However, it iswell known thatother factors, such as antigen accessibility (i.e., membranevs. cytoplasm, physiological, and "binding-site" barriers)and distribution among cells within a tumor are criticalfactors influencing every form of targeted therapy, par-ticularly those that depend on adequate intracellulardelivery of a product to individual cells (48). For example,the binding-site barrier could potentially impede tumorpenetration when antigen expression is high. However, ifthe payload could be released from the conjugate afterlocalizing in the tumor, such as with the CL2A-linkedconjugates, the drug could diffuse to nontargetedbystander cells, thereby enhancing its efficacy range. Thismechanism also is thought to aid the efficacy of otherCL2A–SN-38 conjugates that we examined using poorlyinternalizing antibodies, such as anti-CEACAM5 (26) andthe anti-CEACAM6 used herein. Conjugates based on

milatuzumab rely more on the antibody’s direct interac-tion with the tumor cell, taking advantage of CD740srapid internalization and reexpression that can compen-sate for its lower abundance on the surface of cells.Naturally, this advantage would be reduced when CD74is highly scattered within the tumor, and without directbinding to the tumor antigen to encourage retention, thebenefit of the drug’s slow release from the conjugatewould be lost. These observations suggest a preassess-ment of the distribution of CD74within solid tumors maybe required before selecting a CD74-targeted agent. Aprevious review of human gastrointestinal tumors by ourgroup suggests that they often have a high level of expres-sion with good uniformity (19).

We previously evaluated a "CL2E-like" linker that wascoupled at the 20-hydroxyl position of SN-38, similar tothe CL2A linker, but that antibody conjugate lacked suf-ficient antitumor activity and was not pursued (Unpub-lished Data). Given the exceptional internalization prop-erties of milatuzumab, we revisited the SN-38-linkerchemistry, hypothesizing that a more serum-stable linkermight be preferred with such a rapid internalizing anti-body. To release SN-38 in an active form,we surmised thatif the leaving group was phenolic, this could promotecyclization, and therefore the CL2E-linker was designedto join at the phenolic 10-position of SN-38.We included acathepsin B cleavage site in the CL2E linker to cleave themonocarbamate derivative of SN-38 and N,N0-dimethy-lethylenediamine from the antibody-linker, but for theSN-38 to be active, cyclization was still required. In vitrostudies proved the CL2E-linked SN-38 was highly stablein serum, but under lysosomal conditions (pH 5.0 and inthe presence of cathepsin B), active SN-38 was releasedwith a half-life of approximately 11 hours, similar to therelease rate measured for CL2A-linked SN-38 at lysosom-al pH (i.e., pH 5.0).

The CL2E-linked SN-38 conjugate had a similar IC50 asthe CL2A conjugate in the Raji cell line, which was con-sistent with the view that if rapidly internalized, bothconjugates would release the active form of SN-38 atapproximately the same rate. However, as already men-tioned, the in vitro activity of the CL2A conjugate isinfluenced largely by the release of SN-38 into the media,and does not necessarily reflect uptake by the intactconjugate, whereas cytotoxicity of the CL2E-linked con-jugate reflected internalization of the intact conjugate(presumably by selective binding to CD74). When theCL2E-linked conjugate was found to be much less potentin the solid tumor cell lines than the CL2A conjugate, thissuggested that the lower surface expression of CD74 onthe solid tumor cell lines reduced the internalization ofSN-38 via milatuzumab binding. However, when in vivostudies in Raji also showed the milatuzumab–CL2A–SN-38was superior to theCL2E conjugate, other factors had tobe affecting CL2E-based conjugate’s efficacy.

