A Bispecific Molecule Targeting CD40 and Tumor Antigen ...€¦ · Research Article A Bispecific Molecule Targeting CD40 and Tumor Antigen Mesothelin Enhances Tumor-Specific Immunity

Research Article

A Bispecific Molecule Targeting CD40 and TumorAntigen Mesothelin Enhances Tumor-SpecificImmunityShiming Ye1, Diane Cohen1, Nicole A. Belmar1, Donghee Choi1, Siu Sze Tan1, Mien Sho1,Yoshiko Akamatsu1, Han Kim1, Ramesh Iyer2, Jean Cabel2, Marc Lake2, Danying Song2,John Harlan2, Catherine Zhang1, Yuni Fang1, Alan F.Wahl1, Patricia Culp1,Diane Hollenbaugh1, and Debra T. Chao1

Abstract

Agonistic CD40 monoclonal antibodies (mAb) have dem-onstrated some clinical activity, but with dose-limiting toxic-ity. To reduce systemic toxicity, we developed a bispecificmolecule that wasmaximally active in the presence of a tumorantigen and had limited activity in the absence of the tumorantigen. LB-1 is a bispecific molecule containing single-chainFv domains targeting mouse CD40 and the tumor antigenmesothelin. LB-1 exhibited enhanced activity upon binding tocell-surface mesothelin but was less potent in the absence ofmesothelin binding. In a mouse model implanted with syn-geneic 4T1 tumors expressing cell-surface mesothelin, LB-1demonstrated comparable antitumor activity as an agonisticCD40mAbbut did not cause elevation of serum cytokines andliver enzymes, as was observed in anti-CD40–treated mice.

The results from our study of LB-1 were used to develop ahuman cross-reactive bispecific molecule (ABBV-428) thattargeted human CD40 and mesothelin. ABBV-428 demon-strated enhanced activation of antigen-presenting cells and Tcells upon binding to cell-surface mesothelin, and inhibitionof cultured or implanted PC3 tumor cell growth after immuneactivation. Although expression of cell-surface mesothelinis necessary, the bispecific molecules induced immune-mediated antitumor activity against both mesothelinþ andmesothelin� tumor cells. ABBV-428 represents a class ofbispecific molecules with conditional activity dependent onthebindingof a tumor-specific antigen, and such activity couldpotentially maximize antitumor potency while limiting sys-temic toxicity in clinical studies.

IntroductionCD40 is a key costimulatorymolecule that functions as amaster

switch for both innate and adaptive immune systems (1–3). Anagonistic CD40monoclonal antibody (mAb) can directly activateand "license" antigen-presenting cells (APC) to prime effectivecytotoxic T-cell responses with limited help from CD4þ Tcells (4–6). Agonist anti-CD40 has been explored in phase Iclinical trials (7–10). Although they have shown efficacy, systemicdose-limiting toxicities, including elevated liver enzymes andcytokine release syndrome (7, 8), have prevented CD40 agonistsfrom achieving full antitumor potency in patients. To lower thesystemic toxicity, intratumoral injection of agonistic anti-CD40

has been studied (11, 12) and elicits antitumor response withlimited systemic toxicity in preclinical studies (13–15). However,due to the logistical complexities of intratumoral dosing in theclinical setting, it is desirable to engineer a tumor-targeted mol-ecule that provides full antitumor potency but exhibits limitedtoxicity, thus enabling systemic dosing.

Tumor-targeted activation has been tested in variousmolecularplatforms to reduce systemic toxicity (16–18). For example,tumor-targeted anti-CD3 bispecific molecules have been investi-gated in preclinical as well as in clinical studies (19, 20). A similartumor-targeting strategy has also been used to generate IL2immunocytokine to achieve tumor-localized activity of IL2 (21).Probody is another tumor-targetedmolecular platform that keepsthe active domain masked by a peptide until it is cleaved withinthe tumor microenvironment by tumor-specific proteases (22). Atumor-targeted CD40 molecule has been tested by conjugating aCD40 agonist mAb chemically with a tumor-homing pep-tide (23). However, there are limited reports about recombinantbispecific molecules targeting both CD40 and tumor antigens toachieve maximal activity in the presence of the tumor-associatedantigen (TAA) and minimal activity in its absence, therefore,inducing conditional CD40 activation. Such a bispecificmoleculewould limit CD40 activation in normal tissues with little or noTAA expression, thus reducing the likelihoodof systemic toxicitiesas a result of CD40 activation. Mesothelin (MSLN) is a target thatexhibits tumor-specific expression. MSLN is a GPI-anchored cell-surface molecule and has low expression on a few normal tissues,such as mesothelial cells lining the pleura, peritoneum, andpericardium (24). In contrast, MSLN has high expression on

1AbbVie Biotherapeutics Inc., Redwood City, California. 2AbbVie Inc., NorthChicago, Illinois.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

J. Cabel is retired.

Current address for A.F. Wahl: Ambrx, San Diego, California; current address forP. Culp: Maverick Therapeutics, Inc., Brisbane, California; and current address forD. Hollenbaugh: Good Therapeutics, Inc., Seattle, Washington.

Corresponding Author: Shiming Ye, AbbVie Biotherapeutics Inc., 1500 SeaportBoulevard, Redwood City, CA 94063. Phone: 650-454-2746; Fax: 650-399-8746; E-mail: [email protected]

Cancer Immunol Res 2019;7:1864–75

doi: 10.1158/2326-6066.CIR-18-0805

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mesotheliomas and carcinomas of the pancreas, lung, and ova-ry (25). Overexpression of MSLN in cancers can elicit humoraland cellular immune responses, likely due to the limited expres-sionof this protein innormal tissues (26–28). Therefore, targetingMSLN together with agonist anti-CD40 may help to enhance theimmunogenicity of MSLN-expressing tumors by inducing activeimmune responses against the tumor rather than normal tissues.

LB-1 andABBV-428 are bispecificmolecules targetingmouse orhuman CD40 and MSLN with single-chain fragment variable(scFv) domain converted from variable chains of an agonisticanti-CD40 and anti-MSLN. The current study provides preclinicalevidence demonstrating the conditional activity of this bispecificmolecule and supports the development of an MSLN-targetedCD40 immune therapy to treat patients with MSLNþ cancers.

