Harnessing the Fcm Receptor for Potent and Selective Cytotoxic Therapy of Chronic Lymphocytic Leukemia

Published OnlineFirst October 24, 2014; DOI: 10.1158/0008-5472.CAN-14-2030

Cancer Therapeutics, Targets, and Chemical Biology Research

Bereng ere Vire1, Martin Skarzynski1, Joshua D. Thomas2, Christopher G. Nelson2, Alexandre David3, Georg Aue1, Terrence R. Burke Jr2, Christoph Rader4,5,6, and Adrian Wiestner1

Abstract Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcmR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcm). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM-derived protein scaffold (Fcm) and linked it with the cytotoxic agent monomethylauristatin F. This Fcm–drug conjugate was selectively toxic for FcmR- expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcm–drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcm–drug conjugate in immunodeficient NOD/SCID/IL-2Rgnull (NSG) mice engrafted with peripheral blood cells from patients with leukemia. Three intravenous injections of the Fcm–drug conjugate over a 10-day period were well tolerated and selectively killed the human CLL cells but not the coengrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcmR. FcmR- targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof of concept for FcmR as a valuable therapeutic target in CLL and for IgM-based antibody–drug conjugates as a new targeting platform. Cancer Res; 74(24); 7510–20. 2014 AACR.

Introduction normal cells. The Food and Drug Administration (FDA) On the basis of their ability to selectively deliver highly approval of brentuximab vedotin for the therapy of Hodgkin lymphoma and anaplastic large cell lymphoma in 2011 and of cytotoxic drugs into tumor cells, antibody–drug conjugates þ (ADC) are among the most promising next-generation anti- trastuzumab emtansine for HER2 metastatic breast cancer in body therapeutics for cancer therapy (1, 2). The promise of 2013 were milestones that established the therapeutic utility of ADCs is the targeted delivery of a potent cytotoxic drug ADCs (3, 4). Brentuximab vedotin consists of a chimeric selectively into tumor cells, thereby minimizing toxicity toward mouse/human anti-human CD30 monoclonal antibody (mAb) in IgG1 format as the carrier protein conjugated to monomethylauristatin E (MMAE) as the cytotoxic payload (5). MMAE is a synthetic antitubulin agent active at subnanomolar 1Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland. 2Chemical Biology Laboratory, Molecular Discovery concentrations. Each MMAE drug is linked to the antibody Program, Center for Cancer Research, National Cancer Institute, NIH, molecule through a linker that harbors a valine–citrulline– 3 Frederick, Maryland. Laboratory of Viral Diseases, National Institute of para-aminobenzylcarbamate (Val–Cit–PABC) linker that is Allergy and Infectious Diseases, NIH, Bethesda, Maryland. 4Experimental Transplantation and Immunology Branch, Center for Cancer Research, cleaved by lysosomal proteases such as cathepsin B. The linker National Cancer Institute, NIH, Bethesda, Maryland. 5Department of Can- also contains a maleimide group that reacts with thiol groups cer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, in the IgG1 hinge region. This random conjugation results in an Florida. 6Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida. ADC mixture with an average drug-to-antibody ratio (DAR) of 4:1 (range, 0:1 to 8:1). Trastuzumab emtansine is based on the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). humanized anti-human HER2 mAb trastuzumab randomly conjugated to an average of 3.5 maytansinoid drugs through B. Vire and M. Skarzynski contributed equally to this article. the e-amino group of lysine (Lys) using a noncleavable linker Corresponding Authors: Adrian Wiestner, Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Building 10, CRC (6). This new generation of ADCs has demonstrated two 3-5140, 10 Center Drive, Bethesda, MD 20892-1202. Phone: 301-594- important treatment advances; first, patients who relapse or 6855; Fax: 301-496-8396; E-mail: [email protected]; and Christoph are refractory to first-line therapy can be rescued using tar- Rader, Department of Cancer Biology, Department of Molecular Thera- peutics, The Scripps Research Institute, Scripps Florida, 130 Scripps Way geted cytotoxic drug delivery; and second, an ADC can replace #2C1, Jupiter, FL 33458. Phone: 561-228-2053; Fax: 561-228-2169; systemic cytotoxic therapy demonstrating higher efficacy with E-mail: [email protected] lower toxicity (7, 8). doi: 10.1158/0008-5472.CAN-14-2030 Chronic lymphocytic leukemia (CLL), the most common 2014 American Association for Cancer Research. leukemia in Western countries, is characterized by the