One possibility was that the linker design in CL2E–SN-38 left the 20-position of the drug underivatized, render-ing the lactone group susceptible to ring opening. Studies

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with irinotecan have shown the carboxylate formof SN-38is only 10% as potent as the lactone form (49). The CL2A-linked SN-38 is derivatized at the 20-hydroxyl position, aprocess that stabilizes the lactone group in camptothecinsunder physiologic conditions (50). Because the in vitrostability studies and the analysis of serum stability wereconducted under acidic conditions, we do not have adirect measure of the carboxylate form of SN-38 in eitherof these conjugates, but it is reasonable to suspect thatdestabilization of the lactone ring could have contributedto CL2E’s diminished efficacy in vivo. Another explana-tion for the different activity of the CL2A- and CL2E-linked SN-38 conjugates may be related to the multipleroles that CD74 plays in cell biology. For example, inantigen-presenting cells, it may have a more dominantrole in processing antigenic peptides, where is solidtumors, its role might be related more to survival. Thiscould affect intracellular trafficking andprocessing, there-by affecting the conjugate’s potency.

In conclusion, in vitro and in vivo results indicate that themilatuzumab–doxorubicin conjugate is superior to theCL2A–SN-38 conjugate in the Raji lymphoma cell line,which may reflect the improved serum stability of thedoxorubicin conjugate compared with the CL2A-linkedSN-38. The serum-stable CL2E-linked SN-38 conjugatewas again found to be inferior to the less stable CL2A-linked SN-38 (31), and therefore it seems that at least withSN-38, a linker that allows thedrug to be released in serum(half-life �1 day) is preferred. Finally, antigen accessibil-ity seems to have a dominant role in defining milatuzu-mab–CL2A–SN-380s potencywhenmeasuredagainst con-jugates preparedwith other internalizing (hRS7) or poorlyinternalizing antibodies (hMN15) that were more acces-

sible (surface expressed) and abundant. We suspect thisfinding is universal for targeted therapies, but these stud-ies have at least shown that the unique internalizationproperties of a CD74-targeted agent can provide signifi-cant efficacy even when surface expression of the targetantigen is minimal.

Disclosure of Potential Conflicts of InterestS.V. Govindan is Senior Director of Conjugation Chemistry at Immu-

nomedics andhas ownership interest (including patents) in the same. T.M.Cardillo is the Director, Pre-Clinical Development in Immunomedics, Inc.R.M. Sharkey is Senior Director of Regulatory and Scientific Affairs ofImmunomedics, Inc. D.M. Goldenberg is officer and board member ofImmunomedics, Inc. and has ownership interest (including patents) in thesame.Nopotential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: T.M. Cardillo, D.M. GoldenbergDevelopment of methodology: S.V. Govindan, D.V. GoldAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.V. Govindan, T.M. Cardillo, F. Tat, D.V. GoldAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):S.V.Govindan,T.M.Cardillo, R.M. Sharkey,F. Tat, D.V. Gold, D.M. GoldenbergWriting, review, and/or revision of the manuscript: S.V. Govindan, T.M.Cardillo, R.M. Sharkey, D.M. GoldenbergStudy supervision: T.M. Cardillo, D.M. Goldenberg

AcknowledgmentsThe authors thank Dr. Jennifer Pickett for conducting in vitro stability

studies on themilatuzumab–doxorubicin conjugate andMr. R.Arrojo,Ms.A. Nair, and Ms. N. Sathyanarayan for expert technical assistance.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 3, 2012; revised February 7, 2013; accepted February8, 2013; published OnlineFirst February 20, 2013.

References1. Morton PA, Zacheis ML, Giacoletto KS, Manning JA, Schwartz BD.

Delivery of nascent MHC class II-invariant chain complexes to lyso-somal compartments and proteolysis of invariant chain by cysteineproteases precedes peptide binding in B-lymphoblastoid cells. JImmunol 1995;154:137–50.

2. Roche PA, Cresswell P. Invariant chain association with HLA-DRmolecules inhibits immunogenic peptide binding. Nature 1990;345:615–8.

3. Roche PA, Teletski CL, Stang E, Bakke O, Long EO. Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapidinternalization. Proc Natl Acad Sci U S A 1993;90:8581–5.