Materials and MethodsCells and reagents

HEK-293 (human embryonic kidney; ATCC#CRL-1573), PC-3(human prostate adenocarcinoma; ATCC# CRL-1435), andmouse syngeneic tumor cell line 4T1 (mouse mammary carcino-ma; ATCC#CRL-2539) cells were purchased fromATCC andwerestably transfected to expressmouseor humanCD40orMSLN. Theparental cell lines were authenticated by ATCC and were notauthenticated in the past year. Human CD40 or MSLN gene wascloned into a CHEF1 expression vector (29). One microgram ofvector DNA was transfected into 2.5 � 105 parental cells byFuGENE 6 transfection reagent according to the vendor's instruc-tion (Promega). The cells stably expressing CD40 or MSLN wereselected in the medium containing G418 (0.5 mg/mL, ThermoFisher). All cell lines were tested negative for Mycoplasma andbanked after purchasingor geneticmodifications, andmaintainedin culture for nomore than 10 passages frommaster cell banking.Fresh human peripheral blood mononuclear cells (PBMC) fromhealthy donors (about 109 per donor) were purchased fromAllCells. Donor virus testing results of human immunodeficiencyvirus, hepatitis B virus, and hepatitis C virus were negative.

Antibody against humanMSLN(cloneK1)waspurchased fromSanta Cruz Biotech, CD86 (clone FUN-1) from BD Biosciences,and CD83 (clone HB15e) from eBioscience. The following anti-mouse antibodies were purchased from BD Biosciences: CD45(clone 30-F11), B220 (clone RA3-6B2), CD19 (clone 1D3), CD86(clone GL1), Ly6C (clone AL-21), Ly6G (clone 1A8), and CD11b(clone M1/70). The following molecules were produced atAbbVie (30): agonist anti-mouse CD40 (1C10), agonist anti-human CD40, and anti-MSLN (human and murine cross-reactive), as well as tumor-targeted bispecific molecules LB-1or ABBV-428 with specificities to murine or human CD40 andMSLN. All constructs were subcloned into pHybE vectorsdeveloped at AbbVie (31) and transiently transfected to 293-6Ecells for production. The Fc function was monitored by achromium-51(Cr51) release assay as described previously (32).

AnimalsSix- to 8-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ

(NSG) and BALB/cJ mice (The Jackson Laboratory) werehoused under SPF conditions in an AAALAC accredited facility.Mice were acclimated for 7 days prior to use and were kept on a12/12-hour light/dark cycle with food and water given adlibitum. All animal procedures were reviewed and approvedby the internal Institutional Animal Care and Use Committee(IACUC).

Monitoring immune cell activation in vitroB cells were enriched using a column-free negative selection kit.

Murine B cells were isolated using an EasySep mouse B-cellenrichment kit (STEMCELL Technologies) from single-cell sus-pensions, whichwere prepared by pressing BALB/cmouse spleensthrough 100-mmnylon cell strainer (VWR) using the plunger endof a syringe in RPMI-1640 with 10% FBS. Human B cells wereisolated from human PBMCs using EasySep human B-cell enrich-ment kits (STEMCELL Technologies) according to the vendor'sinstruction. B cellswere cultured in fully definedAIMV serum-freeculture medium (Thermo Fisher Scientific). Human immaturedendritic cells (DC) were derived frommonocytes enriched fromPBMCs by EasySep humanmonocyte enrichment kit (STEMCELLTechnologies) andmaintained in ultralow attachment polystyreneplates (Corning) in StemSpan serum-free medium (STEMCELLTechnologies) supplemented with GM-CSF (10 ng/mL) and IL4(20 ng/mL; PeproTech) at 37�C, 5%CO2 for 6 days. PurifiedB cellsor immature DCs (5� 105/mL) were treated when cultured aloneor coculturedwithequalnumbersof irradiatedHEK293cells or4T1cells with or without MSLN expression. In some experiments,recombinant human MSLN (PeproTech) were mixed in the cellculture at 300 nmol/L. After 48 hours, cells were collected andstained for activation markers with fluorophore-conjugatedantibodies (listed above). The staining was performed onice for 15 minutes followed by washing for 3 times. Theflow-cytometry data were acquired using an LSRFortessa (BDBiosciences) and analyzed using FlowJo (TreeStar Inc.). Thesupernatants of DC cultures (10 mL) were harvested andassayed for IL12p70 production by AlphaLISA (PerkinElmer).B-cell proliferation was determined after treatment for 6 daysby adding 0.5 mCi/well H3TdR (NEN) for the last 16 hours ofculture and measuring H3TdR incorporation as counts perminute by Wallac 1450 MicroBeta scintillation counter.

Human T cells (5 � 104) were purified from PBMCs withEasySep human T-cell enrichment kit (STEMCELL Technologies),cocultured with autologous monocyte-derived DCs (moDC;5 � 103) prepared as described above, and HEK293 cells(5 � 103) with or without MSLN expression. T-cell activationwas monitored by IFNg production in the coculture under dif-ferent treatments for 48 hours. IFNg production wasmeasured byELISPOT assay. Briefly, cells were washed 4 times in Dulbecco'sphosphate-buffered saline (DPBS) and plated in an anti-IFNg–coated PVDF 96-well plate for 24 hours (R&D Systems). IFNgELISPOT was developed per the manufacturer's instructions andwas quantified by a C.T.L. Elispot plate reader (Cellular Technol-ogy Ltd) using ImmunoSpot Version 5.0 software.

