preclinical development of the anti-lag-3 antibody regn3767: … · lag-3 suppresses...

Large Molecule Therapeutics

Preclinical Development of the Anti-LAG-3Antibody REGN3767: Characterization andActivity in Combination with the Anti-PD-1Antibody Cemiplimab in Human PD-1xLAG-3–Knockin MiceElena Burova, Aynur Hermann, Jie Dai, Erica Ullman, Gabor Halasz, Terra Potocky,Seongwon Hong, Matt Liu, Omaira Allbritton, AmyWoodruff, Jerry Pei, Ashique Rafique,William Poueymirou, Joel Martin, Douglas MacDonald,William C. Olson, Andrew Murphy,Ella Ioffe, Gavin Thurston, and Markus Mohrs

Abstract

In the tumor microenvironment, multiple inhibitorycheckpoint receptors can suppress T-cell function, therebyenabling tumor immune evasion. Blockade of one of thesecheckpoint receptors, PD-1, with therapeutic antibodies hasproduced positive clinical responses in various cancers;however, the efficacy of this approach can be furtherimproved. Simultaneously targeting multiple inhibitorycheckpoint receptors has emerged as a promising therapeuticstrategy. Here, we report the development and characteriza-tion of REGN3767, a fully human IgG4 antibody targetingLAG-3, another inhibitory receptor on T cells. REGN3767binds human and monkey LAG-3 with high affinity andspecificity and blocks the interaction of LAG-3 with itsligand, MHC class II. In an engineered T-cell/antigen-presenting cell bioassay, REGN3767 alone, or in combina-

tion with cemiplimab (REGN2810, human anti-PD-1 anti-body), blocked inhibitory signaling to T cells mediated byhLAG-3/MHCII in the presence of PD-1/PD-L1. To test thein vivo activity of REGN3767 alone or in combination withcemiplimab, we generated human PD-1xLAG-3 knockinmice, in which the extracellular domains of mouse Pdcd1and Lag3 were replaced with their human counterparts. Inthese humanized mice, treatment with cemiplimab andREGN3767 showed increased efficacy in a mouse tumormodel and enhanced the secretion of proinflammatorycytokines by tumor-specific T cells. The favorable pharma-cokinetics and toxicology of REGN3767 in nonhuman pri-mates, together with enhancement of antitumor efficacy ofanti-PD-1 antibody in preclinical tumor models, support itsclinical development.

IntroductionTherapeutic mAbs targeting inhibitory receptors such as cyto-

toxic T-lymphocyte–associated protein 4 (CTLA-4) or pro-grammed cell death 1 (PD-1) show impressive clinical activitywith an acceptable benefit risk ratio in several tumor types (1–4).However, sustained responses are only achieved in a minority ofpatients, suggesting that combination approaches may berequired to overcome tumor immune escape mechanisms (5).Antibodies blocking the interaction of the inhibitory receptorlymphocyte-activation gene 3 (LAG-3) with its ligand, MHC classII, may invigorate the immune response to cancer, especially in

combination with antibodies blocking the PD-1/PD-L1 axis(reviewed in refs. 6–8).

The inhibitory receptor LAG-3, a CD4-like molecule, isexpressed on activated CD4þ and CD8þ T cells, a subset ofregulatory T cells (Treg), natural killer (NK) cells, B cells, andplasmacytoid dendritic cells (DC; refs. 8–11). LAG-3 suppressesT-cell activation, proliferation, and homeostasis (11, 12) and hasbeen reported to play a role in Treg-suppressive function (13).LAG-3 binds to MHC class II, which is expressed on DCs, macro-phages, B cells, and epithelial cancer cells, and serves to presentpeptides to CD4þ T cells (14–16). LAG-3 has been shown to beexpressed on dysfunctional T cells in chronic viral infec-tions (17, 18) and during tumor progression in patients withcancer (19, 20). CD8þ T cells coexpressing PD-1 and LAG-3display severely impaired effector functions in a murine modelof self-antigen tolerance (21), and antigen-specific T cells inhuman ovarian cancer are negatively regulated by PD-1 andLAG-3 (20). Multiple reports demonstrate that a large fractionof PD-1–expressing CD8þ and CD4þ tumor-infiltrating lympho-cytes (TIL) coexpress LAG-3, supporting thedominanceof PD-1 inmodulating antitumor T-cell responses, as well as a direct role ofLAG-3 in further suppressing the activity of a subset of PD-1–expressing T cells (18, 20–24). Mice deficient for both PD-1 andLAG-3 show spontaneous autoimmunity and lethality, not seen

Regeneron Pharmaceuticals, Inc., Tarrytown, New York.

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

Corresponding Author: Markus Mohrs, Regeneron Pharmaceuticals, Inc., 777Old Saw Mill River Road, Tarrytown, NY 10591. Phone: 914-847-7000; Fax: 914-847-7453; E-mail: [email protected]

Mol Cancer Ther 2019;18:2051–62

doi: 10.1158/1535-7163.MCT-18-1376

�2019 American Association for Cancer Research.

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in either single knockout (22, 25). In addition, dual blockade ofPD-1 and LAG-3 with antibodies synergistically inhibits tumorgrowth in preclinical tumormodels (22, 23, 26). Thus, it stands toreason that antagonistic anti-LAG-3 antibodies may improveefficacy in combination with anti-PD-1 therapies in theclinic (7, 8, 27–30). Currently, there are several LAG-3–targetingtreatments in various phases of clinical development (31–34). Inparticular, combination therapy of anti-LAG-3 (BMS-986016)plus anti-PD-1 (nivolumab) has shown promising clinical effi-cacy in patients with melanoma (33, 34).

