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BISPECIFIC ANTIBODIES FOR USE IN STEM CELL TRANSPLANTATION

20170298148 · 2017-10-19

    Inventors

    Cpc classification

    International classification

    Abstract

    New monoclonal antibodies for use in pre-treatments prior to stem cell transplantations are disclosed. The antibodies may be used to kill malignant cells and/or stem cells prior to stem cell transplantation. The antibodies can be used for treating hematologic diseases and hematological malignancies, such as leukemia and MDS. The antibodies of the invention might be multi- or bi-specific, such as BiTEs.

    Claims

    1.-95. (canceled)

    96. A therapeutic agent comprising: (a) one or more than one binding moiety with specificity for CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells; and (b) one or more than one binding moiety with specificity for one or more than one type of CD3+ effector cell.

    97. The therapeutic agent according to claim 96, wherein the therapeutic agent is an antibody or antigen binding fragment thereof.

    98. The therapeutic agent according to claim 96, wherein the CD3 protein is selected from the group of proteins comprising the amino acid sequence of GenBank database accession number NP_000723.1 (CD3 delta), GenBank database accession number NP_000724.1 (CD3 epsilon) and GenBank database accession number NP_000064.1 (CD3 gamma).

    99. The therapeutic agent according to claim 96, wherein the CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells comprise cancer stem cells and/or non-cancer stem cells.

    100. The therapeutic agent according to claim 96, wherein the CD34 protein comprises the amino acid sequence of GenBank database accession number NP_001020280.1.

    101. The therapeutic agent according to claim 96, further comprising one or more than one binding moiety with specificity for CD133+ hematopoietic stem cells and/or CD133+ hematopoietic progenitor cells.

    102. The therapeutic agent according to claim 101, wherein the CD133 protein comprises the amino acid sequence of GenBank database accession number NP_001139319.1.

    103. The therapeutic agent according to claim 96, wherein the one or more than one binding moiety with specificity for CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells: (a) has specificity for greater than 50% of daughter cells of the CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells; (b) does not have specificity for daughter cells of the CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells; or (c) has specificity for less than or equal to 50% of daughter cells of the CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells.

    104. The therapeutic agent according to claim 97, wherein the antibody or antigen binding fragment comprises a framework region sequence comprising: TABLE-US-00012 (4C8 anti-CD34 antibody heavy chain constant region) SEQ ID NO: 1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK; (4C8 anti-CD34 antibody light chain constant region) SEQ ID NO: 2 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC; and (anti-CD34-HC-anti-CD3-scFv heavy chain constant region) SEQ ID NO: 64 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK.

    105. The therapeutic agent according to claim 96, wherein the binding moiety with specificity for CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells comprises one or more than one complementarity determining region (CDR) sequence selected from the group consisting of: TABLE-US-00013 SEQ ID NO: 3 (4C8 anti-CD34 antibody heavy chain  CDR1): GYTFTNYGMN; SEQ ID NO: 4 (4C8 anti-CD34 antibody heavy chain  CDR2: WINTNTGEPKYAEEFKG; SEQ ID NO: 5 (4C8 anti-CD34 antibody heavy chain  CDR3): GYGNYARGAWLAY; SEQ ID NO: 6 (4C8 anti-CD34 antibody light chain CDR1): RSSQTIVHSNGNTYLE; (4C8 anti-CD34 antibody light chain CDR2): QVSNRFS;  and (4C8 anti-CD34 antibody light chain CDR3): FQGSHVPRT.

    106. The therapeutic agent according to claim 96, wherein the binding moiety with specificity for CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells comprises a heavy chain variable region comprising the amino acid sequence of: TABLE-US-00014 (a) SEQ ID NO: 14 (4C8 anti-CD34 antibody heavy chain): QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGW INTNTGEPKYAEEFKGRFALSLDTSVSTAYLQINSLKAEDTAVYFCARGY GNYARGAWLAYWGQGTLVTVSS; or (b) SEQ ID NO: 15 (mMY10 anti-CD34 antibody heavy chain): EVQLVESGGGLVQPGGSLRLSCAVSGFSLTSHGVHWVRQAPGKGLEWLGV IWGAGRTDYNAAFISRLSISRDISKSQVYLQMNSLRAEDTAVYYCARNRY ESYFDYWGQGTLVTVSS.