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accumulation of mature monoclonal B cells in the blood, bone more readily undergo apoptosis. FcmR is overexpressed on the marrow, spleen, and lymph nodes. The median age at diagnosis CLL B cells compared with B cells from normal donors and is 75 years, precluding use of allogeneic hematopoietic stem compared with the nonclonal T cells in the blood of patients cell transplantation, the only curative treatment option, in the with CLL. FcmR may play a role in the pathogenesis of CLL, majority of patients. Current first-line treatment is chemoim- possibly by contributing to concomitant BCR and Toll-like munotherapy with an anti-CD20 mAb and an alkylating agent receptor (TLR) activation that could enhance leukemic cell with or without the addition of a purine analog (9–11). One of proliferation and survival (26, 31, 32). However, exactly what the most commonly used regimens for younger patients is the functional role, if any, FcmR has in the pathogenesis of CLL combination of fludarabine, cyclophosphamide, and rituximab remains to be defined. (FCR; refs. 11, 12). Although initial response rates are high, Fc receptors are highly effective in targeting specific mole- most patients relapse. FCR is less active in high-risk disease, cules for binding to and internalization into select target cells. less tolerable in elderly or frail patients, and increases the risk The existence of a variety of distinct Fc receptors with restrict- of infections. Fludarabine and cyclophosphamide are also toxic ed cellular expression provides the basis for selective targeting for normal T cells and myeloid cells, leading to often long- approaches. For example, FcgRIII on dendritic cells has been lasting cytopenias. In contrast, the anti-CD20 mAbs rituximab, used for internalization of vaccine components (33). However, ofatumumab, and obinutuzumab selectively target B cells (13). delivering cytotoxic agents through Fc receptor–mediated These mAbs induce cell death mostly through immunologic internalization as anticancer therapy has not been established. effector mechanisms such as complement-dependent cytotox- In previous work, we have shown that FcmR on CLL cells is icity and antibody-dependent cellular cytotoxicity (13, 14). As functional, rapidly internalizes IgM, and delivers it to the single agents, the efficacy of anti-CD20 mAbs in CLL is limited lysosome where it is degraded (26). In fact, more than 50% of and they are used primarily in combination regimens. Notably, IgM bound to the CLL cell surface is internalized via FcmR CD20 is expressed at higher levels on normal B cells than on within 1 minute, and internalization is complete by 5 minutes. CLL cells (15), and anti-CD20 mAbs effectively kill normal B We hypothesized that the overexpression of FcmR on CLL cells cells, potentially contributing to life-threatening viral, bacte- and its ability to internalize bound IgM could be used for rial, and fungal infections (16). Recently, kinase inhibitors that targeted delivery of antileukemic therapy. target B-cell receptor (BCR) signal transduction have shown Here, we describe the derivation of an FcmR-targeting strat- impressive clinical activity in CLL (17–19). However, while well egy that can deliver a cytotoxic payload selectively into CLL tolerated as single agents, these inhibitors achieve mostly cells and show that it has antitumor activity against primary partial responses and have to be taken continuously. In fact, human tumor cells in vivo. On the basis of the work by even after years of treatment with a kinase inhibitor, residual Kubagawa and colleagues (23), we knew that binding of IgM disease is easily detected in virtually all patients and resistant to FcmR critically depends on the integrity of sequences in the clones emerge that lead to relapse. Thus, there is a great clinical Fc region of IgM (Fcm) in particular the Cm2-4 domains. We need for targeted cytotoxic agents that could be combined with therefore engineered a protein scaffold consisting of Fcm and kinase inhibitors to eradicate the disease. chose a site-directed conjugation technology we have previ- ADCs are also currently being tested as treatment options ously developed (34, 35). In this approach, the introduction of a for CLL. A phase II clinical trial (NCT01461538) is investigating C-terminal selenocysteine (Sec) makes site-specific as opposed þ brentuximab vedotin in CD30 malignancies, including to random conjugation of the payload possible, thereby pre- CLL. In addition, a recently launched phase I clinical trial serving the structure of critical protein domains. (NCT01290549) is based on an analogous ADC that targets CD79B of the BCR complex in non-Hodgkin lymphoma and Materials and Methods CLL. Notably, none of the cell-surface antigens targeted by Supplementary Materials and Methods describe the (i) ADCs in clinical trials are overexpressed in CLL. On the cloning, expression, and purification of Fcµ-Sec; (ii) conju- contrary, CD79B was not detected on the tumor cells of 43% gation of Fcµ-Sec; (iii) SDS-PAGE, Western blotting, and of the patients with CLL tested, while it is expressed on normal gel filtration chromatography; (iv) synthesis of MMAF com- B cells (20), and CD30 is expressed at higher levels on activated pounds 1a and 1b; and (v) comparison of treatment naïve and normal B and T cells compared with CLL cells (21, 22). Thus, previously treated patients. the current panel of clinically investigated ADCs for CLL does not bypass immunosuppression, underscoring the need for Primary cells and cell lines pursuing new ADCs that selectively target CLL. With written informed consent, peripheral blood was col- The Fc receptor for IgM (FcmR), also known as TOSO or Fas lected from patients with treatment-na€ve CLL at the Clinical apoptotic inhibitory molecule 3 (FAIM3), is highly expressed on Center, National Institutes of Health (Bethesda, MD; www. CLL cells (23–26). FcmR is an approximately 60-kDa type I clinicaltrials.gov identifier NCT00923507). Peripheral blood single-pass transmembrane protein whose expression in nor- mononuclear cells (PBMC) were isolated by gradient centri- mal human cells and tissues is virtually restricted to the fugation (Lymphocyte Separation Medium; MP Biomedicals) lymphoid lineage (23, 27). FcmR-deficient mice are viable and and cultured in serum-free AIM-V medium (Life Technolo- exhibit normal development (28–30). However, they have gies). Mantle cell lymphoma (MCL) cell lines Mino, JeKo-1, and reduced numbers of marginal zone B cells (29), and FcmR- HBL-2 were grown in RPMI-1640 medium (Life Technologies) deficient B cells are less responsive to BCR stimulation and and HeLa cells in DMEM (Lonza) supplemented with 10% (v/v)

