A Chemo-enzymatically Linked BispecificAntibody Retargets T Cells to a Sialylated Epitopeon CD43 in Acute Myeloid LeukemiaLina Bartels1,2,3, Greta de Jong1,2,3,4,5, Marijn A. Gillissen1,2,3,4,5, Etsuko Yasuda1,Veronika Kattler1, Camille Bru1, Christien Fatmawati1, Susan E. van Hal-van Veen1,Madalina G. Cercel1, Gemma Moiset1,2,3,4,5, Arjen Q. Bakker1, Pauline M. van Helden1,Julien Villaudy1, Mette D. Hazenberg2,3,4,5, Hergen Spits1,2,3, and Koen Wagner1
Abstract
Acute myeloid leukemia (AML) is a high-risk disease with apoor prognosis, particularly in elderly patients. Because cur-rent AML treatment relies primarily on untargeted therapieswith severe side effects that limit patient eligibility, identifi-cation of novel therapeutic AML targets is highly desired. Werecently described AT1413, an antibody produced by donor Bcells of a patient with AML cured after allogeneic hematopoi-etic stem cell transplantation. AT1413 binds CD43s, a uniquesialylated epitope on CD43, which is weakly expressed onnormalmyeloid cells and overexpressed onAML cells. Becauseof its selectivity for AML cells, we considered CD43s as a targetfor a bispecific T-cell–engaging antibody (bTCE) and gener-ated a bTCE by coupling AT1413 to two T-cell–targetingfragments using chemo-enzymatic linkage. In vitro, AT1413bTCE efficiently induced T-cell–mediated cytotoxicity towarddifferent AML cell lines andpatient-derivedAMLblasts,where-as endothelial cells with low binding capacity for AT1413
remained unaffected. In the presence of AML cells, AT1413bTCE induced upregulation of T-cell activation markers, cyto-kine release, and T-cell proliferation. AT1413 bTCE was alsoeffective in vivo. Mice either coinjected with human peripheralblood mononuclear cells or engrafted with human hemato-poietic stem cells [human immune system (HIS) mice] wereinoculated with an AML cell line or patient-derived primaryAML blasts. AT1413 bTCE treatment strongly inhibited tumorgrowth and, in HIS mice, had minimal effects on normalhuman hematopoietic cells. Taken together, our results indi-cate that CD43s is a promising target for T-cell–engagingantibodies and that AT1413 holds therapeutic potential in abTCE-format.
Significance: These findings offer preclinical evidence forthe therapeutic potential of a bTCE antibody that targets asialylated epitope on CD43 in AML.
IntroductionAcute myeloid leukemia (AML) is a high-risk hematologic
malignancy, with high mortality rates especially in elderlypatients (1, 2). With a median age of 68 years at diagnosis, AMLis predominantly a disease of the elderly population (3). FormostAML subtypes, current standard of care has not improved over thepast three decades and still relies on chemotherapy and allogeneichematopoietic stem cell transplantation (HSCT; ref. 4). The severe
side effects and comorbidities of these treatments limit theirapplicability especially in older patients (>60 years) and/orpatients with poor physical fitness (5, 6). This poses an unmetmedical need and highlights the need for better tolerated, broadlyapplicable AML treatments. To develop new therapeutic optionsfor patients with AML, the identification of novel AML-specifictargets is essential.
Earlier, we have examined whether patients with AML suc-cessfully treated with allogeneic HSCT generate AML-specificantibodies (7–9). Allogeneic HSCT can evoke an immuno-therapeutic graft-versus-leukemia reaction, which includes aB-cell response and the generation of tumor-specific anti-bodies (10). From a number of allogeneic HSCT–treated pati-ents with AML in durable remission we immortalized B cells,to generate clonal lines of donor-derived B cells, which pro-duce antibodies reacting with autologous and allogeneic AMLtumor samples (11, 12).
One of these antibodies is AT1413, which recognizes a uniquesialylated epitope on CD43 (CD43s) and binds to all AML andmyelodysplastic syndrome (MDS) blast samples that we tested(n ¼ 80) including the patient's own blasts (8). These samplesrepresent all World Health Organization 2008 types of AML andMDS. AT1413 also binds to healthy granulocytes andmonocytes,but to a lower extend compared with AML orMDS blasts from thesame donor, and weakly binds endothelial cells. No binding to
1AIMM Therapeutics, Amsterdam, the Netherlands. 2Department of Experimen-tal Immunology, Amsterdam University Medical Center, Location AMC, Amster-dam, the Netherlands. 3Amsterdam Infection and Immunity Institute, Amster-dam, the Netherlands. 4Cancer Center Amsterdam, Amsterdam, the Nether-lands. 5Department of Hematology, Amsterdam University Medical Center,Location AMC, Amsterdam, the Netherlands.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
other normal cells was detected in an IHC screen containingsamples of multiple tissues (8).
AT1413 induces antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) invitro and inhibited growth of an AML cell line in an in vivomouse model of AML, suggesting that AT1413 may haveimpacted the favorable clinical outcome of the patient it wasisolated from.
Because of the preferential reactivity of AT1413 with AMLcells, we reasoned that a bispecific T-cell–engaging antibody(bTCE) based on AT1413 could have increased cytotoxicpotency and therapeutic potential. Many bTCEs are in clinicaldevelopment, and two bTCEs have reached market approval(13). One of these, blinatumomab, is used to treat patientswith relapsed or refractory B-cell precursor acute lymphaticleukemia and is undergoing clinical testing for other types ofleukemia. Hence, the bTCE concept is clinically validated forhematologic malignancies (14, 15).