    107. The therapeutic agent according to claim 101, wherein the binding moiety with specificity for CD133+ hematopoietic cells and/or CD133+ hematopoietic progenitor cells comprises a heavy chain variable region comprising the amino acid sequence of: TABLE-US-00015 (a) SEQ ID NO: 16 (202 anti-CD133 antibody heavy chain 47): LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMG WINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATD YGDYFDYWGQGTTLTVSSAKTTPPSVTSGQ; (b) SEQ ID NO: 17 (202 anti-CD133 antibody heavy chain 48): LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMG WINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATD YGDYFDYWGQGTTLTVSSAKTTPPSVTSGQAGQ; (c) SEQ ID NO: 18 (202 anti-CD133 antibody heavy chain 49): PEVMLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMG WINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATD YGDYFDYWGQGTTLTVSSAKTTPPSVTSGQAGQ; (d) SEQ ID NO: 19 (202 anti-CD133 antibody heavy chain 50): LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMG WINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATD YGDYFDYWGQGTTLTVSSAKTTPPSVTSGQAGQ; (e) SEQ ID NO: 20 (202 anti-CD133 antibody heavy chain 51): LEVHLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWM GWINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCA TDYGDYFDYWGQGTTLTVSSAKTTPPSVTSGQAGQ; (f) SEQ ID NO: 21 (202 anti-CD133 antibody heavy chain 52): LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWM GWINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCA TDCGDYFDYWGQGTTLTVSSAKTTPPSVTSGQAGQ; (g) SEQ ID NO: 22 (202 anti-CD133 antibody heavy chain 53): LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMG WINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATD YGDYFDYWGQGTTLTVSSAKTTPPSVTSGQAGQ; (h) SEQ ID NO: 23 (202 anti-CD133 antibody heavy chain 54): LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMG WINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATD YGDYFDYWGQGTTLTVSSAKTTAPSVTSGQAGQ; or (i) SEQ ID NO: 24 (202 anti-CD133 antibody heavy chain 55): LEVQLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMG WINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATD YGDYFDYWGQGTTLTVSSAKTTAPSVTSGQAGQ.

    108. The therapeutic agent according to claim 96, wherein the binding moiety with specificity for CD34+ hematopoietic stem cells and/or CD34+ hematopoietic progenitor cells comprises a light chain variable region comprising the amino acid sequence of: TABLE-US-00016 (a) SEQ ID NO: 25 (4C8 anti-CD34 antibody light chain): DVLLTQSPLSLPVTLGQPASISCRSSQTIVHSNGNTYLEWFQQRPGQSPR LLIYQVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP RTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC; or (b) SEQ ID NO: 26 (mMY10 anti-CD34 antibody light chain): DIQMTQSPSSLSASVGDRVTITCRSSQNLVHSNGNTYLHWYQQKPGKAPK LLIYKVSNRFSGVPDRFSGSGSGTEFTLTISSLQPEDFATYYCSQSTHVP LTFGQGTKVEIKR.

    109. The therapeutic agent according to claim 101, wherein the binding moiety with specificity for CD133+ hematopoietic stem cells and/or CD133+ hematopoietic progenitor cells comprises a light chain variable region comprising the amino acid sequence of: TABLE-US-00017 (a) SEQ ID NO: 27 (202 anti-CD133 antibody light chain 47): AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVGYMYWYQQKPGSSP KPWIYRPSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK; (b) SEQ ID NO: 28 (202 anti-CD133 antibody light chain 48): AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSP KPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK; (c) SEQ ID NO: 29 (202 anti-CD133 antibody light chain 49): AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSP KPWIYRPSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK; (d) SEQ ID NO: 30 (202 anti-CD133 antibody light chain 50): AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGQPP RLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQYHS YPPTFGAGTKLEIK; (e) SEQ ID NO: 31 (202 anti-CD133 antibody light chain 51): AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSP KPWIYRPSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK; (f) SEQ ID NO: 32 (202 anti-CD133 antibody light chain 52): AQAAELDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSPP KPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK; (g) SEQ ID NO: 33 (202 anti-CD133 antibody light chain 53): AQAAELDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSP KPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK; (h) SEQ ID NO: 34 (202 anti-CD133 antibody light chain 54): AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSP KPWIYRPSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK; or (i) SEQ ID NO: 37 (202 anti-CD133 antibody light chain 55): AQAAELDIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSP KPWIYRPSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHS YPPTFGAGTKLELK.