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fetal bovine serum (FBS; HyClone), 2 mmol/L L-glutamine, Ex vivo cytotoxicity assays 1,000 U/mL penicillin G, and 100 mg/mL streptomycin (all Cytotoxicity toward primary malignant B cells and primary from Life Technologies). normal T cells ex vivo was measured by flow cytometry using TO-PRO-3 and Annexin V staining. Briefly, cryopreserved Confocal immunofluorescence microscopy PBMC samples from 30 different patients with CLL (5 105 We monitored Fcm–Sec internalization as previously cells in 100-mL serum-free AIM-V medium) were incubated described (26). HeLa cells stably transfected with FcmR were with 100 nmol/L compound 1a, Cm2–Cm3–Cm4–Sec/com- grown on coverslips, incubated for 15 minutes at 4 C with pound 1b conjugate, Cm2–Cm3–Cm4–Sec alone, or were left Cm3–Cm4–Sec, washed with ice-cold PBS, and incubated at untreated for 1 hour at 37C, washed and then cytotoxicity 37 C with DMEM (Lonza) for 30 or 120 minutes. Cells were against malignant B cells and normal T cells was evaluated by then fixed with 3% (w/v) paraformaldehyde (Electron Micros- flow cytometry using PE-conjugated Annexin V, a PE–Cy5– copy Sciences), washed with PBS, and incubated in Staining conjugated mouse anti-human CD19 mAb, a FITC-conjugated Buffer [0.05% (w/v) saponin, 10 mmol/L glycine, and 5% (v/v) mouse anti-human CD3 mAb (all from BD Biosciences), and FBS in PBS] for 15 minutes. Cells were costained with rabbit TO-PRO-3 (Invitrogen) according to the manufacturer's anti-human LAMP-1 polyclonal antibodies (pAb; Abcam) for 1 instructions. hour, washed twice with PBS, and incubated with donkey anti- human Fcm pAbs conjugated to DyLight 488 and donkey anti- Circulatory half-life rabbit IgG pAbs conjugated to Cy5 (all from Jackson Immu- Two groups of two NOD/SCID/IL-2Rgnull (NSG) mice (JAX noResearch Laboratories). Cells were then washed twice with strain 5557; The Jackson Laboratory) were injected i.v. (tail PBS and labeled for 5 minutes at room temperature with vein) or i.p. with 5 mg/kg of the Fcm–carrier protein (Cm2–Cm3– 1 mg/mL Hoechst 33258 (Life Technologies) diluted in Staining Cm4–Sec). One control mouse was injected with PBS. Blood Buffer. Subsequently, coverslips were washed twice with PBS, was collected via the tail vein at 0.5, 24, 48, and 72 hours after mounted with Fluoromount-G (SouthernBiotech), and visual- injection and serum was isolated. Sera were diluted 25-fold in ized by confocal microscopy. Images were acquired using a 1% (w/v) BSA in PBS and analyzed by sandwich ELISA. To do Leica TCS SP5 laser scanning confocal microscope (LAS AF this, 100 ng of rabbit anti-human Fcm pAbs (Jackson Immu- software) using the HCX PLAPO 63X objective (numerical noResearch Laboratories) was coated on a 96-well Costar 3690 aperture, 1.4). Images were processed with Adobe Photoshop plate (Corning). After blocking with 3% (w/v) BSA in PBS, the and analyzed using the same settings. diluted sera or purified Cm2–Cm3–Cm4–Sec as standard were added to duplicate wells, washed 10 times with PBS, and Flow cytometry incubated with a mouse anti-His6 mAb conjugated to horse- Mino, JeKo-1, and HBL-2 cells were stained with Cm3–Cm4– radish peroxidase (GenScript). All steps were carried out for 1 Sec/DyLight 488 or Cm2–Cm3–Cm4–Sec/DyLight 488. FcmR hour at 37C. The plate was washed 10 times with PBS and expression in cryopreserved PBMC samples from 30 patients colorimetric detection was performed using 2,20-azino-bis(3- with CLL was measured with a mouse anti-human FcmR mAb ethylbenzthiazoline)-6-sulfonic acid (ABTS; Roche) as sub- (Abnova) and a FITC-conjugated goat anti-mouse secondary strate according to the manufacturer's instructions. Absor- antibody (Sigma-Aldrich) as previously described (26). B cells bance values were measured at 405 nm using a Victor3 plate and normal T cells were distinguished with an APC-conjugated reader (PerkinElmer). mouse anti-human CD19 mAb and a PE-conjugated mouse anti-human CD3 mAb (BD Biosciences), respectively. All sam- NSG/CLL xenograft model ples were analyzed using a FACSCanto II flow cytometer The NSG/CLL xenograft model was first introduced by (Becton-Dickinson) and FlowJo software (TreeStar). Bagnara and colleagues (36). We used a modified protocol established in our laboratory that replicates important aspects In vitro cytotoxicity assays of CLL biology and that we have successfully used in drug Cytotoxicity toward MCL cell lines in vitro was measured by studies (37). Mice were housed and handled in accordance with using the CellTiter 96 AQueous Cell Proliferation Assay (Pro- the guidelines set by the Animal Care and Use Committee of mega). Briefly, Mino or HBL2 cells were distributed in a 96-well the National Heart, Lung, and Blood Institute (Bethesda, MD). flat-bottomed plate (Corning) at a density of 5 104 cells per All animal experiments were carried out on an approved well. Subsequently, compound 1a (0.001–1,000 nmol/L) or animal protocol. Thirty NSG mice were preconditioned with Fcm–Sec protein selectively conjugated to compound 1b (1– an i.p. injection of 25-mg/kg busulfan (Otsuka America Phar- 100 nmol/L) were added to the cells. In parallel, unconjugated maceutical) and on the following day (day 1) i.v. injected with Fcm–Sec protein (1–100 nmol/L) was also tested. The cells were 5 107 PBMC from 1 out of 4 patients with CLL. All mice were exposed to the compound 1a and 1b reagents for 1 hour at bled on day 4 and PBMC were isolated by gradient centrifu- 37 C, washed three times with RPMI-1640 medium, and incu- gation using Lymphocyte Separation Medium. On day 4, 6, and bated for an additional 71 hours at 37 C. After adding 20-mL 8, different cohorts of mice were i.v. injected with 10 mg/kg Assay Reagent to each well, the cells were further cultured for 3 Cm2–Cm3–Cm4–Sec carrier protein or Cm2–Cm3–Cm4–Sec/ hours at 37 C and the absorbance at 490 nm was measured compound 1b conjugate or an equal volume of PBS. On day using a SpectraMax M5 microplate reader with SoftMax Pro 11, retro-orbital puncture bleeds and spleens were harvested software. Data were computed as mean SD of triplicates. from all mice. PBMC were isolated as above. Spleens were

