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[(we)-4.4(re)-471(euthanize)-4.4(d)-464.1(by)-464(c)-4.4(e)1.3(rvical)]TJ
12.7232 0 TD
.0147 Tc
[(disloc)-4.8(ation,)-466.8(and)-470.2(d)2.1(ea)-4.8(th)-470.2(wa)-4.8(s)]TJ
-12.7232 -1.1116 TD
.2948 Tc
[(v)276.4(e)281(r)282.2(i)276.3()282.4(e)275.3(db)276.4(yr)282.2(e)275.3(s)282.4(p)276.4(i)282.1(r)276.5(a)281(t)282.1(o)276.4(r)282.2(y)-5.8(a)281(n)276.4(dc)281(a)275.3(r)]TJ
12.6022 0 TD
.0151 Tc
[(dia)-4.4(c)-280.9(arre)-4.4(st.)-282.1(F)2.6(em)-4.5(ale)-280.9(B)1.5(A)-4.1(L)1.3(B/c)]TJ
-12.6022 -1.1174 TD
.0149 Tc
[(mice)-494.2(\(6)-493(we)-4.6(eks)-487.1(o)-3.5(ld\))-487.3(w)-4.3(ere)-488.5(p)-3.5(urcha)-4.6(s)2.5(e)-4.6(d)-487.3(from)-488.5(C)-4.5(LEA)-493.9(Japa)-4.6(n)]TJ
0 -1.1116 TD
.0129 Tc
[(\()-5.4(T)-.9(ok)-5.5(yo)-5.5(,)-393.7(J).5(ap)-5.5(an)-5.5(\).)-393.7(Th)-5.5(e)-392.5(a)-6.6(nim)-6.7(a)-.9(l)-5.6(s)-391.2(we)-6.6(re)-398.3(hou)-5.5(se)-6.6(d)-391.4(u)-5.5(nde)-6.6(r)-391.3(s)-5.3(pe)-6.6(ci)-5.3(c)]TJ
T*
.0127 Tc
[(p)-5.7(a)-1.1(th)-5.7(og)-5.7(en-)-5.6(f).1(r)-5.6(e)-1.1(e)-283.3(c)-6.8(ond)-5.7(it)-5.8(io)-5.7(ns)-5.5(.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 62.022 105.9023 Tm
0 Tc
[(Hybridoma)-336.9(production)]TJ
/F3 1 Tf
9.843 0 0 9.843 72 89.4614 Tm
[(To)-228(develop)-229(mAbs)-230.9(against)-229(hTIGIT,)-232.1(we)-228.9(employed)-225.6(the)-229.2(CBIS)]TJ
-1.0137 -1.1174 TD
(method.)Tj
7.3823 0 0 6.5614 94.1102 82.885 Tm
(\(16\))Tj
9.843 0 0 9.843 108.5102 78.463 Tm
[(In)-215.3(brief,)-211.8(two)-211.7(BALB/c)-219.4(mice)-215.4(were)-214.9(immunized)-210.8(with)]TJ
-4.723 -1.1116 TD
[(CHO/hTIGIT)-328.2(cells)-324.7(\(1)]TJ
/F5 1 Tf
8.9851 0 TD
()Tj
/F3 1 Tf
.72 0 TD
(10)Tj
7.3823 0 0 6.5614 167.4141 71.8866 Tm
(8)Tj
9.843 0 0 9.843 171.0992 67.5212 Tm
[(\))-323.6(b)0(y)-319(the)-321.4(intraperitoneal)-327(route)-323.5(to-)]TJ
-11.0817 -1.1116 TD
[(gether)-429.5(with)-426(Imject)-430.9(Alum)-421.3(\(Thermo)-431.7(Fisher)-428(Scientic,)-425.8(Inc.\).)]TJ
25.504 67.9242 TD
