( 12 ) United States Patent

US010392438B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 ,392 ,438 B2 Bennett et al. ( 45 ) Date of Patent : Aug. 27 , 2019

(54 ) BISPECIFIC ANTIBODIES 2002/ 0039995 Al 4 / 2002 Gao 2008 /0269466 Al 10 /2008 Humphreys 2010 /0150914 A1 6 /2010 Wang et al . ( 71 ) Applicant: Pfizer Inc. , New York , NY (US ) 2010 /0256340 A1 10 /2010 Brinkmann 2012 /0009621 A11 /2012 Yamasaki ( 72 ) Inventors : Eric M . Bennett, Arlington , MA (US ) ; 2014 /0200331 A1 * 7 /2014 Corper ...... CO7K 16 / 36 Nathan Higginson - Scott, Boston , MA 530 / 387 . 3 (US ) ; Lioudmila Tchistiakova , Stoneham , MA (US ); Kimberly A . FOREIGN PATENT DOCUMENTS Marquette , Somerville , MA (US ) ; WO 199852976 11 / 1998 Janet E . Paulsen , Londonderry , NH WO 200034317 6 / 2000 (US ) ; Ruth E . Gimeno , Carmel , IN WO 2009089004 7 / 2009 (US ) wo 20110117653 9 / 2011 WO 2011143545 11 / 2011 ( 73 ) Assignee : PFIZER INC ., New York , NY (US ) WO 20130096291 6 / 2013 wo 2014081955 Al 5 / 2014 ( * ) Notice : Subject to any disclaimer, the term of this WO 2014150973 9 / 2014 patent is extended or adjusted under 35 WO 2015173756 A2 11/ 2015 U . S .C . 154 (b ) by 0 days . OTHER PUBLICATIONS ( 21) Appl . No. : 15 / 309, 879 Davies et al : “ Fab Assembly : An analysis of different Ch1 :CL May 13, 2015 combinations” , Progress in Immunology VI : Sixth International (22 ) PCT Filed: Congress of Immunology, pp . 145 - 149 , 1986 . ( 86 ) PCT No. : PCT/ IB2015 / 053537 Klein et al: “ Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies " , MABS , vol. 4 , No . 6 , pp . $ 371 (c )( 1 ), 653 -663 , 2012 . ( 2 ) Date : Nov . 9 , 2016 Lewis et al : “Generation of bispecific IgG antibodies by structure based design of an orthogonal Fab interface ” , Nature Biotechnol (87 ) PCT Pub . No. : W02015 /173756 ogy , vol. 32 , No. 2 , pp . 191 - 198 , 2014 . Lewis et al: “ Supplemental Information : Generation of bispecific PCT Pub. Date : Nov . 19 , 2015 IgG antibodies by structure -based design of an orthogonal Fab interface ” , Nature Biotechnology , vol. 32 , No . 2 , 2014 doi: 10 . 1038 / (65 ) Prior Publication Data nbt. 2797 . US 2018/ 0179285 A1 Jun. 28 , 2018 Liu et al: “ A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electro Related U .S . Application Data static Steering Mechanism ” , Journal of Biological Chemistry , vol . 290 , No. 12 , pp . 7535 - 7562 , 2015 . (60 ) Provisional application No .62 /159 , 201 , filed on May Liu et al : “ Disulfide bond structures of IgG molecules: Structural 8 , 2015 , provisional application No. 62 / 150 ,680 , filed variations, chemical modifications and possible impacts to stability on Apr. 21 , 2015 , provisional application No . and biological function ” , MABS , vol . 4, No . 1, pp . 17 -23 , 2012 . 61/ 994 ,720 , filed on May 16 , 2014 . Luo et al: “ Design and Applications of Bispecific Heterodimers : Molecular Imaging and beyond ” Molecular Pharmacuetics, vol . 11 , (51 ) Int. CI. No . 6 , pp . 1750 - 1761, 2014 . CO7K 16 / 00 ( 2006 .01 ) Muller et al: “ The first constant domain ( CH1 and CL ) of an CO7K 16 / 28 ( 2006 .01 ) antibody used as heterodimerization domain for bispecific miniantibod COOK 16 / 24 ( 2006 .01 ) ies ” , FEBS Letters, vol. 422 , No. 2 , pp . 259 - 264 , 1998 . A61P 27/ 16 ( 2006 .01 ) International Search Report and the Written Opinion , PCT/ IB2015 / (52 ) U . S . CI. 053537 , dated Dec . 2 , 2015 ; 23 pages. CPC ...... CO7K 16 / 2863 ( 2013 .01 ); A61P 27 / 16 ( Continued ) ( 2018 . 01 ) ; CO7K 16 /00 (2013 . 01 ) ; CO7K 16 / 24 ( 2013 . 01 ) ; C07K 16 / 244 (2013 . 01 ) ; COZK Primary Examiner - Sean E Aeder 2317 / 24 ( 2013 .01 ) ; CO7K 2317/ 31 ( 2013 . 01 ) ; CO7K 2317/ 35 (2013 .01 ) ; CO7K 2317/ 522 (57 ) ABSTRACT ( 2013 .01 ) ; COZK 2317 / 55 (2013 .01 ); COOK The present invention relates to engineered heteromultim eric proteins , and more specifically , to methods for produc 2317/ 75 ( 2013 .01 ); CO7K 2317/ 92 (2013 . 01 ) ing and purifying heterodimeric proteins, such as bispecific (58 ) Field of Classification Search antibodies and . Methods for producing and purifying such None engineered heterodimeric proteins and their use in diagnos See application file for complete search history. tics and therapeutics are also provided . The present inven References Cited tion also relates to a humanized antibody that specifically (56 ) binds human TrkB and methods for producing and using the U . S . PATENT DOCUMENTS antibody to , inter alia , treat a hearing loss disorder . 5 , 731, 168 A 3 / 1998 Carter et al . 7 , 183 ,076 B2 2 / 2007 Arathoon et al . 24 Claims, 62 Drawing Sheets 7 , 750 , 122 B2 7 / 2010 Cho et al . Specification includes a Sequence Listing . US 10 ,392 ,438 B2 Page 2