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homogenized using the gentleMACS Dissociator (Miltenyi and Fcm–stop are anticipated to form mixed multimers. The Biotec). The homogenate was filtered through a 70-mm nylon respective Fcm–Sec proteins were generated in human embry- cell strainer (BD Biosciences) and washed with ACK Lysing onic kidney (HEK) 293E cells and purified using the His6 tag. Buffer (Quality Biological) to remove erythrocytes and plate- Because of pentamerization and hexamerization of IgM in the lets. PBMC and splenocytes were stained with a mouse anti- absence of the J chain (38, 39), Fcm comprises 10 and 12 human CD45 mAb conjugated to V450, a mouse anti-human polypeptide chains, respectively. Thus, based on a 10:1 ratio CD19 mAb conjugated to PE-Cy5, a mouse anti-human CD3 of terminated polypeptide chains to polypeptide chains having mAb conjugated to FITC, PE-conjugated Annexin V (all from a C-terminal Sec residue, the dominant product of our expres- BD Biosciences), and TO-PRO-3 (Life Technologies), and then sion cassette contains a single Sec (Fig. 1A and B). analyzed by flow cytometry as described above. Normalized Both Coomassie staining and Western blotting after reduc- titers of human lymphocyte populations in PBMC isolated on ing SDS-PAGE revealed an approximately 50-kDa Cm2–Cm3– days 4 and 11 were determined with 5.0- to 5.9-mm AccuCount- Cm4–Sec monomer. In contrast, after nonreducing SDS-PAGE, Blank Particles (Spherotech) according to the manufacturer's high molecular weight multimers similar to whole human IgM instructions. were visualized (Supplementary Fig. S1). Using gel filtration chromatography (data not shown), Cm2–Cm3–Cm4–Sec multi- Results mers revealed a broad peak corresponding to 500 to 600 kDa, Generation and characterization of Fcm–Sec likely reflecting a mixture of pentamers (500 kDa) and To be able to target a payload to FcmR, we first generated hexamers (600 kDa). The Cm3–Cm4–Sec monomer revealed IgM-derived protein scaffolds consisting of two (Cm3–Cm4) or the expected lower molecular weight (40 kDa; Supplemen- three (Cm2–Cm3–Cm4) of the C-terminal constant domains of tary Fig. S1A), with a diminished propensity to form stable and human IgM. In the expression plasmids, the Fcm-encoding defined multimers (Supplementary Fig. S1B and data not sequence was followed by a TGA stop codon, a sequence shown). Indicating successful incorporation of Sec (35), the encoding a hexa-histidine (His6) tag, a TAA stop codon, and His6 tag was detected in both purified Fcm–Sec proteins finally a downstream Sec incorporation sequence (SECIS). In (Supplementary Fig. S1C). the presence of SECIS, the TGA stop codon instructs the Binding, internalization, and trafficking of human IgM by translational insertion of Sec (Fig. 1A and B). Because termi- HeLa cells transiently transfected with human FcmR cDNA was nation at the TGA codon competes with and dominates over previously shown by flow cytometry and confocal immuno- Sec insertion, the expression cassette yields a mixture of fluorescence microscopy (26). We confirmed that purified approximately 10% Fcm–Sec and approximately 90% Fcm–stop. Cm3–Cm4–Sec selectively bound to FcmR-expressing, but not As we have previously demonstrated for Fcg–Sec (35), Fcm–Sec wild-type HeLa cells, followed by rapid internalization and

Sec-His Figure 1. Fcm–Sec engineering and 6 structural formulas of auristatin F Cµ3 Cµ4 Sec derivatives. Schematic of the A -His6 SECIS Fcm–Sec expression cassettes, which encode two (A) or three (B) C-terminal constant domains of the heavy chain of human IgM (gray). A C-terminal Sec (red) followed by a His6 tag (black) was introduced by combining a TGA codon (red) with a 30-untranslated 0 region (3 -UTR) that contains a Sec-His6 SECIS element. The dominant Cµ2 Cµ3 Cµ4 Sec product of these expression B -His6 cassettes is pentameric and SECIS hexameric (shown) proteins with a single Sec-His6–displaying C-terminus. C, we synthesized a tertiary butyl ester of the tubulin polymerization inhibitor MMAF (compound 1a) and its maleimidocaproyl derivative (compound 1b) for site-speciﬁc conjugation to the unique selenol C group in Fcm–Sec.