4. Binsky I, Haran M, Starlets D, Gore Y, Lantner F, Harpaz N, et al. IL-8secreted in a macrophage migration-inhibitory factor- and CD74-dependent manner regulates B cell chronic lymphocytic leukemiasurvival. Proc Natl Acad Sci U S A 2007;104:13408–13.

5. Naujokas MF, Morin M, Anderson MS, Peterson M, Miller J. Thechondroitin sulfate form of invariant chain can enhance stimulation ofT cell responses through interaction with CD44. Cell 1993;74:257–68.

6. Leng L, Metz CN, Fang Y, Xu J, Donnelly S, Baugh J, et al. MIFsignal transduction initiated by binding to CD74. J Exp Med 2003;197:1467–76.

7. Burton JD, Ely S, Reddy PK, Stein R, Gold DV, Cardillo TM, et al. CD74is expressedbymultiplemyelomaand is apromising target for therapy.Clin Cancer Res 2004;10:6606–11.

8. Stein R, Qu Z, Cardillo TM, Chen S, Rosario A, Horak ID, et al.Antiproliferative activity of a humanized anti-CD74 monoclonal anti-body, hLL1, on B-cell malignancies. Blood 2004;104:3705–11.

9. Wraight CJ, van Endert P, Moller P, Lipp J, Ling NR, MacLennan IC,et al. Humanmajor histocompatibility complex class II invariant chain isexpressed on the cell surface. J Biol Chem 1990;265:5787–92.

10. Hansen HJ, Ong GL, Diril H, Valdez A, Roche PA, Griffiths GL, et al.Internalization and catabolism of radiolabelled antibodies to the MHCclass-II invariant chain by B-cell lymphomas. Biochem J 1996;320(Pt1):293–300.

11. Qu Z, Ma H, Consoino K, Li X, Goldenberg DM. Internalization andcytotoxic effects of a humanized anti-CD74 antibody, LL1. Proc AmAssoc Cancer Res 2002;43:255.

12. Fr€olichD, Blabetafeld D, Reiter K,GieseckeC, DaridonC,Mei HE, et al.The anti-CD74 humanized monoclonal antibody, milatuzumab, whichtargets the invariant chain of MHC II complexes, alters B-cell prolif-eration, migration, and adhesion molecule expression. Arthritis ResTher 2012;14:R54.

13. Griffiths GL, Mattes MJ, Stein R, Govindan SV, Horak ID, Hansen HJ,et al. Cure of SCIDmice bearing humanB-lymphoma xenografts by ananti-CD74 antibody-anthracycline drug conjugate. Clin Cancer Res2003;9:6567–71.

14. Sapra P, Stein R, Pickett J, Qu Z, Govindan SV, Cardillo TM, et al. Anti-CD74 antibody-doxorubicin conjugate, IMMU-110, in a human

Govindan et al.

Mol Cancer Ther; 12(6) June 2013 Molecular Cancer TherapeuticsOF10

Research. on December 30, 2020. © 2013 American Association for Cancermct.aacrjournals.org Downloaded from

Published OnlineFirst February 20, 2013; DOI: 10.1158/1535-7163.MCT-12-1170

multiple myeloma xenograft and in monkeys. Clin Cancer Res 2005;11:5257–64.

15. Michel RB, Rosario AV, Andrews PM, Goldenberg DM, Mattes MJ.Therapy of small subcutaneous B-lymphoma xenografts with antibo-dies conjugated to radionuclides emitting low-energy electrons. ClinCancer Res 2005;11:777–86.

16. Chang CH, Sapra P, Vanama SS, Hansen HJ, Horak ID, GoldenbergDM. Effective therapy of human lymphoma xenografts with a novelrecombinant ribonuclease/anti-CD74 humanized IgG4 antibodyimmunotoxin. Blood 2005;106:4308–14.

17. Kaufman J, Niesvizky R, Stadtmauer EA, Chanan-Khan A, Siegel D,Horne H, et al. First trial of humanized anti-CD74monoclonal antibody(MAb), milatuzumab, in multiple myeloma. ASH Annual MeetingAbstracts 2008;112:3697.