( 56 ) References Cited Schimmang et al: “ Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss ,” Development, vol . 130, pp . OTHER PUBLICATIONS 4741- 4750 , 2003 . Abhinandan et al: “ Analysis and prediction of VH /VL packing in Smith et al : " Predicting the Tolerated Sequences for Proteins and antibodies ” , Protein Engineering, Design & Selection , vol. 23 , No . Protein Interfaces Using RosettaBackrub Flexible Backbone Design ,” 9 , pp . 689 -697 , 2010 . PLoS One , vol. 6 , No . 7 , e20451 ,. Corrada et al : “ Energetic and Dynamic Aspects of the Affinity Songsivilai et al: “ Bispecific antibody: a tool for diagnosis and Maturation Process: Characterizing Improved Variants from the treatment of disease , " Clinical Exp Immunology , vol. 79 , pp . Bevacizumab Antibody with Molecular Simulations, ” Journal of 315 - 321 , 1990 . Chemical Information and Modeling , vol. 53 , pp . 2937 - 2950 , 2013 . Wise et al: “ Resprouting and Survival of Guinea Pig Cochlear Dani et al: “MODIP revisited : re -evaluation and refinement of an Neurons in Response to the Administration of the Neurotrophins automated procedure for modeling of disulfide bonds in proteins, " Brain - Derived Neurotrophic Factor and Neurotrophin - 3 , ” Journal of Protein Engineering, vol . 16 , No. 3 , pp . 187 - 193 , 2003 . Comparative Neurology , vol. 487 , pp . 147 - 165 , 2005 . Das et al: “ Macromolecular Modeling with Rosetta ,” Annual Review Zhang et al: “ TM - align : a protein structure alignment algorithm of Biochemistry , vol. 77 , pp . 363 - 82 , 2008 . based on the TM - score ," Nucleic Acids Research , vol. 33, No. 7, pp . Eswar et al : “ Tools for comparative protein structure modeling and 2302 - 2309 , 2005 . analysis , ” Nucleic Acids Research , vol . 31 , No. 13 , pp . 3375 - 3380 , Zhang et al : “ Cyclic -AMP Response Element- Based Signaling 2003 . Assays for Characterization of Trk Family Tyrosine Kinases Modu Feige et al: “ An Unfolded CH1 Domain Controls the Assembly and lators, ” Neurosignals , vol. 15 , pp . 26 -39 , 2006 - 2007 . Secretion of IgG Antibodies , ” Molecular Cell, vol. 34 , pp . 569 -579 , Kleywegt et al: “ Detection , delineation , measurement and display of 2009 . cavities in macromolecular structures, " ACTA Crystallographica Friedman et al : “ Neurotrophin Signaling via Trks and p75 , " Experi Section D , vol. 50 , No . 2 , pp . 178 - 185 , 1994 . mental Cell Research , vol. 253 , pp . 131 - 142 , 1999 . Lewis et al . “ Generation of bispecific IgG antibodies by structure Kostelny et al: “ Formation of a bispecific antibody by the use of based design of an orthogonal Fab interface” 2014 , vol. 32 , No . 2 , leucine zippers , ” Journal of Immunology , vol. 148 , pp . 1547 - 1553 , pp . 191- 202. 1992 . Mazor et al. “ Improving target cell specificity using a novel Krivov et al: “ Improved prediction of protein side -chain conforma monovalent bispecific IgG design ” MAbs. 2015 ;7 ( 2 ): 377 -89 . tions with SCWRL4 ,” Proteins, vol . 77 , pp . 778 -795 , 2009 . Regula et al . " Variable heavy - variable light domain and Fab -arm Laskowski: “ PDBsum : summaries and analyses of PDB structures ,” CrossMabs with charged residue exchanges to enforce correct light Nucleic Acids Research , vol. 29 , No . 1 , pp . 221 - 222 , 2001 . chain assembly .” Protein Engineering, Design and Selection , 2018 , Liberman and Kujawa : “ Adding Insult to Injury : Cochlear Nerve vol. 31 , No. 7 - 8, pp . 289 - 299 . Degeneration after “ Temporary 'Noise - Induced Hearing Loss, ” Jour Schaefer et al. “ Immunoglobulin domain crossover as a generic nal of Neuroscience , vol. 29 , No. 45 , pp . 14077 -14085 , 2009 . approach for the production of bispecific IgG antibodies. ” PNAS, Meltser et al : “ TrkB -Mediated Protection against Circadian Sensi 2011, vol. 108 , No. 27 , pp . 11187 - 11192. tivity to Noise Trauma in the Murine Cochlea, ” Current Biology , Shen et al. “ Conjugation site modulates the in vivo stability and vol. 24 , pp . 658 -663 , 2014 . therapeutic activity of antibody -drug conjugates .” Nature Biotech Qian et al: “Novel Agonist Monoclonal Antibodies Activate TrkB nology , 2012, vol. 30 . No . 2, pp . 184 - 189 . Receptors and Demonstrate Potent Neurotrophic Activities , ” Jour Strop et al . "Generating Bispecific Human IgG1 and IgG2 Anti nal of Neuroscience , vol . 26 , No. 37 , pp . 9394 - 9403 , 2006 . bodies from Any Antibody Pair” J . Mole . Biol. , 2012 , vol. 420 , pp . Saphire et al: " Crystal Structure of a Neutralizing Human IgG 204 - 219 . Against HIV - 1 : A Template for Vaccine Design , ” Science , vol. 293 , pp . 1155 - 1159 , 2001. * cited by examiner U . S . Patent Aug. 27 , 2019 Sheet 1 of 62 US 10 , 392 ,438 B2

when AAAAANNN Lc2 2-CHCL DLC1 4.CHCLH1c2 A FIG.1B FUNCTIONALANTIBODYNON- FIG.1D FUNCTIONALANTIBODYSEMI- HC3-CHIC bindingNon- Lc2th arm CHOLI3-