    110. The therapeutic agent according to claim 96, wherein the binding moiety with specificity for one or more than one type of CD3+ effector cell comprises one or more than one complementarity determining region (CDR) sequence selected from the group consisting of: TABLE-US-00018 (1252 anti-CD3 antibody heavy chain CDR1): GFTFDDYT; (1268 anti-CD3 antibody heavy chain CDR1): GFTFDDFT; (1284 anti-CD3 antibody heavy chain CDR1): GFTFDDYT; (1300 anti-CD3 antibody heavy chain CDR1): GFTFRSYA; (1316 anti-CD3 antibody heavy chain CDR1): GFTFRSYG; (1330 anti-CD3 antibody heavy chain CDR1): GYTFTRYT; (1254 anti-CD3 antibody heavy chain CDR2): ISWNSGSI; (1270 anti-CD3 antibody heavy chain CDR2): ISWNSGSI; (1286 anti-CD3 antibody heavy chain CDR2): ISWNSGSI; (1302 anti-CD3 antibody heavy chain CDR2): VYYDGNNQ; (1318 anti-CD3 antibody heavy chain CDR2): IYYDGKNK; (1331 anti-CD3 antibody heavy chain CDR2): INPSRGYT; SEQ ID NO: 36 (1256 anti-CD3 antibody heavy chain CDR3): AKDNSGYGHYYYGMDV; SEQ ID NO: 37 (1272 anti-CD3 antibody heavy chain CDR3): AKDNSGYGYYYYGMDV; SEQ ID NO: 48 (1288 anti-CD3 antibody heavy chain CDR3): AKDNSGYGHYYYGMDV; SEQ ID NO: 39 (1304 anti-CD3 antibody heavy chain CDR3): ARGPGYNWLDP; SEQ ID NO: 40 (1320 anti-CD3 antibody heavy chain CDR3): ARGPGYNWLDP; SEQ ID NO: 41 (1332 anti-CD3 antibody heavy chain CDR3): ARYYDDHYCLDY; (1260 anti-CD3 antibody light chain CDR1): QSVSSN; (1276 anti-CD3 antibody light chain CDR1): HSVSRN; (1292 anti-CD3 antibody light chain CDR1): QSVSSN; (1308 anti-CD3 antibody light chain CDR1): QSVSRN; (1324 anti-CD3 antibody light chain CDR1): QRISSN; SEQ ID NO: 44 (1334 anti-CD3 antibody light chain CDR1): LSCRASQSVSY; (1262 anti-CD3 antibody light chain CDR2): GAS; (1335 anti-CD3 antibody light chain CDR2): DTS; (1264 anti-CD3 antibody light chain CDR3): QHYINWPLT; (1280 anti-CD3 antibody light chain CDR3): QQYNNWPLT; (1296 anti-CD3 antibody light chain CDR3): QHYINWPLT; (1312 anti-CD3 antibody light chain CDR3): QQYNNWPLT; (1328 anti-CD3 antibody light chain CDR3): QQHHNWPLT; (1336 anti-CD3 antibody light chain CDR3): QQWSSNPLT; and any combination thereof.

    111. The therapeutic agent according to claim 96, wherein the binding moiety with specificity for one or more than one type of CD3+ effector cell comprises a heavy chain variable region comprising the amino acid sequence of: TABLE-US-00019 (a) (anti-CD3 heavy chain) SEQ ID NO: 43 DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTL TVSS; (b) (253 anti-CD3 heavy chain) SEQ ID NO: 44 QVQLVQSGAEVRKPGASVRVTMHWVRQAPGHGLEWIGYINPSRGYTNYNQKFKDRVT MTTDKSFSTAIMDLRSLRSDDSAVYYCARYYDDHYCLDYWGQGTTVTVSSSCKASGYT FTRY; (c) (1250 anti-CD3 antibody heavy chain): SEQ ID NO: 45 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSI GYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDV; (d) (1266 anti-CD3 antibody heavy chain): SEQ ID NO: 46 EVQLVESGGGLVQPGGSLRLSCAATGFTFDDFTMHWVRQAPGKGLEWVSGISWNSGSI GYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSGYGYYYYGMDVWG QGTTVTVSS; (e) (1282 anti-CD3 antibody heavy chain). SEQ ID NO: 47 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSI GYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDVWG QGTTVTVAS; (f) (1298 anti-CD3 antibody heavy chain): SEQ ID NO: 48 QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVAMVYYDGN NQYYADSVRGRFTISRDNSKNTLYLQMNSLRADDTAVYFCARGPGYNWLDPWGQGTL VTVSS; (g) (1314 anti-CD3 antibody heavy chain): SEQ ID NO: 49 QVQLVESGGGVVQPGRSLRLACVASGFTFRSYGMHWVRQAPGKGLQWVAMIYYDGK NKYYADSVRGRFTISRDNSKNTLYLQMNNLRVEDTAMYFCARGPGYNWLDPWGQGTL VTVSS; or (h) (1329 anti-CD3 antibody heavy chain): SEQ ID NO: 50 DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGY TNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTV TVSS.