We have previously generated cemiplimab, a human anti-PD-1antibody blocking PD-1/PD-L1–mediated T-cell inhibition (35).Here we describe the generation of REGN3767, a fully humanhigh-affinity anti-LAG-3 antibody, using VelocImmunemice con-taining human immunoglobulin gene segments (36, 37). In vitro,REGN3767 binds LAG-3 with high affinity and blocks LAG-3/MHC class II–driven T-cell inhibition. In vivo, REGN3767enhances the antitumor activity of the anti-human PD-1 antibodycemiplimab against syngeneic colorectal carcinomas in micehumanized for the extracellular domains of PD-1 and LAG-3.The preclinical results presented here supported the initiation ofclinical trials using REGN3767 in monotherapy or in combina-tion with cemiplimab in patients with solid tumors.

Materials and MethodsAntibodies

To generate anti-human LAG-3 antibodies, VelocImmunemice, carrying genes encoding human immunoglobulin heavyand kappa light chain variable regions (36, 37), were immunizedwith recombinant human LAG-3–mFc protein (Regeneron), con-taining the extracellular domain of LAG-3 (amino acids 1–450)and the Fc portion of mouse IgG2a. Hybridomas were generatedfrom splenocytes and supernatants were screened for binding toHEK293 cells transfected with human LAG-3. REGN3767 wasengineered as a human IgG4 containing a serine to prolinesubstitution (S228P) in the hinge region to minimize half-antibody formation (38). To reduce binding to Fcg receptors,REGN3767 also contains three amino acid substitutions(P236VA238) derived from the lower hinge region of IgG2 repla-cing four amino acids (E236FLG239) in the corresponding lowerhinge region of IgG4 (39). The amino acid sequences of the heavyand light chains of REGN3767 (GenBank accession numbers:MN200290 and MN200291) are shown in SupplementaryFig. S1. The hIgG4 (S228P) anti-human PD-1 antibody cemipli-mab (REGN2810), that binds to both human and monkey PD-1proteins andblocks PD-1 interactionswith PD-L1 andPD-L2,wasdescribed previously (35).

REGN3767 binding to human LAG-3 proteinsBinding kinetics of REGN3767mAb to LAG-3 proteins at 25�C

and pH 7.4 was measured in Surface Plasmon Resonance-Biacorestudies by first capturing REGN3767 on a CM5 (Biacore) surfacechip precoated with anti-human kappa goat polyclonal F(ab0)2Ab (GE Life Sciences) and then injecting various concentrations ofLAG-3 proteins over the surface. Following capture, hLAG-3.mmH (myc-myc-polyhistidine tag), rLAG-3.mmH, or mLAG-3.mmH at concentrations ranging from 50 to 0.78 nmol/L, orhLAG-3.hFc protein ranging from 25 to 0.39 nmol/L were indi-vidually injected over the REGN3767-captured surfaces. The

kinetic parameters were obtained by globally fitting the data toa 1:1 binding model using Biacore T200 Evaluation.

Cell–cell adherence assayThe ability of REGN3767 to block the binding of hLAG-3 to

humanMHC II–positive (Raji) B cells ormurineMHC II–positive(A20) B cells was assessed using a cell–cell adherence assay.Fluorescently labeled Raji or A20 cells were examined for adher-ence to HEK293/hLAG-3 cells in the presence or absence ofREGN3767. The parental HEK293 cell line has no detectableexpression of human LAG-3 as determined by flow cytometry.To confirm expression ofMHC II, Raji cells were stainedwith anti-humanHLA-DR (clone Tu36, BD Biosciences) and A20 cells werestainedwith anti-mouse I-A/I-E (cloneM5/114.15.2, BioLegend).Raji or A20 cells were labeled with Calcein AM (Life Technolo-gies). Human Fc block (BDPharmingen)was added to prelabeledRaji cells andmouse Fc block (BDPharmingen)was added to A20cells at a final concentration of 10 mg/mL. HEK293 parental andHEK293/hLAG-3 cells were added at 1.2 � 104 cells/100 mL into96-well plates and cultured overnight. REGN3767 or the isotypematched control antibody REGN2759 (Regeneron)were added atincreasing concentrations for 1 hour, followedby incubationwith1.2 � 105 labeled Raji or A20 cells for 1 hour. Nonadherent cellswere removed bywashing. Relative fluorescence units of adherentlabeled Raji or A20 cells were measured at an excitation/emissionwavelength of 485 nm/535 nm on a VICTOR X5 plate reader.

Generation of human PD-1xLAG-3 knockin miceVelociGene technology was used to generate human

PD-1xLAG-3 knockin mice as described previously (40). Briefly,a Lag3-targeting vector was engineered that replaced 1750 bp ofthe extracellular portion of the mouse Lag3 gene (containingexons 2–4) with the corresponding 1741 bp region of the humangene (exons 2–4). Dual humanized mouse embryonic stem cells(ESC)were created by electroporation of the Lag3-targeting vectorinto C57BL/6N mouse ESCs that contained a humanized Pdcd1gene encoding the extracellular portion of human PD-1 and thetransmembrane and intracellular portion of mouse Pdcd1 (35).Correct gene targeting in ESC clones was identified by a loss ofallele assay (41). Dual humanized ESC clones were used toimplant female mice to generate a litter of pups containing bothhumanized genes (i.e., Lag3 and Pdcd1).

Tumor challenge experimentsMC38.Ova cells were described previously (35) and authen-

ticated by short tandem repeat profiling in 2016 (IDEXXBioResearch). MC38.Ova cells (5 � 105) were injected subcu-taneously in the hind flank of 8–10 weeks old female humanPD-1xLAG-3 knockin mice. Tumors were measured semiweeklyusing a caliper and reported as mm3 (length � width2/2). In aprophylactic tumor model, antibodies were administered intra-peritoneally in 200 mL starting on day 3 after tumor implan-tation, and then twice a week for 2 weeks. In an establishedtumor model, mice were randomized on day 10–11 afterimplantation when tumors reached 100 mm3. Mice were trea-ted with antibodies on the randomization day and then twice aweek for 2 weeks. Mice were euthanized when the tumorvolumes reached 2,000 mm3 or when tumors ulcerated. Theprotocol was approved by the Regeneron PharmaceuticalsInstitutional Animal Care and Use Committee.