CHOL4- DLC2 CHCLHC22- bindingNon- ARX 1 arm HC .1AFIG PRODUCTSPECIFICDESIREDBI- .1CFIG ANTIBODYFUNCTIONALSEMI- win 2000

HC1WCHCL-1- WinLC1 1-CHCLHC

id . ii i L U . S . Patent Aug . 27, 2019 Sheet 2 of 62 US 10 , 392, 438 B2

190 180

180

170 160 FIG.2 160 SGLYSLSSVVHTFPAVLQS-SWwwanNSGALTSGTSGGTAALGCLVKDYFPEPVTVASTKGPSVFPLAPSSKS- 237 150 230 NNKYAASSYLSLTPEQWKSHRSYSCQVTHAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS- 220 KTHEPKSC-DTYICNVNHKPSNTKVDKKVVTVPSSSLGT-Q 140 CH1DomainNumbering 140 CLDomainNumber22

130 210 215

200 120 120 ?????: Lambda: At EG-STVEKTVAPTECS 114 111 AAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKOSTYSLSSTLTLSKADYEKHKVYACEVTH 200 QGLSSPVTKSFNRGEC atent Aug . 27 , 2019 Sheet 3 of 62 US 10 , 392 ,438 B2

MABCD REDUCING

FIG.3 MABCDEF NON-REDUCING

OvYy 188kDa 98kDa 49kDa 28kDa U . S . Patent Aug . 27, 2019 Sheet 4 of 62 US 10 , 392 ,438 B2

0 1144658.0 160000 144434.0 :Us

1 FIG.4B Cys_1 L121513.0 140000 121290,0 0,144627 160000 120000 1444010 1007 100000 121198.0 1214730 mass Cys3a 150000 120000140000 100000 FIG.4A CysBeta 98083.0 100000 TTTTT FIG.4C1007.

goingenannanT 0,100423189 23413.0 50000 U . S . Patent Aug. 27 , 2019 Sheet 5 of 62 US 10 ,392 , 438 B2

emperaturmass ngtinderoicingmass BAN 160000 160000 0144654, 0144720. 144543.0 1144609.0 144429,0 144318.0 140000 140000 akisaganansamenweringtergantungpadamengineeringpresentecontinuetornarentregadeponerovergethereretsetingimusedphilangan

Osmanganmentegainimesegaranpenangananngpaninginmgataongnarinignatingnannigeriaandinteringthegrowthmemoriesaroundimagehommeengelvaneenoverdensenestet 120000 120000

Cys_3b Cys_6

100% 100000 1007 100000

FIG.4D FIG.4E U . S . Patent Aug . 27, 2019 Sheet 6 of 62 US 10 , 392 ,438 B2

)L176(Ser Val(H190)

FIG.5A Ser(H188)

Val(L133) U . S . Patent Aug . 27, 2019 Sheet 7 of 62 US 10 , 392 ,438 B2

Asp(L176)

)H190{Ser)H188(Ser

pal &tot . FIG.5B Ser(L133)

Lys(H124)

" YAK SOOME U . S . Patent Aug . 27, 2019 Sheet 8 of 62 US 10 , 392 ,438 B2

Lys(L176) Val(H190)

)H188(Gly 3. FIG.5C 3.6

Ser(L133) Glu(H124) U . S . Patent Aug . 27, 2019 Sheet 9 of 62 US 10 , 392, 438 B2

mass masspe 48500 48500 48287 Ab2_H+Ab1L48259 48415 Ab2Fab 48207 Ab2Fab 48191 perempuan 48000 menghitunganparang48000

bebepensagensparapintarungehuyo 47586 Latiquemenanganpendapatan47500 . WW W 47500 Ab1Fab 47381 ? ???????????????????????????????? Ab1H+Ab2_L 47294* tildigpompoenpoweringmentalitet 47327 1007Ab1FabAb1_ H+Ab2_L47361 47000 47000

FIG.6A FIG.6B U . S . Patent Aug. 27 , 2019 Sheet 10 of 62 US 10 , 392, 438 B2

ass mmaandia mass

25157 25028 2888 |53073 Galactose(Gal)

Wh punyang Antibody1FC(-K) Antibody2FC(-K) 54000 GOF FcCombined 54000 3559 53500 5 53500 53397 mpinganparaaprengulanganpengeloladefaptuluilamutasdfelgwaponglalakingnapapangnanayparangnaman 53235 GOF GRU 53235GOF 53073 53000 5307353397 0 53000

www w wwwwwwwwwwomen 52500 100m, f0-52500 FIG.7A100% FIG.7B atent Aug . 27 , 2019 Sheet 11 of 62 US 10 , 392 ,438 B2

*20

70*15Time(min)

Lilin ) 280nm , MAU( Absorbance FIG.8A 20 AVANNY 75** Time(min) YLLIVRA Whemahandanhanteerthanthailandannat

ATA 10

finnakanakinnitmund

tendarehandannataduan winnen ) 280nm , MAU( Absorbance U . S . Patent Aug . 27, 2019 Sheet 12 of 62 US 10 , 392 ,438 B2

? -Peak2B Acidiccharge species

wwwtwit. ter manen

FIG.8B %3 Peak2A Basiccharge species Time(min)

?????????

? ????????????????????????

) 280nm , MAU( Absorbance atent Aug . 27 , 2019 Sheet 13 of 62 US 10 , 392 ,438 B2

mass ssa mass 48500 tarpeidergawenaganagannyamanglendingarsasponpation 48500 48500 48286 ngampangabanglanatinangatingpama Ab2Fab48205 Ab2Fab48206 Ab2Fab48206 48000 rrrrr 48000 48000

47500 hingamingmga 47500 Ab1Fab apayangdippedpampang 47500 Ab1Fab 47380 Ab1_H+Ab2L47327 47381 Ab1_H+Ab2L47326 ning FIG.9A1002Ab1_H+Ab247380 47000 1007 47000 1002 47000

FIG.9B FIG.9C U . S . Patent Aug. 27, 2019 Sheet 14 of 62 US 10 , 392 ,438 B2

ass 048228. massapque

.04775347653.0 47634.0 mpampangangampananngpangarapmagandanganoluntarytreatmentmanagertipongthatmagangpenaynatataykinantotngmatang 47409.0 47429.0 FabAb3 W ishini 470004750048000 Ab3Fab Ab3L Ab3L 47294.0 CCL20L474Ab3H CCL2OH046871. 4750047000*46500- CCL2OH 4800047500470004650046000 046754. .046770 FabCCL20 ang FabCCL20 46719.0 parang46500 46734.0 hapedagangarapetyaptopstiltotototoyottengary namanangmangpanahongUgamanparang -M1C5xAb3