    112. The therapeutic agent according to claim 96, wherein the binding moiety with specificity for one or more than one type of CD3+ effector cell comprises a light chain variable region comprising the amino acid sequence of: TABLE-US-00020 (a) (anti-CD3 light chain)  SEQ ID NO: 65 DDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVP YRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK; (b) (253 anti-CD3 antibody light chain)  SEQ ID NO: 51 EIVLTQSPATLSLSPGERATLSCSASSSVSYMNWYQQKPGQAPRRWIYDTSKLASGIPAR FSGSGSGTDFTLTISSLEPEDFATYYCQQWSSNPFTFGGGTKVEIKR; (c) (1258 anti-CD3 antibody light chain):  SEQ ID NO: 52 AEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIP ARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIK; (d) (1274 anti-CD3 antibody light chain):  SEQ ID NO: 53 EIVMTQSPATLSVSPGERATLSCRASHSVSRNSAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAIYYCQQYNNWPLTFGGGTKVEIK; (e) (1290 anti-CD3 antibody light chain):  SEQ ID NO: 54 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIK; (f) (1306 anti-CD3 antibody light chain):  SEQ ID NO: 55 EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTDFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVVIK; (g) (1322 anti-CD3 antibody light chain):  SEQ ID NO: 56 EIVMTQSPATLSVSPGERATLSCRASQRISSNLAWYQQKPGQAPRLLIYGASTRATGSPA RFSGSGSGTDFTLTISSLQSEDVAVYYCQQHHNWPLTFGGGTKVEIK; or (h) (1333 anti-CD3 antibody light chain):  SEQ ID NO: 57 DIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPA RFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK.

    113. The therapeutic agent according to claim 96, wherein the binding moiety with specificity for one or more than one type of CD3+ effector cell comprises a heavy chain variable region comprising the amino acid sequence of: TABLE-US-00021 (a) (anti-CD3 heavy chain) SEQ ID NO: 43 DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTL TVSS; (b) (253 anti-CD3 heavy chain) SEQ ID NO: 44 QVQLVQSGAEVRKPGASVRVTMHWVRQAPGHGLEWIGYINPSRGYTNYNQKFKDRVT MTTDKSFSTAIMDLRSLRSDDSAVYYCARYYDDHYCLDYWGQGTTVTVSSSCKASGYT FTRY; (c) (1250 anti-CD3 antibody heavy chain): SEQ ID NO: 45 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSI GYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDV; (d) (1266 anti-CD3 antibody heavy chain): SEQ ID NO: 46 EVQLVESGGGLVQPGGSLRLSCAATGFTFDDFTMHWVRQAPGKGLEWVSGISWNSGSI GYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSGYGYYYYGMDVWG QGTTVTVSS; (e) (1282 anti-CD3 antibody heavy chain). SEQ ID NO: 47 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSI GYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDVWG QGTTVTVAS; (f) (1298 anti-CD3 antibody heavy chain): SEQ ID NO: 48 QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYAMHWVRQAPGKGLEWVAMVYYDGN NQYYADSVRGRFTISRDNSKNTLYLQMNSLRADDTAVYFCARGPGYNWLDPWGQGTL VTVSS; (g) (1314 anti-CD3 antibody heavy chain): SEQ ID NO: 49 QVQLVESGGGVVQPGRSLRLACVASGFTFRSYGMHWVRQAPGKGLQWVAMIYYDGK NKYYADSVRGRFTISRDNSKNTLYLQMNNLRVEDTAMYFCARGPGYNWLDPWGQGTL VTVSS; or (h) (1329 anti-CD3 antibody heavy chain): SEQ ID NO: 50 DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGY TNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTV TVSS. and a light chain variable region comprising the amino acid sequence of: TABLE-US-00022 (a) (anti-CD3 light chain)  SEQ ID NO: 65 DDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVP YRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK; (b) (253 anti-CD3 antibody light chain)  SEQ ID NO: 51 EIVLTQSPATLSLSPGERATLSCSASSSVSYMNWYQQKPGQAPRRWIYDTSKLASGIPAR FSGSGSGTDFTLTISSLEPEDFATYYCQQWSSNPFTFGGGTKVEIKR; (c) (1258 anti-CD3 antibody light chain):  SEQ ID NO: 52 AEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIP ARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIK; (d) (1274 anti-CD3 antibody light chain):  SEQ ID NO: 53 EIVMTQSPATLSVSPGERATLSCRASHSVSRNSAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAIYYCQQYNNWPLTFGGGTKVEIK; (e) (1290 anti-CD3 antibody light chain):  SEQ ID NO: 54 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIK; (f) (1306 anti-CD3 antibody light chain):  SEQ ID NO: 55 EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTDFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVVIK; (g) (1322 anti-CD3 antibody light chain):  SEQ ID NO: 56 EIVMTQSPATLSVSPGERATLSCRASQRISSNLAWYQQKPGQAPRLLIYGASTRATGSPA RFSGSGSGTDFTLTISSLQSEDVAVYYCQQHHNWPLTFGGGTKVEIK; or (h) (1333 anti-CD3 antibody light chain):  SEQ ID NO: 57 DIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPA RFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK.