Burova et al.

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Flow cytometric assessment of tumoral T-cell activationTumors were harvested and processed using GentleMacs Cell

Disruptors (Miltenyi) to generate single-cell suspensions. Lym-phocytes were enriched by Percoll (GE Healthcare) gradientcentrifugation, stained with fluorescently labeled antibodies, andanalyzed on a FACS Canto Flow Cytometer (BD Biosciences).Ex vivo TILs stimulation was performed with 1 mg/mL Ova257-264MHC I peptide and 5 mg/mL Ova323-339 MHC II peptide (Invivo-Gen) in the presence of Brefeldin A (1 mg/mL) for 4 hours at 37�C.Draining lymph nodes (dLN) were processed into single-cellsuspension by mechanical dissociation. Cells were stimulatedwith phorbol 12-myristate 13-acetate (PMA; 20 ng/mL) andionomycin (500 ng/mL) for 4 hours in the presence of BrefeldinA (1 mg/mL), and then processed with the Cytofix/Cytoperm Kit(BDBiosciences) for intracellular cytokine staining. The followingantibody clones were used: mCD45 (30-F11, BioLegend), mCD4(GK1.5, BioLegend), mCD8a (53-6.7, BioLegend), mIFNg(XMG1.2, eBioscience), mTNFa (MP6-XT22, BioLegend), hPD-1(EH12.2H7, BioLegend), hLAG-3 (3DS223H, eBioscience),mouse PD-1 (J43, eBioscience), and mouse LAG-3 (C9B7W,BioLegend).

Cytokine quantification in mouse serum and in spleenSpleens were harvested from tumor-bearing mice and manu-

ally dissociated into single-cell suspensions. Spleen homogenateswere clarified by centrifugation and the protein content wasquantified using a BCA Assay (Pierce). A total of 50 mg ofsupernatant from a spleen sample and 25 mL of serum were usedin duplicates to measure cytokine concentrations by the V-PLEXProinflammatory Panel 1 mouse kit according to the manufac-turer's instructions (Meso Scale Discovery).

Pharmacokinetics studies in cynomolgus monkeysFemale cynomolgusmonkeys (5 animals/dose group) received

a single intravenous infusion (1, 5, or 15mg/kg) or subcutaneousinjection (1 or 15 mg/kg) of REGN3767. Blood samples werecollected for measurement of functional REGN3767 concen-trations and antidrug antibody (ADA) in serum at predose andpostdose at various times for up to 56 days. Concentrations offunctional REGN3767 in serum were determined using ELISA.This procedure employed microtiter plates coated with arecombinant protein containing the extracellular domain ofhuman LAG-3 and utilized REGN3767 as the reference stan-dard. REGN3767 captured on the plate was detected using abiotinylated mouse anti-human IgG4 mAb REGN1298 (Rege-neron), followed by NeutrAvidin conjugated with horseradishperoxidase.

ResultsIn vitro characterization of REGN3767 as a single agent and incombination with anti-PD-1 antibody cemiplimab

Clone REGN3767 was selected from a panel of human anti-bodies generated by immunization of VelocImmune mice trans-genic for human variable regions (36, 37). REGN3767 boundspecifically to human LAG-3, with a KD of 3.22 nmol/L and0.13 nmol/L for monomeric and dimeric recombinant proteins,respectively. No cross-reactivity to mouse and rat LAG-3 wasdetected, consistent with low amino acid sequence identitybetween the extracellular regions of human and mouse (70%)or rat (67%) LAG-3. REGN3767 bound HEK293 cells engineered

to overexpress human or cynomolgus monkey LAG-3 with asimilar EC50 value of 1.1 nmol/L and 0.8 nmol/L, respectively(Fig. 1A). Flow cytometry confirmed REGN3767 binding toLAG-3 on activated primary human and cynomolgus monkeyCD4þ and CD8þ T cells (Supplementary Fig. S2A and S2B).REGN3767 bound activated CD8þ T cells, expressing either lowor high level of early activation marker CD69, with comparableEC50 values of 0.65–1.3 nmol/L and 0.7–1.0 nmol/L for humanand cynomolgus monkey donors, respectively (SupplementaryFig. S2C and S2D). The staining intensity was lower on activatedCD4þ T cells compared with CD8þ T cells in both species,suggesting lower LAG-3 expression; this observation is in linewith published studies (42, 43). In summary, REGN3767 exhib-ited similar EC50 values for binding to human and cynomolgusmonkey LAG-3 overexpressed on HEK293 cells or expressedendogenously on activated primary cells.

Human LAG-3 binds both human andmouseMHC II (15, 16),so the ability of REGN3767 to block these interactions wasassessed using a cell–cell adherence assay. A similar assay waspreviously used to demonstrate MHC II interactions withCD4 (14) and to identify the domains of LAG-3 responsible forbinding toMHC II (16). REGN3767 blocked the binding ofMHCII–positive human Raji B cells and murine A20 B cells, respec-tively, to HEK293/hLAG-3 cells with EC50 values of 4.2 and7.1 nmol/L, respectively (Fig. 1B). Binding of HEK293/hLAG-3to Raji cells or A20 cells was reversed when anti-MHC class IIantibody (anti-human HLA-DR or anti-mouse HLA I-A/I-E,respectively) was added to the system (Supplementary Fig. S3),suggesting that MHC class II plays a major role in this process.Neither Raji nor A20 cells displayed significant adherence toparental HEK293 cells, demonstrating that LAG-3 expression onHEK293 is required for adherence.