NEGATIVE 46000 wwwwwwwwww FIG.10A1001C5XAb3-M1 46000 CCS 1007FIG.10B U . S . Patent Aug . 27, 2019 Sheet 15 of 62 US 10 , 392 ,438 B2

ass ass

gedeponeranganlainyangmempunyaitaynaparangmanlangangnatingpampangangailanganyangandam Ab3L47675.0 Ab3Fab 47469.0 0-fragraparpelgapapapapapunapangwagenpolyhyenyetimpaguapeganganpoliproponglangangmgapinakamahtanganpenangananpreparingprogramyangmemeriamm Ab3Fab CCL2OHI 475004800047000 47449.046913.0 CCL2OLCCL2OHAb3HL Ab3? CCL2OL 46783.0 4600046500470004750048000 CCL20Fab46768.0 CCL20Fab TO 46733.0 46500 NEGATIVE C5%Ab3-M2 anah 7 FIG.11A1007C5xAb3-M2 46000 FIG.11B1007 bancaU . S . Patent com Aug . 27, 2019 Sheet 16 of 62 US 10 , 392 ,438 B2 FIG . 12A Ab3 Absorbanceat280nm

handhaayAntuanandina inchiriat de hora na bahwasaba 13 14 15 16 17 Time (min ) 13 14 15 16 17 FIG . 12B 80 % C5xAb3- M2 NEGATIVE C5xAb3- 12 Absorbanceat280nm VVV where vanpenggunaan pengurangangarapenerangan perpaparampangangaphaengegangenen photopompong p inagpampumputing gjennom 13 14 15 16 17 Time( min ) 13 14 15 16 17 FIG . 12C

C5xAb3- M4 C5XA3-M1 NEGATIVE Absorbanceat280nm i weberler wwwwww p agpapagampang matricularitadanhanhand parama 13 14 15 16 17 Time (min ) 13 14 15 16 17 atent Aug . 27 , 2019 Sheet 17 of 62 US 10 , 392 ,438 B2

FIG . 13

ESTE ARROSSENSES vaatit

WAS

ESTA

Route SEN r. .. U . S . Patent Aug . 27, 2019 Sheet 18 of 62 US 10 , 392 ,438 B2

S .

2 SPILS

1 U . S . Patent Aug . 27, 2019 Sheet 19 of 62 US 10 , 392 ,438 B2

+ 2 FIGY . 15

* *

2 W0.

18 58 : ** 20 tutti $ U . S . Patent Aug . 27, 2019 Sheet 20 of 62 US 10 , 392 ,438 B2

FIG . 16

BA Sa

2. 29 ST WATAN " . SER 502 4 2015 29ers . . U . S . Patent Aug . 27 , 2019 Sheet 21 of 62 US 10, 392, 438 B2

FIG . 17A C5 Fab Deconvolute - 5 46758 . 0 1007 ( ??? “ ?5 "}

47084. 0 45500 46000 46500 47000 47500 48000 48500 mass

Ab3 Fab FIG . 17B 47413. 0 1 Deconvolute - 6 (??? “ Ab3 ” )

47637 . 0 47739. 0 0 - 3 ?????????????????????????????????????????? ?????? ???? ?????? ????? ??????????? 45500 46000 46500 47000 47500 48000 4 8500 mass FIG , 17C C5 Fab Deconvolute - 1 46758 ( ??? “ Ab3 5 - M1 NEGATIVE " ) Ab3? Ab3 Fab % 46875?5 . ??? 47413 Ab3L 47295 47084 47637 47738 9 - ???????????? ??????????????????? ????? ????????? ???? ?????????????? 45500 46000 46500 47000 47500 48000 48500 mass atent Aug . 27, 2019 Sheet 22 of 62 US 10, 392, 438 B2

? . FIG? . , . . , . 17D C5 Fab Deconvolute - 2 46773 1 (??? “ Ab3 ?5 - M1" } Ab3 Fab 47432

47462 ??03 47298) 47758 3 ???????????????? ??????? ??????? ?????????????????? ???????????????? ????????? ????? 45600 46000 46500 47000 47500 48000 48500 ?ass

????? C5 Fab ???? : 46815 100 Deconvolute - 3 Ab3 Fab 47456 Ab3 H C5H C5 L Ab3L 146932 47339 / 47680 \ 47782 3 ?????????? ???????????? ???????? ??? ? ????????????? ???????????????? ??????? ????????? 45500 46000 46500 47000 47500 48000 48500 mass

FIG . 17F C5 Fab 100 , Deconvolute - 4 46803 Ab3 Fab 47444 Ab3Hc5 C5H 46920 47326Ab3L 4766847770 45500 46000 46500 47000 47500 48000 48500 ?ass U . S . Patent Aug. 27 , 2019 Sheet 23 of 62 US 10 , 392, 438 B2

FIG . 18A | Deconvolute - 1 (AKA " Ab3 C5- M1 NEGATIVE " )

Absorbance(mAU,280nm)

mong munagampananampanan naming mingg agaminining ng gamangangagemangmanguagmamangmangangamninginning in an insig in inanana nanning R 13 14 15 16 17 Time (min ) FIG . 18B | Deconvolute - 2 (AKA " Ab3 C5 -M1 " )

Absorbance(MAU,280nm)

obocongecoran ????????? ????????? ??????? ? ???????? ?????????????? ?????????? 13 15 Time (min ) atent Aug . 27 , 2019 Sheet 24 of 62 US 10 , 392 ,438 B2

FIG . 18C Deconvolute - 3

Absorbance(MAU,280nm) .

wwwwwww ????????????????????????????????????????????????? ?????? ??????????????? ?????????????????????????????????? ???????????? 2L LSL SL L LL Time (min ) FIG . 18D Deconvolute - 4

Absorbance(MAU,280nm) êtywowany

N damanstendurmatoare13interna sakoneranderendimento14 montato dans15 tamatisations bonentwurstmundustes16 terminatawatankwadon17 en bruin Time (min ) U . S . Patent Aug. 27, 2019 Sheet 25 of 62 US 10 , 392 ,438 B2

FIG . 18E Deconvolute - 5 (AKA " C5" )

Absorbance(MAU,280nm)

? ??? ???????? ??????? ??? ????? ? ? ?? ?? ???????? ?????????? ?????? ???????????? ????????????? ??????????? ???????? 12 13 14 15 16 17 Time (min ) FIGNO . 18F Deconvolute - 6 (AKA " Ab3 ") .WWW Absorbance(MAU,280nm)

henkinematecanduantumatontamin binnen banatuantenmukaiseden treenkomstenmetrimahinanthinaikinan horwachsenetinantiemstromalactonealocation - 13 14 15 16 17 Time (min ) U . S . Patent Aug . 27, 2019 Sheet 26 of 62 US 10 , 392 ,438 B2

FIG . 19A - Ab3- S1XC5 - T4

molel°Ckcal/Cp(

w 50 55 60 65 70 75 80 85 90 Temperature ( °C )

. .