In vitro tumor cell lysis assayTo set up the in vitro tumor lysis assay, the human prostate

cancer cell line PC3 was transfected to express different amountsof human MSLN and coexpress green fluorescence protein (GFP)by FuGENE 6 as described previously. In parallel, PC3 cells werealso transduced with a NucLight Red Lentivirus reagent (EssenBioScience) to express red fluorescence protein (dsRed). MSLNexpression on PC3 stable cells was evaluated byQIFIKIT (Agilent)according to themanufacture's instruction, andby IHCusing anti-5B2 (Thermo Fisher) on formalin-fixed paraffin-embedded cellpellets after antigen retrieval and detection by DAB with second-ary polymer kit (DAKO). Based on MSLN expression, the PC3stable cells were defined as PC3-MSLNhi, PC3-MSLNmed, andPC3-MSLNlow according to IHC score 4þ, 3þ, and 2þ. PC3 cells

CD40 Activation by a Tumor-Targeted Bispecific Molecule

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expressing GFP or dsRed with differing expression of MSLN wereplated at 5� 103 cells per well in a 96-well plate for 24 hours priorto the addition of 5 � 103 moDCs and 5 � 104 T cells. The cellswere then treated with antibodies or bispecific molecules (5mg/mL) and incubated for 6 to 7 days inside an IncuCyte ZOOMLive-Cell Analysis System (Essen BioScience), during which liveimages were acquired every 4 to 6 hours to quantify the number ofGFP or dsRed-positive cells. Results were then converted to per-centage of starting seed count and graphed using GraphPad Prism.

Measurement of antitumor activity in animal modelsBALB/cJmice were implanted subcutaneously in the right flank

with 2.5 � 105 4T1 or 4T1-MSLN cells coexpressing GFP. Whenpalpable tumors had formed, mice were randomly assigned todifferent groups based on mean tumor volume (from 50 to100 mm3) and were treated once a week with control murineimmunoglobulin G1 (muIgG1; 5 mg/kg), anti-MSLN muIgG1(5 mg/kg), anti-muCD40 1C10 muIgG1 (1.25, 2.5, or 5 mg/kg),or the surrogate bispecific molecule, LB-1(1.25, 2.5, or 5 mg/kg).

To establish a prophylactic PC3 tumor model, immune-deficient NSG mice were inoculated subcutaneously with a mix-ture of cells including 1 � 107 PC3 or PC3-MSLN tumor cells,1� 106 T cells, and 5� 105moDCs in 100mLDPBS. At the time ofinoculation, mice were dosed intraperitoneally (i.p.) with a mAbagainst human cytomegalovirus as an isotype control, parentalagonistic anti-CD40 or ABBV-428 at 1 mg/kg.

For both prophylactic and syngeneic models, tumor size wasmeasured approximately every 3 to 4dayswith electronic calipers,and tumor volumes calculated using the formula: V ¼ 1/2 �length � width � height. Mice were euthanized when tumorvolumes reached a maximum of 2,500 mm3 or if animal healthwas compromised, per IACUC approved protocol.

In vivoMSLNexpression, liver toxicity, and cytokine productionTo monitor MSLN expression in the 4T1-MSLN model, tumor

tissues were harvested at randomization and were minced in PBSwith a scalpel to obtain�1 to 3 mm3 pieces. Minced tissues weretransferred into 50-mL conical tubes and were digested with10 mL of 0.05% trypsin-EDTA (Thermo Fisher) in each tube ona nutating platform (18 rpm) at 37 C for 30 minutes. Enzymaticdigestion was stopped by adding FBS to 20%. Single-cell suspen-sions were prepared after carefully triturating and flowingthrough a 70-mm cell strainer. MSLN expression was measuredby flow-cytometry analysis on GFPþ cells after stained by fluor-ophore-conjugated anti-MSLN (clone K1). Soluble MSLN in thebloodwasmeasured by ELISA usingMaxDeluxe Set (BioLegend).

Blood samples were collected from mice 24 hours after i.p.dosing. Directmeasurement of alanine aminotransferase (ALT) inblood was performed by comprehensive blood profiling rotor(ABAXIS) per the manufacturer's instructions. Mouse sera wereprepared and mouse cytokines/chemokines in 25 mL serumsamples were assayed using the MILLIPLEX MAP Mouse Cyto-kine/Chemokine Multiplex Assay kit (Millipore), measured on aBio-Rad Bio-Plex 200 system, and analyzed by Bio-Plex Data ProSoftware (Bio-Rad).

Mouse livers harvested at the endof the studywere fixed in 10%formalin and embedded in paraffin, and sections were stainedwith hematoxylin and eosin (H&E) to assess hepatocyte mor-phology and tumor metastasis. Immune cell infiltration wasevaluated by IHC staining with anti-CD45 (clone 30-F11; BD)and rabbit polyclonal anti-IBA1 (Wako) after the antigen retrieval

process. The staining was detected using Rat on Mouse or RabbitHRP-polymer (Biocare Medical) and Envisionþ (DAKO) kitsaccordingly. Images were captured by Aperio AT2 Scanner (Leica)and analyzed by HALO imaging analysis system.

Measurement of immune cell activation in animal modelsIn a subset of mice, blood, spleen, tumor, and tumor-draining

lymph node samples were collected 24 hours after treatment forimmune phenotyping. B-cell counts in blood were measured byflow cytometry using Trucount tubes (BD Biosciences) per themanufacturer's instructions. Draining lymph nodes and spleenswere pressed through 100-mmnylon cell strainer (VWR) using theplunger end of a syringe in RPMI-1640 with 10% FBS. Tumorswere processed using a gentleMACS mouse tumor dissociationkit and gentleMACS Octo Dissociator with Heaters under37C_m_TDK-2 program (Miltenyi Biotec). Red blood cells fromblood and spleen cell suspensions were lysedwith Red BloodCellLysing Buffer Hybri-Max (Sigma-Aldrich). All murine immunecells collected were blocked with 10 mg/mL mouse BD Fc Block(BD Biosciences) for 15 minutes and stained with fluorophore-conjugated antibodies for flow-cytometry analysis as describedabove.

To measure T-cell responses to recall antigens, mouse spleenswere harvested at the end of the study. CD3þ T cells were purifiedby negative selection using EasySep mouse T-cell isolation kit(STEMCELL Technologies), and 1 � 105 T cells were culturedalone or cocultured for 24 hours with 5� 105 irradiated parental4T1 or 4T1-MSLN cells in RPMI-1640 with 10% FBS and 50ng/mL rIL2 (R&DSystems) in triplicatewells in 96-well U-bottomplates (Sigma-Aldrich). Production of IFNg was measured byELISPOT as described above.