[(The)-368.5(procedure)-370.4(included)-368.5(three)-370.8(additional)-371(immunization)-369.9(fol-)]TJ
0 -1.1174 TD
[(lowed)-399.3(by)-393.9(a)-399.7(nal)-397.2(booster)-394.7(intraperitoneal)-401.9(injection)-398.7(adminis-)]TJ
0 -1.1116 TD
[(tered)-365(2)-358.2(days)-362.5(before)-360.2(spleen)-360.2(cells)-365(were)-358.9(harvested.)-368.1(Harvested)]TJ
T*
[(spleen)-314.1(cells)-313.2(were)-312.8(subsequently)-314.9(fused)-311.7(with)-310.8(P3U1)-310.4(cells)-313.2(using)]TJ
T*
[(PEG15)-7.4(00)-474.5(\(Roche)-482.2(Diagnostics,)-479.5(Indianapolis,)-479.9(IN\).)-479.8(T)15(h)0(e)-452.6(h)16.2(y)16.2(-)]TJ
0 -1.1174 TD
-.0158 Tc
[(b)-15.8(r)10.8(ido)-15.8(m)9.6(as)-408.4(wer)-15.8(e)-399.3(grow)-15.8(n)-399(i).3(n)-408.6(R)-.6(PMI)-408.5(m)-.8(e)-15.8(d)9.6(ium)-409.8(s)-15.8(u)11(pple)-15.8(m)8.4(ented)-408.6(w)-15.8(i)9.8(t).3(h)]TJ
0 -1.1116 TD
[(1)-15.8(0)10.8(%)-196.5(h)-15.8(e)9.6(a)-.8(t-i)-15.8(n)10.7(act)-15.8(i)10.5(v).4(ate)-15.8(d)-186.2(FB)-15.8(S)-185.9(\().4(T)-15.8(h)9.6(ermo)-201.2(Fis)-15.8(h)11(er)-201.2(Scie)-15.8(n)9.6(t).3(i)-15.8(c)9.8(,)-197.8(Inc)-15.8(.)13.1(\))-15.8(,)-187.3(100)]TJ
T*
-.0159 Tc
[(U)-15.9(/)9.7(mL)-438.6(peni)-15.9(c)9.4(ill)-15.9(i)10.4(n).3(,)-439.8(100)]TJ
/F10 1 Tf
8.9909 0 TD
0 Tc
(l)Tj
/F3 1 Tf
.5357 0 TD
-.0159 Tc
[(g/)-15.9(m)9.3(L)-438.6(stre)-15.9(p)9.5(t).2(omy)-15.9(c)9.5(in,)-439.8(0).3(.25)]TJ
/F10 1 Tf
10.1428 0 TD
0 Tc
(l)Tj
/F3 1 Tf
.5414 0 TD
-.0153 Tc
[(g/mL)-438(am-)]TJ
-20.2108 -1.1174 TD
-.0157 Tc
[(p)-15.7(h)10.9(ote)-15.7(r)9.8(ici)-15.7(n)-363.6(B)-380.7(\().5(N)-15.7(a)8.9(cala)-15.7(i)-364.8(T)-15.7(e)8.5(squ)-15.7(e)9.7(,)-376.2(I).5(n)-15.7(c)9.7(.\),)-376.2(a)-.7(n)-15.7(d)-369.2(hypox)-15.7(a)9.7(n).5(th)-15.7(i)10.8(n).5(e,)-382(ami-)]TJ
0 -1.1116 TD
0 Tc
[(no)26.6(p)16.2(t)16.1(er)25.5(i)16.1(n)16.2(,)-423.9(t)16.1(h)16.2(y)16.2(m)15(id)26.5(i)16.1(n)16.2(e)-422.7(f)16.2(o)16.2(r)-421.5(se)25.6(l)16(e)15(c)15(t)0(i)26.3(o)16.2(n)-421.6(\()16.2(T)15(h)16.2(er)25.5(m)15(o)-421.6(F)16.3(i)16.1(s)16.4(h)0(e)25.4(r)-421.5(S)16.4(c)15(i)16.1(e)15(nt)26.5(i)16()16.3(c,)]TJ
T*
-.0157 Tc
[(I)-15.7(n)10.9(c.\).)-284.1(Cult)-15.7(u)10.8(r).5(e)-282.9(s).7(up)-15.7(e)9.7(rnat)-15.7(a)9.6(nts)-281.6(w)-15.7(e)8.9(r).5(e)-282.9(s).7(cr)-15.7(e)9.8(e)-.7(ned)-281.7(b).5(y)-281.7().6(o)-15.7(w)-272.2(cytome)-15.7(t)9.6(ry.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 589.4927 Tm
0 Tc
[(Flow)-338.7(cytometry)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 573.0518 Tm
[(Cells)-243.9(were)-243.7(harvested)-243.7(after)-244(brief)-242.9(exposure)-241.3(to)-244.2(0.25%)-239.3(trypsin)]TJ
-1.0137 -1.1116 TD
[(and)-304(1)-162.4(m)0(M)-298.1(ethylenediaminetetraacetic)-314.4(acid)-300.6(\(Nacalai)-302.6(Tesque,)]TJ
T*
[(Inc.\).)-231.2(They)-231.4(were)-232.2(washed)-228.5(with)-230.2(0.1%)-226.7(bovine)-232.5(serum)-226.5(albumin)-229.2(in)]TJ
0 -1.1174 TD
[(phosph)-6.4(ate-buffered)-213.7(saline)-210.6(and)-206.1(treated)-213(with)-207.1(primary)-211.8(mAbs)-207.9(for)]TJ
0 -1.1116 TD
[(30)-336.3(minutes)-332.7(at)-331.8(4)]TJ
/F5 1 Tf
6.4048 0 TD
()Tj
/F3 1 Tf
.3974 0 TD
[(C.)-333.9(Then,)-332.7(the)-332.9(cells)-336.2(were)-335.9(treated)-334(with)-333.8(Alexa)]TJ
-6.8022 -1.1116 TD
[(Fluor)-322.2(488-conjugated)-318.7(anti-mouse)-325.6(IgG)-315.2(\(1:1000;)-320.1(Cell)-317.9(Signal-)]TJ
T*
[(ing)-302.9(Technology,)-300.3(Inc.\).)-294.6(Fluorescence)-306.8(data)-300.6(were)-295.6(collected)-302.9(us-)]TJ
0 -1.1174 TD
[(ing)-458.4(the)-459.6(EC800)-457(Cell)-461.9(Analyzer)-456.8(\(Sony)-460.3(Corp.\))-459.2(and)-459.5(analyzed)]TJ
0 -1.1116 TD
[(using)-281.9(FlowJo)-283.7(\(BD)-281.6(Biosciences,)-286.1(Franklin)-280.8(Lakes,)-288.7(NJ\).)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 459.3259 Tm
[(Determination)-337.6(of)-336.7(binding)-332.2(afnity)-334.4(by)-332.7(ow)-335.5(cytometry)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 442.8849 Tm
[(CHO)11.2(/hT)12.7(IGI)13.2(T)-290.3(was)-287.4(sus)14.4(pen)12.8(ded)-280.9(in)-290.3(100)]TJ
/F10 1 Tf
14.7276 0 TD
(l)Tj
/F3 1 Tf
.5472 0 TD
[(L)-290.3(o)0(f)-290.1(ser)13(ial)12.6(ly)-284.5(dil)13.7(ute)12.7(d)]TJ
-16.2885 -1.1116 TD
[(ant)12.7(i-h)13.9(TIG)11.9(IT)-464.1(mAb)11.9(s,)-459.4(and)-459.5(Ale)11.8(xa)-464.1(Flu)14(or)-462.9(488)14(-co)12.9(nju)13.9(ga)13.9(ted)-459.6(ant)12.7(i-)]TJ
T*
[(mou)12.7(se)-487(IgG)-482.2(\(1:)13.9(200)14(;)-496.6(C)0(e)12.9(l)0(l)-486.3(Sig)14(nal)12.7(ing)-481.5(Tec)10.5(hno)14(log)13.9(y,)-488.3(Inc)18.6(.\))-488.3(was)]TJ
T*
[(add)12.8(ed.)-271.7(Flu)14(ore)12.8(sce)11.8(nce)-270.6(dat)12.7(a)-278.7(wer)12(e)-278.7(col)12.7(lec)11.5(ted)-269.5(usi)14(ng)-278.7(the)-269.5(BD)-274.8(FAC)12.2(-)]TJ
0 -1.1174 TD
[(SLy)12.9(ric)-217.7(\(BD)-212.5(Bio)12.8(sci)12.9(enc)11.7(es\))13(.)-222.3(The)-213(dis)14(soc)13(iat)12.6(ion)-216.5(co)13.9(nst)14(ant)-217.7(\()]TJ
/F4 1 Tf
20.9999 0 TD
(K)Tj
/F3 1 Tf
7.3823 0 0 6.5614 526.2802 397.4172 Tm
(D)Tj
9.843 0 0 9.843 531.5526 399.0613 Tm
[(\))-219.9(was)]TJ
-22.1979 -1.1116 TD
[(cal)11.5(cul)12.7(ate)11.5(d)-450.4(b)0(y)-445.7(tt)13.9(ing)-441.2(bin)13.8(din)13.8(g)-450.3(iso)14(the)12.7(rm)13.9(s)-450.2(t)0(o)-445.8(bui)13.8(lt-)13.8(in)-445.8(one)12.8(-si)14.1(te)]TJ
T*
[(bin)13.9(din)13.9(g)-294.8(mod)12.7(els)-286.6(in)-284.5(Gra)12(phP)14.1(ad)-291.3(PRI)13.1(SM)-290.9(8)-289.1(\(Gr)13.2(aph)12.8(Pad)-286.5(Sof)14.2(twa)11.8(re,)]TJ
0 -1.1174 TD
[(Inc)12.9(.,)-271.8(La)-269.5(Jol)14(la,)-266.1(CA\))12.1(.)]TJ
/F7 1 Tf
8.9663 0 0 8.9663 313.0582 340.1574 Tm
(Results)Tj
/F9 1 Tf
0 -1.8336 TD
[(Establishment)-337.8(of)-336.7(the)-328.5(anti-hTIGIT)-340.8(mAbs)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 307.2755 Tm
-.0156 Tc
[(To)-448.7(es)-15.6(t)11.1(a)-.6(bl)-15.6(i)10.7(s).8(h)-448.7(a)-.6(nt)-15.6(i)10.7(-hTI)-15.6(G)10.2(IT)-449.9(mAbs)-15.6(,)-434.6(w)-.3(e)-449.8(emp)-15.6(l)10.9(oyed)-448.7(t)-15.6(h)10.9(e)-449.8(CBIS)]TJ
-1.0137 -1.1116 TD
0 Tc
[(me)24.2(t)16.1(h)16.2(o)16.2(d)-381.2(\(F)26.8(i)16.1(g)16.2(.)-383.6(1)16.2(\))16.2(.)-383.6(F)16.3(i)16.1(r)16.2(s)0(t)26.7(,)-377.8(we)-367.1(im)25.2(m)15(u)16.2(n)16.2(i)16(ze)24.3(d)-381.2(t)16.1(w)15.3(o)-381.2(m)15(i)16(c)15(e)-382.4(w)15.3(i)16(th)-370.9(C)15.2(H)15.3(O)15.3(/)]TJ
0 -1.1174 TD
-.0157 Tc
[(h)-15.7(T)9.7(IGI)-15.7(T)-272.5(ce)-15.7(l)9.6(l).4(s)-287.3(a)-.7(nd)-287.5(pro)-15.7(d)10.9(u).5(ced)-287.5(hy)-15.7(b)10.9(r).5(id)-15.7(o)10.9(mas)-287.3(by)-287.5(fus)-15.7(i)11(n).5(g)-287.5(s).7(pl)-15.7(e)9.6(nocy)-15.7(t)10.8(es)]TJ
0 -1.1116 TD
-.0155 Tc
[(w)-15.5(i)10.1(th)-535(P)-15.5(3)11.3(U1)-535(myel)-15.5(o)11(m)-.5(a)-536.1(cel)-15.5(l)10.8(s).9(.)-537.3(N)-.2(ext)-15.5(,)-521.3(w)-15.5(e)-526.6(selec)-15.5(t)9.8(ed)-535(hy)-15.5(b)11.1(r).7(id)-15.5(o)11.1(mas)]TJ
T*
-.0159 Tc
[(w)-15.9(h)9.8(ose)-346.5(s).5(upe)-15.9(r)9.6(n).3(ata)-15.9(n)9.5(ts)-339.3(w)-15.9(e)8.7(re)-340.7(po)-15.9(s)10.9(i).2(ti)-15.9(v)10.6(e)-340.7(for)-345.3(CHO/h)-15.9(T)9.5(IGI)-15.9(T)-330.3(cells)-345.1(and)]TJ
0 -1.1174 TD
-.0158 Tc
[(n)-15.8(e)9.6(gat)-15.8(i)10.5(ve)-582.5(fo)-15.8(r)-570.9(C)-.6(HO-)-15.8(K)10(1)-581.3(cel)-15.8(l)10.5(s)-581.2(by)-587.1(ow)-582.2(c)-.8(y)-15.8(t)10.7(omet)-15.8(r)10.7(y).4(.)-583.7(T)-.8(he)-582.5()-15.8(r)11(st)]TJ
0 -1.1116 TD
0 Tc
[(sc)25.6(r)16.2(e)15(en)25.4(i)16.1(n)16.2(g)-375.5(a)15(p)16.2(p)0(r)26.6(o)16.2(a)15(c)15(h)-375.5(i)16.1(d)16.2(en)25.4(t)16.1(i)16(e)25.6(d)-369.7(s)0(t)26.7(r)16.2(o)16.2(n)0(g)-359.3(s)0(i)26.7(g)16.2(n)16.2(a)15(ls)-359.2(fr)26.7(o)16.2(m)-376.7(C)15.2(H)15.3(O)15.3(/)16.1(hT)25.4(I)16.2(-)]TJ
T*
-.0157 Tc
[(G)-15.7(I)10.1(T)-271.4(cell)-15.7(s)-259.7(and)-270.2(w)-.4(ea)-15.7(k)-255.2(o)-15.7(r)-259.8(n).5(o)-270.2(s).7(ig)-15.7(n)10.9(a)-.7(ls)-270(f)-15.7(r)11(om)-271.5(CHO-)-15.7(K)10.1(1)-270.2(cell)-15.7(s)-259.7(in)-270.2(11)-270.2(of)]TJ
T*
[(9)-15.7(5)10.9(8)-391.2(w)-15.7(e)8.9(l).3(ls)-396.7(\(1.1%\))-15.7(.)-383.1(The)-398.1(s).7(econ)-15.7(d)-380.8(sc)-15.7(r)9.8(eeni)-15.7(n)10.8(g)-391.2(a)-15.7(p)9.7(pro)-15.7(a)9.7(c)-.7(h)-391.2(i)-15.7(d)10.8(e)-.7(nti)-15.7()10.9(ed)]TJ
0 -1.1174 TD
-.0159 Tc
[(s)-15.9(t)10.8(ro)-15.9(n)10.7(g)-437.5(si)-15.9(g)10.6(n).3(als)-443(fro)-15.9(m)-428.3(N)-.6(K)-438.3(cel)-15.9(l)10.4(s)-437.3(u)-15.9(s)10.9(ing)-443.2(2)-437.5(of)-437.4(t)-15.9(h)10.6(e)-438.6(1).3(1)-437.5(h)-15.9(y)10.7(bri)-15.9(d)10.6(o).3(ma)]TJ
0 -1.1116 TD
0 Tc
[(su)26.8(p)16.2(e)15(rn)26.6(a)15(t)16.1(an)25.4(t)16(s)-490.5(de)25.4(t)16.1(e)15(rm)25.4(i)16.1(n)16.2(e)15(d)-496.4(i)16.1(n)-490.7(t)16.1(h)0(e)-481.4(p)16.2(re)25.5(v)16.2(i)16(o)16.2(u)0(s)-480.1(s)16.4(te)25.3(p)-490.7(\()16.2(18)26.6(%)15.1(\))16.2(.)-493(A)15.3(ft)26.5(e)15(r)]TJ
T*
-.0158 Tc
[(l)-15.8(i)10.5(mit)-15.8(i)10.5(ng)-368.2(dilu)-15.8(t)10.7(i).2(on)-368.2(an)-15.8(d)-352.1(s)-15.8(e)9.8(v).4(er)-15.8(a)9.7(l)-362.6(a)-15.8(d)9.6(dit)-15.8(i)10.5(onal)-368.4(s)-15.8(c)9.8(r).4(een)-15.8(i)10.7(ngs)-15.8(,)-354.2(T)-.8(gMa)-15.8(b)9.6(-2)]TJ
0 -1.1174 TD
0 Tc
[(\(I)26.7(g)16.2(G)]TJ
7.3823 0 0 6.5614 331.0298 174.1039 Tm
(1)Tj
9.843 0 0 9.843 334.6015 175.748 Tm
-.0158 Tc
[(,)-209.3(k).4(app)-15.8(a)9.6(\),)-209.3(whic)-15.8(h)-197.7(r).4(eac)-15.8(t)9.5(e)-.8(d)-207(m)-.8(os)-15.8(t)-196.5(s).6(tr)-15.8(o)10.8(ngl)-15.8(y)-196.7(w)-.5(ith)-212.7(CHO/h)-15.8(T)9.6(IGIT)-15.8(,)]TJ
-2.1887 -1.1116 TD
0 Tc
[(wa)24.6(s)-265.9()16.3(n)16.2(a)15(ll)26.3(y)-266(e)15(s)16.4(t)0(a)25.3(b)16.2(l)16(i)16.1(sh)26.8(e)15(d)16.2(.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 138.7842 Tm
[(Flow)-338.7(cytometric)-331.3(analyses)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 122.3433 Tm
.0131 Tc
[(T)-6.5(g).5(Ma)-6.4(b-)-5.2(2)-558.2(r)-5.2(ec)-6.4(ogn)-5.3(iz)-6.4(ed)-564(CHO)-6.1(/).4(h)-5.3(T)-.7(I)-5.2(G)-.4(IT)-565.2(bu)-5.3(t)-564.1(not)-564.1(CH)-6.1(O-)-5.2(K1)]TJ
-1.0137 -1.1174 TD
[(\()-5.2(F).6(i)-5.4(g).5(.)-197.7(2)-5.3(A\).)-197.7(A)-6.1(ls)-5.1(o,)-197.7(Tg)-5.3(Ma)-6.4(b-2)-201.1(re)-6.4(act)-5.4(ed)-195.3(w)-6.1(i).4(t)-5.4(h)-195.3(en)-5.3(do)-5.3(ge)-6.4(nou)-5.3(s)-195.1(h)-5.3(TIG)-6.1(I).5(T)]TJ
0 -1.1116 TD
.0128 Tc
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