2 Ab3 - S1xC5 - T9

Cp(kcal/mole°C

Owntimer .. . p e ngertian85 peminating90 50 55 60 Temperature65 70 75 (80 °C ) FIG1 . . 19C - Ab3 -S1xC5 -S1rev

Cp(kcal/mole°C

* * * * * ???????? ????????????? ?? ?????????????? 50 55 60 Temperature65 70 75 (°C80 ) 85 90 U . S . Patent Aug. 27, 2019 Sheet 27 of 62 US 10 , 392 ,438 B2

FIG . 19D U wwww Ab3 -S1xC5 - T1

Cp(kcal/molel°C Sillanperint Aternate NH

50 55 60 65 70yung 75 ga 80 85 90 Temperature (°C ) FIG . 19E .

. - Ab3- S1xC5 - T2 . ATTRATTENTVISTA Cp(kcalmolel°C ilominus h UVO

w 50 55 60 65 70 75 80 85 90 Temperature (°C ) FIG . 19F Ab3 -S1xC5 - T3

Cp(kcal/molelºc

50 55 60 65 70 75 80 85 90 Temperature (°C ) U . S . Patent Aug . 27, 2019 Sheet 28 of 62 US 10 , 392 ,438 B2

FIG . 19G WA -- Ab3 -S1xC5

Cp(kcal/moleC

minimamente sem aprenen porno pornogramapanese porn masyaranmagane pasaportes para 50 55 60 65 70 75 80 85 90 Temperature (°C )

FIG . 19H mwema. Ab3XC5 - S1rev

Cp(kcal/molel°C

????? ????????? 50 55 0 Temperature65 70 75 (80°C ) 85 90 FIG . 191 Ab3xC5

°Cmole/kcalCp(

50 55 60 65 70 75 80 85 90 Temperature (°C ) U . S . Patent Aug. 27, 2019 Sheet 29 of 62 US 10, 392, 438 B2

22 HER Ab3 Fab Ab3 -S1xC5 - T1 C5 Fab 47428 1007 46694

invisiem 50184 46728 47653 + - 50203 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mass 100 47428Ab3 Fab Ab3 -S1xC5 - T2 FIG90 , 20B C5 Fab 46703 50184 46146 47652 - 50203 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mass 1007 C5 Fab 47427 46810 Ab3 Fab Ab3- S1xC5 - T3 SI

50184 AIMER 47652 50222 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mass 47427 100 , Ab3 Fab Ab3- S1XC5 -T4 FIG . 20D C5 Fab 46761 miniinitie 50183 46145 47652 50223 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mass U . S . Patent Aug . 27, 2019 Sheet 30 of 62 US 10 , 392 ,438 B2

. LT Ab3 Fab Ab3 -S1xC5 - T9 47427 1001

C5 Fab 46767 50164 46146 47652 50204 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mass 47426 100 , C546767 Fab Ab3 Fab Ab3- S1xC5 - S1rey FIG . 20F

50183 146146 50163 | 50203 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mass C5 Fab 46753 : 100mg Ab3 -S1xC5 FIG . 20G Ab3 Fab 47426 46873 i Ab3 H 50183 46717C5L 47652 V50221 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mas 47406 1007 C5 Fab Ab3 Fab Ab3xC5 - S1rev 46899 : 46767 Ab3 H C5 L 47276 : 47276 C5 H i 47632 50202 Ab3L 0899 47704 | 50221 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 U . S . Patent Aug . 27, 2019 Sheet 31 of 62 US 10 , 392 ,438 B2

C5 Fab Ab3xC5 100 , 46753ons Asics FIG. 201

Ab3 H -C5 L 46869 Ab3 Fab 47406

10718 1 47631 50201 46718 19 47704 50220 46000 46500 47000 47500 48000 48500 49000 49500 50000 50500 mass U . S . Patent Aug. 27 , 2019 Sheet 32 of 62 US 10 ,392 ,438 B2

Ab3- S1XC5 - T1

helenietmeermed FIG . 21A (MAU,280nm)

Absorbance mele IC N ARCHIOSO inimene tidnini sinni etaan t toimi kesimittainen 16 Time (min ) 18 Ab3- S1XC5 - T2 FIG . 21B

ishoWALLSLLLLLL ww wwww MAU , Time( min ) 805 Ab3 -S1xC5 - 73 601 40 FIG . 21C 20 ARRERA WW 16 Time€( ( min ) ) 1818 MAU , 1001hahahahahahahahhahandaantuunah Ab3 - S1XC5 - T4 FIG . 21D 25 ALAN WALAUDONUMOWAPO Re nown www . mini b 16 Time (min ) 18 MAU . Ab3 -S1xC5 - T9 FIG . 21E

NORINONDO SUUS LE 16 Time( mini mau ; Time( min ) Ab3 -S1xC5 - Sirev FIG . 21F

mininginimuisinuniningniningning i n winninginn 16 Timene( minmin ) ) 118 U . S . Patent Aug . 27 , 2019 Sheet 33 of 62 US 10 ,392 ,438 B2

Ab3- S1XC5 FIG . 21G (MAU,280nma Absorbance A 14 other 16 the Time (min ) 18 maus Ab3xC5 - S1rev FIG. . . 21H on

or phoneortwolitakallid

my 16 Time( min )

maun wwwwww Ab3xC5 FIG . 211 OD8888 maintenukaa w 16 Time (min ) 18 MAU , C5 monospecific * FIG . 21J

* wwwww wwwwwww ment demanera aperture enpresarenerancanegara dentro sono interiorme en momento per la non force 16 Time ( min ) 18 MAUT wwwwwwwwwwwwwwwwwwwwwwwwww Ab3 monospecific FIG . 21K wwwwww E ine mahutattakaaalaanbaatamakatakas 14 16 Time (min ) 18 U . S . Patent Aug . 27, 2019 Sheet 34 of 62 US 10 , 392 ,438 B2

2-Fab

-CHCL2-CHCL

A2-CL

XN - D

FIG.22 1 1-CH3Y2 .

IV . Set1-CH2 |1H3 . LII

LII-HA . . .

......

- - -

-1 ä 1-CHCL

1-Fab U . S . Patent Aug. 27, 2019 Sheet 35 of 62 US 10 ,392 ,438 B2

FIG . 23

- 1 chimeric TOA1 * * Chimeric TOA1 A1D - - * * * bkgd OD450 TTT

10 -12 10 -11 10 - 10 10 - 9 10- 8 [Ab ] U . S . Patent Aug. 27 , 2019 Sheet 36 of 62 US 10 , 392, 438 B2

FIG . 24

havininin evininky !

chimeric TOA1 . 7 - chimeric TOA1A1D - - * * - bkgd

450OD

10Income - 12 10 -11 10 -8 10 -10 10 -9 [Ab ] U . S . Patent Aug. 27 , 2019 Sheet 37 of 62 US 10 , 392, 438 B2

FIG . 25

siensiiniii

- - * * - - -- Chimeric TOA - 1 - - - muVHUVL v1. 0 inning tomarmuVHUVL v1. 1 mane muVHIHUVL v2. 0 khuVH v1 .0 /muVL OpticalDensity450 viivit mert huVH V1 .0 /huVL v1. 0 of bio TOA - 1 only - - Xn - bkgd w ww .

10 - 10 10 - 9 10 -8 - 10 - 7 10 - 6 TOA - 1 Ab ( M ) U . S . Patent Aug . 27, 2019 Sheet 38 of 62 US 10 , 392 ,438 B2

FIG .1 26g

YOY memohon chiTOA - 1

+ - huVH v1 .0 /huVLV1 . 1 with mom huVH v1. 0 /huVL v2 . 0 mer1 warung ByVH v1 . 1 /muVL mement finahuVHv1 . 1 /huVL V1. 0 OpticalDensity450 - - - huVH v1. 1/ huVL v1. 1 - * - bio chiTOA - - * - - bkgd

A .

.. 10 - 10 10 -9 10 - 8 10 -7 10 - 6 TOA - 1 M U . S . Patent Aug . 27, 2019 Sheet 39 of 62 US 10 , 392 ,438 B2

FIG . 27

. chiTOA - 1 more on ww huVH vl. 1 /huVL 12 .01 medlem huVH v2 .0 /muVL maraton huVH v2 . 0 /huVL v1. 0 named Fawwa huVH v2 . 0 /huVLv1 . 1 OpticalDensity450 * huVH v2 . 0 /huVL v2 .0 anot. com bio chiTOA * * * * - bkgd

mana*

10- 10 10 - 9 10 - 8 107 10 -6 TOA - 1 [M AR ] U . S . Patent Aug . 27, 2019 Sheet 40 of 62 US 10 , 392 ,438 B2

FIG . 28

www est u win W • - • * * - chiTOA - 1 * ** * ** ** chiTOA - I ( CM ) were huTOA - 1 v1. 0 / 1 . 0 sorum Informer huTOA - 1 v1 . 0 /11 OpticalDensity450 www .bean HUTOA - 1 v1. 0 / 1 .2 ( (K428 ) ne biom huTOA - 1 v1. 0 / 1 . 3 ( A43S ) Watoto www farm huTOA - I v1. 0 / 1 . 4 ( 449K )

2 often som bio chiTOA - I ww - bkgd

You said . . . * * Oxwwwww va 10 - 10 10 -8 10 -7 Antibody [ M ] U . S . Patent Aug . 27, 2019 Sheet 41 of 62 US 10 , 392 ,438 B2

FIG .. 29

muTOA - 1 mulgG . . Chi TOA - 3 VH1. 0 / L1. 0 V VH1. 0NL2 . 0 Luciferaseactivitycps • HulgG

10 10 10 10 -2 10 -4 100 101 102

ab ugVY /mi

: U . S . Patent Aug. 27 , 2019 Sheet 42 of 62 US 10 , 392 ,438 B2

hlgG Humanized TOA - 1 FIG . 30A 0 ,0 7 .3 73 . 03 .09 27 0 .8 2. 4 7. 3 ( nM ) rhu TrkB - CRE cells P - Trk P - PLCX1 P - AKT P - ERK1/ 2 WE ROS Actin

higg Humanized TOA - 1 FIG . 30B 0 .0 7 .3 73 .03 .09 . 27 0 .8 24 7- 3 (nM ) rmu TrkB - CRE cells P - Trk SER 2012 P -PLCK1 P - AKT

WAAAA! P - ERK1/ 2 . ** * Actin .

higo : wana HumanizedS . TOA " Wind wo n tenitorimin FIG . 30C 0 10 . 1 10 100 0 0 , 1 10 100 (RM ) Differentiated P -PLC Human SH - SY5Y P -AKT Neuroblastoma Cells P- Erk172

what BDNF (nM ) ] MTOA - 1 (nM ) FIG . 30D 0 0 . 3 0 . 8 2 . 4 7 . 3 22 0 0 . 3 0 . 8 2 . 4 7 . 3 22 rhu TrkB -CRE cells P . Trk P- PLOM ------P -ERK1 / 2 Actin U . S . Patent Aug. 27 , 2019 Sheet 43 of 62 US 10 ,392 ,438 B2

KASAWARSAA D _ _ _ _ DISODDDDDDDDDDDDD_

www.

3 ara

. AborBDNF(A) huBDNFwithCompetitionofhuTOA1for biotinylated- Doo iaIrrelevantAb.-*biohuTOA1@25ng,mil inonimninininsanl bindingtohuTrkB mining serveringHUTOA-1 ahahahahahhahanan s S

SLEDiesesercioklantenserviciosesdeLEDLEDERENboekenen 10.000 FIG.31 222222* SSSSSSSSSSSSSSSSSC000000000000 pecccc

ooe

0.1001000 .COSCADA 000000000 abugimi CompetitionELISAwith Anti-TrkBAbtorhFc S24 20000otococcoSOOOOOOOOOOOOOOOOOOAAAQCGACACO

0.010

)control % ( Binding LLLLLLL ...... 41 . . . . LTO sanaciaCOROC SOROCCOO U . S . Patent Aug. 27, 2019 Sheet 44 of 62 US 10 , 392 ,438 B2

FIG . 32 hu TrkB expressing cells huTrkA expressing cells mehmanti- TrkA panti- Trka Ghanayabbit IgG ?? th - rabbit IgG muTOA - 1 ?? muTOA - 1 OD450 mu IgG OD450 ?? XX ?? mu IgG

?? 0.3333 0.1111 0.0002 0.0000 1.0000 0.0370 0.0123 0,0041 SPRE0.0014 0 .0005 0.0001 0.0000 ab ugimi hutrka (d5 TrkB ) expressing cells huTKA (d4 TrkB ) expressing cells mamanti- Trka A anti- Trka SVUSNA * abbit IgG ) w fabbit lgG Rise MUTOA - 1 N TAS muTOA - 1 450OD mu lgG OD450 22 : u lg OSTI D o 10.00 3.33 0.37 0.12 0.04 0.01 0.00 1.0000 0.3333 0.1111 0.0370 0.Wo0123 0 .0041 0.0014 0.0005 0.0002 0.0001 0.0000 0.0000 ab ugim, ab ugF/ ml . * huTrkB ( d5 TIKA ) expressing cells huTrKB ( 04 TrkA ) expressing cells anti- Trka manti- Trka heyabbit IgG ist rabbit IgG 2012 MUTOA - 1 muTOA - 11 38 OD450 OD450 ts mu IgG mu IgG ???????? ??? ? ??SA?? V?????? 29: 1.0000 0.3333 0.1111 0.0370 0.0123Do 0.To0041 0 .0014 0.0005 0.0002 0.0001 0.0000 00000 Pa ab ug/ ml atent Aug . 27 , 2019 Sheet 45 of 62 US 10 , 392 ,438 B2

101 wwwww

w . 100 HumanizedTOA-1bindstocatTrkB wwwwwwww 103102101100 ug/ml HumanziedTOA-1bindstoDogTrkB 101 mlug/

W Mouse-TIKB Dog-TIKB Mouse-TXB cat-TIKB 102

O V

-103 1.0T 2.0 RLU

. 101

100

MouseTOA-1bindstocatTrkB 103102101100 ug/ml MouseTOA-1bindstoDogTrkB 101 mlug/

Mouse.This cat-Tk MouseTIKS TIKBDog- 102

1 Y * Y

onde 103 1.5mm 1.0 RLU Limi RLU

FIG.33 U . S . Patent Aug . 27, 2019 Sheet 46 of 62 US 10 , 392 ,438 B2

wowerplannenHumanizedTOA-1(TrkA) HumanizedTOA-1(TrkB) TrkC)TOA-1(Humanized- MurineTOA-1(Trka) MurineTOA-1(TrkC) ChimericTOA-1(Trka) ChimericTOA-1(TrkC) Background(TrkB) Background(TrkC) numeroMurineTOA-1(TrkB) montrefemmesChimericTOA-1(TrkB) -Background(TrkA)

hryA1

* - ve - - -

: - ' -...... * . . " - . . T . c . : : : . .* . *...... * . . * . . . . 4 . . . . . : + : ......

. ! : - w in ...... - . -- ! * - * * . - - * . . . - - ...... : : . .

. , 1 , . , . v 3 : - ...... vir. . . . iii . W S . , , . . * * . , * * , , , , . . ARAT

TrkA,TrkCBindingELISA ????????????????????????? ?????? YRRA ATY W . V . 2 * * * . . . wis th i W X " - . . .

* * .. . 12 : 47 : . . * nM * . i ...... www ...... S ...... -

...... " ! !! . . . . . " Y . ! ! " T UN . 1 . 1 . 1

v . . . is . . . . * ......

i 0.1 3.5 450 OD FIG.34 U . S . Patent Aug. 27, 2019 Sheet 47 of 62 US 10 ,392 , 438 B2 FIG . 35

. Antibody : Humanized. . TOA - 1 293 -Control

. CellCount . 293 -p75NTR Yhtey WS w

LabelingPA i Intensity

Antibody : anti- p75 -NTR :

L Lilienteerumustenburg LOVY CellCount KA WWW ats preslepen to the point get 4

SILabeling Intensity U . S . Patent Aug. 27 , 2019 Sheet 48 of 62 US 10 , 392 ,438 B2

10.0

.0031610032103 p75NTRAntibodyanti-: ml]/ugAntibody[

03.0 293-TrkBcells cells293-P75NTR+ parental293-cellsA 0ab poJOS ab2ºNo

H0.0000 1.2000 1.0000 0.8000 0.6000 0.4000 0.2000 36FIG. OD450

10.0

3.16

1.00

0.32 1HumanizedTOA-Antibody: ml]ug/Antibody[ 0.10

.03 cells293-TrkB P75NTRcells-293 parentalcells-293 NO ab20 No

1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 OD450 atent Aug . 27 , 2019 Sheet 49 of 62 US 10 ,392 , 438 B2

CellLine P-ERK1/2 Agonist pTrk P-PLCy1 Actin chiTOA1 HUTOA1 rhuTrkC-CRE mTOA1 3- NT higo chiTOA1

FIG.37 rhuTrkB-CRE MTOA1 BDNF higo CHITOA1 huTOA1 rhuTrKA-CRE MTOA1 NGF

chiTOA1 huTOA1 HEK-293 MTOAT

.'" . parental KA. BDNF A 904 U . S . Patent Aug. 27 , 2019 Sheet 50 of 62 US 10 , 392 ,438 B2

FIG . 38

TAM1638 hulgG BDNF Luciferaseactivity induction)(Fold

0000OOOOOOOOOOO 10- 2 100 102 104 NM U . S . Patent Aug. 27, 2019 Sheet 51 of 62 US 10 ,392 ,438 B2