Statistical analysisData were analyzed using GraphPad Prism 7 software. Statis-

tical differences were determined to be significant at P � 0.05using one-way (treatment) ANOVA and Duncan multiple rangetests as appropriate. Data were presented as mean � SEM. In allfigures: �, P � 0.05.

ResultsGeneration of bispecific molecules LB-1 and ABBV-428

LB-1 is a recombinant bispecific molecule targeting mouseCD40 and MSLN, and ABBV-428 is an equivalent version target-ing human CD40 and MSLN. Both LB-1 and ABBV-428 containscFv domains constructed from the variable regions of anti-CD40andanti-MSLNas shown in Fig. 1A. Eachmolecule is composedofa homodimer of 2 identical chains covalently linked by disulfidebonds. Each chain incorporates CD40 and MSLN targeting scFvdomains at the N- and C- termini, respectively. The CD40 andMSLN domains are on the opposite ends of each chain, separatedby a hinge region, the Fc part of an antibody, and a short aminoacid linker. The calculatedmolecular weight of LB-1 or ABBV-428is approximately 160 kDa, slightly larger than a typical IgGantibody having a molecular weight of approximately150 kDa (33). Binding of LB-1 or ABBV-428 to CD40 and MSLNwasmeasuredbyflow-cytometry analysis, and results showed thatbinding of the scFv domains within the bispecific molecule totheir target antigens was similar to that of the correspondingparental anti-CD40 and anti-MSLN, except for LB-1, whichshowed lower binding potency (by EC50, half-maximal effective

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concentrations), but similar maximal binding (by MFI, meanfluorescence intensity) to 1C10, the parental anti-mouse CD40(Supplementary Table S1; Supplementary Fig. S1). LB-1 containsa mouse IgG1 constant region, and ABBV-428 contains a humanIgG1 constant region consisting of a V273Emutation that did notinduce antibody-dependent cell-mediated cytotoxicity (ADCC)on CD40þ cells (Supplementary Fig. S1C). Thus, ABBV-428 isfunctionally similar to LB-1 containing themouse IgG1backbone.

LB-1 induces antitumor responses with limited toxicityTo determine the MSLN-dependent conditional activity of

LB-1, mouse B cells were evaluated for activation by LB-1 in thepresence or absence of cell-surface–expressed MSLN. As shownin Fig. 1B, when B cells were cultured withmouse 4T1 tumor cellswithout MSLN expression, LB-1 was less effective than parentalanti-CD40 (1C10; here on referred to as 1C10) in stimulatingB-cell expression of activation markers CD23 and CD86. How-ever, when B cells were cocultured with 4T1 stably overexpressingMSLN (4T1-MSLN), LB-1 was more potent than parental anti-CD40 in activating B cells. Although LB-1 maintained baselineactivity in the absence of MSLN, enhanced CD40 activation uponMSLN binding suggests that LB-1 exhibited MSLN-dependentactivity.

To test whether the MSLN-dependent activity of LB-1 couldprovide a better therapeutic index, LB-1 was evaluated in ananimal model carrying 4T1-MSLN tumors. Like many MSLN-positive human cancers, established 4T1-MSLN syngeneic tumormaintained heterogeneous MSLN expression (SupplementaryFig. S2A), and soluble MSLN in circulation could be detected(Supplementary Fig. S2B), probably due to MSLN shedding fromtumor cells.

In the 4T1-MSLN tumor model, LB-1 was as potent as 1C10 ininhibiting tumor growthwhen dosed at 2.5 or 5.0mg/kg, whereasanti-MSLN or a control antibody with the same mouse IgG1isotype had no antitumor activity (Fig. 2A). Antitumor activitywas not further enhanced when treated with a 1C10 and anti-MSLN combination (Supplementary Fig. S2C). Although both1C10 and LB-1 showed antitumor activity at the dose of 5mg/kg,1C10 resulted in elevated serum ALT, whereas LB-1 maintainedthe same ALT concentrations as the control (Fig. 2B). Livers wereharvested at the conclusion of the study to evaluate inflammationby IHC staining for CD45 (pan-immune cell marker) andIBA1 (ionized calcium-binding adaptor molecule-1, a pan-macrophage marker, as well as an activation marker). Increasedparenchymal andperivascular CD45þ and IBA1þ cells in the liverswere observed in the 1C10-treated mice compared with isotypecontrol-treated mice. In contrast, livers from LB-1–treated micedid not show increased inflammation and had similar basalstaining in the parenchyma as animals treated with controlantibody (Fig. 2C and D; Supplementary Fig. S3A and S3B).Serum cytokines were also monitored 24 hours after dosing(Fig. 2E). Cytokines, including IL6, TNFa, KC (CXCL1), IP-10(CXCL6), and MIG (CXCL9), were significantly elevated in micedosed with 1C10 at 5mg/kg. However, at the same dose, LB-1 didnot enhance cytokine release in the circulation comparedwith thecontrol.

In the 4T1 tumor model without MSLN expression, LB-1 didnot show antitumor activity or systemic toxicity (SupplementaryFig. S4), suggesting that LB-1 only had antitumor activity in micecarrying MSLNþ tumors. The systemic presence of soluble MSLNin 4T1-MSLN model had minimal impact on the conditionalactivity of LB-1. Taken together, these data suggest that LB-1 could

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Figure 1.

LB-1 enhances B-cell activation in the presence of cell-surface–expressed MSLN. A, Schematic diagram of our tumor-targeted bispecific molecule. LB-1 (orABBV-428) is a bispecific molecule with an scFv domain targeting CD40 at N-terminus and an scFv domain targeting MSLN at C-terminus flanking mouse IgG1 Fc(or human IgG1 Fc carrying V273E for ABBV-428). B, B cells were cocultured with 4T1 cells without MSLN expression (4T1) or with MSLN expression (4T1-MSLN),and treated with 1C10, LB-1, or an irrelevant murine antibody as negative control. Expression of CD23 and CD86 on B cells was measured by flow cytometry48 hours later, indicated by mean fluorescence intensity (MFI). Data are representative of three independent experiments.






Figure 2.