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Fcµ-Sec LAMP1 merge/Hoechst

Figure 2. Binding, internalization, and trafficking of Fcm–Sec. HeLa 0 min cells stably transfected with human FcmR cDNA were incubated with 25 mg/mL Cm3– Cm4–Sec (Fcm–Sec) for 30 minutes at 4C. After washing, the cells were kept at 4C or were incubated at 37C for 30 and 120 minutes. The cells were then fixed, permeabilized, and costained with rabbit anti-human LAMP1 pAbs followed by donkey anti-rabbit IgG 30 min pAbs conjugated to Cy5 (red) and donkey anti-human Fcm pAbs conjugated to DyLight 488 (green). Nuclei were stained with Hoechst 33258 (blue). Stained cells were visualized by confocal immunofluorescence microscopy. Note the colocalization (yellow) of Fcm–Sec and lysosomal marker LAMP1 following internalization. 120 min Scale bars, 10 mm.

trafficking to lysosomes (Fig. 2). Thus, as described previously potent than the freely diffusing drug. Under the same conditions, (23), the Fc fragment of IgM is sufficient for FcmR binding, the Fcm–Sec/MMAF conjugate killed Mino and HBL-2 cells fi fi internalization, and traf cking. We also con rmed the binding with IC50 values of 43 nmol/L and 223 mmol/L, respectively of both purified Fcm–Sec proteins to the FcmR-expressing MCL (Fig. 3B). Thus, FcmR-positive Mino cells were approximately cell line, Mino, using Fcm–Sec proteins that we labeled in a 5,200 times more sensitive to targeted MMAF than FcmR-neg- Sec-dependent reaction with a maleimide derivative of DyLight ative HBL-2 cells. Taking the different sensitivities to free drug 488. Two other MCL cell lines, JeKo-1 and HBL-2, which do not into account, we observed a 1,000-fold increase in potency when express FcmR, did not bind Fcm–Sec/DyLight 488 (Fig. 3A and FcmR, the target of the carrier protein, was present. data not shown). Ex vivo and in vivo investigations of Fcm–drug conjugate Generation and in vitro investigations of Fcm–drug Next, we tested whether the Fcm–Sec/MMAF conjugate can conjugate selectively kill primary CLL cells ex vivo.FcmR expression on As the cytotoxic payload, we chose the tubulin polymeriza- cryopreserved PBMC from 30 patients with CLL (Supplemen- tion inhibitor monomethylauristatin F (MMAF). MMAF differs tary Table S1) was measured by flow cytometry. In patients from MMAE (the drug component of brentuximab vedotin) by with CLL, there are very few or no normal B cells in circulation; þ having a C-terminal phenylalanine (40). As free drug control, therefore, >99% of CD19 cells are CLL cells (31). As expected, þ þ we synthesized a tertiary butyl ester of MMAF (MMAF-tBu; virtually all CLL cells (CD19 ) and much fewer T cells (CD3 ) compound 1a), which freely diffuses into cells through the expressed FcmR (Fig. 4). These findings are consistent with plasma membrane (40), and for Fcm–Sec conjugation, we previously published observations that FcmR is overexpressed synthesized a maleimide derivative with a stable alkyl linker in CLL and that some T cells, especially when activated, can (maleimidocaproyl–MMAF–tBu; compound 1b; Fig. 1C). also express FcmR (23), albeit at low cell-surface levels (24). To test the potency and specificity of free versus conjugated FcmR expression was equally high in treatment-na€ve and drug, we used MCL cell lines Mino (FcmR-positive) and HBL-2 previously treated patients (Supplementary Fig. S4), which (FcmR-negative; Fig. 3A). After exposure for 1 hour and chase indicates for wide clinical applicability of the FcmR-targeting incubation for 71 hours, the free drug potently killed both Mino strategy. and HBL-2 cells with IC50 values of 0.26 and 1.35 nmol/L, respec- The same 30 CLL PBMC samples were then treated in tively (Fig. 3A). As expected, the Fcm–drug conjugate was less parallel with 100-nmol/L free MMAF or the Fcm–Sec/MMAF

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A B

FcµR

p97

Free drug Fcµ-Sec/drug IC = 0.26 nmol/L IC = 43 nmol/L 50Mino 50Mino IC = 1.35 nmol/L IC50 = 223,000 nmol/L 50HBL-2 HBL-2 Cytotoxicity (%) Cytotoxicity (%) Cytotoxicity

Concentration (nmol/L) Concentration (nmol/L)

Figure 3. In vitro cytotoxicity of Fcm–drug conjugate. A, FcmR is expressed in MCL cell line, Mino, but not HBL-2, as previously shown (26). Western blot analysis of cell lysates probed with anti-FcmR and p97 for loading control (inset). Viability of Mino (red circles) and HBL-2 cells (purple squares) exposed to free MMAF compound 1a was determined using a colorimetric assay. B, the same experiment was carried out with MMAF compound 1b conjugated to Cm2–Cm3–Cm4–Sec (Fcm–Sec/drug). conjugate for 1 hour, washed, and then analyzed for cell stantial support to the idea that Fcm–drug conjugates can be death 47 hours later by ﬂow cytometry using TO-PRO-3 and used to selectively deliver cytotoxic payloads to target cells Annexin V staining. Clearly, malignant B cells were effec- without harming bystander cells (Fig. 5B). The Fcm–drug tively killed by both free and targeted MMAF, whereas conjugate was equally effective against tumor cells with normal T cells were only killed by free MMAF (Fig. 5A). In high-risk molecular features (expression of an unmutated fact, the average viability of CLL B cells was reduced from IGVH gene and/or a deletion of chromosome 17p) and those >90% in controls to <20% in Fcm–drug–treated cultures, without these characteristics (Fig. 5C and D). while T-cell viability was unchanged. The Fcm–Sec carrier In preparation for the investigation of the Fcm–drug conju- protein alone did not affect cell viability. This exquisitely gate in vivo, we assessed the circulatory half-life of the Fcm–Sec selective cytotoxicity and the stark differential expression of carrier protein in NSG mice. After i.v. or i.p. injection of 100 mg FcmR between malignant B cells and T cells provide sub- (5 mg/kg) Fcm–Sec in 100-mL isotonic saline, the serum

A B P < 0.0001 100 100 100 T cells CLL cells 80

40 5.3% 97.6% 20 Count (% Count (% of max)

0 0 Specific expression (%) 100 2 103 104 105 100 2 103 104 105 0 T cells CLL cells FcµR (FITC)

Figure 4. Cell-surface expression of FcmR on PBMC samples from patients with CLL. PBMC samples from 30 untreated patients with CLL were analyzed for þ cell-surface expression of FcmRbyﬂow cytometry. A, histograms comparing FcmR expression (red) by CD19 malignant B cells (right) and autologous þ þ CD3 T cells (left) of a representative sample. The percentages of FcmR cells are indicated in red. The isotype control is shown in blue. B, the speciﬁc þ þ expression of FcmR by CD3 T cells and CD19 malignant B cells is plotted for all 30 PBMC samples (for color-code and patient characteristics; see Supplementary Table S1). A paired two-tailed t test was used to calculate P.