It has been suggested that the target of a bTCE needs to belocated sufficiently close to the cell membrane, to form a stablecytolytic synapse between the target tumor cell and the T cell (16).AT1413 binds a sialylated epitope in the extracellular domain ofCD43 (8). Being highly glycosylated, this extracellular domain isdescribed to protrude away from the plasma membrane in a rod-like manner with a length of about 45 nm (17). While otherCD43-targeting antibodies bind to membrane-distal CD43regions, the CD43s epitope was mapped within a membrane-proximal part of the extracellular domain (8). This further encour-aged us to generate a bTCE against CD43s as this favorablelocation of the CD43s epitope will likely support the bTCE'sefficacy and potency (16).
To prepare bTCEs, antibodies or antibody fragments havesuccessfully been equipped with single-chain variable fragments(scFv) derived from established CD3e-binding antibodies (18–21).To convert AT1413 into a bTCE, we chose to chemo-enzymaticallylink a CD3e-binding scFv, derived from the antibody UCHT1(22), to the C-termini of the AT1413 IgG heavy chains in aC-to-C orientation. C-to-C fusion of the IgG and UCHT1 scFv isachieved by combining sortase transpeptidation and click chem-istry (23–25). To abolish interactionwith other non-T-cell immuneeffector cells, we removed Fc-gamma receptor (FcgR) interactionsites by introducing two point mutations G236R and L328Rinto the AT1413 heavy chains (AT1413-Fc0), as described previ-ously (26). The resulting conjugation product is a bispecific anti-body, in which two CD3e-binding scFv are coupled to one IgGmolecule. Here, we describe the generation of an AT1413 bTCE anddemonstrate that this bTCE efficiently induces T-cell–mediatedlysis of CD43s-expressing AML cells in vitro and in vivo.
Materials and MethodsProtein expression and purification
S. aureus sortase A (residues 82–249) with an N-terminalHis6-tag was expressed and purified as described previously(27). The E. coli strain expressing S. aureus sortase A was a giftfrom Hidde Ploegh (Harvard Medical School, Boston, MA).
C-terminally LPETGGH6-tagged (tag sequence: GGGGSLPET-GGHHHHHH) antibodies were cloned, expressed, and purifiedas described previously (24).
The UCHT1 scFv sequence was derived from the antibodyUCHT1 (22, 28), by fusing the UCHT1 heavy and light chain
variable regions, with the heavy chain variable domain orientedN-terminal to the light chain variable domain and a (G4S)3-linkersequence in between both domains, and adding a C-terminalLPETGGH6-tag. The construct was cloned into a pXC19 vector(Lonza), using synthetic codon-optimized open reading frames(GeneArt) encoding thehumanVK3 leader sequence. LPETGGH6-tagged UCHT1 scFv was stably expressed in CHO-GS cells andpurified by immobilized metal affinity chromatography (IMAC)and size exclusion chromatography (SEC). IMAC was conductedon HisTrap excel and SEC on HiLoad Superdex 200 16/600 (GEHealthcare) columns using an €AKTA Explorer 10S System (GEHealthcare).
Generation, purification, and stability of bTCEAT1413bTCE andAT1002bTCEmoleculeswere generated and
purified as described previously (25).bTCE stability was assessed by incubation of 0.1 mmol/L bTCE
in PBS or IMDMþ 8% FBS at 37�C for up to 21 days followed bySDS-PAGE and flow cytometric analysis.
Primary cell isolation and cell cultureStudy protocols were approved by the institutional Medical
Ethical Committee of Amsterdam UMC, location AMC (Amster-dam, the Netherlands). Human peripheral blood mononuclearcells (PBMC) were obtained from buffy coats of healthy blooddonors (Sanquin) and AML blasts from peripheral blood (PB) orbonemarrow (BM) of patientswithAMLbyficoll density gradientcentrifugation, after obtaining written informed consent, andcryopreserved or used directly.
For cytotoxicity assays, PBMCs and AML blasts were thawedand rested in IMDMMedium (Gibco) supplementedwith 8%FBS(Gibco) and 100 IU/mL penicillin and 100 mg/mL streptomycin(PS; Roche) overnight.
CD8-positive T cells for T-cell cultures were isolated fromPBMCs by FACS on a FACSAria System (BD Biosciences). T cellswere expanded in the presence of 1 eq irradiated (25 Gy) JY cellsand 10 eq irradiated (100 Gy) PBMCs with 0.5 mmol/L phyto-haemagglutinin (BioTrading) in Yssel medium (29) containing1% human serum (ATCC), 0.1 nmol/L recombinant human IL2(Sigma), and PS. Medium was renewed every 3–4 days and freshIL2 was added.
Cell lines were obtained from DSMZ (SH-2, Molm13,BV173, and Kasumi3) or ATCC (THP-1, HL-60, Jurkat, andU266) and endothelial cells from Lonza (HAEC and HUVEC)and were maintained as recommended by the supplier. JurkatTCRa�/� cells were a gift from Ton Schumacher (NetherlandsCancer Institute, Amsterdam, the Netherlands). Cell linesand endothelial cells were tested monthly for Mycoplasma byPCR. Cell lines were authenticated using short tandem repeatanalysis.