Tumor-targeted bispecific molecule LB-1 demonstrates antitumor efficacy with limited toxicity in a mouse syngeneic tumor model. BALB/cJ mice withestablished MSLN-expressing 4T1 tumors were dosed i.p. with LB-1, anti-muCD40 1C10, anti-MSLN, or an irrelevant murine antibody as control onceevery week at the indicated doses. A, Antitumor efficacy was assessed by measuring tumor volumes from each group. For each data point, mean andSEM were plotted. Statistical difference was calculated by one-way ANOVA, and presented as � , P � 0.05 (n ¼ 8). Data are representative of twoindependent experiments. B, Systemic toxicities were monitored by measuring circulating liver enzyme ALT 24 hours after dosing. Data arerepresentative of three independent experiments and were plotted with mean and SEM. Statistical difference was calculated by ANOVA with Duncanmultiple range test and presented as � , P � 0.05 (n ¼ 5). At the end of the study, liver inflammation was monitored by IHC for parenchymal andperivascular CD45þ leukocytes (C) and quantified by HALO imaging analysis (D). Data were plotted with mean and SEM. Statistical difference wascalculated by ANOVA with Duncan multiple range test and are presented as � , P � 0.05 (n ¼ 4). E, Cytokine and chemokine release was monitoredby measuring IL6, TNFa, KC, IP10, and MIG in the circulation 24 hours after dosing. For each bar, mean and SEM were plotted. Statistical differencewas calculated by ANOVA with Duncan multiple range test and presented as �, P � 0.05 (n ¼ 8).

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achieve antitumor potency with reduced systemic toxicity in atumor model expressing cell-surface MSLN.

LB-1 demonstrated enhanced immune activity within tumorTo understand the mechanisms mediating the antitumor

potency with limited toxicity by LB-1 in vivo, immune cell acti-

vation was monitored on cells isolated from blood, spleen,draining lymph nodes, as well as tumors, 24 hours after dosing.In Fig. 3A,more than 90% reduction of peripheral B cells (B220þ)in the blood was observed in the mice treated with 1C10. B-cellreductionby LB-1was about 50%comparedwith the1C10 group,probably due to the lower baseline activity of LB-1 as indicated by

Figure 3.

LB-1 induces tumor-specific immune activation. BALB/cJ mice with established MSLN-expressing 4T1 tumors (n¼ 10) were dosed i.p. with LB-1, anti-muCD401C10, or control (Ctrl) antibody once weekly at 5 mg/kg. 24 hours after the first dose, 5 animals per group were sacrificed. B cells in the blood (A) were counted,and B220þ B cells from spleen (B) and draining lymph nodes (C) were assessed for expression of CD86 by flow-cytometry analysis, indicated by MFI. MDSCs(CD11bþLy6GþLy6Clow) from blood (D) and tumor (E) were quantified and indicated as percentage of total CD45þ cells collected. F, At the end of study (day 31),T cells were purified from spleen and either cultured alone or rechallenged with 4T1 or 4T1-MSLN cells in the presence of 50 ng/mL of IL2 for 24 hours. T-cellactivation was measured by counting colonies of ELISPOT representing IFNg secretion. The data shown are representative from three individual studies. For eachbar, mean and SEMwere plotted. Statistical difference was calculated by ANOVAwith Duncanmultiple range test and presented as � , P� 0.05 (n¼ 5).






in vitro assays (Fig. 1B). The in vivo activity of LB-1 was furthercompared with 1C10 on B cells, as well as on myeloid-derivedsuppressive cells (MDSC; CD11bþLy6GþLy6Clow). Treatmentwith 1C10 enhanced activation marker CD86 expression on Bcells from the spleen and tumor-draining lymph nodes. LB-1treatment induced less B-cell activation in the spleen comparedwith 1C10 (Fig. 3B). However, within tumor-draining lymphnodes, LB-1 was as effective as 1C10 in upregulating CD86 onB cells (Fig. 3C). The differential activity of LB-1was also observedon MDSCs from circulation versus tumors. In the circulation, theproportion of MDSCs within CD45þ cells was reduced by 1C10,but not by LB-1 (Fig. 3D). Within tumor, the proportion ofMDSCs was reduced by both LB-1 and 1C10 treatment(Fig. 3E). These data suggest that LB-1 was less active in peripheraltissues with limited MSLN expression and was as potent as 1C10within tumor microenvironment after engaging with MSLN.

To evaluate whether LB-1 could influence the generation oftumor-specific T cells in vivo after 4 weeks with weekly treatment,CD3þ T cells isolated from spleens were cocultured with irradi-ated 4T1-MSLN or 4T1 parental cell lines, and IFNg secretion wasmeasured by ELISpot. As shown in Fig. 3F, T cells fromboth LB-1–and 1C10-treatedmice producedmore IFNg than did T cells fromcontrol-treated mice in all groups. Upon rechallenge with4T1-MSLN cells, T cells from LB-1 treated mice produced moreIFNg than those from1C10-treatedmice.When rechallengedwithparental 4T1 cells without MSLN expression, T cells from bothLB-1– and 1C10-treated mice enhanced IFNg production simi-larly. These data suggest that LB-1 could initiate T-cell responsesagainst MSLN, as well as other antigens on the tumor cells. Thus,the antitumor immune response induced by LB-1 within thetumor microenvironment was not restricted to MSLN.