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P < 0.0001 P = 0.32 A 100 100

80 80

60 60

P = 0.56 40 P = 0.92 40 Dead T cells (%) Dead T cells Dead CLL cells (%) Dead CLL 20 20

0 0 Control Fcµ-Sec Free drug Fcµ-Sec/drug Control Fcµ-Sec Free drug Fcµ-Sec/drug

B C P = 0.23 D P = 0.29 100 100 100 R 2 = 0.9627 80 P < 0.0001 80 80

60 60 60

40 40 40 Dead cells (%) Dead cells 20 20 20 Dead CLL cells (%) Dead CLL Dead CLL cells (%) Dead CLL

0 0 0 0 20406080100 no del17p del17p M-CLL U-CLL + FcµR cells (%) FISH IGHV

Figure 5. Ex vivo cytotoxicity of Fcm–drug conjugate. PBMC from 30 patients with CLL were left untreated (control) or treated with (i) 100 nmol/L Cm2–Cm3–Cm4– Sec (Fcm–Sec), (ii) 100 nmol/L free MMAF compound 1a (free drug), or (iii) 100 nmol/L MMAF compound 1b conjugated to Cm2–Cm3–Cm4–Sec þ (Fcm–Sec/drug). Cell viability was determined by ﬂow cytometry using TO-PRO-3 staining. A, Fcm–Sec/drug conjugate-mediated cytotoxicity of CD19 þ malignant B cells (left) and autologous CD3 T cells (right) in PBMC samples from patients with CLL (Supplementary Table S1). Paired two-tailed t tests were used to calculate P. B, cytotoxicity of the Fcm–Sec/drug conjugate and expression of FcmR are highly correlated (Spearman correlation, r ¼ 0.8431 and P < 0.0001). C and D, the Fcm–Sec/drug conjugate is equally effective against tumor cells with adverse prognostic features. C, comparison of cytotoxicity against cells with or without a deletion of chromosome 17p (del17p and no del17p, respectively). D, comparison of cytotoxicity against cells of the IGHV-mutated (M-CLL) and of the IGHV-unmutated CLL subtype (U-CLL).

concentration was measured by a sandwich ELISA. Regardless terminally bled by retro-orbital puncture and spleens were of the route of injection, the peak serum concentration ranged harvested. PBMC prepared from mouse blood before (day 4) between 8 and 10 mg/mL and the circulatory half-life was and 72 hours after the last injection of the Fcm–drug conjugate approximately 18 hours (Supplementary Fig. S2). In compar- (day 11) were stained with anti-human CD45 (to identify ison, the reported circulatory half-life of mouse IgM in mice human cells), CD19, and CD3 mAbs to distinguish malignant þ þ þ þ ranges between 24 and 30 hours (41). Thus, despite their B cells (CD45 CD19 ) and normal T cells (CD45 CD3 ). The differences in molecular weight and composition, Fcm–Sec viability of the xenografted human cells was assessed by flow and IgM share similar circulatory half-lives. cytometry using Annexin V and TO-PRO-3 staining. We chose the recently described NSG/CLL xenograft model To quantify the reduction in tumor burden on treatment to evaluate the activity of the Fcm–drug conjugate in vivo.In with the Fcm–Sec/MMAF conjugate, we measured a number of these immunodeficient mice, xenografted human CLL cells different endpoints. First, we determined the leukemic cell and the coinjected autologous T cells engraft in the murine count in the blood using flow cytometry and counting beads. blood and spleen (36, 37). Thirty NSG mice were precondi- There was a substantial decrease in circulating malignant B tioned with busulfan and on the following day (day 1) i.v. cells in treated mice compared with controls (Fig. 6A). The injected with 5 107 PBMC (typically composed of 90% average leukemic cell count over the treatment period of 1 tumor cells, and up to 10% T cells). Each set of mice was week decreased by 74.4 6.6 compared with untreated mice injected with cells from 1 out of 4 different patients with CLL (P < 0.0001). In contrast, there was no change in the number of (Supplementary Table S1). On days 4, 6, and 8 half of the mice circulating T cells (Fig. 6A). To be able to quantify the tumor were i.v. injected with 10 mg/kg Fcm–Sec/MMAF conjugate burden in the tissue, we determined the number of live and the other half were left untreated. On day 11, mice were malignant B cells relative to the total number of nucleated

7516 Cancer Res; 74(24) December 15, 2014 Cancer Research

Targeted Cytotoxic Drug Delivery through the Fcm Receptor

P = 0.96 A P < 0.0001 P = 0.23 B P < 0.0001

80 20 10 2.0

8 60 15 1.5 6 40 10 1.0 4 Live T cells/ Live T 1 µL blood 1 µL 20 blood 1 µL 0.5 Live T cells/ Live T 5 Live CLL cells/ 2 Live CLL cells/ total spleen cells (%) total spleen cells (%) 0 0 0 0.0 Control Fcµ-Sec/ Control Fcµ-Sec/ Control Fcµ-Sec/ Control Fcµ-Sec/ drug drug drug drug