    114. The therapeutic agent according to claim 96, wherein the one or more than one binding moiety of (a) and the one or more than one binding moiety of (b) are attached by a peptide linker.

    115. The therapeutic agent according to claim 114, wherein the peptide linker comprises from about 1 to about 50 amino acids.

    116. The therapeutic agent according to claim 114, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of: TABLE-US-00023 (a) SEQ ID NO: 58 GGGGSGGGGSGGGGS (b) SEQ ID NO: 59 (253 anti-CD3 linker 1) NSTYRVVSVLTVLHQDWLNGKEYKCK (c) SEQ ID NO: 60 (253 anti-CD3 linker 2) FQNALLVRYTKKVPQVSTPTLVEVS (d) SEQ ID NO: 61 (253 anti-CD3 linker 3) ASADDAKKDAAKKDDAKKDDAKKDL (e) SEQ ID NO: 62 (202 anti-CD133 antibody linker): SSGGGGSGGGGGGSSRSS;  and (f) any combination of (a)-(e).

    117. The therapeutic agent according to claim 96, wherein the therapeutic agent comprises the amino acid sequence of: TABLE-US-00024 (a) (anti-CD34-HC-anti-CD3-scFv) SEQ ID NO: 63 QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGW INTNTGEPKYAEEFKGRFALSLDTSVSTAYLQINSLKAEDTAVYFCARGY GNYARGAWLAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYT MHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQL SSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGS GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRW IYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLT FGAGTKLELK.

    118. A pharmaceutical composition comprising an effective amount of the therapeutic agent according to claim 96 and a pharmaceutically-acceptable diluent, carrier or excipient.

    119. A method of reducing the number of hematopoietic stem cells, hematopoietic progenitor cells and/or hematopoietic cancer stem cells in a subject in need thereof, comprising administering to the subject an effective amount of the therapeutic agent according to claim 96.

    120. The method according to claim 119, further comprising the step of administering to the subject an effective amount of one or more than one chemotherapeutic agent, radiotherapy and/or immunotherapy.

    121. The method according to claim 119, wherein the subject is receiving and/or will receive hematopoietic stem cell transplantation (HSCT).

    122. A method of treating a subject for a disease or condition that would benefit from a reduction of hematopoietic stem cells, hematopoietic progenitor cells and/or hematopoietic cancer stem cells in the subject, comprising administering to the subject an effective amount of the therapeutic agent according to claim 96.

    Description

    [0393] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

    [0394] FIG. 1: CD34/CD3 bispecific antibodies selectively kill CD34+ hematopoetic stem cells in a dose-dependent manner.