ABBV-428 exhibits MSLN-dependent activity in vitroHaving demonstrated that LB-1 with conditional CD40 activity

could achieve antitumorpotencywith limited toxicity in ananimalmodel, we generated ABBV-428, a therapeutic candidate withspecificity to human CD40 and MSLN. The conditional activityof ABBV-428 on CD40 activation was tested in human B cells(Fig. 4A) andmoDC (Fig. 4B) cultured in the presence of HEK293cells with or without MSLN expression. Consistent with knownCD40 biology (1–3), agonistic anti-CD40 induced B-cell andmoDC activation, whereas ABBV-428 induced minimal B-cellactivation and undetectable IL12p70 production from moDCs incocultures with HEK293 cells without MSLN expression. In con-trast, when immune cells were cocultured with HEK293 cellsexpressingMSLN, ABBV-428 induced B-cell proliferation and IL12p70 production frommoDCs (Fig. 4A and B; Table 1). TheMSLN-dependent activity of ABBV-428 was further confirmed by mea-suring the expression of activation markers on B cells and moDCsafter treatments (Table 1). In the absence ofMSLN, ABBV-428 wassignificantly less potent than the anti-CD40 in the induction ofactivationmarkers, as indicatedbyahigherEC50. In thepresence ofMSLN-expressing cells, ABBV-428 elicited significantly increasedCD86 and CD83 expression on B cells and CD83 expression onmoDCs. The MSLN-dependent activity of ABBV-428 requiredinteraction with MSLN expressed on cell surface, as soluble MSLNwas not able to drive activation in the culture without cell-surfaceMSLN (Table 1). While in cultures containing MSLN-expressingcells, soluble MSLN at high concentration (300 nmol/L) reducedactivity of ABBV-428, possibly through preventing ABBV-428 frombinding to cell-surface MSLN. These results demonstrated that

binding to cell-surfaceMSLNallowedABBV-428 to enhanceCD40activation in APCs such as B cells and moDCs.

ABBV-428 induces T-cell activation and prevents tumor growthThe physiologic consequence of antigen presentation is T-cell

activation. Thus, ABBV-428was tested for the induction ofMSLN-dependent T-cell activation. In an in vitro coculture systemmixingT cells with autologous moDCs and PC3 cells or PC3 cellstransfected to express high MSLN (PC3-MSLNhi), ABBV-428increased IFNg production only in cultures containingPC3-MSLNhi cells, whereas anti-CD40 stimulated IFNg produc-tion in both cultures containing either PC3 or PC3-MSLNhi

cells (Fig. 5A).The impact of ABBV-428 on tumor growth in vivo was evalu-

ated. PC3 cells or PC3-MSLNhi cells, together with autologousmoDCs and T cells, were inoculated into NSG mice. Mice weretreated immediately after inoculation. Tumor growth was mon-itored over 3 weeks. Compared with isotype control, ABBV-428reduced the growth of MSLNþ PC3 tumors, but not MSLN�

tumors (Fig. 5B). In contrast, anti-CD40 reduced the growth ofboth MSLNþ and MSLN� tumors. These data suggest that ABBV-428 could only activate moDCs in an MSLN-dependent mannerto stimulate the cytotoxicity of autologous T cells toward allogenictumor cells.

The amount of MSLN expression is critical for the efficacy ofABBV-428

Because MSLN expression was required for ABBV-428 activity,we next tested whether different amounts of MSLN expressionmay differentially impact ABBV-428–induced immune cell acti-vation and subsequent tumor growth inhibition. We, therefore,made several PC3 cell lines stably expressing different amounts ofMSLN as determined by quantifying the number of cell-surfacemolecules and assessed in parallel by IHC (SupplementaryFig. S5). The cell lines with differing expression of MSLN werenamed as PC3-MSLNhi, PC3-MSLNmed, PC3-MSLNlow accordingto IHC score 4þ, 3þ, and 2þ, respectively. Each of these PC3MSLN-expressing cell lines was then transfected to express GFPand cultured with moDCs and autologous T cells under differenttreatments. Cell growth was monitored and analyzed on thefluorescence images collected by IncuCyte. Similar to anti-CD40,ABBV-428 inhibited the growth of PC3-MSLNhi andPC3-MSLNmed tumor cells (Fig. 6A). Unlike anti-CD40, whichinhibited the growth of tumor cells independent of MSLN expres-sion, ABBV-428 was not able to inhibit the growth of PC3-MSLNlow cells (Fig. 6A). These results suggest that ABBV-428 wasactive only whenMSLN expression was above a certain threshold.

Tumors are heterogeneous, and thus, tumor cells with high orlowMSLN expressionmay be present within the same tumor. Wenext tested the activity of ABBV-428 in cocultures ofmoDCs and Tcells with a mixture of parental PC3 cells coexpressing dsRed andPC3-MSLNhi cells coexpressing GFP. Consistent with in vivo data,ABBV-428 inhibited the growth of PC3-MSLNhi cells similarly toanti-CD40 (Fig. 6B), but unlike anti-CD40, ABBV-428 did notrestrict the growth of parental PC3 cells (Fig. 6C).When amixtureof PC3 cells and PC3-MSLNhi cells was cultured withmoDCs andT cells, ABBV-428was able to inhibit the growth of both cell types,independent of MSLN expression (Fig. 6D). The data suggest thatABBV-428 required a certain threshold of MSLN expression forinitial immune activation, butMSLNwas not necessary as a targetfor tumor cell killing.

Ye et al.





DiscussionLB-1 and ABBV-428 were designed as tumor-targeted, agonistic

anti-CD40 bispecific molecules to avoid systemic toxicity whentreating solid tumors expressingMSLN. It is postulated that MSLN-dependent CD40 activity could reduce toxicity resulting fromCD40 systemic activation (23, 24). MSLNþ tumors, such as meso-thelioma, ovarian, and pancreatic cancers, usually have a micro-environment associated with a more suppressive phenotype con-taining few tumor-infiltrating lymphocytes (TIL; refs.34,35).Directactivation of CD40 might be able to reprogram suppressive phe-notypes, enhance antigen presentation, and elicit subsequent T-cellresponses (36–40). Therefore, MSLN-targeted CD40 therapy mayhave potential to restore immune responses against those tumorswhichmight not respondwell to therapies directly targeting T cells.

LB-1 and ABBV-428 were selected from a panel of moleculeswith different bispecific formats through in vitro assays for con-ditional activity. Proof-of-concept studies using LB-1 providedin vivo evidence of the conditional activity which improved thetherapeutic index. Similar to LB-1, ABBV-428 also demonstratedin vitro conditional activities and was expected to achieve tumorantigen–driven CD40 activation to improve the safety profilewhile maintaining potent antitumor activity as a therapeuticcandidate.