18. Ishigami S, Natsugoe S, Tokuda K, Nakajo A, Iwashige H, Aridome K,et al. Invariant chain expression in gastric cancer. Cancer Lett 2001;168:87–91.

19. Gold DV, Stein R, Burton J, Goldenberg DM. Enhanced expression ofCD74 in gastrointestinal cancers and benign tissues. Int J Clin ExpPathol 2010;4:1–12.

20. Zheng YX, Yang M, Rong TT, Yuan XL, Ma YH, Wang ZH, et al. CD74and macrophage migration inhibitory factor as therapeutic targets ingastric cancer. World J Gastroenterol 2012;18:2253–61.

21. KitangeGJ,CarlsonBL, SchroederMA,Decker PA,Morlan BW,WuW,et al. Expression of CD74 in high grade gliomas: a potential role intemozolomide resistance. J Neurooncol 2010;100:177–86.

22. Goldenberg DM, Zagzag D, Heselmeyer-Haddad KM, Berroa GarciaLY, Ried T, Loo M, et al. Horizontal transmission and retention ofmalignancy, as well as functional human genes, after spontaneousfusion of human glioblastoma and hamster host cells in vivo. IntJ Cancer 2012;131:49–58.

23. Ioachim HL, Pambuccian SE, Hekimgil M, Giancotti FR, Dorsett BH.Lymphoid monoclonal antibodies reactive with lung tumors. Diagnos-tic applications. Am J Surg Pathol 1996;20:64–71.

24. Young AN, Amin MB, Moreno CS, Lim SD, Cohen C, Petros JA, et al.Expression profiling of renal epithelial neoplasms: a method for tumorclassification and discovery of diagnostic molecular markers. AmJ Pathol 2001;158:1639–51.

25. Choi JW,KimY, Lee JH, KimYS.CD74expression is increased in high-grade, invasive urothelial carcinoma of the bladder. Int J Urol2013;20:251–5.

26. Govindan SV, Cardillo TM, Moon SJ, Hansen HJ, Goldenberg DM.CEACAM5-targeted therapy of human colonic and pancreatic cancerxenografts with potent labetuzumab–SN-38 immunoconjugates. ClinCancer Res 2009;15:6052–61.

27. Moon SJ, Govindan SV, Cardillo TM, D'Souza CA, Hansen HJ, Gold-enberg DM. Antibody conjugates of 7-ethyl-10-hydroxycamptothecin(SN-38) for targeted cancer chemotherapy. J Med Chem 2008;51:6916–26.

28. Mathijssen RH, van Alphen RJ, Verweij J, LoosWJ, Nooter K, Stoter G,et al. Clinical pharmacokinetics and metabolism of irinotecan (CPT-11). Clin Cancer Res 2001;7:2182–94.

29. Rivory LP.MetabolismofCPT-11. Impact on activity. AnnNYAcadSci2000;922:205–15.

30. Cardillo TM, Govindan SV, Sharkey RM, Trisal P, Goldenberg DM.Humanized anti-Trop-2 IgG–SN-38 conjugate for effective treatmentof diverse epithelial cancers: preclinical studies in human cancerxenograft models and monkeys. Clin Cancer Res 2011;17:3157–69.

31. Sharkey RM, Govindan SV, Cardillo TM, Goldenberg DM. Epratuzu-mab–SN-38: a new antibody–drug conjugate for the therapy of hema-tologic malignancies. Mol Cancer Ther 2012;11:224–34.

32. Stein R,MattesMJ,Cardillo TM,HansenHJ,ChangCH,Burton J, et al.CD74: a new candidate target for the immunotherapy of B-cell neo-plasms. Clin Cancer Res 2007;13:5556s–63s.

33. Goldenberg DM. Epratuzumab in the therapy of oncologicaland immunological diseases. Expert Rev Anticancer Ther 2006;6:1341–53.