The potential of REGN3767 in combination with cemiplimabto rescue T-cell activity was evaluated in in vitro functional assays.In an engineered T-cell/antigen-presenting cell (APC) luciferase–based bioassay (Supplementary Fig. S4; Supplementary Materialsand Methods), activation of engineered JRT3.T3.5 T cells isdriven by engagement of a TCR (Ob2F3) with its cognate peptideMBP85-99 presented on MHC II by engineered HEK293 cells thatserve as APCs (44). JRT3.T3.5 T cells were additionally engineeredto express chimeric human LAG-3 and human PD-1. Chimerichuman LAG-3 was constructed of the human LAG-3 extracellulardomain and the cytoplasmic domain of the human inhibitoryreceptor CD300a, which contains immunoreceptor tyrosine-based inhibition motifs. APC express human MHC II with orwithout exogenously expressedhumanPD-L1. In both T-cell/APCformats, REGN3767 was titrated in the presence of 30 nmol/L ofcemiplimab. In the absence of PD-1/PD-L1 signaling, REGN3767restored T-cell activity with an EC50 value of 4.7 nmol/L; theactivity was unaffected by the presence of cemiplimab, asexpected. When the bioassay included the PD-1/PD-L1 signalingaxis, REGN3767 increased T-cell activation when combined withcemiplimab.

In an additional in vitro functional assay of allogeneic T cells:peripheral blood mononuclear cell (PBMC) mixed lymphocytereaction (MLR), cemiplimab or REGN3767, used as single agentsaugmented IFNg release by the alloreactive T cells (Fig. 1C).Combination of cemiplimab and REGN3767 further enhancedT-cell reactivity in the presence of a T-cell receptor stimulus. Toshow that REGN3767 alone, or in combinationwith cemiplimab,does not induce nonspecific T-cell activation, we performed

Characterization of Human Anti-LAG-3 Antibody REGN3767

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proliferation and cytokine release assay in PBMC ex vivo. Theaddition of cemiplimab and/or REGN3767 had no effect onPBMC proliferation and did not induce significant productionof IFNg , TNFa, IL2, and IL10 from freshly isolated or preacti-vated PBMC. Finally, we tested the ability of REGN3767 toinduce antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) in vitro. REGN3767at concentrations ranging from 95 fmol/L to 100 nmol/L didnot mediate ADCC or CDC activity against Jurkat/hLAG-3target cells in the presence of human NK cells or human serumcomplement, respectively. Taken together, these data suggestthat REGN3767 and cemiplimab treatments could synergisti-

cally enhance effector T-cell responses without nonspecific T-cell activation.

REGN3767 enhances the antitumor activity of cemiplimab inhuman PD-1xLAG-3 knockin mice by promoting T-cell–mediated immunity

We tested the antitumor activity of REGN3767 as a single agentor in combination with Regeneron's clinical anti-PD-1 antibodycemiplimab in a mouse MC38.Ova colon carcinoma model.Because cemiplimab and REGN3767 do not cross-react withmouse PD-1 and LAG-3, respectively, we genetically engineereddual human PD-1xLAG-3 knockin mice to express the

Figure 1.

Binding characteristics and in vitroactivity of anti-human LAG-3antibody REGN3767. A,REGN3767 displays dose-dependent binding to HEK293cells expressing human orcynomolgus monkey (mf) LAG-3.HEK293WT (left), HEK293/hLAG-3 (middle), and HEK293/mfLAG-3 (right) cells wereincubated with REGN3767 (blackcircles) or isotype control (IsoC)mAb REGN2759 (gray triangles)precomplexed with Alexa 647 Fabanti-hIgG. The x-axis indicates theantibody (Log10) concentration,and the y-axis indicates thegeometric median fluorescentintensity (MFI) of Alexa 647 cellstaining. B, REGN3767 antibodyblocks adherence of Raji and A20cells expressing endogenous MHCII to HEK293 cells expressinghuman LAG-3. Raji cells or A20cells fluorescently labeled withCalcein AMwere added toHEK293/hLAG-3 or HEK293 wtcells that had been pretreatedwith anti-LAG-3 antibodyREGN3767 (black circles) orisotype control REGN2759antibody (gray triangles). Thex-axis indicates the antibody(Log10) concentration, and they-axis indicates the fluorescenceintensity given in relativefluorescence units (RFU). C,Combination blockade withcemiplimab and REGN3767enhances CD4þ T-cell function inan allogeneic MLR. IFNg secretionin culture was assayed aftercoculturing 105 CD4þ T cells(initially exposed to PBMCs) withfreshly Mitomycin C–treated 2�105 PBMCs with or without testantibodies for 4 days.

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extracellular domains of human PD-1 and human LAG-3 aschimeric proteins containing the corresponding mouse trans-membrane and cytoplasmic domains from the respective mouseloci (Supplementary Fig. S5A). As shown in SupplementaryFig. S5B,PD-1xLAG-3dual knockinmice faithfully express humanPD-1 and LAG-3 proteins on activated T cells. The absence ofstaining with anti-mouse LAG-3 and anti-mouse PD-1 antibodiesconfirmed that full-lengthmouse PD-1 and LAG-3were deleted inPD-1xLAG-3 dual knockin mice. Of note, as human PD-1 bindsboth human and mouse PD-L1 with similar affinity, and humanLAG-3 binds both human and mouse MHC II (15, 16, 45),humanization of PD-L1 and MHC II are not required. Homozy-gous human PD-1xLAG-3 dual knockin mice displayed a normallife span and no overt signs of autoimmunity for at least 1 year,whereas PD-1xLAG-3-knockout mice develop strain and tissue-specific autoimmunity (22, 25).

In MC38.Ova tumors in PD-1xLAG-3–knockin mice, PD-1and LAG-3 coexpression was largely limited to CD4þ and CD8þ

TILs, while splenic T cells predominantly expressed PD-1 butnot LAG-3 (Supplementary Fig. S5C), consistent with previousreports (22). Approximately 47% of CD8þ and 50% of CD4þ

TILs cells were double positive, supporting the hypothesisthat these two receptors could directly cooperate in T-cellsuppression.