Flow cytometryFlow cytometry was conducted on a FACS Canto II (BD Bio-
sciences) or a FACS LSR Fortessa X-20 (BD Biosciences). Datawere processed with FlowJo 10.1 (FlowJo, LLC) and Prism8.0.1 (GraphPad Software, Inc.).
FACS binding assays were carried out in 384-well plates in1% BSA (Roche) in PBS (Gibco) at 4�C. Cells were harvested,washed, and incubated with antibody, scFv, or bTCE at 12,000cells per well for 30 minutes. After washing, surface boundantibody and bTCE were detected with AF647 goat anti-human
A T-Cell Engager Targeting CD43s on Acute Myeloid Leukemia
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IgG HþL (Thermo Fisher Scientific) and UCHT1 scFv withAF647 anti-PentaHis (Qiagen) by incubating for 30 minutesin the dark. Cells were washed and resuspended in 10 nmol/L40,6-diamidino-2-fenylindole (DAPI; Sigma) directly beforecytometric analysis.
In vitro cytotoxicity assaysCytotoxicity was assessed by a modified Calcein AM retention
assay adjusted for T-cell engagement (30). PBMCs or T-cell–depleted PBMCs were added at an effector-to-target (E:T) ratioof 10:1 (unless otherwise indicated) to target cells before incu-bation at 37�C and 5 % CO2 for 6–44 hours.
Whole-blood assays were performed likewise, but by addingPB from healthy volunteers instead of PBMCs to target cellsbefore incubating for 5 hours at 37�C and 5 % CO2. Leukocyteswere stained with anti-human CD66b PE and CD14 APC-Cy7(BioLegend) and live cells with an eFluor660 Viability Marker(eBioscience). Red blood cells were lysed by adding BD FACSLysing Solution (BD Biosciences), incubating for 15 minutes at22�C, centrifuging, discarding supernatant, and repeating thisstep once. Accudrop beads were added and samples analyzed asabove.
T-cell activation assaysAML target cells were allowed to opsonize AT1413 bTCE
or control as during cytotoxicity assays and were incubatedwith PBMCs (E:T ratio 10:1 or 2:1) or T cells (E:T ratio of 1:1),for 24–48 hours at 37�C and 5% CO2. T cells were stained withanti-human CD4 PE-Cy7, CD8a APC-Cy7, CD25 APC, CD45ROFITC (all BioLegend), CD69 PE/APC (BioLegend/BD Bios-ciences), CD107a APC (BD Biosciences), and/or CD197 (CCR7)PE (BD Biosciences) for 30 minutes in the dark or during incu-bation (CD107a). Cells were washed, resuspended in 10 nmol/LDAPI, and analyzed by flow cytometry. T cells were selected asDAPI-negative, gated for CD4- or CD8-positive, where applicablefor CD45RO- and CD197-positive or -negative, and for CD69-,CD25-, or CD107a-positive cells. Statistical testingwas performedwith Prism 8.0.1.
IFNg ELISAAML target cells were incubated with AT1413 bTCE or control
and T cells for 48 hours as for T-cell activation assays. Cell-freesupernatants were collected and diluted 4–10� with ELISA/ELISPOT Diluent (Thermo Fisher Scientific). ELISAs were per-formed with a Human IFN gamma ELISA Ready-SET-Go! Kit(Thermo Fisher Scientific) in a 384-well format. Absorbance at450 nmwasmonitored with an EnVision 2104Multilabel Reader(PerkinElmer).
Proliferation assaysCultured T cells were washed with PBS, stained with 5 mmol/L
Celltrace CFSE in PBS at 37�C and 5% CO2 for 20 minutes andwashed with IMDM medium þ 8% FBS. In 96-well U-bottomplates, 25,000 target cells perwellwere preincubatedwithAT1413bTCE or control in IMDM medium þ 8% FBS for 15 minutes at22�C. T cells were added (E:T ratio 1:1) and samples wereincubated for 5 days at 37�C and 5% CO2; then stained withAPC-Cy7 anti-human CD8a as described for T-cell–activationassays, washed, resuspended in 10 nmol/L DAPI, and analyzedby flow cytometry.
In vivo experimentsEthical consideration. All animal protocols were carried out inaccordance with Dutch and European laws. Experiments wereapproved by the AMC Animal Experiments Commission (Dier-experimentencommissie) and conducted according to AMC'sinstitutional guidelines.
Generation of human immune system mice. Human immunesystem (HIS) mice were generated using NOD.Cg-Prkdcscid
Il2rgtm1Wjl/SzJ (NSG; The Jackson laboratory) mice. Briefly,NSG newborns before 5 days of age were irradiated with1 Gy using a Cs137 source and injected within 24 hours with5 � 104 Lin�CD34þCD38� human stem cells obtained fromfetal liver.
Efficacy studies against SH-2. At 7–8 weeks, 12 female NSG mice(19.9 � 1.5 g) were injected intravenously in the tail vein with10 � 106 luciferase/ZsGreen-labeled SH-2 cells in 100 mL of PBS(day 0). Starting after 15 days, tumor growth was assessed weeklyby bioluminescence imaging (BLI) using a Photon Imager (Bio-space Lab). Mice were injected intraperitoneally with VivoGloLuciferin, In Vivo Grade (3.75 mg, Promega) 15 minutes beforeimage acquisition. During the acquisition, the mice were anes-thetized using Isoflurane and maintained on a thermally con-trolled pad. Mice were weighted twice per week. Mice losingmorethan 15 % of their maximum weight were humanely sacrificed.