C 40 40 20 20 0 0 –20 –20 –40 –40

Change in –60 Change in –60 T cell count (%) tumor burden (%) –80 –80 –100 –100 Blood Spleen Blood Spleen CLL cells T cells

Figure 6. In vivo cytotoxicity of the Fcm–Sec/drug conjugate. Thirty NSG mice were injected i.v. on day 1 with 5 107 PBMC from four different patients with CLL and treated on days 4, 6, and 8 with 10 mg/kg Fcm–Sec/drug conjugate in PBS or with PBS alone (control) by i.v. injection. On day 11, the absolute þ þ numbers of live (TO-PRO-3–negative) CD19 malignant B cells and autologous CD3 T cells in blood and spleen were quantified by flow cytometry. Two-way ANOVA was used to compare cell numbers in treated and control mice. A, the leukemic cell count in the blood of the xenografted NSG mice was þ significantly reduced by Fcm–Sec/drug, while there was no change in T-cell numbers. B, live CD19 malignant B cells relative to the total number of live nucleated cells in the spleen were also greatly reduced by Fcm–Sec/drug. Again, T cells were unchanged. Note that while the human cells engraft in the spleen, the majority of nucleated cells are of murine origin. C, the column graph summarizes the effect of the Fcm–Sec/drug conjugate on tumor burden (left) and T cells (right). Shown is the mean SEM. cells in single-cell suspensions prepared from spleens. The natural ligand and not a mAb. In other respects, our Fcm–drug majority of these nucleated cells are murine cells that have conjugate is built on important design principles shared with been used successfully as internal reference in other studies the recently FDA-approved ADCs brentuximab vedotin and (36, 37). The tumor burden in spleens decreased on average trastuzumab emtansine (1–8). Specifically, the target antigen by 64.9 9.7 on treatment with the Fcm–Sec/MMAF conjugate FcmR is overexpressed on tumor cells and rapidly internalized (P < 0.0001) but again there was no effect on T-cell numbers upon ligand binding; the Fcm carrier protein was engineered (Fig. 6B). The unconjugated Fcm–Sec carrier protein alone had with a C-terminal Sec residue for site-specific conjugation to no effect on the viability of CLL cells nor T cells (Supplementary the potent antitubulin agent MMAF; finally, use of a nonclea- Fig. S3A and S3B). Taken together, these in vivo studies revealed vable linker that can minimize systemic drug release is possible substantial reductions of the total tumor burden in both blood because internalized FcmR travels to the lysosome where the (74%) and spleens (65%) of mice treated with Fcm–drug carrier protein is degraded releasing the cytotoxic payload. conjugate (Fig. 6C). Consistent with the desired selectivity of The resulting Fcm–Sec/MMAF conjugate selectively killed this targeted approach, there was no effect on T cells neither in FcmR-expressing malignant B cells in vitro, ex vivo, and in vivo. blood nor spleen (Fig. 6C). Notably, none of the mice showed Accordingly, our study provides proof-of-concept for FcmRas signs of toxicity, and the viability of murine blood cells was not a therapeutic target in CLL and for Fcm carrier proteins as reduced in treated as compared with control mice (data not new targeting devices. shown). As the carrier protein, we tested two formats of the Fc fragment of IgM carrying a C-terminal Sec residue. Both Cm3–Cm4–Sec and Cm2–Cm3–Cm4–Sec were expressed, puri- Discussion fied, and conjugated in high yield, and both mediated selective FcmR is overexpressed in CLL and mediates the rapid drug delivery in vitro. However, for the subsequent ex vivo and in internalization of IgM by malignant B cells and its trafficking vivo studies, we focused on the larger Cm2–Cm3–Cm4–Sec, as it to lysosomes (24–26). In this study, we used an Fcm–drug more closely resembled IgM with respect to the formation of conjugate to exploit FcmR for targeted drug delivery. In con- stable pentamers and hexamers, which are required for high- trast to conventional ADCs, our targeting platform is a recom- affinity binding to the target molecule (23). Despite their binant protein scaffold designed to mimic binding of the differences in molecular weight and composition, Cm2–Cm3–

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Vire et al.