34. Goldenberg DM, Morschhauser F, Wegener WA. Veltuzumab (human-ized anti-CD20monoclonal antibody): characterization, current clinicalresults, and future prospects. Leuk Lymphoma 2010;51:747–55.

35. Stein R, Qu Z, Chen S, Rosario A, Shi V, Hayes M, et al. Character-ization of a new humanized anti-CD20 monoclonal antibody, IMMU-106, and its use in combination with the humanized anti-CD22 anti-body, epratuzumab, for the therapy of non-Hodgkin's lymphoma. ClinCancer Res 2004;10:2868–78.

36. Sharkey RM, Juweid M, Shevitz J, Behr T, Dunn R, Swayne LC, et al.Evaluation of a complementarity-determining region-grafted (human-ized) anti-carcinoembryonic antigen monoclonal antibody in preclin-ical and clinical studies. Cancer Res 1995;55:5935s–45s.

37. Hansen HJ, Goldenberg DM, Newman ES, Grebenau R, Sharkey RM.Characterization of second-generationmonoclonal antibodies againstcarcinoembryonic antigen. Cancer 1993;71:3478–85.

38. Blumenthal RD, Leon E, Hansen HJ, Goldenberg DM. Expressionpatterns of CEACAM5 and CEACAM6 in primary and metastaticcancers. BMC Cancer 2007;7:2.

39. Stein R, Basu A, Chen S, Shih LB, Goldenberg DM. Specificity andproperties of MAb RS7-3G11 and the antigen defined by this pancar-cinoma monoclonal antibody. Int J Cancer 1993;55:938–46.

40. de Claro RA, McGinn K, Kwitkowski V, Bullock J, Khandelwal A,Habtemariam B, et al. U.S. Food and Drug Administration approvalsummary: brentuximab vedotin for the treatment of relapsed Hodgkinlymphoma or relapsed systemic anaplastic large-cell lymphoma. ClinCancer Res 2012;18:5845–9.

41. Govindan SV, Goldenberg DM. Designing immunoconjugates forcancer therapy. Expert Opin Biol Ther 2012;12:873–90.

42. LoRusso PM, Weiss D, Guardino E, Girish S, Sliwkowski MX. Trastu-zumab emtansine: a unique antibody–drug conjugate in developmentfor human epidermal growth factor receptor 2-positive cancer. ClinCancer Res 2011;17:6437–47.

43. Polson AG, Ho WY, Ramakrishnan V. Investigational antibody–drugconjugates for hematological malignancies. Expert Opin InvestigDrugs 2011;20:75–85.

44. Pro B, Perini GF. Brentuximab vedotin in Hodgkin's lymphoma. ExpertOpin Biol Ther 2012;12:1415–21.

45. Sapra P, Hooper AT, O'Donnell CJ, Gerber HP. Investigational anti-body drug conjugates for solid tumors. Expert Opin Investig Drugs2011;20:1131–49.

46. Teicher BA, Chari RV. Antibody conjugate therapeutics: challengesand potential. Clin Cancer Res 2011;17:6389–97.

47. Sharkey RM, Karacay H, Govindan SV, Goldenberg DM. Combinationradioimmunotherapy and chemoimmunotherapy involving different orthe same targets improves therapy of human pancreatic carcinomaxenograft models. Mol Cancer Ther 2011;10:1072–81.

48. Thurber GM, Schmidt MM, Wittrup KD. Antibody tumor penetration:transport opposed by systemic and antigen-mediated clearance. AdvDrug Del Rev 2008;60:1421–34.

49. Giovanella BC, Harris N, Mendoza J, Cao Z, Liehr J, Stehlin JS.Dependence of anticancer activity of camptothecins on maintainingtheir lactone function. Ann N Y Acad Sci 2000;922:27–35.

50. Zhao H, Lee C, Sai P, Choe YH, Boro M, Pendri A, et al. 20-O-acylcamptothecin derivatives: evidence for lactone stabilization. J OrgChem 2000;65:4601–6.

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