At day 19, mice were randomized and allocated to groups of 6mice each. Mice were injected intravenously with 5 � 106 PBMCand directly given the first dose of 40 mg bTCE in 100 mL PBS i.p.Subsequent bTCE doses were biweekly injected intravenouslyfrom day 22 to day 33. Blood samples were collected from thesubmandibular vein. Mice were sacrificed on day 36 or day 40 bycervical dislocation after deep isoflurane anesthesia. The druginjection and measurements were performed by techniciansblinded to the group allocation. Tumor growth inhibition wascalculated as the median of tumor size of the AT1413 bTCE–treated groupdivided by the one from the control group at the endof the experiment.
Studies with HIS-NSG mice were conducted in the same way,but without PBMC injection and with the following variations.At 12–13 weeks of age, 7 female HIS-NSG mice (20.3 � 1.4 g)were injected intravenously with 8 � 106 luciferase/ZsGreen-labeled SH-2 cells. Weekly BLI was started on day 12. Mice weretreated biweekly with 40 mg of bTCE (4 mice AT1413 bTCEgroup, 3 mice control group) from day 23 to 40 after tumorengraftment. Blood samples were collected on day 30, day 40,and at sacrifice (day 43). Statistical testing was performed inPrism 8.0.1.
Efficacy studies against primary AML samples. At 4–7 weeks, 12females NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3,CSF2)10-7Jic/JicTac (hGM-CSF/hIL-3 NOG; Taconic; 16.2 g � 0.9 g)were injected intravenously with AML blasts isolated from the PBof a patient with AML (BL059); T cells were depleted using a CD3MicroBeads MACS Kit (Miltenyi Biotech) and 1.2 � 106 cells/mouse were injected intravenously. The engraftment of the AMLcells was followed through time by blood sampling and FACSanalysis for CD45þCD33þ cells. When AML blasts could bereliably detected in the blood (�1% of live cells), mice wereinjected with PBMC and treated as described above.
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ResultsAT1413 bTCE is assembled by sortase-catalyzedtranspeptidation and click chemistry
C-to-C fusion of AT1413-Fc0 and UCHT1 scFv into abTCE-format was accomplished by the combination of sortasetranspeptidation and click chemistry (Fig. 1A; refs. 23–25).Methyltetrazine (MeTz) and trans-Cyclooctene (TCO) were usedas complementary click chemistry handles (Fig. 1B; ref. 31).To prepare AT1413 bTCE, AT1413-Fc0-MeTz was incubatedwith UCHT1-TCO; the reaction product was purified by SEC(Supplementary Fig. S1A).
Coupling of AT1413-Fc0-MeTz andUCHT1-TCO scFv by TCO-tetrazine cycloaddition proceeded nearly quantitatively as indi-cated by reducing and nonreducing SDS-PAGE (SupplementaryFig. S1B). The integrities of AT1413 bTCE, as well as its compo-nents, AT1413-Fc0 andUCHT1 scFv,weremaintained for up to21days when incubated in PBS at 37�C (Supplementary Fig. S2).
Analogously, a control bTCE was prepared from the antibodyAT1002 that recognizes an irrelevant target (influenza group 2HA-protein; ref. 24).
AT1413 bTCE binds AML and T-cell receptor complex–expressing cells
To confirm that AT1413bTCE retained dual binding capacity toCD43s- and CD3e-expressing cells, binding to the SH-2 AML andJurkat T-cell lines was compared with that of bTCE components,AT1413-Fc0 and UCHT1 scFv, by flow cytometry. Comparison ofmedian fluorescence intensity signals for AT1413-Fc0 andAT1413 bTCE on SH-2 cells indicated only a slight signal reduc-tion for the latter (Supplementary Fig. S3). Both, AT1413 bTCEand the control bTCE, bound Jurkat cells with similar half-maximal concentrations as unmodified UCHT1 scFv. TCRa�/�
Jurkat cells, devoid of CD3e surface expression, were used toconfirm binding specificity toward CD3e as part of the TCRcomplex. Complete lack of bTCE and UCHT1 scFv binding toTCRa�/� Jurkat cells was observed.
bTCE binding to SH-2 and Jurkat cells wasmaintained for up to21 days of incubation at 37�C in PBS or IMDM medium (Sup-plementary Fig. S4).
The binding of AT1413 bTCE on SH-2 and Jurkat cells indicatesthat the bTCE retained the binding capacities of both compo-nents, AT1413-Fc0 IgG and UCHT1 scFv.
AML cell lines and primary AML, but not primary endothelialcells, are lysed by AT1413 bTCE–redirected T cells
AT1413 bTCE was tested for inducing T-cell–mediated lysis ofCD43s-expressing SH-2 cells in an in vitro Calcein AM retentionassay (30). Using PBMCs as effector cells at an E:T ratio of 10:1,concentration-dependent target cell lysis was observed with EC50
values of 30–60 pmol/L (Fig. 2A; Table 1). Neither the controlbTCE, nor UCHT1 scFv or AT1413-Fc0 induced target cell death.Incubation of TCRa�/� Jurkat cells with AT1413 bTCE and PBMCdid not induce target cell lysis, assuring target cell specificity(Fig. 2A). At decreased E:T ratios of as low as 0.5:1, AT1413bTCE–induced SH-2 target cell lysis was retained, albeit withgradually reduced efficacies (Supplementary Fig. S5).