    [0395] A. Graphs represents frequencies of 7AAD+ cells (necrotic) gated from lymphocytes/CD3+/CD34+ cells. Y-axis side scatter and X-axis 7AAD intensity. Upper left graph shows the background of dead cells (7AAD+) after overnight culture. The three lower lines represents cells incubated with CD34/(−), CD3/(−) and CD34/CD3 respectively in the increasing concentrations, 0.1, 1.0 and 10 ug/ml. B. Graphs represent frequencies of 7AAD+ cells (necrotic) gated from lymphocytes/CD3−/CD19+ cells and lymphocytes/CD3−/CD56+ representivaly. The Cells where treated with increasing concentrations, 0.1, 1.0 and 10 ug/ml of CD34/CD3 bispecific antibodies.

    EXAMPLES

    Example 1

    [0396] A patient suffering from AML is about to undergo allogeneic HSCT. The patient is >70 years with fungal infection and has little chance to survive standard chemotherapy given as conditioning regimen. Before the transplantation the patient needs to undergo a preconditioning to minimize the risk of relapse due to survival of malignant cancer cells, minimize risk for rejection and make space for the new hematopoietic stem cells. In addition, the pre-conditioning will also influence the risk of GVHD. Instead of using the common methods of high dose chemotherapy and irradiation, the patient is treated with antibody-based immunotherapy using a CD34/CD3 BiTE (e.g., the agent of Example 5, below) in combination with or without mild chemotherapy/radiation and with or without T-cell antibodies. This milder variant of pre-conditioning will likely reduce the risk of acute GVHD and the patient will have very little toxicity resulting from the treatment. Due to the target antigen of the antibody-based immunotherapy, a putatively better anti-tumor effect will be achieved against cancer cells with undifferentiated phenotype.

    Example 2

    [0397] A patient suffering from MDS has already received an allogeneic HSCT but has relapsed in the underlying malignant disease. The patient is eligible for a re-transplantation if the patient will be in hematological remission, i.e. no detection of malignant cells in the bone marrow. Patients considered for a re-transplantation are usually in much worse shape clinically than patients prior to primary HSCT. Instead of using the common methods of heavy chemotherapy as induction therapy, these patients greatly benefit from a milder and more efficient treatment in order to achieve remission. Before the re-transplantation the patient is treated with infusion(s) of a CD34/CD3 BiTE (e.g., the agent of Example 5, below) with chemotherapy and with or without T-cell antibodies. This will minimize the risk of another relapse due to survival of malignant cancer cells, and minimize risk for GVHD.

    Example 3

    [0398] A patient suffering from a non-malignant disease, e.g. Severe aplastic anemia (SAA), Wiskott Aldrich Syndrome, Hurlers Syndrome, FHL, CGD, Kostmanns syndrome, Severe immunodeficiency syndrome (SCID), other autoimmune disorders, inborn errors of metabolism or other immunodeficiencies, are eligible for a HSCT to exchange their non-functional hematopoietic cell compartment. Without treatment, the mortality rate for both of these conditions is extremely high. Before transplantation, a milder pretreatment compared to patients with malignant disease is used. While patients with non-malignant disease won't benefit from GVHD or GVL (Graft-versus-leukemia) (which could be beneficial in for example leukemia), an even milder pre-conditioning is warranted in non-malignant diseases. Instead of using conventional chemotherapy with or without irradiation the patient is instead pretreated with a CD34/C3 specific BiTE (e.g., the agent of Example 5, below), T-cell antibodies and very mild chemotherapy. This will make the risk for GVHD decrease further, reduce toxicity and possibly open up the use of allogeneic HSCT in other non-malignant diseases, which are not currently treatable, such as various autoimmune diseases.

    Example 4

    [0399] A patient suffering from MDS is eligible for HSCT. The malignant cells of the patient express CD34. While using the CD34/CD3 specific BiTE (e.g., the agent of Example 5, below) in the pre-conditioning together with standard reduced protocol with chemotherapy treatment prior to HSCT, there will be a reduced risk that the patient will relapse in the underlying malignancy after HSCT. This will be of pivotal importance since relapse of MDS is the most common cause of death after allogeneic HSCT.

