FS118, a Bispeciﬁc Antibody Targeting LAG-3 and PD-L1, … · PD-L1 mAb2 was generated for use as a FS118 surrogate in murine tumor models because FS118 has no functional activity

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

FS118, a Bispecific Antibody Targeting LAG-3 and PD-L1,Enhances T-Cell ActivationResulting in Potent AntitumorActivity A C

Matthew Kraman, Mustapha Faroudi, Natalie L. Allen, Katarzyna Kmiecik, Daniel Gliddon, Claire Seal,Alexander Koers, Mateusz M. Wydro, Sarah Batey, Julia Winnewisser, Lesley Young, Mihriban Tuna,Jacqueline Doody, Michelle Morrow, and Neil Brewis

ABSTRACT◥

Purpose: Although programmed death-ligand 1 (PD-L1) anti-body–based therapy has improved the outcome of patients withcancer, acquired resistance to these treatments limits their clin-ical efficacy. FS118 is a novel bispecific, tetravalent antibody(mAb2) against human lymphocyte activation gene-3 (LAG-3)and PD-L1 with the potential to reinvigorate exhausted immunecells and overcome resistance mechanisms to PD-L1 blockade.Here, using FS118 and a murine surrogate, we characterized theactivity and report a novel mechanism of action of this bispecificantibody.

Experimental Design: This study characterizes the bindingactivity and immune function of FS118 in cell lines andhuman peripheral blood mononuclear cells and further inves-tigates its antitumor activity and mechanism of actionusing a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb2).

Results: FS118 demonstrated simultaneous binding to LAG-3and PD-L1 with high affinity and comparable or better activitythan the combination of the single component parts of the mAb2

in blocking LAG-3- and PD-L1–mediated immune suppressionand enhancing T-cell activity. In syngeneic tumor mouse models,mLAG-3/PD-L1 mAb2 significantly suppressed tumor growth.Mechanistic studies revealed decreased LAG-3 expression onT cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb2, whereas LAG-3 expression increased upon treat-ment with the combination of mAbs targeting LAG-3 and PD-L1.Moreover, following binding of mLAG-3/PD-L1 mAb2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood.

Conclusions: This study demonstrates a novel benefit of thebispecific approach over a combination of mAbs and supports thefurther development of FS118 for the treatment of patients withcancer.

IntroductionImmunotherapy has significantly improved the clinical outcome of

patients with cancer. Targeting of immune checkpoint inhibitors suchas programmed cell death protein 1 (PD-1) or its ligand (PD-L1) shiftsimmune cells within the tumor microenvironment (TME) from anexhausted or immunosuppressive state to a proinflammatory statefavoring antitumor response. Unfortunately, only a limited proportionof patients benefit from these therapies due to the development ofresistance mechanisms within the TME.

PD-1, present on the surface of immune cells, has a role in down-regulating immune responses and promoting self-tolerance by sup-pressing T-cell inflammatory activity. This prevents autoimmunediseases, but it can also prevent the immune system from killingcancer cells. PD-L1 is expressed by a broad range of both nonhema-topoietic and immune cells. It has also been detected in a variety ofsolid tumors (1, 2) on both tumor cells and host immune cells. A wide

range of tumors upregulate PD-L1 in response to proinflammatorycytokines such as IFNg (3, 4). Engagement of PD-L1 with PD-1 onactivated tumor-infiltrating lymphocytes (TILs) can deliver inhibitorysignals to protect the tumor from immune destruction (5, 6).

Preclinical (6–10) and clinical data (11–13) have demonstrated thatblockade of PD-1/PD-L1 interaction can restore functional T-cellresponses in several clinical settings, providing clinical benefit topatients with advanced cancer. Immune checkpoint blockade (ICB)has been integrated in treatment regimen of patients with advanced,relapsed/refractory and,more recently, in patients with earlier stages ofdisease (14–19).

Currently, several PD-L1 antibodies are in development orapproved for various cancer indications. Although these agents pro-vide encouraging responses, only a small percentage of patientsrespond to these treatments with many showing innate and adaptiveresistance to therapy (20, 21). Emerging data suggest that amechanismof adaptive resistance to PD-1 or PD-L1 therapy is the upregulation ofadditional checkpointmolecules such as lymphocyte activation gene-3(LAG-3; refs. 22–27).

LAG-3 is expressed on the surface of B cells, natural killer cells,plasmacytoid dendritic cells, gd T cells, and activated T cells where italso functions as a negative regulator of T-cell function (28–30).Expression of LAG-3 on exhausted T cells or regulatory T cells (Tregs)in patients' tumorsmay also be a key factor responsible for resistance totherapies targeting PD-1 or PD-L1 (31, 32). LAG-3 is upregulated onboth human andmouse TILs and several studies blocking LAG-3 haveshown enhanced antitumor T-cell responses (33–36). Several LAG-3–targeting molecules are in clinical development, but while these arewell-tolerated, only moderate responses as monotherapies have beenreported (37–39).

F-star Therapeutics Ltd, Cambridge, United Kingdom.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M. Kraman, M. Faroudi and N.L. Allen contributed equally to this article.

Corresponding Author: Michelle Morrow, F-Star Therapeutics Ltd, CambridgeCB22 3AT, United Kingdom. Phone: þ44 (0) 1223 948098; Fax: þ44 (0) 1223497461; E-mail: [email protected]

Clin Cancer Res 2020;26:3333–44

doi: 10.1158/1078-0432.CCR-19-3548

�2020 American Association for Cancer Research.

AACRJournals.org | 3333

on October 20, 2020. © 2020 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 16, 2020; DOI: 10.1158/1078-0432.CCR-19-3548

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-19-3548&domain=pdf&date_stamp=2020-6-16

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-19-3548&domain=pdf&date_stamp=2020-6-16

http://clincancerres.aacrjournals.org/

The combination of LAG-3 and PD-1 mAbs has demonstratedsynergistic improvement of the antitumor response in murine tumormodels compared with blocking either one alone (36, 40). Clinicalactivity of the combination has also been established with a LAG-3–blocking mAb (relatlimab) plus PD-1 mAb (nivolumab) combina-tion (22) and a number of LAG-3–targeting approaches are beinginvestigated in clinical trials. In a small cohort of heavily pretreatedmelanoma patients, the majority of whom were PD-1/PD-L1 relapsedor refractory, LAG-3 expression enriched for responsiveness to thecombination of relatlimab plus nivolumab, with an approximatelythreefold increase in overall response rate where TIL LAG-3 expres-sion was ≥1%.

FS118 was developed as a human tetravalent bispecific antibody(mAb2) that is a full-length human IgG1 PD-L1 mAb with a distinctLAG-3–binding capability introduced into the fragment crystallizable(Fc) region (named Fcab: Fc with antigen binding; ref. 41). It isanticipated that such a bispecific antibody could provide the benefitof ICB combination approaches with additional efficacy and safetybenefits through more efficient recruitment and activation of tumor-specific T cells. To minimize antibody-dependent cell-mediated cyto-toxicity, additional mutations [leucine-to-alanine amino acid substi-tutions (LALA) at positions 1.3 and 1.2 (IMGT)] were engineered intothe CH2domain to decrease Fcg receptor binding (42). Further to that,FS118 has been through a standard manufacturing developmentprocess and the expression titer of the FS118 clonal manufacturingcell line is within the industry standards for mammalian expressionsystems. Fcabs are designed to retain key structural and functionalproperties of the wild-type Fc fragments, retain FcRn binding, and canbe purified by protein A resins (43). The activity of FS118 wascharacterized with in vitro pharmacologic studies using cell lines andprimary human immune cells. To further understand the in vivomechanism of action of FS118, a surrogate mouse bispecific antibody(mLAG-3/PD-L1 mAb2) was created (41) and tested in vitro andin vivo. Both the human FS118 and mouse surrogate mLAG-3/PD-L1mAb2 demonstrated simultaneous binding and neutralization ofhuman or mouse LAG-3 and PD-L1 inhibitory signaling that werecomparable with the combination of LAG-3 and PD-L1 single anti-body treatment. In vivo mouse studies using two different murinegenetic backgrounds demonstrate significant tumor growth inhibition

(TGI). Moreover, the mLAG-3/PD-L1 mAb2 exhibited a novel mech-anism of blocking immune-mediated suppression in vivo comparedwith the administration of a single antibody.

Materials and MethodsCell line generation

Human embryonic kidney (HEK) 293 cell lines (Thermo FisherScientific) ectopically expressing human or mouse PD-L1 or LAG-3were generated (HEK293-hLAG-3, HEK293-mLAG-3, HEK293-hPD-L1, and HEK293-mPD-L1). In addition, lentivirus constructswere created by cloning human or mouse LAG-3 and PD-L1 intolentiviral vectors [Lenti-X expression vector (pLVX); Clontech,631253] and viral particles were generated using the Lenti-X HTXPackaging System (Clontech, 632180). The chicken ovalbumin (OVAaa 323–339)-specific DO11.10 murine T-cell hybridoma cell lineexpressing endogenous PD-1 (Jewish National Health) or the LK35.2B cell line were transduced with the LAG-3 or PD-L1 lentiviralparticles, respectively, to ectopically express the receptors on the cellsurface: DO11.10-mLAG-3, DO11.10-hLAG-3, LK35.2-mPD-L1, andLK35.2-hPD-L1. The cell lines were also transduced with emptyparticles (pLVX): DO11.10(pLVX) and LK35.2(pLVX). Raji cells werepurchased from ATCC. All cell lines were tested for Mycoplasmacontamination internally every 4 weeks using a R&D MycoProbeMycoplasma Detection Kit, and were authenticated by IDEXX BioA-nalytics providing a short tandem repeat profile, IMPACTPCRprofile,and testing for interspecies contamination.

Antibody generation and nomenclatureAll antibodies, except the commercial rat mouse IgG LAG-3

antibody clone C9B7W (BioXCell), were fully human IgG1 antibodiesand contained the LALA (amino acid) mutation in the Fc region.

FS118 was constructed by combining a human LAG-3 Fcab (41)with the fragment antigen-binding (Fab) domains of an IgG1 humanPD-L1 (hPD-L1) mAb (“hPD-L1 mAb”). During the drug discoveryprocess, the optimal pairing of Fab and Fcab was selected using theDO11.10 T-cell activation system described below. A mouse LAG-3/PD-L1 mAb2 was generated for use as a FS118 surrogate in murinetumor models because FS118 has no functional activity in the mouse.The mLAG-3/PD-L1 mAb2 was constructed by combining a mouseLAG-3 Fcab with the Fab domains of an IgG1 mouse cross-reactivePD-L1 mAb (G1-AA/S1, sequence from clone YW243.55.S1, Genen-tech patent publication US20100203056A1; “mPD-L1 mAb”). Tocharacterize the properties of the human and surrogate Fcabs in themAb2 format, a “mock” LAG-3 mAb2 (LAG-3/mock mAb2) wasgenerated and contained the human or the mouse LAG-3 Fcab anda Fab region, which can bind to a nonrelevant antigen, the FITC (clone4420; refs. 41, 44). The human and mouse LAG-3/mock mAb2 arecalled “hLAG-3/mock mAb2” and “mLAG-3/mock mAb2,” respec-tively. Other comparator antibodies included G1-AA/S1 (“mPD-L1mAb”), the human IgG1 isotype control (anti-FITC, G1-AA/4420),and rat monoclonal mouse LAG-3 antibody [clone C9B7W, BioXCell;“mLAG-3mAb (Rat IgG)”]. The sequence of the Fab domain of the ratmouse LAG-3 mAb was used to generate a chimeric mouse LAG-3antibody with a human IgG1 Fc backbone (“mLAG-3 mAb”).

Binding association assaysBiacore T200 or Biacore 3000 Instruments (GE Healthcare) were

used to assess the binding affinity and kinetics of FS118 or mLAG-3/PD-L1 mAb2 to recombinant human or mouse LAG-3 or PD-L1,respectively, by surface plasmon resonance (SPR).

Translational Relevance

Despite advances with therapies targeting the programmedcell death protein 1 (PD-1) or its ligand (PD-L1), many patientswith cancer are refractory to or relapse following treatment.Resistance to anti–PD-1 treatment is associated with upregula-tion of other checkpoint inhibitor receptors such as LAG-3(lymphocyte activation gene-3). FS118, currently being evalu-ated in a phase I clinical trial in patients with advanced malig-nancies (NCT03440437), is a tetravalent bispecific antibodytargeting LAG-3 and PD-L1, two immune checkpoint moleculesthat promote tumor escape from immune surveillance. We havecharacterized both the in vitro and in vivo functional activity ofFS118 and find that this bispecific antibody can overcome PD-L1and LAG-3 immune suppressive signals. We report a potentialnovel mechanism of action not observed with the combinationof single PD-L1 and LAG-3 antibodies. Our results indicate thatFS118 represents a possible novel approach to overcome some ofthe mechanism of resistance to PD-L1 blockade.

Kraman et al.

Clin Cancer Res; 26(13) July 1, 2020 CLINICAL CANCER RESEARCH3334




For the human protein studies, FS118 or control human PD-L1mAb were captured on a sensor chip by immobilized protein L(Thermo Fisher Scientific), and various concentrations of humanLAG-3-Fc (R&D Systems) or human PD-L1-Fc (R&D Systems)were flowed across the chip. Simultaneous binding of FS118 to humanLAG-3 and PD-L1 was also assessed by SPR using LAG-3 and PD-L1Fc fusion proteins. Human PD-L1-Fc was directly immobilized on thecarboxymethyl dextran-5 (CM5) sensor chip surface, and FS118 or thecomparator human PD-L1 mAb, was captured by bound PD-L1-Fc.Then the LAG-3-Fc was flowed across the bound antibody.

For the mouse protein studies, recombinant mouse LAG-3-Fc(R&D Systems) or mouse PD-L1-Fc (R&D Systems) were directlyimmobilized onto a CM5 sensor chip followed by an injection ofserially diluted mLAG-3/PD-L1 mAb2 to generate binding curves andequilibrium KD values. Finally, to demonstrate mLAG-3/PD-L1mAb2

could simultaneously bind both mouse LAG-3 and PD-L1, mPD-L1-Fc was covalently linked to the surface of a CM5 sensor chip. ThemLAG-3/PD-L1 mAb2 flowed across at a fixed concentration, andthen mLAG-3-Fc was applied.

In vitro T-cell function assaysAntigen-specific DO11.10 T-cell activation assay

An IL2 release assay utilizing both the DO11.10 T cells andLK35.2 B cell lines was set up to assess the function of mAb2 andcontrol antibodies. DO11.10 T cells transduced either with human,mouse LAG-3 construct, or an empty vector, and LK35.2 B cellstransduced either with human, mouse PD-L1, or empty vectorconstruct were used in the assay. Briefly, serial dilutions of mAb2

or control antibody were prepared in media (DMEM, 10% FBS, and1 mmol/L sodium pyruvate). Diluted mAb2 or control antibodieswere added to a mixture of DO11.10 T cells (2 � 104 cells) andLK35.2 B cells (antigen-presenting cells, 2� 104 cells) in presence ofOVA peptide (H-ISQAVHAAHAEINEAGR-OH, Pepscan; 1 mmol/L) and incubated for 24 hours. Supernatants were collected andassayed with a mouse IL2 ELISA Kit (eBioscience or R&D Systems)following the manufacturer's instructions. Plates were read on aplate reader using Gen5 Software (BioTek).

Staphylococcal enterotoxin B assayFor the Staphylococcal enterotoxin B (SEB) assay, human peripheral

blood mononuclear cells (PBMCs) were isolated from leukocyte cones(anonymized samples from consenting donors at NHS Blood andTransplant Service) by layering over Ficoll-Paque Plus per the man-ufacturer's instructions (GE Healthcare). CD4þ T cells and CD14þ

monocytes were isolated by magnetic selection (Miltenyi IsolationKits, Miltenyi Biotec). CD4þ T cells were activated with aCD3/CD28Beads (Thermo Fisher Scientific) for 8 days prior to freezing. Autol-ogous monocytes were cultured and differentiated into immaturedendritic cells (iDCs) for 7 days and then frozen. For coculture andstimulation, cells were thawed and then cocultured (1� 105 T cells plus1� 104 iDC) for 4 days with SEB (Sigma) at a single concentration (0.1ng/mL) in the presence of various concentration of test antibodies.Culture supernatants were harvested and analyzed for IFNg release byELISA (Human IFN-gamma Quantikine ELISA Kit, R&D Systems).Assays were performed in triplicate.

Mouse tumor modelThree mouse colon carcinoma cell lines, MC38 (NCI), MC38.OVA

(an engineered MC38 cell line expressing ovalbumin), and CT26(ATCC) were used. Tumor model studies used 8- to 10-week-oldfemale C57BL/6 mice (Jackson Laboratory) or 8- to 11-week-old

female Balb/c mice (Charles River Laboratories). Experiments wereconducted under a United Kingdom Home Office Project Licenseand approved by an Animal Welfare and Ethical Review Boardin accordance with the United Kingdom Animal (Scientific Proce-dures) Act 1986 and with EU Directive EU 86/609. For TGI studies,mice were inoculated subcutaneously with 1 � 106 MC38 cells inC57BL/6 mice or 1 � 105 CT26 cells in Balb/c mice. When tumorswere palpable, 6 days postinoculation (CT26 tumor–bearing mice) or8 days postinoculation (MC38 tumor–bearing mice), respectively, theanimals received an intraperitoneal injection of the test antibodiesevery 3 days for a total of three administrations. Tumor volume (mm3)was measured throughout the study using calipers. The followingformula was used to calculate tumor volume: (length � width2)/2. Inthe study where combination therapy was investigated, animalsreceiving a single test antibody treatment were coinjected with acontrol antibody [10 mg/kg rat (HRP-1, BioXCell) or human IgG1

(G1-AA/4420 isotype control)] to ensure that mice received the sameamount of total antibody as the mice treated with combination ofsingle antibodies.

For the tumor immune-profiling study, C57BL/6micewere injectedsubcutaneously with 1.5 � 106 MC38.OVA or MC38 cells. When thetumor reached a size of approximately 65 mm3, the animals weretreated with a single intraperitoneal dose of the test antibodies. Micewere sacrificed at different time points postdosing, then tumors andspleens were collected, processed, and analyzed by flow cytometry.Serum was isolated from peripheral blood to assess the level of solubleLAG-3 (sLAG-3) and PD-L1 proteins.

T-cell depletion in a murine MC38 tumor modelC57BL/6 mice were injected subcutaneously with 1 � 106 MC38

tumor cells (NIH). Two days after inoculation, the first dose of10 mg/kg CD8-depleting antibody (clone 2.43, IgG2b, 2BScientific)was given intraperitoneally. Depleting antibody (10 mg/kg) was given1 day prior to dosing with 10 mg/kg mLAG-3/PD-L1 mAb2 or10 mg/kg IgG1 isotype control (G1-AA/4420) for a total of threeadministrations, and a last time at day 11, 1 day after the last dose withmLAG-3/PD-L1 mAb2. Tumor volume (mm3) was measuredthroughout the study using calipers. The following formula was usedto calculate tumor volume: (length � width2)/2.

Flow cytometrySingle-cell suspensions of splenocytes were obtained following

mechanical dissociation through a 70-mm nylon cell strainer andtreatment with red blood cell lysis (RBC Lysis Buffer, Miltenyi Biotec).Mouse tumor tissues were mechanically dissociated with the Gentle-MACSMouse Tumor Dissociation Kit (Miltenyi Biotec) following themanufacturer's instructions. Cell suspensions were washed with RPMI(Gibco) and filtered through 70-mm cell strainer (BD Falcon).

Cells were then stained with a viability Dye Live/Dead e780(eBioscience) or fixable Near-IR Dead Cell Stain (Life Technologies)and then blocked with anti-CD16/32 (eBioscience) before stainingwith fluorescence-conjugated antibodies in flow cytometry–stainingbuffer. For surface staining the following antibodies were used: CD45-BV510 and CD45-Alexa 700 (clone 30-F11) from BioLegend andeBioscience, respectively; CD8a-BUV395 and CD8a-BUV737 (clone53-6.7) from BioLegend and BD Bioscience, respectively; CD4-BV786and CD4-BUV395 (clone RM4-5) from eBioscience and BD Biosci-ence, respectively; CD3e-BUV737 and CD3e-Alexa 488 (clone 145-2C11) from BD Bioscience and eBioscience, respectively; LAG-3-PE(cloneC9B7W) fromBDBiosciences; andPD-L1-Alexa488 [cloneG1-AA/S1 conjugated with Alexa488 using the Lightning Link Rapid

Characterization of a LAG-3/PD-L1 Bispecific Antibody

AACRJournals.org Clin Cancer Res; 26(13) July 1, 2020 3335




Alexa488 Labeling Kit (Innova Bioscience)]. Intracellular stainingwas performed using the FoxP3/Transcription Factor StainingBuffer Set (eBioscience). The following antibodies were used forintracellular staining: Foxp3-APC and FoxP3-PerCP-Cy5.5 (cloneFJK-16s) from eBioscience and Invitrogen, respectively, and Ki-67-PE-Cy7 (clone SolA15) from eBioscience. Data were acquired on aLSRFortessa (BD Biosciences) and analysis performed using FlowJo(Tree Star, v10).

Measurement of total sLAG-3 and PD-L1 receptorsMeso scale discovery (MSD) technology platform was used to

measure total sLAG-3 from the serum of tumor-bearing mice.Briefly, biotinylated goat polyclonal antibodies against-mouseLAG-3 (R&D Systems) were captured on MSD streptavidin platesand washed three times with PBS/0.05% Tween. Serum samples andstandards were added to the plates and incubated. Followingwashes, sulfo-tagged detection goat polyclonal anti-mouse LAG-3antibody was added and incubated. The plates were then washedand read on the MSD instrument. Total mouse soluble PD-L1concentrations in the mouse serum samples were determined usingthe mouse PD-L1/B7-H1 PicoKine ELISA Kit (Boster) following themanufacturer's instructions.

Statistical analysisStatistical analyses were performed with GraphPad Prism 5 soft-

ware. Nonparametric testing was performed and means � SEM ormeans � SD are shown on all graphs.

Statistical analysis of TGISTATA/IC 15.1 software was used to implement a mixed model for

analysis of the tumor growth rate. Statistical significance was shownpairwise for growth rates over the full time of study using the mixed-model analysis comparing all groups. A separate model was fitted toeach pair of treatments of interest. The model was

Log10 volumeð Þ ¼ AþB� day � start dayð Þþ"

A and B are the intercept and slope, respectively; they are differentfor each mouse, and include a fixed effect for the group and a randomeffect for the animal:

A¼A0þA1Tþ "A

B¼B0þB1Tþ"B

T is a dummy variable representing the treatment groupwith value 0in one group and 1 in the other. The random effects are distributedwith a normal distribution:

"A�N 0;sAð Þ; "B�N 0;sBð Þ

where sA and sB are the SDs of the interanimal variability in theintercept and slope, respectively. The intra-animal variability is alsonormally distributed with SD s:

" �N 0; sð Þ

For each pair of treatments, the model above was fitted to thedata. The model parameters were reported, together with their 95%confidence intervals. For A1 and B1, the (two-sided) P value for adifference from zero was calculated; a P value below 0.05 isstatistically significant evidence for a difference between the treat-ment groups.

ResultsSimultaneous high-affinity binding ofFS118 to LAG-3 and PD-L1

FS118, hLAG-3/PD-L1 mAb2, bound to recombinant humanLAG-3-Fc with a KD value of 0.056 nmol/L (Table 1). The bindingaffinity of FS118 for human PD-L1-Fc (KD ¼ 1 nmol/L) was similarto that of the hPD-L1 mAb composing the Fab part of the mAb2

(KD ¼ 1.2 nmol/L), indicating that introduction of a LAG-3–binding site into the CH3 domain of the antibody did not affectPD-L1 binding. Concurrent dual-target binding (LAG-3 and PD-L1) by FS118 was also confirmed by SPR where FS118 binding toPD-L1-Fc did not interfere with the bispecific molecule binding toLAG-3-Fc (Fig. 1). FS118 specifically bound to PD-L1 and LAG-3because binding to closely related proteins, PD-L2 and CD4, wasnot observed (Supplementary Fig. S1).

The ability of FS118 to bind to the native form of cell surface–expressed proteins was assessed by flow cytometry. The resultsdemonstrated strong binding of FS118 to human LAG-3 (EC50 ¼3.2 nmol/L) and human PD-L1 (EC50 ¼ 3.2 nmol/L) expressedon the surface of HEK293 cells (Table 1; Supplementary Fig. S2).Binding of FS118 to LAG-3 and PD-L1 in a concentration-dependent manner was also confirmed on CD3/CD28-activatedhuman CD4þ T cells by flow cytometry (Supplementary Fig. S2).

Moreover, the ability of FS118 to block binding of LAG-3 and PD-L1 to their respective ligandswasmeasured. FS118 inhibited binding ofLAG-3 to Raji cells endogenously expressing MHC class II (MHCII).In the sameway, FS118 also inhibited PD-L1 binding to PD-1 or CD80(Supplementary Fig. S3).

FS118 overcame PD-L1- and LAG-3–mediated inhibition of T-cellactivation

FS118 was tested in a LAG-3/PD-L1–dependent T-cell assay whereOVA323–339-specific mouse DO11.10 T cells expressing human LAG-3, or nonexpressing control cells were incubated with LK35.2 B cells(antigen-presenting cells) expressing, or not, human PD-L1 in pres-ence of OVA peptide and isotype control or test antibodies. Theaddition of FS118 to the cultured cells reversed T-cell inhibitionmediated by LAG-3 or PD-L1 (Fig. 2A and B; SupplementaryTable S1). FS118 demonstrated greater activity than the combinationof its component parts hLAG-3/mock mAb2 plus hPD-L1 mAb (EC50

¼ 0.61 nmol/L and 2.07 nmol/L, respectively), when both humanLAG-3 and human PD-L1 were overexpressed (Fig. 2C). When onlyhuman LAG-3 or human PD-L1 was overexpressed, FS118 showedsimilar activity compared with the single or the combination compo-nent antibodies.

FS118 and single-component antibodies were also tested in aprimary SEB-activated human T-cell assay. The magnitude of theeffect of test antibodies in the SEB T-cell assay was donor dependent(Fig. 2D). However, FS118 and the comparators, hPD-L1 mAb andhLAG-3/mock mAb2 plus hPD-L1 mAb, increased IFNg levels abovethat of the isotype control in all donor samples. FS118 demonstratedsimilar or, in some donors, enhanced T-cell activation compared withthe single-agent combination. The potency values (EC50) of FS118were on average, 0.125 nmol/L. FS118 was also able to enhance CD8þ

T-cell cytokine production and cytotoxicity in an antigen recall assay,as measured by intracellular IFNg and CD107a staining (Supplemen-tary Fig. S4).

Overall, these data demonstrate that FS118 can reverse PD-L1-and LAG-3–mediated inhibition of T-cell activation and effectorfunction.

Kraman et al.





Generation and characterization of mouse LAG-3/PD-L1surrogate mAb2

As FS118 showed no functionality in a mouse LAG-3 and PD-L1assay system (data not shown), a murine surrogate mAb2 (mLAG-3/PD-L1 mAb2) was generated to evaluate the potential for a mLAG-3/PD-L1 mAb2 to mediate an antitumor response in mouse tumormodels. The binding and function of mLAG-3/PD-L1 mAb2 in mouse

systems was compared with that of FS118 in human systems (Table 1;Supplementary Fig. S5).

In a SPR assay, mLAG-3/PD-L1 mAb2 bound to immobilizedmLAG-3-Fc fusion protein with a KD of 0.98 nmol/L and to mPD-L1-Fc with a KD of 0.09 nmol/L. Simultaneous binding of LAG-3and PD-L1 was also demonstrated by SPR (SupplementaryFig. S5A).

Figure 1.

Simultaneous binding of FS118 to immobilized human PD-L1-Fc and human LAG-3-Fc in solution as detected by SPR. Human PD-L1-Fc recombinant protein wascaptured directly to the SPR chip. FS118 or the anti-human PD-L1mAb (hPD-L1mAb)were injected in the firstmobile phase followedby the addition of human LAG-3-Fc recombinant protein.

Table 1. Binding and functional activity of FS118 and mLAG-3/PD-L1 mAb2.

Binding to immobilizedtarget proteins by SPR LAG-3 KD (nmol/L) PD-L1 KD (nmol/L)

Concurrent LAG-3 andPD-L1 binding

Human target proteinsFS118 0.056 1.0 Yes

Mouse target proteinsmLAG-3/PD-L1 mAb2 0.98 0.09 Yes

Binding to HEK293 cellsoverexpressing targetproteins LAG-3 EC50 (nmol/L) PD-L1 EC50 (nmol/L)

Human target proteinsFS118 3.179 3.19

Mouse target proteinsmLAG-3/PD-L1 mAb2 2.27 11.9

Functional activity inDO11.10 T-cell activationassay LAG-3 EC50 (nmol/L) PD-L1 EC50 (nmol/L)

LAG-3 andPD-L1 EC50 (nmol/L)

Human target proteinsFS118 1.25 0.47 0.75

Mouse target proteinsmLAG-3/PD-L1 mAb2 2.1 0.2 5.8






In addition, mLAG-3/PD-L1 mAb2 bound to mouse LAG-3or mouse PD-L1 overexpressed on HEK293 cells in a concentra-tion-dependent manner (EC50 values of 2.3 and 12 nmol/L,respectively; Table 1; Supplementary Fig. S5B).

The functional activity of mLAG-3/PD-L1 mAb2 was evaluatedin the DO11.10 T-cell activation system. When both mouse LAG-3and mouse PD-L1 are expressed in the same assay format asdescribed in Fig. 2A–C, mLAG-3/PD-L1 mAb2 activated T cells

Figure 2.

FS118 has functional activity in vitro in a T-cell assay. A–C, FS118 has functional activity in vitro in an antigen-specific T-cell assay. DO11.10 OVA–specific T cellsoverexpressing human LAG-3 (hLAG-3) or empty vector (pLVX) were mixed with either wild-type LK35.2 cell lines [antigen-presenting cells (APC)] or LK35.2 cellsoverexpressinghumanPD-L1 [APC (PD-L1)] in presence of theOVA324–339 peptide andwith various concentration of antibodies for 24 hours. Culture supernatantwascollected and assessed for secreted levels of mouse IL2 by ELISA. LAG-3–mediated inhibition assay (A), PD-L1–mediated inhibition assay (B), and both LAG-3- andPD-L1–mediated inhibition assay (C). Assayswere performed in duplicate and data are presented as mean� SEM. D, CD4þ T cells were activated and were culturedwith iDCs and SEB in the presence of FS118 or other test antibodies over a range of concentrations from0.00128 nmol/L to 100 nmol/L. IFNg secreted into the culturesupernatant was quantified by ELISA. Representative graphs from assays performed in triplicate and data are presented as mean � SEM.

Kraman et al.





in a dose-dependent manner comparable with the combinationof the mouse LAG-3 (mLAG-3/mock mAb2) and PD-L1 mAb(Supplementary Fig. S5C). The single-agent controls (mouseLAG-3 or mouse PD-L1 mAb alone) did not increase T-cellactivation when both mouse targets were overexpressed. (Supple-mentary Fig. S5C).

While the binding affinity of mLAG-3/PD-L1 mAb2 for recom-binant mouse LAG-3 is approximately 10-fold lower than FS118affinity for human LAG-3 and binds to mouse PD-L1 with approx-

imately 10-fold higher affinity than FS118 for human PD-L1,functional activity of mLAG-3/PD-L1 mAb2 and FS118 againsteach single target (LAG-3 or PD-L1) within their correspondingspecies (mouse or human) was found to be similar (Table 1).Although the functional activity of FS118 (EC50 ¼ 0.61 nmol/L)was greater than mLAG-3/PD-L1 mAb2 (EC50 ¼ 5.8 nmol/L) whenboth LAG-3 and PD-L1 were expressed, this difference was within10-fold and therefore mLAG-3/PD-L1 mAb2 was considered asuitable surrogate of FS118 for mouse studies.

Figure 3.

mLAG-3/PD-L1 mAb2 inhibits growth of MC38 and CT26 tumors. Female C57/BL6 or Balb/c mice were subcutaneously injected with MC38 (A and C) or CT26tumor cells (B). Following tumor formation, mice were administrated intraperitoneally with either IgG1 control (10 mg/kg) or anti-mLAG-3/PD-L1 mAb2 at 1, 3, 10, or20mg/kg at day0, 3, and 6 postrandomization (A), or IgG1 control (10mg/kg) or anti-mLAG-3/PD-L1mAb2 at 10mg/kg at day0, 3, and 6postrandomization (B), or atotal of 20mg/kg test antibody (eg, 10mg/kgantibodyAþ 10mg/kg antibodyBor IgG1 control) at day8, 11, day 14postinoculation (C). Arrows indicate dayof dosing.Groups consisted of 9–24 mice. A mixed-model analysis was used to determine significance (�� , P < 0.01; �� , P < 0.001).






mLAG-3/PD-L1 mAb2 inhibited tumor growth in vivoTo test whether mLAG-3/PD-L1 mAb2 has antitumor activity,

tumor-bearing mice were administered with mLAG-3/PD-L1 mAb2

or IgG1 control. mLAG-3/PD-L1 mAb2 had significant antitumoractivity in both the C57BL/6 MC38 and Balb/c CT26 tumor models(Fig. 3A and B, respectively). In all studies performed, mLAG-3/PD-L1 mAb2 was well-tolerated at all dose levels tested with no signs ofweight loss or toxicity.

A dose–response experiment was performed in the MC38 tumormodel to understand the optimal dose level for further investigation.Significant differences in mean tumor volume were observed betweenthe IgG1 control and mLAG-3/PD-L1 mAb2–treated MC38-bearingmice at the 3, 10, or 20 mg/kg dose levels but not at 1 mg/kg (Fig. 3A;Supplementary Fig. S6A). A dose of 3 mg/kg was determined to be theminimal dose level required to drive this effect. Subsequent TGI studiesused 10 mg/kg as an optimal dose level to ensure maximal antitumoreffects.

Antitumor activity was observed with treatment ofmLAG-3/PD-L1mAb2 and compared with mouse LAG-3 (clone C9B7W) and PD-L1(clone G1-AA/S1) mAb given alone or in combination. mLAG-3/PD-L1mAb2 eliminated the tumor in 6 of 8mice and slowed tumor growthin the remaining 2 mice (Fig. 3C; Supplementary Fig. S6B). Thecombination of the test antibodies did not induce any complete tumorregressions, although it did slow tumor growth (SupplementaryFig. S6B). Although the administration of the individual mouseLAG-3 antibody ormouse PD-L1 antibody did slow the tumor growth,it did not suppress it to the extent of either the combination or themAb2. Therefore, in theMC38model, mLAG-3/PD-L1mAb2 inducedan antitumor immune response as potent as the combination of PD-L1and LAG-3mAbs. The antitumor effect of the mAb2 was confirmed tobe CD8þ T-cell mediated as the mAb2 failed to control tumor growthwhen CD8þ T cells were depleted (Supplementary Fig. S7A).

mLAG-3/PD-L1 mAb2 decreased LAG-3 and free PD-L1 surfaceexpression on T cells

Studies were carried out to investigate the pharmacodynamic effectsof a single dose of the mouse LAG-3/PD-L1 mAb2 on tumor immunecell infiltrates and on splenic T cells in a mouse tumor model.

Administration of mLAG-3/PD-L1 mAb2, single agents alone or incombination resulted in no or only marginal change in the proportionof CD4þ or CD8þ T cells in TILs (Fig. 4A and B) and splenic cells(Supplementary Fig. S8A).While therewas no significant change in thefrequencies of Ki-67–positive (proliferating) CD4þ or CD8þ T cells inthe tumor (Supplementary Fig. S8B), treatment with mLAG-3/PD-L1mAb2, the combination of single antibodies or the mouse PD-L1 mAbincreased the percentage of proliferating CD4þ T cells in the spleen at96 hours postdosing compared with control IgG1 (SupplementaryFig. S8C). In a separate experiment, this effect was confirmed to beindependent of CD8þ T cells (Supplementary Fig. S7B). PD-L1 andLAG-3 antibodies were used to determine the expression of PD-L1 andLAG-3 on tumor-infiltrating T cells. Characterization by flow cyto-metry demonstrated that the mouse PD-L1 flow cytometry antibodycompeted with mLAG-3/PD-L1 mAb2, and therefore measures freePD-L1 surface expression. The mouse LAG-3 antibody (cloneC9B7W) used to detect cell surface LAG-3 was noncompetitive withmLAG-3/PD-L1 mAb2 and therefore detected total LAG-3 (free andbound) cell surface receptor. Treatment with mLAG-3/PD-L1 mAb2

resulted in a transient decrease over several days in the percentage ofLAG-3þ T cells in tumor and spleen, while the combination or themouse PD-L1 mAb induced a transient increase of the frequency ofLAG-3þT cells (Fig. 4C–E; Supplementary Fig. S9A). In themLAG-3/

PD-L1 mAb2 treatment group, LAG-3 expression on CD4þ and TregTILS was reduced at 24 hours and persisted for 96 hours (Fig. 4Cand E; Supplementary Fig. S9A). LAG-3 expression onCD8þTILs wasalso reduced 8 hours posttreatment with mAb2 and remained low for96 hours (Fig. 4D; Supplementary Fig. S9A). The decrease in LAG-3expression could not be attributed to a loss of T-cell populationsbecause overall proportion of T cells did not alter following treatment(Fig. 4A and B), nor could the decrease in LAG-3 expression beattributed to internalization of the mAb2 as this did not showsignificant internalization over a 3-hour period in an in vitro assay(Supplementary Fig. S10).

In contrast, treatment with mouse PD-L1 mAb alone appeared toincrease expression of LAG-3 on CD4þ and CD8þ T cells in both thetumor and spleens (Fig. 4C–E; Supplementary Fig. S9A).

Similarly, an increase of free mouse PD-L1 was observed whenmicewere treated with mLAG-3/mock alone. Interestingly, it was observedthat when mouse PD-L1 antibody was administered, LAG-3 expres-sion increased and whenmouse LAG-3mAb treatment was given, freePD-L1 appeared to increase (Fig. 4F and G; Supplementary Fig. S9B).

Treatment with the combination ofmLAG-3/mock plusmouse PD-L1 mAb appeared to induce an overall increase in total surface LAG-3(Fig. 4C and D). Total LAG-3 surface level was more difficult toascertain because all the commercially available PD-L1 mAbs testedcompeted with mAb2 for PD-L1 binding, although it appeared thatcombination treatment might increase available PD-L1 in this setting.

Taken together, these data show that treatment with single dose ofmLAG-3/PD-L1 mAb2 prevented the compensatory upregulation ofthe LAG-3 receptor on T cells in TILs observed with the combinationtreatment.

mLAG-3/PD-L1mAb2 shed cell surface LAG-3 andPD-L1 into theblood of tumor-bearing mice

LAG-3 is known to undergo a rapid cleavage event from the cellsurface of activated T cells by the transmembrane metalloproteasesADAM 10 and ADAM 17 (45). Therefore, total sLAG-3 (free andbound to antibody) was measured in the serum of treated mice. Asingle 10 mg/kg treatment with mLAG-3/PD-L1 mAb2 or mLAG-3/mock plus PD-L1 significantly increased sLAG-3 in the serum up tofourfold over negative controls. This increase persisted up to 96 hoursfollowing dosing (Fig. 5A). Similarly, the levels of total soluble PD-L1following treatment with mLAG-3/PD-L1 mAb2 or mLAG-3/mockplus PD-L1 increased compared with isotype control–treated mice,although this was not apparent until 48 hours posttreatment (Fig. 5B).These results suggest that the mLAG-3/PD-L1 mAb2 reduced LAG-3cell surface expression by accelerating its shedding from the cell surfaceof T cells.

DiscussionDespite significant advances made by PD-1:PD-L1 blockade

therapy, a large proportion of patients do not derive clinical benefitfrom these therapies. An underlying resistance mechanism is theupregulation of checkpoint inhibitors such as LAG-3 (22, 23, 27).FS118 is a tetravalent bispecific antibody that was selected on thebasis of its ability to overcome the immunosuppressive signalingmediated by both LAG-3 and PD-L1 in patients with cancer. Here,we have reported characterization of the activity of FS118 andunveiled a novel mechanism of action utilizing a surrogate mousebispecific antibody.

We have demonstrated that FS118 simultaneously bound to LAG-3and PD-L1, blocked LAG-3:MHCII, PD-L1:PD-1, and PD-L1:CD80

Kraman et al.





interactions and as a result reversed T-cell inhibition. In a humanprimary T-cell activation assay, FS118 reversed immune suppressionto enhance T-cell activation and was more potent than the combina-tion of individual components of FS118 in some of PBMC donorstested. FS118 enhanced cytokine production by both CD4þ (in a SEBassay) andCD8þT cells (in an antigen recall assay), two central playersof the antitumor response. These results demonstrated that the LAG-3–targeting Fcab and PD-L1 Fab regions of FS118 can work in synergyto overcome LAG-3- and PD-L1–mediated inhibition of T-cell acti-vation. The stronger T-cell response observed in PBMCdonors treated

with the bispecific over the combination may be explained by theavidity effects of themolecule created by the dual cis-binding to a singlecell or trans-binding between two cells resulting in long-lastingblocking of immunosuppressive signals.

Amouse surrogate of FS118 (mLAG-3/PD-L1mAb2)was generatedto characterize functional activity in vitro and in vivo.mLAG-3/PD-L1mAb2 demonstrated comparable binding properties and in vitrofunctional activity against mouse LAG-3 and PD-L1 when comparedwith FS118. The antitumor efficacy of the surrogate mAb2 wasevaluated in MC38 and CT26 syngeneic mouse tumor models. The

Figure 4.

Decreased mLAG-3 and free mPD-L1 from the surface of T cells after treatment with mLAG-3/PD-L1 mAb2. Mice with subcutaneous MC38.OVA tumors wereadministrated with one intraperitoneal injection of anti-mLAG-3/PD-L1 mAb2 or control antibodies. At designated time points following single dosing of testantibodies, mice were culled, and tumors processed for flow cytometry. Proportion of CD4þ (A) and CD8þ (B) T cells within the CD45þ population of TILs asdetermined by flow cytometry. Proportion of totalmLAG-3 expression onCD4þ TILs (C), CD8þ TILs (D), and CD4þ FoxP3þ regulatory TILs (E) as determined by flowcytometry. Proportion of freemPD-L1 expression onCD4þTILs (F) and CD8þ TILs (G) asmeasured by flow cytometry. Representative graphs from assays performedin triplicate and data are presented as mean � SD. Statistical significance (P value) was determined using one-way ANOVA with Tukey correction for multiplecomparisons. Significant P values are labeled with one or more “� ,” denoting � , P <0.05; �� , P <0.01; ��, P <0.001; �� , P <0.0001.






results showed the potential to elicit CD8þ T-cell–dependent antitu-mor immune response that was greater than mouse PD-L1 or mouseLAG-3 antibody monotherapy and was at least as effective as thecombination of mouse LAG-3 plus mouse PD-L1 mAbs. These datademonstrate that the mLAG-3/PD-L1 mAb2 can provide dual block-ade of LAG-3 and PD-L1 in vivo and enhance the antitumor immuneresponse.

Treatment with mLAG-3/PD-L1 mAb2 was shown to reduceLAG-3 expression on the cell surface of CD4þ and CD8þ T cells inTILs and splenic T cells. This effect was not observed with PD-L1blockade alone and therefore suggests that LAG-3 downregulationwas due to the LAG-3–targeting moiety in the mAb2 format. Thiscontrasts with treatment with mouse LAG-3 antibody thatincreased surface expression of PD-L1 and treatment with mousePD-L1 antibody that caused an increase in surface LAG-3. The ideathat single targeting of a checkpoint inhibitor receptor could resultin the upregulation of an additional checkpoint receptor has beenpreviously reported in murine tumor models (24, 26). This obser-vation could allude to the numbers of patients who become refrac-tory to current monospecific immune therapy as a reflexive immunecompensatory response (22, 23). Here, following dosing withmLAG-3/PD-L1 mAb2, total LAG-3 was rapidly reduced from thesurface of the T cells in tumor and spleen limiting the impact of thecompensatory upregulation of LAG-3 on T-cell activation andenhancing the antitumor response. The cross-talk between CD4þ

and CD8þ T cells for suppression of tumor growth is a hallmarkfeature of the immune system. While the antitumor effect in MC38tumor mouse model was driven by cytotoxic CD8þ T cells, mLAG-

3/PD-L1 mAb2 increased proliferation of peripheral CD4þ cells.This could reflect the activity of CD4þ T cells to provide helpersignals to tumor -specific CD8þ T cells and subsequently augmentantitumor immunity.

Although the mechanism regulating LAG-3 expression is notfully understood, one could hypothesize that mLAG-3/PD-L1 mAb2

altered the cellular trafficking and/or the shedding of LAG-3from the cell surface, potentially via the metalloproteases ADAM10and ADAM17 (45). In unstimulated T cells, LAG-3 exists as anintracellular pool that is localized to the surface upon stimula-tion (46). Cross-linking of LAG-3 through cis- or trans-binding ofthe mAb2 could potentially regulate LAG-3 surface expression byaltering the signaling pathway that controls its translocation fromearly/recycling endosomal compartments to the cell surface with aputative impact on PD-1 expression and function as well. Indeed, astudy by Huang and colleagues suggested that LAG-3 and PD-1shared intracellular localization and trafficking pathway, andthat LAG-3 may enable the rapid translocation of PD-1 fromearly/recycling endosomal compartments to the immunologicalsynapse (40).

Our data revealed that the action of the mLAG-3/PD-L1 mAb2 onLAG-3 cell surface expression correlated with an increased level ofsLAG-3 in the serum of the treated animals suggesting that themLAG-3/PD-L1mAb2 accelerated the shedding ofmouse LAG-3 from the cellsurface into the blood. Similarly, the combination treatment increasedsLAG-3 into the blood following treatment.However, if this increase ofsLAG-3 resulted from the shedding of cell surface LAG-3, it did notreduce drastically or prevent the increased cell surface LAG-3

Figure 5.

Soluble mLAG-3 andmPD-L1 levels after treatment withmLAG-3/PD-L1 mAb2. Fold change of serum soluble mLAG-3 (A) andmPD-L1 (B) levels in mice treatedwiththe mLAG-3/PD-L1 mAb2 (1, 3, or 10 mg/kg), the combination of the mLAG-3/mock mAb2 plus anti-mPD-L1 mAb (1, 3, or 10 mg/kg) or the IgG1 isotype controlantibody (10 mg/kg). Representative graphs from assays performed in triplicate and data are presented as mean � SEM. � , no data for this group at 72 hours.

Kraman et al.





expression observed upon combination therapy. Here, sLAG-3 ishypothesized to be a potential surrogate indicator of T-cell activationin response to LAG-3/PD-L1 mAb2 treatment. sLAG-3, through itsbinding to MHCII, has been reported to stimulate antigen-presentingcells such as macrophages and dendritic cells to activate T-cellresponses and enhance tumor-specific cytotoxic T cells (47, 48). Therelevance of this mechanism in human cancers is supported byevidence that the presence of sLAG-3 positively correlates withimproved survival in patients with breast cancer and gastric can-cer (42, 49). In addition to PD-L1 and LAG-3 inhibition, FS118 maypromote the recognition and elimination of the tumor by bridging theimmune effector cells and the cancer cells favoring a directed and localresponse.

In conclusion, these preclinical results demonstrate that LAG-3/PD-L1 mAb2 have the potential to drive a potent antitumor responseand unveil a differentiated mechanism of action compared with thecombination therapy. These data support the evaluation of FS118in patients with cancer.

Disclosure of Potential Conflicts of InterestD. Gliddon is an employee/paid consultant for and holds ownership interest

(including patents) in F-star Biotechnology. S. Batey is an employee/paid con-sultant for F-star Biotechnology. L. Young holds ownership interest (includingpatents) in F-star. N. Brewis is an employee/paid consultant for, reports receivingcommercial research grants from and reports receiving other commercial researchsupport from, and holds ownership interest (including patents) in F. star. Nopotential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: M. Kraman, D. Gliddon, M.M. Wydro, J. Winnewisser,M. Tuna, M. Morrow, N. BrewisDevelopment of methodology:M. Kraman, N.L. Allen, K. Kmiecik, C. Seal, S. BateyAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): M. Kraman, N.L. Allen, K. Kmiecik, A. Koers, M.M. Wydro,J. WinnewisserAnalysis and interpretation of data (ie, statistical analysis, biostatistics,computational analysis): M. Kraman, M. Faroudi, N.L. Allen, K. Kmiecik,C. Seal, A. Koers, L. Young, J. Doody, M. Morrow, N. BrewisWriting, review, and/or revision of the manuscript: M. Kraman, M. Faroudi,N.L. Allen, D. Gliddon, A. Koers, S. Batey, J. Winnewisser, M. Tuna, J. Doody,M. Morrow, N. BrewisAdministrative, technical, or material support (ie, reporting or organizing data,constructing databases): M. KramanStudy supervision: M. Kraman, D. Gliddon, M. Tuna, J. DoodyOther (antibody generation): M.M. Wydro

AcknowledgmentsThe authors would like to thank the F-star protein sciences team; F-star in vivo

team; F-star drug discovery team; Cristian Gradinaru for statistical analyses; AlisonMcGhee for critical review; Babraham BSU staff members for animal husbandry andtechnical assistance; and Dr. Sarah Burl for article editing and review.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 28, 2019; revised February 22, 2020; accepted March 27, 2020;published first April 16, 2020.

References1. Grosso J, InzunzaD,WuQ, Simon J, Singh P, ZhangX, et al. Programmed death-

ligand 1 (PD-L1) expression in various tumor types. J Immunother Cancer 2013;1:P53.

2. Zou W, Chen L. Inhibitory B7-family molecules in the tumour microenviron-ment. Nat Rev Immunol 2008;8:467–77.

3. Abiko K, Matsumura N, Hamanishi J, Horikawa N, Murakami R, Yamaguchi K,et al. IFN-g from lymphocytes induces PD-L1 expression and promotes pro-gression of ovarian cancer. Br J Cancer 2015;112:1501–9.

4. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immuneevasion. Nat Med 2002;8:793–800.

5. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD-1, a novelmember of the immunoglobulin gene superfamily, upon programmed cell death.EMBO J 1992;11:3887–95.

6. Hirano F, Kaneko K, TamuraH, DongH,Wang S, IchikawaM, et al. Blockade ofB7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeuticimmunity. Cancer Res 2005;65:1089–96.

7. Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4 combi-nation blockade expands infiltrating T cells and reduces regulatory T andmyeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A 2010;107:4275–80.

8. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement ofPD-L1 on tumor cells in the escape from host immune system and tumorimmunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 2002;99:12293–7.

9. Okudaira K, Hokari R, Tsuzuki Y, Okada Y, Komoto S, Watanabe C, et al.Blockade of B7-H1 or B7-DC induces an anti-tumor effect in amouse pancreaticcancer model. Int J Oncol 2009;35:741–9.

10. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1)pathway to activate anti-tumor immunity. Curr Opin Immunol 2012;24:207–12.

11. Brahmer JR, Tykodi SS, Chow LQM, HwuW-J, Topalian SL, Hwu P, et al. Safetyand activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl JMed 2012;366:2455–65.

12. Weber JS,D'Angelo SP,MinorD,Hodi FS, GutzmerR,Neyns B, et al. Nivolumabversus chemotherapy in patients with advanced melanoma who progressed afteranti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label,phase 3 trial. Lancet Oncol 2015;16:375–84.

13. Robert C, Long GV, Brady B, Dutriaux C,MaioM,Mortier L, et al. Nivolumab inpreviously untreated melanoma without BRAF mutation. N Engl J Med 2015;372:320–30.

14. Gettinger S, Rizvi NA, Chow LQ, Borghaei H, Brahmer J, Ready N, et al.Nivolumab monotherapy for first-line treatment of advanced non–small-celllung cancer. J Clin Oncol 2016;34:2980–7.

15. Rizvi NA, Hellmann MD, Brahmer JR, Juergens RA, Borghaei H, Gettinger S,et al. Nivolumab in combination with platinumbased doublet chemotherapy forfirst-line treatment of advanced non–small-cell lung cancer. J Clin Oncol 2016;34:2969–79.

16. Amy LC, Edward BG. The ascent of immune checkpoint inhibitors: is theunderstudy ready for a leading role? Cancer Biol Med 2017;14:341.

17. Reck M, Rodríguez-Abreu D, Robinson AG, Hui R, Cso��szi T, F€ul€op A, et al.Pembrolizumab versus chemotherapy for PD-L1–positive non–small-cell lungcancer. N Engl J Med 2016;375:1823–33.

18. Stenehjem DD, Tran D, Nkrumah MA, Gupta S. PD1/PDL1 inhibitors for thetreatment of advanced urothelial bladder cancer. Onco Targets Ther 2018;11:5973–89.

19. Velcheti V, Chandwani S, Chen X, Pietanza MC, Burke T. First-line pembro-lizumabmonotherapy inmetastatic PD-L1 positive non-small cell lung cancer: areal-world analysis of time on treatment in US community oncology practices.Ann Oncol 2018;29:x18.

20. Shen X, Zhao B. Efficacy of PD-1 or PD-L1 inhibitors and PD-L1 expressionstatus in cancer: meta-analysis. BMJ 2018;362:k3529.

21. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, et al. PD-1 andPD-L1 checkpoint signaling inhibition for cancer immunotherapy: mechanism,combinations, and clinical outcome. Front Pharmacol 2017;8:561.

22. Ascierto PA, Melero I, Bhatia S, Bono P, Sanborn RE, Lipson EJ, et al. Initialefficacy of anti-lymphocyte activation gene-3 (anti–LAG-3; BMS-986016) incombination with nivolumab (nivo) in pts with melanoma (MEL) previouslytreated with anti–PD-1/PD-L1 therapy. J Clin Oncol 2017;35:9520.

23. Thommen DS, Schreiner J, Muller P, Herzig P, Roller A, Belousov A, et al.Progression of lung cancer is associated with increased dysfunction of T cellsdefined by coexpression of multiple inhibitory receptors. Cancer Immunol Res2015;3:1344–55.

24. Koyama S, Akbay EA, Li YY, Herter-Sprie GS, Buczkowski KA, RichardsWG, et al. Adaptive resistance to therapeutic PD-1 blockade is associated






with upregulation of alternative immune checkpoints. Nat Commun 2016;7:10501.

25. Jenkins RW, Barbie DA, Flaherty KT. Mechanisms of resistance to immunecheckpoint inhibitors. Br J Cancer 2018;118:9–16.

26. Huang R-Y, Francois A, McGray AR, Miliotto A, Odunsi K. Compensatoryupregulation of PD-1, LAG-3, and CTLA-4 limits the efficacy of single-agentcheckpoint blockade in metastatic ovarian cancer. Oncoimmunology 2017;6:e1249561.

27. Shayan G, Srivastava R, Li J, Schmitt N, Kane LP, Ferris RL. Adaptiveresistance to anti-PD1 therapy by Tim-3 upregulation is mediated by thePI3K-Akt pathway in head and neck cancer. Oncoimmunology 2017;6:e1261779.

28. Durham NM, Nirschl CJ, Jackson CM, Elias J, Kochel CM, Anders RA, et al.Lymphocyte activation gene 3 (LAG-3)modulates the ability of CD4T-cells to besuppressed in vivo. PLoS One 2014;9:e109080.

29. Grosso JF, Kelleher CC, Harris TJ, Maris CH, Hipkiss EL, De Marzo A,et al. LAG-3 regulates CD8þ T cell accumulation and effector functionin murine self- and tumor-tolerance systems. J Clin Invest 2007;117:3383–92.

30. Workman CJ, Rice DS, Dugger KJ, Kurschner C, Vignali DAA. Phenotypicanalysis of the murine CD4-related glycoprotein, CD223 (LAG-3). Eur J Immu-nol 2002;32:2255.

31. Taube JM, Young GD, McMiller TL, Chen S, Salas JT, Pritchard TS, et al.Differential expression of immune-regulatory genes associated with PD-L1display in melanoma: implications for PD-1 pathway blockade. Clin CancerRes 2015;21:3969–76.

32. Huang C-T, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, et al. Role ofLAG-3 in regulatory T cells. Immunity 2004;21:503–13.

33. Matsuzaki J, Gnjatic S, Mhawech-Fauceglia P, Beck A, Miller A, Tsuji T, et al.Tumor-infiltratingNY-ESO-1–specificCD8þT cells are negatively regulated byLAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U S A 2010;107:7875–80.

34. Demeure C, Wolfers J, Martin-Garcia N, Gaulard P, Triebel F. T Lymphocytesinfiltrating various tumour types express the MHC class II ligand lymphocyteactivation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell–cellcontacts. Eur J Cancer 2001;37:1709–18.

35. Wang Y, Dong T, Xuan Q, Zhao H, Qin L, Zhang Q. Lymphocyte-activationgene-3 expression and prognostic value in neoadjuvant-treated triple-negativebreast cancer. J Breast Cancer 2018;21:124.

36. Woo S-R, Turnis ME, Goldberg M V., Bankoti J, Selby M, Nirschl CJ, et al.Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cellfunction to promote tumoral immune escape. Cancer Res 2012;72:917–27.

37. Long L, Zhang X, Chen F, Pan Q, Phiphatwatchara P, Zeng Y, et al. Thepromising immune checkpoint LAG-3: from tumor microenvironment tocancer immunotherapy. Genes Cancer 2018;9:176–89.

38. Brignone C, Escudier B, Grygar C, Marcu M, Triebel F. A phase I pharma-cokinetic and biological correlative study of IMP321, a novel MHC class IIagonist, in patients with advanced renal cell carcinoma. Clin Cancer Res2009;15:6225–31.

39. Puhr HC, Ilhan-Mutlu A. New emerging targets in cancer immunotherapy: therole of LAG3. ESMO Open 2019;4:e000482.

40. Huang R-Y, Eppolito C, Lele S, Shrikant P, Matsuzaki J, Odunsi K. LAG3 andPD1 co-inhibitory molecules collaborate to limit CD8þ T cell signaling anddampen antitumor immunity in a murine ovarian cancer model. Oncotarget2015;6:27359–77.

41. Everett KL, KramanM,Wollerton FPG, Zimarino C, Kmiecik K, GasparM, et al.Generation of Fcabs targeting human and murine LAG-3 as building blocks fornovel bispecific antibody therapeutics. Methods 2019;154:60–9.

42. Arduin E, Arora S, Bamert PR, Kuiper T, Popp S, Geisse S, et al. Highly reducedbinding to high and low affinity mouse Fc gamma receptors by L234A/L235Aand N297A Fc mutations engineered into mouse IgG2a. Mol Immunol 2015;63:456–63.

43. Lobner E, Traxlmayr MW, Obinger C, Hasenhindl C. Engineered IgG1-Fc–onefragment to bind them all. Imunnol Rev 2016;270:113–31.

44. Kranz DM, Voss EW. Partial elucidation of an anti-hapten repertoire in BALB/cmice: comparative characterization of several monoclonal anti-fluorescyl anti-bodies. Mol Immunol 1981;18:889–98.

45. Li N, Wang Y, Forbes K, Vignali KM, Heale BS, Saftig P, et al. Metalloproteasesregulate T-cell proliferation and effector function via LAG-3. EMBO J 2007;26:494–504.

46. Woo S-R, Li N, Bruno TC, Forbes K, Brown S, Workman C, et al. Differentialsubcellular localization of the regulatory T-cell protein LAG-3 and the core-ceptor CD4. Eur J Immunol 2010;40:1768–77.

47. Avice M-N, Sarfati M, Triebel F, Delespesse G, Demeure CE. Lymphocyteactivation gene-3, aMHCclass II ligand expressed on activatedT cells, stimulatesTNF-alpha and IL-12 production by monocytes and dendritic cells. J Immunol1999;162:2748–53.

48. Casati C, Camisaschi C, Rini F, Arienti F, Rivoltini L, Triebel F, et al. Solublehuman LAG-3 molecule amplifies the in vitro generation of Type 1 tumor-specific immunity. Cancer Res 2006;66:4450–60.

49. Li N, Jilisihan B, Wang W, Tang Y, Keyoumu S. Soluble LAG3 acts as apotential prognostic marker of gastric cancer and its positive correlation withCD8þT cell frequency and secretion of IL-12 and INF-g in peripheral blood.Cancer Biomarkers 2018;23:341–51.


Kraman et al.




2020;26:3333-3344. Published OnlineFirst April 16, 2020.Clin Cancer Res Matthew Kraman, Mustapha Faroudi, Natalie L. Allen, et al. Enhances T-Cell Activation Resulting in Potent Antitumor ActivityFS118, a Bispecific Antibody Targeting LAG-3 and PD-L1,

Updated version

10.1158/1078-0432.CCR-19-3548doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2020/04/16/1078-0432.CCR-19-3548.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/26/13/3333.full#ref-list-1

This article cites 49 articles, 16 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/26/13/3333.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/26/13/3333To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-19-3548

http://clincancerres.aacrjournals.org/content/suppl/2020/04/16/1078-0432.CCR-19-3548.DC1

http://clincancerres.aacrjournals.org/content/26/13/3333.full#ref-list-1

http://clincancerres.aacrjournals.org/content/26/13/3333.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/26/13/3333


Documents

FS118, a Bispeciﬁc Antibody Targeting LAG-3 and PD-L1, … · PD-L1 mAb2 was generated for use as a FS118 surrogate in murine tumor models because FS118 has no functional activity