In a prophylactic MC38.Ova tumor model, monotherapy withcemiplimab at 10 mg/kg was moderately efficacious, whileREGN3767 dosed at 25 mg/kg had a minimal effect as a mono-therapy (Fig. 2A and C). However, combination treatment pro-longed survival (P < 0.0001) compared with isotype antibodycontrol treatment at the endof the studyonday46. Five of 12micewere tumor free in the combination treatment group, whereasonly 1 of 12 and 2 of 12 mice were tumor free in REGN3767 andcemiplimab monotherapy groups, respectively. In a therapeuticmodel, treatment of 100 mm3 MC38.Ova tumors with cemipli-mab at 10 mg/kg was partially efficacious, while there was nosingle-agent activity of REGN3767 at 25 mg/kg (Fig. 2B and D).Combination therapy showed a significant reduction of tumorgrowth on day 22 relative to isotype control (P < 0.05) and toREGN3767 (P < 0.01) groups.

To determine the relative contribution of cemiplimab andREGN3767 to the combination effect, we tested regimens, inwhich the dose of one antibody was constant and the other wastitrated (Supplementary Table S1).When the anti-PD-1 antibody,cemiplimab, was administered at 10mg/kg (five injectionswithin2 weeks), addition of REGN3767 at increasing doses (5 mg/kg or25 mg/kg) on the same schedule enhanced efficacy. Similarly,when REGN3767 was dosed at 25 mg/kg, lowering cemiplimabdose from10 to 1mg/kg reduced the antitumor activity. Thus, thecoblockade of PD-1 and LAG-3 is superior to either monotherapyand the efficacy is more dependent on anti-PD-1 activity.

Next, we determined the impact of anti-PD-1 and anti-LAG-3treatment on T cells in MC38.Ova tumors. Upon treatment ofmice with 100 mm3 MC38.Ova tumors with a combination ofcemiplimab and REGN3767 the average tumor size was signif-icantly smaller than in the isotype control or anti-LAG-3–treatedgroups (Fig. 3A), and combination treatment, but not mono-therapy, resulted in significant increase of tumor-infiltratingCD4þ T cells (Fig. 3B). To examine antigen-specific T cells intumors, TILs were restimulated ex vivowithOVAMHC I andMHCII peptides followed by intracellular staining for IFNg and TNFa.Neither mono- nor combination therapy had a significant

impact on the frequency of IFNg- or TNFa-producing CD8þ TILs,however, the frequency of IFNg- and TNFa-producing CD4þ

TILs was significantly higher in cemiplimab and dual-anti-body–treated groups as compared with the control treatment(Fig. 3C). As T-cell priming occurs in tumor dLN, we examinedantigen-specific and IFNg-expressing T cells in dLNs. There wasno expansion of OVA-specific CD8þ T cells in dLN as a result ofsingle or dual anti-PD-1 and anti-LAG-3 treatment. However,we observed a significant increase in IFNg-producing CD4þ andCD8þ T cell in dLNs in the dual anti-PD-1 and anti-LAG-3treatment group (Fig. 3D). In contrast to tumors, there were nochanges in TNFa production by T cells in response to single ordual antibody therapy in dLNs. Taken together, these datasuggest that cemiplimab and REGN3767 combination immu-notherapy reduces tumor growth by increasing the proportionof effector T cells both in the tumor and in dLNs. In addition,we detected increased concentrations of IFNg , TNFa, and IL10in serum and spleen lysates of PD-1xLAG-3–knockin micetreated with both REGN3767 and cemiplimab compared withtreatment with either antibody alone (Fig. 4).

To explore the underlying molecular mechanisms of MC38.Ova tumors response to anti-PD-1 and anti-LAG-3 treatment, weconducted RNA sequencing of MC38.Ova tumors in PD-1xLAG-3mice following treatment with one or two doses of cemiplimab,REGN3767, or their combination. Mice were randomized on day10 after MC38.Ova cells implantation when tumors reached100mm3. Treatment with two doses of REGN3767, cemiplimab,or their combination (on randomization day 10 and on day 14)significantly reduced tumor volumes compared with isotypecontrol treatment, while single-dose treatment on day 10 did notshow antitumor activity (Fig. 5A). Both REGN3767 and cemipli-mab promoted robust transcriptional changes in tumors, andcombined PD-1 and LAG-3 blockade resulted in enhancedimmune activation signatures. The combination treatmentresulted in significant upregulation of 1,616 and 2,256 genesafter one or twodoses, respectively (fold changes vs. control group>1.5; Fig. 5B). While the number of upregulated genes withREGN3767 treatment kept increasing (131 genes after one dosevs. 731 genes after two doses), the number of cemiplimab-induced genes decreased (696 upregulated genes after one dosevs. 161 genes after two doses), indicating different kinetics ofresponse to thesemonotherapies. Transcriptional gene expressionprofiles indicative of different TILs subpopulations in the com-bination group showed the enrichment of immune cell signa-tures, including neutrophils, macrophages, NK, and T cells; allsignatures were more pronounced after two doses (Fig. 5C). Thecombination therapy also enhanced immune responses promot-ed by either antibody alone, including genes associated withT cells' activation and effector function. In the T cells signature,a prominent increase in the expression of Cd40lg receptor, whichis involved in costimulatory T-cell receptor signaling, wasobserved in a combination treatment (Fig. 5C). Blocking PD-1and LAG-3 in combination therapy also resulted in upregulationof inhibitory PD-L1 and VISTA ligands that can suppress T-cellresponses, suggesting a potentialmechanismof adaptive immuneescape (Fig. 5D). Cytotoxic Gzma, Gzmb, and Prf1 as well asselected T-cell immunoregulatory and Th1 signaling genes wereupregulated in the combination treatment group, consistent withour finding that combination treatment enhanced the cytotoxicfunction of intratumoral T cells after tumor antigen restimulation(Fig. 3C).






Figure 2.

Antitumor responses of cemiplimab and REGN3767 combination treatment in MC38.Ova tumor model in human PD-1xLAG-3–knockin mice. A and C,Prophylactic cemiplimabþREGN3767 treatment inhibits MC38.Ova tumor growth. On day 0, human PD-1xLAG-3–knockin mice were injected subcutaneouslywith 5� 105 MC38.Ova cells and randomized into four treatment groups. Mice were administered REGN3767 (25 mg/kg; N¼ 12), cemiplimab (10 mg/kg; N¼ 12),REGN3767 (25 mg/kg)þ cemiplimab (10 mg/kg) combination (N¼ 12), or hIgG4 control (25 mg/kg;N¼ 6) by i.p. injection on days 3, 7, 10, 14, and 17. A, Averagetumor volumes (mm3� SEM) in each treatment group. Treatment days are indicated by arrows (top, left graph). Individual tumor volumes in each treatmentgroup were measured on day 22, the last timepoint when all animals in the study were alive (top, right graph). Statistical significance was determined by one-wayANOVAwith Tukey multiple comparison post-test (� , P < 0.05; �� , P < 0.01). Kaplan–Meier survival curves of mice treated with single agents or combinationagents are shown (bottom graph). A log-rank (Mantel–Cox) test revealed that combination therapy or cemiplimab monotherapy significantly prolongedmousesurvival compared with the control group (�� , P < 0.0001; �� , P < 0.01, respectively). C, Individual tumor growth curves in each treatment group. The number oftumor-free (TF) animals on day 32 is shown. B and D, CemiplimabþREGN3767 combination treatment delays growth of established tumors. PD-1xLAG-3–knockin mice were injected subcutaneously with 5� 105 MC38.Ova cells on day 0, randomized into four treatment groups on day 10 (108mm3mean tumorvolume per group) and treated by i.p. injection with cemiplimab (10 mg/kg; N¼ 10), REGN3767 (25mg/kg;N¼ 9), combination cemiplimabþ REGN3767(10 mg/kgþ 25 mg/kg; N¼ 11), or isotype control antibody (25 mg/kg, N¼ 7) on days 10, 14, 17, 22, and 25. B, Average tumor volumes (mm3� SEM) in eachtreatment group (left graph). Individual tumor volumes in each treatment group were measured on day 22, the last timepoint when all animals in the study werealive (right graph). D, Individual tumor growth curves in each treatment group.

Burova et al.





Figure 3.

Combination cemiplimab and REGN3767 treatment results in enhanced cytokine production by tumor-infiltrating T cells. A total of 5 � 105 MC38.Ovacells were injected subcutaneously into female human PD-1xLAG-3–knockin mice. At day 11 postimplantation, mice were randomized into fourtreatment groups with an average tumors size of 100 mm3 (n ¼ 12 per group). Mice were dosed intraperitoneally either with hIgG4 control antibody(25 mg/kg), or cemiplimab (10 mg/kg) or REGN3767 (25 mg/kg), or combination of cemiplimab and REGN3767 (10 mg/kg and 25 mg/kg,respectively). Antibodies were administered on days 11 and 15, and on day 18 postimplantation tumors were harvested, dissociated into single-cellsuspensions, and stained for flow cytometry. Data are representative of two combined experiments. A, Individual tumor volumes in each treatmentgroup were measured on day 18 at harvest. B, Numbers of CD4þ and CD8þ T cells within tumors after treatments on day 18. Tumors were dissociatedinto single-cell suspension, and CD45-enriched fraction was isolated through Percoll gradient centrifugation and further processed for flow cytometry.C, Plots of IFNgþ or TNFaþ cells of total tumor-infiltrating CD8þ or CD4þ T cells, following ex vivo OVA MHC I and MHC II peptide stimulation. D,Plots of IFNgþ cells of total dLN CD4þ and CD8þ T cells. Tumor inguinal dLNs were isolated and stimulated with PMA/ionomycin for 4 hours in thepresence of Brefeldin A, then analyzed for cytokine production by intracellular staining and flow cytometry. Error bars depict SEM. Nonparametricone-way ANOVA with Kruskal–Wallis test was used with Dunn post-test (� , P < 0.05; �� , P < 0.01; �� , P < 0.0001).






Pharmacokinetics and toxicology studies of REGN3767 incynomolgus monkeys

Twenty-five (25) female cynomolgus monkeys (5 animals/dose group) received a single intravenous infusion (1, 5, or15 mg/kg) or subcutaneous injection (1 or 15 mg/kg) ofREGN3767 and animals were followed over the 8-week studyperiod. Single dose pharmacokinetic of functional REGN3767showed linear kinetics with a half-life of 10–12 days (Fig. 6).The immunogenicity of REGN3767 was high, with 5 of 15 and5 of 10 animals showing positive ADA responses followingintravenous and subcutaneous administrations, respectively,but there were no observable adverse effects. Following intra-venous infusion of REGN3767, the pharmacokinetic para-meters were estimated using noncompartmental analysis (Sup-plementary Table S2). The mean Cmax increased in an approx-imately dose-proportional manner across the tested dose levels.Following intravenous infusion of REGN3767, the meanAUCinf increased in a dose-proportional manner as demon-strated by similar AUCinf/dose values. Consistent with this

finding, the mean clearance values were similar for the threedose groups, ranging from 4.11 to 4.47 mL/day/kg. The elim-ination phase t1/2 values for the three dose groups were alsosimilar, ranging from 10.8 to 11.5 days.

In a cynomolgus monkey toxicology study, 5 animals/sex/group were given 0, 2, 10, or 50 mg/kg/week REGN3767 byi.v. infusion for 4 consecutive weeks followed by an 8-weekrecovery period. REGN3767 was well-tolerated at all dose levels.There were no unscheduled deaths during the study and no drug-related clinical signs evident. There were no REGN3767-relatedeffects on body weights, body temperatures, clinical pathology,cardiovascular, or neurological endpoints, with no substantial sexdifferences noted. In addition, there were no REGN3767-relatedchanges in lymphocyte subpopulations in PBMC, organ weights,or findings from the gross andmicroscopic examination of tissuesfrom primary necropsy (day 30) or after the 8-week recoveryperiod (day 85). The no observable adverse effect level for thisstudy was considered to be 50 mg/kg, the highest dose levelevaluated.

Figure 4.

Combined cemiplimab and REGN3767 treatment results in enhancement of cytokine secretion in serum and spleens by therapeutic antibodies. PD-1xLAG-3–knockin mice were injected subcutaneously with 5� 105 MC38.Ova cells. At day 11 postimplantation, mice were randomized into treatment groups with anaverage tumor size of 100mm3. Mice were dosed intraperitoneally either with isotype control antibody (hIgG4) at 25 mg/kg, cemiplimab at 10 mg/kg, REGN3767at 25 mg/kg, or the combination of cemiplimab and REGN3767 at 10 mg/kg and 25 mg/kg, respectively. Antibodies were administered on days 11 and 15, and onday 18 postimplantation serum and spleens were collected. Spleens were dissociated into single-cell suspensions. Levels of serum and spleen cytokines weremeasured with V-PLEX Proinflammatory Panel 1 Mouse Kit (Meso Scale Discovery). Data points represent the medians plotted in a box-and-whiskers plots(Prizm software) of the cytokines levels in serum (A) and spleens (B) in each treatment group at collection timepoint. Results are shown for IFNg , TNFa, and IL10.�� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.

Burova et al.





Figure 5.

Changes in expression of immune-related genes in tumors treated with cemiplimab, REGN3767, and the combination. A,Mean volumes of MC38.Ova tumors inPD-1xLAG-3mice, treated with single dose or two doses of control antibodies, cemiplimab, REGN3767, or cemiplimabþREGN3767. On day 0, MC38.Ova cells(5� 105 cells/mouse) were implanted subcutaneously and mice were randomized on day 10, when tumors reached 100mm3. One group of mice was treated onrandomization day 10 with a single dose of control antibodies, REGN3767, cemiplimab, or their combination, and another group was treated with two doses (onday 10 and day 14). Tumors in a single-dose treatment group were harvested on day 14, and tumors in two doses treatment group were harvested on day 17. Datarepresent analysis of 9–10 mice per each antibody treatment, mean� SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. B, Differentially expressed genes with at least1.5-fold change by RNA seq analysis after single or two doses of single or dual blockade of cemiplimab and REGN3767. C, Heatmap of predefinedmarker genesfor neutrophils, macrophages, NK cells, T cells, and cytokines/cytotoxic genes regulated by combination therapy. For single or two doses points, fold changesrelative to median values for the isotype controls are shown. D, Combination treatment results in increased relative levels of mPD-L1 and mVISTA RNA(normalized to mouse cyclophilin RNA) compared with the single antibody or isotype control group (assigned a value of 1.0). Mice were treated as in A, andtumors were collected after single or two doses of monotherapy or dual therapy of cemiplimab and REGN3767. Tumor RNAwas analyzed by TaqMan real-timePCR. Values are median, plotted in box-and-whiskers plot with whiskers extended fromminimum tomaximum (� , P < 0.05; �� , P < 0.01; ��, P < 0.001;�� , P < 0.0001).






DiscussionBlockade of inhibitory pathways on activated T cells with

mAbs against the immune checkpoint receptors PD-1 andCTLA-4 has shown remarkable antitumor activity in diversehuman cancers. Furthermore, therapeutic cotargeting of PD-1and CTLA-4 showed enhanced clinical activity compared withsingle agents. These successes have led to the exploration ofadditional inhibitory signaling pathways on immune cells.LAG-3 receptor is another inhibitory checkpoint on T cells; itspotential role in cancer is supported by extensive preclinicalstudies (8, 22, 23). Notably, analysis of mouse and humantumors suggests that coexpression of LAG-3 and PD-1 is asso-ciated with T-cell dysfunction (21, 22), indicating that induc-ible LAG-3 expression on activated T cells could be a potentialmechanism of therapeutic resistance to PD-1 checkpoint block-ade (46). These findings have prompted efforts to explore usinganti-LAG-3–blocking antibodies, either alone or in combina-tion with anti-PD-1, in cancers (32–34).

To target LAG-3, we generated a human mAb REGN3767 thatbinds to human and monkey LAG-3 and inhibits the interactionwith its ligand, MHC class II. REGN3767 is engineered as humanIgG4 isotype and contains one mutation in the hinge region tominimize half-antibody formation and three additional pointmutations in Fc region to reduce the potential for inducingantibody-dependent cytotoxicity against LAG-3–expressing cells.We confirmed that REGN3767 does not elicit antibody Fc-mediated ADCC or CDC activity. In cell-based assays, REGN3767rescued T-cell activation as a single agent and enhanced responsesto a clinical anti-PD-1 antibody, cemiplimab, demonstrating thatLAG-3 delivers an independent inhibitory signal to activated Tcells during PD-1 blockade. In an engineered T-cell/APCbioassay,the combination of REGN3767 and cemiplimab was able toovercome the inhibitory effects of MHC II/LAG-3 and PD-L1/PD-1 signaling. In a second assay, REGN3767was able to enhancethe response to cemiplimab of primary CD4þT cells in allogeneicT cell:PBMC MLR assay.

Consistent with in vitro findings, REGN3767 therapy reducedtumor growth in a subset of human PD-1XLAG-3–knockin mice,and improved antitumor efficacy of cemiplimab, thus providingpreclinical proof of concept for dual PD-1 and LAG-3 blockade inpatients with cancer.

The potential mechanism underlying the additive effect ofREGN3767 and cemiplimab was consistent with T-cell activationboth in tumors and the periphery of MC38.Ova-bearing mice.Combination treatment was associated with increased intratu-moral CD4þ and CD8þ T cells producing IFNg , TNFa, as well aselevated IFNg , TNFa, and IL10 levels in blood and spleen.Although IL10 is normally associated with reduced antitumoractivity (47), some reports describe a role of IL10 in expandingtumor CD8þ T cells (48).