AT1413 bTCE was also tested on other cell lines and primaryAMLblast cells. AT1413 binds the AML cell lines THP-1,Molm13,HL-60, and Kasumi3, as well as the B-cell precursor leukemia cellline BV173, although at reduced levels compared with SH-2
cells (8).Notably, AT1413bTCEdemonstrated in vitro cytotoxicityalso toward these cell lines, with EC50 values in the range of 40–250 pmol/L (Fig. 2B; Table 1). Activity of AT1413 bTCE towardprimary AML blast cells was tested on 14 samples of PB or BMfrompatients with various AML subtypes (Fig. 2B; SupplementaryTable S1). AT1413 bTCE induced lysis in all 14 tested AML blastsamples; blasts were lysed with a wide range in efficacy (maximallysis: 9%–92%). No correlations between the extend of maximaltarget cell lysis and AML subtype or AT1413 binding wereobserved in samples, where corresponding data were available(Supplementary Fig. S6A). Consistently, no significant differencesin AT1413 binding were found in a set of 52 blast samples ofvarious AML subtypes (Supplementary Fig. S6B).
To confirm that AT1413 bTCE–induced lysis is mediated byT-cell cytotoxicity, Calcein AM retention assay was performedwith T-cell–depleted PBMCs as effector cells, which abolishedtarget cell lysis (Fig. 2C).
Previously, AT1413 was shown to bind normal endothelialcells, but no triggering of ADCC nor of CDC against endothelialcells was observed (8). Because AT1413 bTCE has a highercytotoxic potency than AT1413, we investigated whether AT1413bTCE triggers T cells to lyse endothelial cells isolated from humanaorta (HAEC) and human umbilical vein (HUVEC). AlthoughAT1413 binds to these endothelial cells at IgG concentrations of�6.7 nmol/L, neither HAEC nor HUVEC were lysed by PBMCs inthe presence of AT1413 bTCE (Fig. 2D).
Expression of T-cell activation markers by AT1413 bTCErequires CD43sþ target cells
To confirm that AT1413 bTCE activates T cells only in thepresence of CD43s-expressing target cells, upregulation of theT-cell activation markers CD69 and CD25 was monitored. In thepresence of SH-2 cells, CD69 expression was observed only on Tcells incubated with AT1413 bTCE, but not with the controlAT1002 bTCE, AT1413-Fc0, or UCHT1 scFv alone (Fig. 3A). Inthe absence of target cells, AT1413 bTCE did not induce CD69expression. CD69 expression on CD4- and CD8-positive T cellsfrom PBMCs of four different donors was compared (Supple-mentary Fig. S7A). While PBMC donors showed variations in themaximal percentage of CD69- CD4-positive and CD8-positive Tcells, potency was similar for all donors.
CD25 expression was also detected on both CD4- and CD8-positive T cells within PBMCs, but percentages of CD25-positive Tcells were lower than those of CD69-positive T cells (Fig. 3B). Inaddition, the EC50 values for CD25 expression (140–170 nmol/Lfor CD4-positive T cells and 170–200 pmol/L for CD8-positive Tcells) were higher than those for CD69 expression. Importantly,no activation markers were induced in PBMC in the absence oftarget cells, which implies that monocytes despite expression oflow levels of CD43s do not activate T cells in the presence ofAT1413 bTCE.
Using previously stimulated, resting CD8-positive T cells,higher CD25-expression levels were observed with AT1413 bTCE,whereas cells treated with the control bTCE remained CD25-negative. In addition to SH-2, other AML cell lines, such asTHP-1 and HL-60, induced CD25 expression when incubatedwith AT1413 bTCE in this setting (Supplementary Fig. S7B).
Expression of CD69 and CD25 was compared for na€�ve,effector (E), central memory (CM), and effector memory (EM)T-cell subsets (Fig. 3C). Both activation markers were signifi-cantly upregulated on CD4 and CD8 T cells across most subsets
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Schematic representation ofAT1413 bTCE generation. A,Schematic representation of thebTCE-format. B, Reactionscheme of the bTCE synthesisinvolving (1) the incorporation oftetrazine/TCO by sortase-catalyzed transpeptidationand (2) a tetrazine-TCO ligation.
Figure 2.
In vitro cytotoxicity of AT1413 bTCE against AML but not endothelial cells. Redirected T-cell–mediated lysis monitored by viable target cell count in Calcein AMretention assay with PBMCs as effector cells (unless otherwise indicated). A, SH-2 AML and Jurkat TCRa�/� target cell lines treated with AT1413 bTCE or controldrugs. B,A panel of selected AML cell lines: THP-1, Molm13, HL-60, and Kasumi3, the CML cell line BV173 and primary AML blasts (donor BL-123) treated withAT1413 bTCE or AT1002 bTCE control. C, SH-2 cells incubated with full or T-cell–depleted PBMCs as effector cells and treated with AT1413 bTCE.D, Endothelialcells (HAEC and HUVEC) treated with AT1413 bTCE or AT1002 bTCE control.
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in AT1413 bTCE compared with AT1002 bTCE–treated sam-ples. However, activation was more pronounced in some sub-sets compared with others. Of CD8 T cells, primarily those ofCM or EM phenotype expressed CD69 and CD25. On CD4 Tcells, CD25 expression showed a similar pattern, although lesspronounced, whereas CD69 expression was also high on na€�veCD4 T cells.