    Example 5

    [0400] We started with 50 ml peripheral blood from a healthy individual pretreated with Granolucyte Colony stimulating Factor (G-CSF, Amgen, Calif., US, 10 micrograms/kg for two consecutive days). This growth hormone promotes in vivo growth of lymphocytes and Hematopoetic stem cells (CD34+ cells). When these increase the growth in the bone marrow cells are pushed out into peripheral blood. This increases the frequency of CD34 Hematopoetic stem cells in peripheral blood often to more than >2%. The blood was centrifuged on a ficoll density gradient (Fresenius Kabi, Norway) in order to remove red blood cells. The remaining white blood cells were collected and washed twice in PBS buffer. White blood cells where then resuspended in complete RPMI growth medium (GIBCO, Germany) containing 10% human heat-inactivated serum, penicillin and glutamine (GIBCO, Germany). Cells were then seeded in 6 well plates at a concentration of 1 million cells/ml medium. CD34/CD3 bispecific antibodies or the relevant controls missing one of the binding sites, CD34/(−) or CD3/(−) (for sequences see Appendix 1) were incubated with the white blood cells over night at 37° C., 5% CO2. After incubation the cells were collected and washed before resuspension with PBS (GIBCO, Germany).

    [0401] In short, for surface staining only, white blood cells were incubated with antibodies for 20 minutes at 4° C. and subsequently stained with dead cell marker 7AAD for 10 minutes at room temperature. Acquisition was performed with the BD LSRII using BD FACS Diva software (BD Biosciences, Franklin Lakes, N.J., USA). Fluorescence-minus-one (FMO) samples were used to obtain proper gating strategies.

    [0402] The following antibodies were used. For Fluorescein isothiocyanate (FITC) anti-CD3 (SK7); FITC anti-CD34 (8G12); FITC anti-CD19 (HIB19); FITC anti-CD56 (NCAM16.2); phycoerythrin (PE) anti-CD3 (SK7); PE anti-CD56 (MY31), PE anti-IL-2 (MQ1-17H12); 7-Amino-Actinomycin D (7AAD); APC anti-CD4 (RPA-T4); APC anti-CD8 (RPA-T8) where bought from BD Biosciences (Franklin Lakes, N.J.); Qdot605 anti-CD3 (UCHT1) and Pacific Orange anti-CD8 (3B5) came from Invitrogen, Eugene, Oreg., USA. FITC anti-TCR PANγδ (IMMU510) and Krome Orange anti-CD4 (13B8.2) was purchased from Beckman Coulter, Fullerton, Calif., USA.

    [0403] Data is described in FIG. 1 that shows one representative experiment out of three. All with similar results with blood from different individuals. The peripheral blood contained between 1 and 2% CD34+ hematopoetic stem cells. The data clearly shows that there is a specific effect of the bispecific antibody CD34/CD3 with specific killing effect against CD34+ cells after the overnight culture. (FIG. 1 A). With the increasing concentration of CD34/CD3 bispecific the percentage of dead cells (7AAD+) increases from background (10.8%) to 32.3%. This is in contrast to cells treated with either CD34+/CD3(−) reagent where there is only an increase to 14.2%. In cells treated with CD34(−)/CD3+ reagent there is an increase to 27.2% dead CD34+ cells. This is higher than the other control but lower than the bispecific CD3+/CD34+ bispecific antibody. The effect of the CD34/CD3 bispecific antibody is also specific against CD34+ cells. FIG. 1 B shows that with increasing concentrations of the bispecific antibody there is no increase in the amount of dead B cells (defined as CD3(−)/CD19+). With the lowest concentration there is 3% dead B-cells and with the highest concentration the dead cells are only 2%. The same situation is seen with NK-cells (defined as CD3(−)/CD56+). With the lowest concentration the frequency of dead cells in 26.8% while in the highest concentration it is 29.8%. So there does not seem to be any specific toxicitiy of the bispecific antibody against non-relevant white blood cells such as B-cells and NK-cells.

    APPENDIX I

    [0404] anti-CD34 antibody sequences derived from US patent US 2010/0311955 A1.
    anti-CD3 scFv sequences based on blinatumomab as described in U.S. Pat. No. 7,635,472.
    anti-CD34-HC-anti-CD3-scFv

    TABLE-US-00011 [SEQ ID NO: 63] QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGW INTNTGEPKYAEEFKGRFALSLDTSVSTAYLQINSLKAEDTAVYFCARGY GNYARGAWLAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYT MHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQL SSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGS GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRW IYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLT FGAGTKLELK anti-CD34-LC [SEQ ID NO: 26] DVLLTQSPLSLPVTLGQPASISCRSSQTIVHSNGNTYLEWFQQRPGQSPR LLIYQVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP RTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSST