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[(te)-15.9(i)9.4(n)-189.8(G-S)-15.9(e)9.7(phar)-15.9(o)10.7(se)-190.9(\()-15.9(G)9.9(E)-191(H)-.6(ea)-15.9(l)9.4(t).2(hca)-15.9(r)9.6(e)-190.9(B)-.7(io)-15.9(s)10.9(c)-.9(ie)-15.9(n)9.5(ces,)-192.1(P).4(i)-15.9(t)10.4(ts)-15.9(b)10.9(urgh)-15.9(,)-175.9(P)-15.9(A)10(\)).3(.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 62.022 325.0771 Tm
0 Tc
[(Preparation)-339(of)-330.4(ADC)-332.3(P38B-DM1)]TJ
/F3 1 Tf
9.843 0 0 9.843 72 308.6361 Tm
-.0159 Tc
[(For)-247.4(t)-15.9(h)10.6(e)-248.5(prep)-15.9(a)9.5(rat)-15.9(i)10.4(on)-247.4(of)-247.4(th)-15.9(e)-238.1(ADC)-248.4(usi)-15.9(n)10.6(g)-247.4(P).4(38)-15.9(B)9.7(,)-244(a)-15.9(z)8.4(ide-)-15.9(m)9.5(odi)-15.9(e)9.7(d)]TJ
-1.0137 -1.1116 TD
-.0155 Tc
[(CCA)-15.5(P)-456.1(p)-15.5(e)9.9(p).7(tid)-15.5(e)-456.6(\()-15.5(F)11.3(NMQQ)-15.5(Q)9.4(\().7(Lys)-15.5([)11.3(Azi)-15.5(d)11(e]\))-15.5(R)10.1(FYEA)-15.5(L)9(HDPN)-15.5(L)9(NE)]TJ
0 -1.1174 TD
.0183 Tc
[(EQRNARI\(Lys[Azide]\)SIRDDPSRR\(Lys[Azide]\))5.7(RR\(Lys)]TJ
0 -1.1116 TD
.0185 Tc
[([Azide]\)RR\(Ly)5.9(s[Azide]\)-C)4.9(ONH2\),)-532.1(where)-530.9(L)-1.1(ys[Azide])-535.5(i)0(s)]TJ
T*
0 Tc
[(5)-18.4(-)-18.3(az)18.5(id)20.7(e)15(-)0(2)20.9(-)0(a)19.7(m)0(i)19.5(n)0(o)-509.1(p)16.2(en)19.7(ta)19.6(no)20.8(ic)-510.3(a)15(c)0(i)19.5(d)0(\))-509(d)0(e)19.7(r)0(i)20.7(v)0(e)25.4(d)-519.5(fr)20.9(om)-510.3(Z)15(3)0(4)20.8(C)-520.5(pe)19.7(p-)]TJ
0 -1.1174 TD
[(ti)20.6(de)]TJ
7.3823 0 0 6.5614 76.4787 258.1795 Tm
-.0181 Tc
[(\(1)-18.1(8)6.6(\))]TJ
9.843 0 0 9.843 91.1622 253.8141 Tm
0 Tc
[(wa)24.6(s)-271.6(u)0(s)21(e)0(d)25.4(.)-274.1(Th)25.4(is)-261.3(CC)18.8(A)15.3(P)-277.4(p)16.2(e)0(p)19.7(t)16.1(id)20.7(e)-273(w)0(a)24.6(s)-277.4(N)15.3(-)0(t)20.7(e)0(r)25.5(m)0(i)19.5(n)16.2(al)19.5(ly)-261.5(m)15(o)0(d)20.8(-)]TJ
-2.9605 -1.1116 TD
[(i)20.9(e)15(d)-306.4(wi)19.8(t)16.1(h)-306.4(di)20.7(s)16.4(u)0(c)19.7(c)15(in)20.7(im)25.2(id)20.7(yl)-290.3(gl)20.7(ut)26.5(ar)19.7(at)25.3(e)-307.5(t)0(o)-290.3(g)0(i)20.7(v)0(e)-291.3(t)0(h)20.7(e)-307.5(C)15.2(C)0(A)18.9(P)-300.4(re)19.7(ag)25.4(en)19.7(t)]TJ
T*
(\()Tj
/F4 1 Tf
.3225 0 TD
(N)Tj
/F3 1 Tf
.6566 0 TD
[(-)16.2(t)0(e)19.6(r)0(m)25.4(i)0(n)20.7(a)15(l)-536.9(s)0(u)21(c)15(ci)19.5(ni)26.5(mi)19.5(d)16.2(y)0(l)-526.5(g)16.2(lu)20.7(ta)25.3(ry)20.9(l)-536.9(p)16.2(ep)19.7(t)16.1(i)0(d)20.7(e)0(\))25.5(.)-539.1(P)16.3(3)0(8)20.8(B)-532(\(4)20.9(.)13.9(5)-156.6(mL)24.2(,)]TJ
-.9791 -1.1174 TD
[(5.)24.3(1)-156.6(m)15(g/)20.7(mL)24.2(;)-381.4(3)0(4)]TJ
/F10 1 Tf
6.0822 0 TD
(m)Tj
/F3 1 Tf
.5414 0 TD
[(M\))-371.7(i)16.1(n)-381.2(ph)20.8(o)16.2(s)0(p)21(h)0(a)25.4(t)0(e)19.6(-)0(b)26.6(u)0(f)20.9(f)0(e)19.7(r)16.2(ed)-372(sa)25.6(li)20.6(ne)-366.2(\(P)21(BS)25.7(\))-381.2(w)0(a)18.8(s)]TJ
-6.6237 -1.1116 TD
[(mi)25.2(xe)19.7(d)-271.8(w)15.3(it)20.6(h)-271.8(2)0(2)]TJ
/F10 1 Tf
5.8922 0 TD
(m)Tj
/F3 1 Tf
.5414 0 TD
-.0192 Tc
[(L)-292.2(o)-19.2(f)-274.8(2)-19.2(7.)-5.3(9)-175.8(m)-19.2(M)-277(C)-19.2(CAP)-290.8(r)-19.2(ea)-19.2(g)6.2(e)-19.2(nt)-291.1(di)-19.2(ss)-19.2(olv)-19.2(e).5(d)-291(i)-3.1(n)-291(d)-19.2(i-)]TJ
-6.4336 -1.1116 TD
0 Tc
[(me)24.2(th)20.7(yl)-157.8(su)21(l)16.1(f)0(o)20.9(x)0(i)26.5(d)0(e)-164.6(a)15(nd)-157.7(in)20.7(cu)25.4(ba)19.7(t)16.1(e)0(d)-164.6(f)16.2(or)-163.4(1)-168.1(h)0(o)26.6(u)0(r)-163.4(a)15(t)-174(r)0(o)26.6(o)0(m)-158.9(t)0(e)19.6(m)15(pe)19.7(ra)25.5(tu)20.7(re)25.5(.)]TJ
1.0137 -1.1174 TD
[(The)-207.3(reaction)-208.4(of)-209.5(antibody)-206.1(with)-207.1(CCAP)-208.8(peptide)-211.9(generated)-210.5(the)]TJ
-1.0137 -1.1116 TD
[(monovalent)-297.1(and)-292.4(divalent)-293.8(antibodies)-295.8(owing)-288.7(to)-296.1(the)-292.6(two)-292.3(modi-)]TJ
T*
[(ed)-246.2(sites)-248.4(of)-244.1(CCAP)-249.1(peptide)-246.4(on)-244.1(Fc.)-242.8(To)-245.3(remove)-246.3(the)-246.5(unmodied)]TJ
T*
[(and)-407.6(the)-407.8(divalent)-408.9(modied)-407.5(antibodies,)-408.6(the)-407.8(reaction)-409.9(mixture)]TJ
0 -1.1174 TD
[(was)-419.8(subjected)-422.4(to)-417(afnity)-423.6(chromatography)-423.4(conjugated)-418(with)]TJ
0 -1.1116 TD
[(Z33)-430.7(afnity)-429.4(peptide)-436.5(\(Acetyl-FNMQQQRRFYEALHDPNL)]TJ
T*
[(NEEQRNARIRSIRDDPSGSGSK-NH2\).)-687.7(The)-673.8(monovalent)]TJ
0 -1.1174 TD
[(modied)-240.5(antibody)-234.9(was)-235.5(recovered)-244.9(by)-232.6(eluting)-239.6(with)-235.9(mild)-236.3(acidic)]TJ
0 -1.1116 TD
[(buffer.)-212.7(Based)-210.3(on)-209.5(ion-exchange)-208(chromatogr)-8.9(aphy,)-204.8(the)-211.9(purity)-208.4(of)]TJ
T*
[(monovalent)-469.9(modied)-465.1(antibody)-471(was)-465.9(estimated)-469.8(to)-463.1(be)-469.9(95%,)]TJ
0 -1.1174 TD
[(leaving)-211.9(the)-211.9(residual)-212.8(contaminant)-213.1(of)-209.5(5%)-210.6(unmodied)-213.9(antibody.)]TJ
1.0137 -1.1116 TD
[(The)-391.6(mod)18.5(ic)12.8(ati)12.6(on)-393.9(of)-399.6(HSA)-390.8(wit)12.8(h)-398.5(DM-)12.2(1)-398.5(was)-391(per)12.8(for)14(med)-391.6(as)]TJ
-1.0137 -1.1116 TD
[(fol)13.9(low)13(s.)-182.9(HSA)-183.4(\(20)-153(mg\))-183.1(dis)14(sol)14(ve)13.9(d)-191.2(i)0(n)-186.6(1.3)17.4(5)-162.4(m)0(L)-188.9(o)0(f)-186.5(5)0(0)-157.7(m)0(M)-182.9(HEP)12.1(ES)]TJ
25.504 67.9242 TD
[(\(pH)-251.8(7.0)11.7(\))-260.3(was)-252.8(red)12.8(uce)11.7(d)-260.3(w)0(i)14.1(t)0(h)-255.8(0.5)-155.4(mM)-257.8(dit)19.5(hio)13.8(thr)13.9(eit)12.6(ol)-255.8(\(DT)11.9(T\).)-248.7(The)]TJ
0 -1.1174 TD
[(par)12.8(tia)12.6(lly)-366.4(red)12.9(uce)11.6(d)-375.5(HSA)-367.7(wa)13(s)-375.3(sup)14.2(pli)13.7(ed)-372(to)-370.9(a)-376.6(H)0(i)-371.8(Tra)11.7(p)-369.7(Cap)11.8(to)-370.9(Q)]TJ
0 -1.1116 TD
[(col)12.7(umn)-741.8(\(GE)-742.6(Hea)10.8(lth)13.7(car)11.7(e)-751(Bio)12.8(sci)12.9(en)13.9(ces)11.8(\))-755.6(e)0(q)13.9(uil)13.7(ibr)13.9(ate)11.5(d)-749.9(wit)12.8(h)]TJ
T*
[(50)-157.7(mM)-217.4(HEP)12.1(ES)-216.3(buf)14(fer)-211.8(\(pH)-211.5(7.)12.7(0\),)-213(was)12.1(hed)-211.8(wit)12.8(h)-220(the)-211.9(sam)11.8(e)-221.1(buf)14(fer)12.9(,)]TJ
T*
[(and)-234.9(elu)12.7(ted)-235(wit)12.8(h)-237.2(buf)14(fer)-234.8(con)12.8(tai)12.6(nin)13.9(g)-237.2(1)-162.4(M)-243.9(NaC)10.9(l.)-235(The)-236.1(elu)12.7(te)13.8(d)-243(HSA)]TJ
0 -1.1174 TD
(\(50)Tj
/F10 1 Tf
1.486 0 TD
(m)Tj
/F3 1 Tf
.5472 0 TD
[(M\))-763.4(was)-759.7(rea)11.7(ct)13.8(ed)-763.6(wit)12.8(h)-767.1(250)]TJ
/F10 1 Tf
12.1127 0 TD
(m)Tj
/F3 1 Tf
.5472 0 TD
[(M)-768(dib)13.8(enz)11.7(ocy)12.8(cl)13.8(ooc)12.8(tyn)13.8(e)]TJ
-14.693 -1.1116 TD
[(\(DB)12.1(CO\))12.1(-PE)13(G4-)13.1(Mal)11.8(eim)17.2(ide)-350.2(\(Cl)12.8(ick)-350.2(Che)11.8(mis)12.8(try)-349(To)13.9(ols)14(\))-358.2(and)-350(in-)]TJ
T*
[(cub)12.8(ate)11.5(d)-490.7(for)-487(2)-490.7(hou)14(rs)-485.8(at)-493.1(a)-491.8(roo)14(m)-491.9(tem)11.5(per)12.8(atu)12.7(re)13.9(.)-493(The)-483.7(DBC)11(O-)]TJ
0 -1.1174 TD
[(mod)12.7(ie)12.8(d)-254.5(HSA)-252.6(for)14(me)12.7(d)-260.3(\()0(7)]TJ
/F10 1 Tf
10.1486 0 TD
(m)Tj
/F3 1 Tf
.5472 0 TD
[(M\))-250.7(was)-247.1(sub)14.2(seq)13(uen)12.8(tly)-245.5(rea)11.7(cte)11.5(d)-254.5(wit)12.8(h)]TJ
-10.6958 -1.1116 TD
[(a)-307.5(2)0(0)15.1(-fo)14(ld)-301.8(ex)13.9(ces)11.8(s)-306.2(o)0(f)-295.9(D)0(M)13.3(1-S)14.2(MCC)-294.3(\()-81.7(Med)17.7(Che)11.8(mEx)11.6(pre)12.8(ss\))-290.9(for)-291.2(12)]TJ
T*
[(hou)14(rs)-272.7(at)-274.2(37)]TJ
/F8 1 Tf
4.4638 0 TD
()Tj
/F3 1 Tf
.3917 0 TD
(C.)Tj
-3.8417 -1.1174 TD
[(After)-335.9(the)-327.1(reaction,)-338.5(the)-327.1(sample)-332.6(was)-333.4(supplied)-332.6(to)-330.6(a)-330.6(H)0(i)-331.5(Trap)]TJ
-1.0137 -1.1116 TD
[(Capto)-376.5(Q)-370.6(column)-366.2(\(GE)-374(Healthcare)-375(Biosciences\))-375.8(equilibrated)]TJ
T*
[(with)-305(50)-169.2(mM)-303.8(HEPES)-302.2(buffer)-301.4(\(pH)-303.6(7.0\),)-303.9(washed)-303.4(with)-305(the)-304.1(same)]TJ
T*
[(buffer,)-218.5(and)-211.8(eluted)-210.8(with)-212.9(buffer)-215(containing)-215.3(1)-168.1(M)-209.3(NaCl.)-218.4(The)-213(drug)]TJ
0 -1.1174 TD
[(\(DM-1\))-348.1(attachment)-351.4(efciency)-348.6(to)-347.9(the)-344.4(HSA)-344.7(\(DAR\))-348(was)-350.7(esti-)]TJ
0 -1.1116 TD
[(mated)-677.3(to)-670.5(be)-677.2(2.9)-668(using)-673.6(matrix-associate)-13.2(d)-669.2(laser)-675.9(desorp-)]TJ
T*
[(tion/ioniza)-9.3(tion)-551.8(time-of-ig)-8.9(ht)-555.3(mass)-559.3(spectroscopy)-557.8(\(autoex)]TJ
0 -1.1174 TD
[(speed)-284.1(TOF;)-282.9(Bruker)-285.2(Daltonics\).)]TJ
1.0137 -1.1116 TD
[(Finally,)-582.7(azide-modied)-584.7(Z33-monovalent-modied)-584.5(anti-)]TJ
-1.0137 -1.1116 TD
[(body)-586.1(\(10)]TJ
/F10 1 Tf
4.0894 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
[(M\))-584.8(and)-586.2(DM-1/DBCO-modied)-591.2(HSA)-586.6(\(20)]TJ
/F10 1 Tf
17.8781 0 TD
(m)Tj
/F3 1 Tf
.5529 0 TD
(M\))Tj
-23.0734 -1.1174 TD
[(were)-428(reacted)-421.4(at)-424(pH)-423.5(7.0)-420.3(for)-417.9(12)-422.7(hours)-420(at)-423.9(room)-420.3(temperature)]TJ
0 -1.1116 TD
[(throug)-6.7(h)-185.4(the)-188.9(click)-186.6(chemistry.)-189.7(The)-190(generation)-193.3(of)-186.5(antibody-HSA)]TJ
T*
[(conjugate)-399.6(was)-391(conrmed)-398.1(by)-388.1(sodium)-393.7(dodecyl)-396.1(sulfatepoly-)]TJ
0 -1.1174 TD
[(acrylamide)-414.6(gel)-407.8(electrophor)-10(esis)-408.5(\(SDS-PAGE\).)-409.4(The)-408.8(reaction)]TJ
0 -1.1116 TD
[(mixture)-373.2(was)-373.8(subjected)-376.3(to)-370.9(a)-370.9(Z33)-367.4(afnity)-371.8(peptide)-373.2(column)-372(to)]TJ
T*
[(remove)-275.1(the)-269.5(excess)-268.9(HSA)-269.8(eluted)-274.1(in)-267.3(the)-269.5(excluded)-271.6(fraction.)-274(The)]TJ
T*
[(bound)-443.3(HSA-monovalent)-447(modied)-442.1(antibody)-442.2(was)-442.9(eluted)-441.2(by)]TJ
0 -1.1174 TD
[(0.1)-166.9(M)-203.6(acetic)-207.3(acid)-202.7(buffer)-203.5(\(pH)-205.7(4.0\))-202.5(and)-200.3(dialyzed)-202.6(in)-203.9(PBS)-205.6(for)-199.1(use.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 313.0582 396.3401 Tm
[(Cell)-336.3(lines)-337.4(and)-329.4(animals)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 379.8991 Tm
[(We)-437.6(previously)-432.7(inserted)-437.4(dPDPN)-433.2(with)-431.8(an)-435.3(N-terminal)-436.2(PA)]TJ
-1.0137 -1.1116 TD
[(tag)-540.2(\(GVAMPGAEDDVV\))]TJ
7.3823 0 0 6.5614 420.8881 373.3228 Tm
(\(19\))Tj
9.843 0 0 9.843 438.4629 368.9574 Tm
[(and)-534.4(a)-537.9(C-terminal)-534.3(RAP)-540.7(tag)]TJ
-12.7405 -1.1174 TD
[(\(DMVN)-9(PGLEDRIE\))]TJ
7.3823 0 0 6.5614 397.1905 362.381 Tm
(\(20\))Tj
9.843 0 0 9.843 409.4928 357.959 Tm
[(-MAP)-449.5(tag)-442.3(\(GDGMVPPGIEDK\))]TJ
7.3823 0 0 6.5614 539.8865 362.381 Tm
(\(21\))Tj
9.843 0 0 9.843 313.0582 347.0172 Tm
[(\(PA-dPDPN-RA)-11.4(P-MAP\))-284.5(in)-290.3(a)-284.5(pCAG-Ble)-296.1(vector)-285.5(\(FUJIFILM)]TJ