Cell surface expression of CD107a throughout the incubationwith bTCE and target cells was also monitored for different T-cellsubsets (Fig. 3D). Consistent with their primary cytotoxic func-tion, mainly CD8 T cells stained CD107a positive. CD107a-positive CD8 T cells spread across the CM, E, and EM phenotypes,whereas na€�ve CD8 T cells, with low levels of preformed cytotoxicgranules remained widely CD107a negative as expected.
AT1413 bTCE induces cytokine production and T-cellproliferation only in presence of CD43sþ tumor cells
Next, we studied the ability of AT1413 bTCE to induce cytokine(IFNg) production, and to stimulate T-cell proliferation. IFNgproduction was only observed when previously stimulated T cellswere used as effector cells and was dependent on AT1413 bTCEand the presence of CD43s-expressing target cells (Fig. 4A). Withthe control bTCE or in the absence of target cells, no IFNg wasdetected. With SH-2 as target cells, IFNg production was observedat lower AT1413 bTCE concentrations than for THP-1 and HL-60cells, consistent with AT1413 binding (Fig. 4B). When coincu-bated with SH-2 cells and AT1413 bTCE over 5 days, T cellsproliferated in a concentration-dependent manner whereas con-trol bTCE–treated T cells remained unaffected (Fig. 4C).
In conclusion, our analyses of T-cell marker expression, cyto-kine production, and T-cell proliferation induced by AT1413bTCE indicate that, in the presence of CD43s-positive target cells,AT1413 bTCE is capable of activating T cells and engaging them tokill CD43s-expressing AML cells.
AT1413 bTCE induces strong tumor growth inhibition of AMLcell line SH-2 in vivo
To test the in vivo efficacy of AT1413 bTCE we used a xenograftNSG mouse model, inoculated with human luciferase/ZsGreen-labeled SH-2 cells and engrafted with human PBMC. Mice weretreated biweekly with AT1413 bTCE or control (AT1002) bTCEwith 2 mg/kg i.v. Treatment with AT1413 bTCE for 15 daysresulted in tumor growth inhibition of 99% (P<0.001) comparedwith AT1002 bTCE–treated mice, demonstrating that AT1413bTCE is efficacious against SH-2 cells in vivo (Fig. 5A). Duringthe course of the experiment, no obvious signs of discomfort orweight loss were observed.
Next, AT1413 bTCE was tested in a HIS-mouse xenograftmodel, to better mimic its effect on human immune cell subsets.Successfully engrafted femaleHIS-NSGmicewere inoculatedwith
luciferase/ZsGreen-labeled SH-2 cells. AT1413 bTCE or controlbTCE treatment (biweekly, 2mg/kg i.v.)was initiated 23days afterAML inoculation. In this model, after 20 days of treatment, BLIrevealed a tumor growth inhibition of 89% (P < 0.01) in AT1413bTCE–treated mice compared with the control bTCE group(Fig. 5B).
To detect whether AT1413 bTCE had an effect on the engraftedhuman hematopoietic cells in vivo, populations of humanhematopoietic stem cells, granulocytes, and monocytes wereanalyzed at sacrifice (after 20 days of treatment with AT1413bTCE). In BM, the percentages of neither human hematopoieticstem cells (CD34þ), nor monocytes (CD14þ), nor granulocytes(CD66bþ) were significantly reduced by AT1413 bTCE (Fig. 5C).In addition, liver, PB, and spleen monocytes were present atcomparable percentages within the human compartment com-pared with the control group. To further investigate a potentiallycytotoxic effect of AT1413 bTCE toward human monocytes orgranulocytes, whole blood from healthy donors was incubatedwithAT1413bTCE in the presence or absence of SH-2 cells in an invitro assay.While up to29%of SH-2 cellswere lysed in this setup at1 nmol/L AT1413 bTCE, cytotoxicity toward monocytes andgranulocytes remained at a background level and was hardlyaffected by SH-2 cell lysis (Supplementary Fig. S8).
Taken together, AT1413 bTCE revealed strong tumor growthinhibition against SH-2 AML cells in vivo. In a HIS-NSG mousemodel, AT1413 bTCE did not show a depletion of the engraftedhuman myeloid cells.
AT1413 bTCE reduces tumor burden in a patient-derivedxenograft AML model
To assess its efficacy in vivo against primary AML blast cells,AT1413 bTCE was tested in a patient-derived xenograft (PDX)mouse model. hGM-CSF/hIL-3 NOG mice were engrafted withpatient-derived (BL059), T-cell–depleted AML blast samples.Greater than or equal to 1% CD45þCD33þ cells were observedon day 49 after engraftment. Treatment was initiated 4 days laterand CD45þCD33þ cells were monitored on day 59.
Onday 59, CD45þCD33þ cells were reduced in AT1413 bTCE–treatedmice by amedian of 89% compared with day 49 (Fig. 6A).In the same period, CD45þCD33þ cells further increased by amedian of 57% in the AT1002 bTCE–treatedmice. At sacrifice, thepercentages of human CD33þ cells within human CD45þ cellswere assessed in PB, BM, liver, and spleen (Fig. 6B). In all threeorgans and PB, CD33þ cells were significantly reduced in AT1413bTCE compared with AT1002 bTCE–treated mice.