Cm4–Sec and IgM shared similar circulatory half-lives (1day), human lymph nodes. Thus, the model allows testing of potency which are relatively short compared with IgG (>7 days), but long and selectivity of the Fcm–drug conjugate in a partially human- compared with scFv and other antibody fragments (<1 hour). ized environment. Furthermore, variability in tumor biology Although providing sufficient time for on-target toxicity, a among individual patients is, at least partially, reproduced circulatory half-life of approximately 1 day may diminish off- by injecting cells from different patients into separate cohorts target toxicity. We conclude that the pharmacokinetic proper- of mice. A caveat of this model is that the survival of the mice ties of Cm2–Cm3–Cm4–Sec are suitable for targeted drug is not determined by tumor progression and that over time delivery. T-cell expansion dominates and can lead to the demise of The rationale for using our Sec technology (34, 35) was to the animals. We therefore chose the impact of the Fcm–drug enable site-specific as opposed to random attachment of the conjugate on tumor burden as the clinical endpoint. drug payload. Site-specific conjugation minimizes interference Here, we demonstrated effectiveness and selectivity of with functional domains of the Fcm scaffold and has been Fcm–drug conjugates by comparing the effect on malignant shown to increase the therapeutic index compared with con- B cells and normal T cells from patients with CLL side-by- ventional drug conjugation strategies (42, 43). Given a Sec side ex vivo and in vivo. With only three injections of the insertion rate of approximately 10% (35), the dominant fraction Fcm–Sec/MMAF over 1 week, we consistently obtained of the purified Fcm–Sec pentamers and hexamers contains one objective responses quantified as >60%reductionintumor C-terminal Sec residue, affording a drug-to-carrier ratio of 1:1. burden. Notably, the tumor samples studied here were Although this proved to be sufficient for killing malignant B primarily from patients with high-risk disease characterized cells in vitro, ex vivo, and in vivo, it is lower than the ratio of 4:1 by advanced Rai stage and the expression of unmutated considered ideal for ADCs generated using non–site-specific IGHV genes, an indicator of more rapid disease progression techniques (2). Although the lower drug-to-carrier ratio may and reduced benefit from standard treatment. The potent decrease the potency of the current Fcm–drug conjugate, activity of Fcm–Sec/MMAF against tumor cells from these improvements in site-specific conjugation technologies that high-risk patients is promising. Using this partially human- increase the number of drug molecules per carrier could ized xenograft model, we could also verify a degree of further increase the potency of the Fcm–drug conjugate. For selectivity in vivo; while CLL B cells were killed, we did not example, we have recently shown that proteins carrying two observe a decrease in the viability of the autologous T cells. C-terminal Sec residues can be conjugated with two drug This is consistent with the factthatthemalignantBcells molecules (44). overexpress the FcmR while the normal T cells of the patients The linker between carrier protein and payload has to be with CLL as well as the normal B and normal T cells of stable to minimize systemic toxicity while at the same time healthy donors have considerably lower expression levels ensuring effective intracellular drug release. Drug release (24, 26). This suggests that FcmR targeting with ADCs will be from noncleavable linkers, such as the alkyl linker, used here less damaging to normal cells and tissues than current and in trastuzumab emtansine requires antibody degrada- treatment options in CLL. tion within lysosomes (45). The use of this technology in the Collectively, our study provides proof-of-concept for the targeting of FcmRismadepossiblebytheeffectivedeliveryof therapeutic targeting of the recently identified FcmR with a the internalized complex to the lysosome where it is degrad- novel IgM-derived protein–drug conjugate. We established the ed (26, 46). utility of lead components and compositions of Fcm–drug FcmR is the only Ig receptor that exclusively binds IgM conjugates that provide opportunities for further optimization (23). In addition, the polymeric Ig receptor (PIGR) and the of FcmR-targeted drug delivery and translation of this approach Fca/m receptor (FCAMR) bind and internalize both IgM and into the clinic. IgA. Whereas binding of IgA and IgM to PIGR requires the J chain, which is not present in our Fcm–drug conjugates, Disclosure of Potential Conflicts of Interest FCAMR can mediate IgA and IgM binding in the absence C. Rader, T.R. Burke Jr, and J.D. Thomas have ownership interest in the U.S. patent application 20100104510. No potential conflicts of interest were disclosed of the J chain (47, 48). As is the case for FcmR, the expression by the other authors. of FCAMR is virtually restricted to specialized immune cells, including follicular B cells and follicular dendritic Authors' Contributions cells of germinal centers (49). Nonetheless, an assessment Conception and design: B. Vire, M. Skarzynski, A. David, T.R. Burke Jr, C. Rader, of on-target and off-target toxicities of Fcm–drug conjugates A. Wiestner Development of methodology: B. Vire, M. Skarzynski, G. Aue, T.R. Burke Jr, in preclinical and clinical investigations will have to take C. Rader, A. Wiestner both FcmR and FCAMR expression by normal cells into Acquisition of data (provided animals, acquired and managed patients, consideration. provided facilities, etc.): M. Skarzynski, C.G. Nelson, G. Aue, C. Rader, A. Wiestner For the in vivo studies, we chose a recently established Analysis and interpretation of data (e.g., statistical analysis, biostatistics, adoptive transfer model of human primary PBMC from computational analysis): B. Vire, M. Skarzynski, C.G. Nelson, A. David, G. Aue, patients with CLL injected into NSG mice, which recapitulates C. Rader, A. Wiestner Writing, review, and/or revision of the manuscript: B. Vire, M. Skarzynski, key aspects of tumor biology as seen in patients (36, 37). Both C.G. Nelson, T.R. Burke Jr, C. Rader, A. Wiestner malignant B cells and the corresponding autologous T cells of Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): M. Skarzynski the patient engraft in the blood and spleen of the mouse, the Study supervision: C. Rader, A. Wiestner latter demonstrating tumor cell aggregates reminiscent of Other (synthesized reagents used in bioconjugation): J.D. Thomas

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Targeted Cytotoxic Drug Delivery through the Fcm Receptor

Acknowledgments The costs of publication of this article were defrayed in part by the The authors thank Dr. William K. Gillette and his team at the Protein payment of page charges. This article must therefore be hereby marked Expression Laboratory, SAIC-Frederick, for custom expression and puriﬁcation advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this of Fcm–Sec, and Dr. David J. Fitzgerald for helpful comments on the article. fact.

Grant Support This work was funded by the Intramural Research Programs of the National Heart, Lung, and Blood Institute and the National Cancer Institute, NIH, and by Received July 9, 2014; revised September 24, 2014; accepted October 13, 2014; the NIH U01 grant CA174844. published OnlineFirst October 24, 2014.

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7520 Cancer Res; 74(24) December 15, 2014 Cancer Research

Harnessing the Fcµ Receptor for Potent and Selective Cytotoxic Therapy of Chronic Lymphocytic Leukemia

Bérengère Vire, Martin Skarzynski, Joshua D. Thomas, et al.

Cancer Res 2014;74:7510-7520. Published OnlineFirst October 24, 2014.

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