0 -1.1116 TD
.013 Tc
[(W)-7.6(a)-.8(k)-5.4(o)-673.5(P)-5.2(u).4(r)-5.3(e)-674.7(C)-6.4(h).4(em)-6.6(ic)-6.5(al)-679.4(Co)-5.4(rp)-5.4(ora)-6.5(ti)-5.5(on)-5.4(\).)]TJ
7.3823 0 0 6.5614 470.3243 340.4409 Tm
.0217 Tc
(\(11\))Tj
9.843 0 0 9.843 489.8833 336.0755 Tm
.0192 Tc
[(CH)5.7(O-)6.6(K1)-667.3(ce)5.4(ll)6.5(s)]TJ
-17.9645 -1.1174 TD
0 Tc
[(\(American)-447.6(Type)-438.7(Culture)-442.1(Collection,)-444.6(Manassas,)-442.3(VA\))-442.8(were)]TJ
0 -1.1116 TD
[(transfected)-644.6(with)-639.1(pCAG-Ble/PA-dPDPN-RAP-)-16(MAP)-638.5(using)]TJ
T*
[(Gen)11.9(e)-324.8(Pul)14(ser)-315.3(Xce)10.8(ll)-319.2(ele)11.5(ctr)12.7(opo)14(rat)12.7(ion)-314.5(sys)14.4(tem)-316.8(\(Bi)12.8(o-R)18.7(ad)-320.1(Lab)11.6(ora)12.8(-)]TJ
T*
[(tor)13.9(ies)12.9(,)-279.9(Inc)18.6(.,)-271.8(Ber)11.8(kel)12.7(ey,)-266(CA\))-264.4(for)-268.2(dev)12.8(elo)12.7(pin)19.6(g)-277.6(CHO)11.2(/dP)14(DPN)12.4(.)]TJ
1.0137 -1.1174 TD
[(CHO/dPDPN)-590.2(and)-580.4(CHO-K1)-586.2(cells)-589.6(were)-583.5(cultured)-587.3(using)]TJ
-1.0137 -1.1116 TD
[(RPMI-1640)-426.3(medium)-425.1(\(Nacalai)-423.5(Tesque,)-428.1(Inc.,)-420.2(Kyoto,)-424.6(Japan\))]TJ
T*
-.0158 Tc
[(s)-15.8(u)11(ppl)-15.8(e)9.5(ment)-15.8(e)9.5(d)-408.6(w)-.5(it)-15.8(h)-398.3(10%)-409.6(h)-15.8(e)9.6(at-)-15.8(i)10.7(nact)-15.8(i)10.5(v).4(at)-15.8(e)9.5(d)-408.6(fet)-15.8(a)9.5(l)-408.7(bov)-15.8(i)10.7(n).4(e)-409.7(s)-15.8(e)9.8(rum)]TJ
0 -1.1174 TD
0 Tc
[(\(T)25.4(h)16.2(e)15(rm)25.4(o)-196.9(F)16.3(i)16(s)16.4(he)25.4(r)-196.9(S)16.3(c)15(i)16.1(en)25.4(t)16.1(i)16.1()0(c)25.6(,)-193.5(I)16.2(n)0(c)25.4(.)13.9(\))16.2(.)-193.5(An)25.7(t)16(i)16.1(bi)26.5(o)16.2(t)16(i)16.1(c)0(s)25.6(,)-193.5(su)26.8(c)15(h)-196.9(a)15(s)-196.7(1)16.2(0)0(0)-140.4(U)15.3(/)16.1(m)15(L)]TJ
0 -1.1116 TD
[(pe)25.4(n)16.2(i)16.1(ci)25.3(l)16.1(l)16.1(i)16(n,)-298.3(1)16.2(0)0(0)]TJ
/F10 1 Tf
5.8403 0 TD
(m)Tj
/F3 1 Tf
.5357 0 TD
-.0158 Tc
[(g/m)-15.8(L)-314.2(s)-15.8(t)10.9(rep)-15.8(t)10.7(omyci)-15.8(n)10.7(,)-324.5(a)-15.8(n)9.6(d)-327.9(25)]TJ
/F10 1 Tf
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