DiscussionWith AT1413 bTCE we have generated a T-cell–engaging anti-
body against CD43s as a potential new drug for the treatment ofAML. Previously we had demonstrated that AT1413 inducedADCC in vitro (5). By converting AT1413 into a bTCE, we expectedto increase its cytotoxic potency. Indeed, AT1413 bTCE inducedSH-2 target cell death with EC50 values of 30–60 pmol/L, whereasnaked AT1413 antibody induced ADCC with EC50 values only inthe low nanomolar range (8). In addition, we demonstratedAT1413 bTCE to induce lysis of other AML cell lines, as well aspatient-derived primary AML blast cells. Consistently, weobserved more efficient tumor growth inhibition in vivo using alower dose of AT1413 bTCE (2 mg/kg) compared with the nakedAT1413 antibody (15 mg/kg). Further, AT1413 bTCE also
Table 1. Summary of in vitro potencies and efficacies of AT1413 bTCE–mediatedtarget cell lysis for different AML target cell lines incubated with PBMCs (E:Tratio 10:1)
On T cells, activation markers are upregulated by AT1413 bTCE only in the presence of target cells. A and B, Percentages of CD69-positive CD8-positive T cells after incubation with AT1413 bTCE, AT1002 bTCE, and anti-CD3/anti-CD28 containing beads, AT1413-Fc0 or UCHT1 scFv (A) and CD25-positive CD4- and CD8-positive T cells after incubation with AT1413 bTCE or AT1002 bTCE in the presence or absence of SH-2 target cells (B). C andD, Percentages of CD69- and CD25-positive (C) and CD107a-positive CD4- and CD8-positive T cells (D) within different T-cell subsets according toCD45RO and CCR7 gating (na€�ve, CM, E, and EM) after incubation with AT1413 bTCE or AT1002 bTCE control. Dot plots, overlay of activation markerexpressing T cells (colored dots) with all T cells (density plot) of the same subset. Statistical analysis, two-way ANOVA (C and D); � , P < 0.05;�� , P < 0.01; ��� , P < 0.001; ���� , P < 0.0001; ns, not significant.
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inhibited growth of patient-derived primary AML blasts in an invivo PDX model. Despite the potency increase, AT1413 bTCE didnot mediate lysis of monocytes or granulocytes, which expressedsome CD43s, in an in vitro cytotoxicity assay in whole blood.Consistently, human monocytes were not depleted by AT1413bTCE in the in vivoHIS-NSGmousemodel.We conclude thereforethat AT1413 bTCE has a very strong tumor reducing activitywithout significantly impacting the normal myeloid cells.
Although thefinal reason for this difference remains elusive, weexpect that the lower level of CD43s expression protects normalmyeloid cells from T-cell–mediated lysis or at least reduces theirsusceptibility. To directly compare lysis of primaryAMLblasts andnormal myeloid cells from the same donor, whole-blood CalceinAM retention assays could be conducted on fresh AML patientsamples, which contain similar amounts of AML blasts comparedwith the normal myeloid populations. In addition to the CD43sexpression level, protection of normal myeloid cells againstT-cell–mediated lysis may explain the difference. For instance,na€�ve bloodmonocytes have beendescribed tobe able to suppressT-cell function (32), which is partially dependent on nitric oxidesynthase (NOS). If this is applicable, AT1413 bTCE–mediatedT-cell cytotoxicity against this monocyte population should beenhanced by the addition of a NOS inhibitor. Finally, we spec-
ulate that the target, CD43s, may be exposed differently on AMLcells compared with normal myeloid cells hampering efficientformation of a cytolytic synapse.
Compared with other bTCE molecules, features of our bTCE-format are, its relatively high molecular weight and its bivalentbinding to the tumor antigen. Unlike smaller bTCE-formats suchas the bispecific T-cell engager or the dual-affinity retargetingprotein formats, our bTCE-format is expected to have a longerin vivo half-life, as it is not prone to renal clearance (33–35).Further advantages of our bTCE-format are the perpetuation ofintegrity and stability of the full-length parent IgG, as well as itsbivalency for tumor cell binding. Particularly if the T-cell–engaging antibody binds a glycopeptide epitope, as in case ofAT1413 bTCE, antibodies often have lower affinities as whentargeting peptide epitopes (36). Thus, the avidity effect ofbivalent target binding gives a valuable addition to its overallfunctional affinity.
Our bTCE-format contains twoCD3e-binding domains. Know-ing that CD3e-targeting antibodies such as UCHT1 and OKT3 aremitogenic toward T cells and observing that this effect is not fullyabrogated by using an antibody with abolished FcgR interaction,one of our initial concerns was whether bivalent binding ofAT1413 bTCE to CD3e could cause CD43s-independent T-cell
Figure 4.
AT1413 bTCE induces IFNg production and T-cell proliferation. A and B, IFNg concentration determined by ELISA in supernatants of cultured CD8-positive T cellsincubated for 48 hours with AT1413 bTCE or AT1002 bTCE and with or without SH-2 target cells (A) or with different AML target cell lines (B). C, Proliferation ofCellTrace CFSE–stained cultured CD8-positive T cells incubated with SH-2 cells and varying concentrations of AT1413 bTCE or 10 nmol/L AT1002 bTCE.
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activation (37). However, AT1413 bTCE neither induced lysis ofCD43s-negative TCRa�/� Jurkat cells nor activation of T cells inthe absence of CD43s-positive target cells. Likewise, the AT1002control bTCE had no effect on either target cell death or T-cellactivation. Hence, triggering of T-cell activation and cytotoxicityby this bTCE-format is dependent on target binding, which isconsistent with previously described T-cell–engaging antibodyformats with bivalent CD3e binding (19, 38).
AT1413 bTCE is the first bTCE generated through chemo-enzymatic linkage of a CD3e-targeting scFv to an IgG by com-bining sortase-catalyzed transpeptidation and click chemistry.