The lower baseline CD40 activity of LB-1 and ABBV-428 in theabsence of MSLN cannot be explained by lower binding potencyon CD40, although here LB-1 showed lower binding on murineCD40 and ABBV-428 showed similar binding to human CD40 asparental anti-CD40. Themolecular structure of LB-1 or ABBV-428is, therefore, hypothesized to mediate conditional activity. LB-1

Figure 4.

ABBV-428 enhances B-cell andmoDC activation through binding to cell-surface–expressed MSLN.A, B cells purified from human PBMCswere cocultured withHEK293 cells (B cellþ 293, left) or 293 cells expressing MSLN (B cellþ 293-MSLN, right). B-cell proliferation was measured by 3H-thymidine incorporation aftertreatment with ABBV-428, anti-CD40, or an irrelevant human antibody as control. CPM, counts per minute. B, DCs derived from purified humanmonocytes(moDC) were cocultured with HEK293 cells (moDCþ 293, left) or 293 cells expressing MSLN (moDCþ 293-MSLN, right). IL12p70 production frommoDCs wasmeasured in supernatant after treatment with ABBV-428, anti-CD40, or control antibodies. Data shown are representative of 10 donors.

Table 1. Summary of cellular assays for ABBV-428 showing MSLN-dependent enhancement of immune cell activations

–MSLN (EC50, nmol/L) þMSLN (EC50, nmol/L)Agonisticassay

Assayreadout ABBV-927 ABBV-428

ABBV-428(þsol MSLN) ABBV-927 ABBV-428

ABBV-428(þsol MSLN)

B cell CD86 0.90 � 0.72 6.0 � 3.8 4.8 � 4.3 0.78 � 0.60 0.006 � 0.012 0.10 � 0.12CD83 0.60 � 0.54 11 � 6.8 12 � 4.7 0.60 � 0.66 0.66 � 0.62 0.18 � 0.18Proliferation 3.6 � 3.2 ¥ ¥ 8.3 � 7.2 0.18 � 0.06 2.4 � 0.96

moDC CD83 0.72 � 0.9 ¥ ¥ 0.82 � 0.24 0.42 � 0.36 2.4 � 2.1IL12 p70 11 � 5.3 ¥ ¥ 9.2 � 7.6 1.44 � 0.96 ¥

NOTE: B cells and moDC were cultured alone (�MSLN) or with MSLN-expressing cells (þMSLN). ABBV-428 was either added alone or premixed with300 nmol/L of soluble MSLN (þsol MSLN). Readouts of in vitro activity were monitored, and the data are indicated as mean � SEM (n ¼ 5). EC50, half maximalconcentration; ¥, infinite.






and ABBV-428 contains scFv domains constructed from thevariable regions of an agonistic CD40 mAb and anti-MSLN. Infunctional assays, the CD40 scFv domain of the bispecific mol-ecule was less effective than an intact CD40 antibody in initiatingCD40 signaling in the absence of MSLN. The structural basis forconditional activation may be related to the structural aspect ofCD40 signaling. CD40 belongs to the TNF receptor super family(TNFRSF). Receptors within this family need to be oligomerizedto initiate intracellular signaling (41). An intact antibody thatbinds to CD40 in a bivalent manner potentiates CD40 multi-merization, required for CD40 activation. It is possible that aCD40 targeting scFvdomainwithin LB-1orABBV-428might bindto CD40 in a different geometry from the bivalent binding of aconventional antibody, such that the bispecificmolecule needs tobind simultaneously to cell-surface MSLN to establish cross-linking, which would in turn multimerize the CD40 moleculesand activate downstream signaling. This idea was supported byour observation that the molecule could only achieve enhancedCD40 activation when it bound to cell-surface MSLN not solubleMSLN. In fact, solubleMSLNathigh concentrations (300nmol/L)reduced conditional activity by preventing the bispecificmoleculefrombinding to the cell-surfaceMSLN. BecauseMSLN shedding iscommon inmanyMSLNþ tumors, soluble MSLN can be detectedin patient serum (26). Although soluble MSLN from patients isusually less than 3 nmol/L (42), and the antigen sink effect ofsolubleMSLNwas not observed in our preclinicalmodel, optimal

dose of ABBV-428 should be explored in future studies in order toovercome the effect of soluble MSLN.

With the MSLN-dependent activity, LB-1 demonstrated anti-tumor potency with limited systemic toxicity in mice carryingsubcutaneous 4T1-MSLN tumors. Subcutaneous 4T1 tumor cellscan metastasize to lung and liver (43), and the lower concentra-tions of liver enzymes and cytokines induced by LB-1 comparedwithCD40mAb inour study suggest that LB-1maintained tumor-specific CD40 activation with minimal damage to adjacenthealthy tissue in the organ with metastasis. To further confirmthe tumor-specific activity of LB-1, additional in vivo studies willbe performed using an orthotopic tumor model or a geneticallyengineered mouse model which carries spontaneous tumor andorgan metastasis.

Although ABBV-428 did not cross-react with mouse CD40 andcould not be evaluated in a syngeneic tumor model for condi-tional activity, ABBV-428 was tested in multiple in vitro assays forfunctional activity uponMSLN binding. The activity of ABBV-428was associatedwithMSLNexpression. InmoDC/T-cell/tumor cellcocultures, ABBV-428 showed better tumor cell growth inhibitionif tumor cells expressed higher MSLN. Higher MSLN expressionensured the functional enhancement of ABBV-428, even whenonly a subset of cells expressed MSLN. When MSLN expressionwas low, even if all cells were MSLNþ, ABBV-428 was not able todrive CD40 activation. This allows ABBV-428 to maintain lowerCD40 activation within normal tissues whereMSLN expression is

Figure 5.