The role of REGN3767 in modulating T-cell responses wasfurther supported by robust transcriptional changes consistentwith T-cell expansion and activation in tumors in response toREGN3767 therapy. Combination therapy elicited additionalimmune-related gene changes, not seen with either antibodyalone. Surprisingly, in addition to T-cell expansion and acti-vation, REGN3767 therapy engaged other types of tumor-associated leukocytes, including macrophages, neutrophils,and NK cells, suggesting that the myeloid compartment maycontribute to the therapeutic response following anti-LAG-3therapy. The physiologic role of LAG-3 on immune cell typesother than T cells, including plasmacytoid DCs, NK cells, and Bcells is poorly understood (8, 10), and may be related toadditional LAG-3 ligands reported recently. It has been sug-gested that LSECtin, a member of the DC-SIGN family, andGalectin-3, both expressed in many tumors, are ligands that canalso regulate LAG-3–expressing CD8þ T cell and NK cells intumor microenvironment (49, 50). Fibrinogen-like proteinFGL1, a liver-secreted protein, is another LAG-3 functionalligand independent of MHC II (51). At this time, it is not clearhow REGN3767 affects LAG-3 interaction with additionalputative ligands. Detailed studies as to when and where theseputative ligands are expressed and how they interact with

0 7 14 21 28 35 42 49 560.01

0.1

1

10

100

1,000

Time (day)

Con

cent

ratio

n (m

g/m

L)REGN3767 1 mg/kg i.v. infusionREGN3767 5 mg/kg i.v. infusionREGN3767 15 mg/kg i.v. infusion

LLOQ = 0.078 mg/mL

REGN3767 15 mg/kg s.c.REGN3767 1 mg/kg s.c.

Figure 6.

Pharmacokinetic parameters forREGN3767 following singleintravenous (i.v.) and subcutaneous(s.c.) administration to cynomolgusmonkeys. Single-dosepharmacokinetic of REGN3767 incynomolgus monkeys shows linearkinetics with a half-life of 10–12 days.ADA impacted REGN3767concentrations in 33% (5/15) and 50%(5/10) of intravenous andsubcutaneous group animals,respectively. ADA-impactedconcentrations were excluded. LLOQ,lower limit of quantification.

Burova et al.





LAG-3 will further facilitate unravelling the mechanisms ofLAG-3 targeting.

Neither REGN3767 nor cemiplimab, as monotherapy or incombination caused nonspecific lymphocyte activation in anex vivo cytokine release assay. Cynomolgus monkey toxicologystudies were performed separately for REGN3767 and cemipli-mab and there were no potential adverse effects related to lym-phocyte-binding patterns of these antibodies.

Our results expand on the existing preclinical data that com-bination blockade of PD-1 and LAG-3 signaling has potential forincreased therapeutic benefit in cancer treatment. REGN3767 incombination with cemiplimab is currently being investigatedclinically in multiple tumor types.

Disclosure of Potential Conflicts of InterestE. Burova has ownership interest (including patents, stock, and stock options)

in Regeneron and is co-inventor on a pending patent application relating to thesubject matter of the manuscript. A. Hermann has ownership interest (includingpatents and stock) in Regeneron. E. Ullman has ownership interest (includingpatents and stock) in Regeneron. G. Halasz has ownership interest (includingpatents) in Regeneron Pharmaceuticals stock options. T. Potocky has ownershipinterest (including patents and stock) in Regeneron. O. Allbritton has ownershipinterest (including stock) in Regeneron. W. Poueymirou is an executive directoranimal production and vivarium operations (paid consulting) at RegeneronPharmaceuticals. J. Martin has ownership interest (including patents and stock)in Regeneron. W.C. Olson is vice president (paid consulting) at RegeneronPharmaceuticals. A. Murphy has ownership interest (including patents, stock,and stock options) in Regeneron. E. Ioffe has ownership interest (includingpatents) in Regeneron, and is co-inventor on a pending patent applicationrelating to the subjectmatter of themanuscript.M.Mohrs has ownership interest(including patents, stock, and stock options) in Regeneron and co-inventor on apending patent application relating to the subject matter of the manuscript. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: E. Burova, A. Hermann, J. Dai, E. Ullman, T. Potocky,W.C. Olson, A. Murphy, E. Ioffe, G. Thurston, M. MohrsDevelopment of methodology: E. Burova, A. Hermann, J. Dai, E. Ullman,T. Potocky, M. Liu, J. Pei, A. Rafique, A. Murphy, E. IoffeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Burova, A. Hermann, J. Dai, E. Ullman,T. Potocky, M. Liu, O. Allbritton, A. Woodruff, J. Pei, W. PoueymirouAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Burova, A. Hermann, J. Dai, E. Ullman, G. Halasz,T. Potocky, S. Hong, M. Liu, O. Allbritton, A. Woodruff, J. Pei, A. Rafique,A. Murphy, G. ThurstonWriting, review, and/or revision of themanuscript: E. Burova, S.Hong,M. Liu,A. Rafique, J. Martin, W.C. Olson, E. Ioffe, G. Thurston, M. MohrsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Burova, A. Hermann, J. Dai, T. Potocky, J. Pei,D. MacDonaldStudy supervision: E. Burova, A. Hermann, J. Dai, J. Martin, W.C. Olson,A. Murphy, E. Ioffe, M. Mohrs

AcknowledgmentsThe authors thank all Regeneron employees who contributed to the

generation and characterization of REGN3767 including R. Leidich,A. Badithe, P. Kruger, C.-J. Siao, A. Mujica, H. Polites, G. Kroog, I. Lowy,and N. Papadopoulos. This work is funded by Regeneron Pharmaceuticals, Inc.

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

ReceivedDecember 17, 2018; revised June 17, 2019; accepted August 2, 2019;published first August 8, 2019.

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Burova et al.




2019;18:2051-2062. Published OnlineFirst August 8, 2019.Mol Cancer Ther Elena Burova, Aynur Hermann, Jie Dai, et al.

Knockin Mice−PD-1xLAG-3Antibody Cemiplimab in Human Characterization and Activity in Combination with the Anti-PD-1 Preclinical Development of the Anti-LAG-3 Antibody REGN3767:

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