This bTCE-format provides a platform for simple conversion ofany recombinantly expressed antibody into a T-cell–engagingformat by adding just the short six amino acid sortase-recognitionmotif (LPETGG) to its heavy chain C-termini. Thus, extensiveantibody engineering, which is often required to prepare anantibody-fusion product, or other bispecific formats (e.g.,knob-into-hole) may be bypassed. This allows for quickscreening of panels of antibody candidates for use as a bTCE.Alternative to T-cell–engaging antibodies, bispecific antibodieswith other modalities (e.g., cytokines) or antibody–drug conju-gates are readily accessible, by coupling a functional group of
Figure 5.
In vivo efficacy of AT1413 bTCE in PBMC coinjected NSG and HIS-NSGmice.A, Tumor growth in PBMC coinjected mice treated with AT1413 bTCE or AT1002 bTCEfor individual mice (left) or as average (right). B, Tumor growth in HIS-NSGmice treated with AT1413 bTCE or AT1002 bTCE as average. Arrows, bTCE injection;suns, BLI. C, Percentages of human stem cells (CD34þ), granulocytes (CD66bþ), and monocytes (CD14þ) in bone marrow or selected organs of HIS-NSGmice atsacrifice. Statistical analysis, two-way repeated measures ANOVA (A); mixed-effects analysis (REML; B); two-way ANOVA (C); �� , P < 0.01;��� , P < 0.001; ns, not significant.
Figure 6.
In vivo efficacy of AT1413 bTCE against primary AML blast samples.A, Human CD33þ cells in the blood of PDXmice on day 59 as percentage of initial numberbefore start of treatment (day 49) in the same animal. B, Percentages of human CD33þ cells within human CD45þ cells in BM, liver, PB, and spleen atsacrifice. Statistical analysis, Mann–Whitney test (A); �� , P < 0.01; error bars, median with 95 % confidence interval; two-way ANOVA (B), significant at P < 0.05for treatment allocation.
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interest to the antibodyC-termini via the presented sortaseþ clickprocedure (39).
To summarize, with AT1413 bTCE we present a bTCE againstCD43s, a novel target overexpressed in AML. Efficacy of AT1413bTCE toward AML cell lines and toward primary AML blasts invitro and in vivo validates both the antibody and the target for T-cellengagement, and confirms the therapeutic potential of AT1413 ina T-cell–engaging format.
Disclosure of Potential Conflicts of InterestM.A.Gillissenhas ownership interest (including stock, patents, etc.) inAIMM
Therapeutics. A.Q. Bakker is a senior scientist at AIMM Therapeutics. H. Spitsreports receiving commercial research grant from, has ownership interest(including stock, patents, etc.) in AIMM Therapeutics, and is a consultant/advisory board member for GSK. K. Wagner has ownership interest (includingstock, patents, etc.) inAIMMTherapeutics.Nopotential conflicts of interest weredisclosed by the other authors.
Authors' ContributionsConception and design: G. de Jong, A.Q. Bakker, M.D. Hazenberg, H. Spits,K. WagnerDevelopment of methodology: L. Bartels, M.A. Gillissen, E. Yasuda, C. Bru,P.M. van Helden, J. Villaudy, K. WagnerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Bartels, G. de Jong, E. Yasuda, V. Kattler, C. Bru,C. Fatmawati, S.E. van Hal-van Veen, M.G. Cercel, J. Villaudy, M.D. Hazenberg,K. WagnerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Bartels, E. Yasuda, C. Fatmawati, J. Villaudy,K. Wagner
Writing, review, and/or revision of the manuscript: L. Bartels, G. de Jong,G. Moiset, P.M. van Helden, J. Villaudy, M.D. Hazenberg, H. Spits, K. WagnerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V. Kattler, C. Fatmawati, G.Moiset, A.Q. Bakker,K. WagnerStudy supervision: A.Q. Bakker, P.M. van Helden, M.D. Hazenberg, H. Spits,K. WagnerOther (in vivo and in vitro experiments): C. Bru
AcknowledgmentsThe authors thank the patients for participating in this study and the Trial
Office of the Amsterdam UMC, location AMC, Department of Hematology forthe provision of the AML samples.Wewould also like to thankAndrea Solar andWouter Pos for their initial contribution to the work on bispecific antibodies atAIMM Therapeutics and Sophie Levie for her assistance in testing AT1413binding to AML blast samples. This study was financially supported by theNetherland's Organization for Scientific Research (NWO), the Dutch CancerSociety (KWF), and Amsterdam UMC, location AMC. Zwaartekracht grant bythe Netherland's Organization for Scientific Research (NWO) to H. Spits(ICI00004 to L. Bartels). Intramural AMC PhD Scholarship (to M. A. Gillissen).KWF Kankerbestrijding to H. Spits and M.D. Hazenberg (CA301006 toG. de Jong and G. Moiset). NWO ZonMW VIDI to M.D. Hazenberg (grant no.91715362).
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.
Received January 23, 2018; revised March 16, 2019; accepted April 30, 2019;published first May 7, 2019.
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2019;79:3372-3382. Published OnlineFirst May 7, 2019.Cancer Res Lina Bartels, Greta de Jong, Marijn A. Gillissen, et al. Cells to a Sialylated Epitope on CD43 in Acute Myeloid LeukemiaA Chemo-enzymatically Linked Bispecific Antibody Retargets T
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