ABBV-428 induced T-cell activation and inhibited the growth of PC3 tumor upon MSLN binding.A, T-cell activation was monitored by coculturing T cells withautologous moDCs and PC3 cells without MSLN expression (TþmoDCþ PC3, left), or PC3 cells with high MSLN expression (TþmoDCþ PC3-MSLNhi, right).T-cell activation indicated by IFNg production was measured by ELISpot 48 hours later. For each data point, mean and SEMwere plotted. B,Antitumor activity ofABBV-428 was monitored in a prophylactic tumor model established in immune-deficient NSGmice through subcutaneous inoculation of a mixture of cells,including T cells, moDCs, and PC3 (left) or PC3-MSLNhi (right) tumor cells. At the time of inoculation, mice were administered a single intraperitoneal (i.p.)injection of isotype control mAb (control IgG), an agonistic anti-CD40, or ABBV-428 at 1 mg/kg. Tumor growth was monitored every 5 days. For each data point,mean and SEMwere plotted. Statistical difference was calculated by one-way ANOVA, and presented as � , P� 0.05 (n¼ 8).

Ye et al.





low, and to initiate more potent CD40 activation in tumors withhigher MSLN expression. Understanding the relationshipbetween ABBV-428 activity and MSLN expression is crucial foridentifying which patients would be most likely to respond tosuch therapy.

Although highMSLN expression was required for ABBV-428 toinitiate immune cell activation, ABBV-428–mediated tumor cellgrowth inhibition was not limited to MSLNþ cells. We observedthat activated immune cells stimulated by ABBV-428 couldinhibit the growth of either MSLNþ or MSLN�tumor cells, aslong as the cells with high MSLN expression were present inthe mixed cell culture. This phenomenon was confirmed withLB-1, the surrogate of ABBV-428. LB-1 inhibited the growth of

4T1-MSLN syngeneic tumors that contained both MSLNþ andMSLN� 4T1 cells. T cells from mice carrying 4T1-MSLN tumorstreated with LB-1 also produced IFNg in response to bothMSLNþ

and MSLN� 4T1 cells in an ex vivo antigen recall assay. Thisindicated that MSLN expression may be required only for CD40activation, and once activated, immune cells can limit tumorcell growth independent of MSLN expression. This featureis different from other tumor-targeting strategies, such asDC-targeted delivery of MSLN, where MSLN functions as avaccine (44), or T-cell–redirected anti-CD3 bispecific mole-cules, which only kill target-positive tumor cells upon directtarget binding (18, 19). Unlike a vaccine strategy or CD3-mediated T-cell activation, CD40 activation by ABBV-428 or

Figure 6.

Higher MSLN expression ensures pan-tumor growth inhibition mediated by ABBV-428–induced immune activation. Autologous moDCs and T cells werecocultured with PC3 cells with differing expression of MSLN and coexpressing a fluorescence protein. After treatment with ABBV-428 or an irrelevant humanantibody as control, tumor cell growth was continuously monitored by the IncuCyte ZOOM Live-Cell Analysis System, collecting fluorescence images every 4 to6 hours for 7 days. Cell growth at each time point was indicated as the percentage of starting cell number (#) and was the average from triplicates. A, Growth ofPC3 cell lines with different MSLN expression. B,Growth of PC3-MSLNhigh cells in cocultures containing 100% PC3-MSLNhigh cells. C,Growth of PC3 cells incocultures containing 100% of PC3. D, Growth of PC3-MSLNhigh cells (left) and PC cells (right) in cocultures containing 50% of PC3-MSLNhigh and 50% of PC3. Thedata are representative from five individual experiments with cells from 5 different donors.






LB-1 within the tumor microenvironment after engaging withMSLN has the potential to initiate immune responses against abroader array of tumor-associated antigens.

The immunememory againstMSLN� 4T1 cells observed in ouranimal studies was less likely due to the baseline CD40 activity ofthe bispecific molecule, or due to the lower expression of MSLNon normal tissues. In mice carrying 4T1 tumors without MSLNstable expression, LB-1 treatment did not reduce tumor growth orsystemic toxicities. These data again confirmed that the lowerexpression of MSLN was not sufficient to trigger robust immuneactivation by LB-1 treatment.

In summary, tumor-targeting anti-CD40 bispecific molecules,such as ABBV-428 and its surrogate LB-1, can achieve tumor-targeted CD40 activation with the potential to improve thera-peutic widow. The preclinical data from the current study warrantfurther clinical development of ABBV-428 and support testingadditional bispecific molecules targeting different antigens withtumor specificity to achieve therapeutic activity across differentsolid tumor indications.

Disclosure of Potential Conflicts of InterestS. Ye has ownership interest (including patents) in AbbVie. D. Cohen has

ownership interest (including patents) in AbbVie. Y. Akamatsu is seniorprincipal research scientist at and has ownership interest (including patents)in AbbVie. M. Lake is Senior Scientist III at AbbVie. P. Culp has ownershipinterest (including patents) in AbbVie. D. Hollenbaugh has ownershipinterest (including patents) in AbbVie. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: S. Ye, N.A. Belmar, A.F. Wahl, D. Hollenbaugh,D.T. ChaoDevelopment of methodology: S. Ye, D. Cohen, M. Sho, J. Cabel, C. Zhang,A.F. WahlAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Ye, D. Cohen, N.A. Belmar, D. Choi, S.S. Tan,H. Kim, R. Iyer, M. Lake, D. Song, J. Harlan, C. Zhang, Y. FangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ye, D. Cohen, D. Choi, S.S. Tan, C. Zhang, Y. Fang,P. Culp, D.T. ChaoWriting, review, and/or revision of the manuscript: S. Ye, D. Cohen, D. Choi,Y. Akamatsu, R. Iyer, J. Harlan, Y. Fang, P. Culp, D.T. ChaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Ye, D. Choi, M. LakeStudy supervision: S. Ye, N.A. Belmar, P. Culp, D.T. Chao

AcknowledgmentsThis study was supported by AbbVie Inc. The authors thank AbbVie Biother-

apeutics Inc. Animal Facility for animal care support, FACS Core Facility for cellsorting, Protein Science Group for protein expression and purification, andSusan Rhodes fromMolecular Cloning Group for generating all constructs usedin the studies.

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 19, 2018; revised April 2, 2019; accepted August 22,2019; published first August 28, 2019.

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2019;7:1864-1875. Published OnlineFirst August 28, 2019.Cancer Immunol Res Shiming Ye, Diane Cohen, Nicole A. Belmar, et al. Mesothelin Enhances Tumor-Specific ImmunityA Bispecific Molecule Targeting CD40 and Tumor Antigen

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