50nM20100. hTrkC-Cre ViVILNIAUSIAI ti NT-3TAM163higg ** * ** * * * LT ATE3 . * 12 W panananananaman

3.5 . . . . hTrkB-Cre ROM4 ? 50nM20100M NA . increase- Fold BDNFTAM-163higG FIG.39 Lii

hTrkA.Cre 50nM20100 NGFTAM-163higg increase - Fold

. ** ** *

o a A o N - o increase - Fold U . S . Patent Aug. 27, 2019 Sheet 52 of 62 US 10 , 392 ,438 B2

ODPORODDODOM . ORRA 102oodtonowe U2OS-hTrkB 101ded ARDRUM

W vom 10-1M100 102 HumanIgG 10-2 NGF 163TAM-V NT3A NT4Y POMOCOUDOUGONDOWODO 101 2.0x10Udy-BDNF 10-3 1,5x10UCH 1000000 U2OS-hTrkC SCORS50000001 10-14100 40FIG. 102 HumanlgG NT4 NGF fo-3162 101 NT3A 163-TAMV+1000000 U2OS-hTIKA 2.0x10007*BDNF tot 5x1000.1 SOOOOOOOOOOoooassociest50000001 10010-10M

HumanIgG&163 -TAMV 10-2 NT3A NT4 NGF 2.0x100d7#BDNF 10-3 SAAAAAAAAAAA www 1.5x100db 1000000 5000000 RLU U . S . Patent Aug . 27, 2019 Sheet 53 of 62 US 10 , 392 ,438 B2

FIG . 41

TAM -163 (nM )

LILIT TILTILIT 10 100 0 032 . 1 32 1 3. 2 10 32 100

pPLCY

PERK1/ 2 U . S . Patent Aug . 27, 2019 Sheet 54 of 62 US 10 , 392 ,438 B2

FIG . 42

Cell Lin Parental hTIKA -Cre hTrkB -Cre hTrkc -cre WYM TAM-163 TAM-163 TAM-163 TAM-163 Agonist higgBDNF 2007 higo 29D7 higGBDNF 2907 higo NT-3 29D7 AN CA Here SILYtis STAR SOLO FEST 1 . p - Trk NASREVODOM

SAS . OKRAS PESME OVOM23 P -PLCg1 SOS400 14

RAMOUR 30- PARATERASA ? - ??? SAASTAS

P -ERK1 / 2 12 SA WA

943 Wherees . .. NA Home Actin ex Saan P OWER SS Concentrations used : 100 nM TAM - 163, 29D7 or higg ; 10 nM NGF or BDNF ; 25 nMNT- 3 U . S . Patent Aug . 27, 2019 Sheet 55 of 62 US 10 , 392 ,438 B2

. ww08 XXXXXXXXXXXXXw Wow BDNFTAM-163hIgG W 2008 FIG.43 1101000.022200110100 www

Conc(nM)0 Ligand PLCY1-PS AKT-P p-Erkin Actin U . S . Patent Aug . 27, 2019 Sheet 56 of 62 US 10 , 392 ,438 B2

WATE BDNF 1560 BDNF BDNF:20M 0 60 TAM-163 TAM-163 FIG.44 SKU higG:100M XXV . 15600 Time(min)03060120O hlgG w higg ascare 0 ha VAATA AKS KNX

200 TAM-163:100nM Ligand (min) Time TrkB EGFR Ligand EGFR TrkB NMDAR1 MAMMA SUAS

hTrkB-Cre SH-SY5Y U . S . Patent Aug . 27, 2019 Sheet 57 of 62 US 10 , 392 ,438 B2

FIGVX . 45

Ligand Time (W ) 0 1 3 5 0 1 3 50 13

WWWXXXXXXX XXXXXXXXX X

ELEEEE Time (h ) o 2 4 6 0 2 4 6 0 2 4 6

Ligand higG TAM - 163 11 BDNF SH -SYSY Time (min ) 0 30 60 120 0 30 60 120 O 30 60 120 EGFR

TAM - 163 : 100 nM higg : 100 nM BDNF$ 249 .00 : 10 nM U . S . Patent Aug. 27, 2019 Sheet 58 of 62 US 10 , 392 ,438 B2

FIG . 46

TAM - 163 100T savanane vnom 293 - control 02 a normativa 293 - p75NTR . CountCell .

100 101 102 103 104 Labeling Intensity Anti -p75NTR 293 -control * * ** * 293- TrkB CellCount per una semana 293- p75NTR

VSINTR

101 102 - 103 104 Labeling Intensity U . S . Patent Aug . 27, 2019 Sheet 59 of 62 US 10 , 392 ,438 B2

FIG . 47

TAM - 163 293 TrkB - - 293- p75N TR secondes 293 - control OD450 within ENNALNAM. :

: 0 . 2 0. 7 2 .1 6 . 7 21. 0 67 . 0 No1ab No2ab 2 SED

www - * * w . . .

a ngawasan ------293 -p75NTR manting - 293 -control E L . . . . . OD450 a finan

PORO! quam magampanan namanya 0. 2 0 .7 2. 1 6 .7 21 . 0 67 .0 No1ab ( antibody, nM ] U . S . Patent Aug . 27, 2019 Sheet 60 of 62 US 10 , 392 ,438 B2

FIG . 48

1 .57 • mouse - TrkB Dog - TrkB Cat- TrkB Lacz OD450nm HA Bird 0. 102 104 100 101 102 U . S . Patent Aug . 27, 2019 Sheet 61 of 62 US 10 , 392 ,438 B2

FIG . 49

hlgG (nM ) TAM - 163 (NM ) www BB 0 7 .3 73 .. 03 . 09 .27 . 8 2. 47 . 3 TINT p - Trk P -PLCy1 P - AKT

p - ERK1/ 2 WWW Actin Patent Aug. 27 , 2019 Sheet 62 of 62 US 10 , 392 ,438 B2

citateares 10

1.0 ASSES BDNF )(nM 01. * 100

32 FIG.50 SUS 10

TAM163- )(nM 2 1.032

.32

.1 Perfect hlgG nM)( 100

ERK1/2P- PLCy1P- p-Trk AKT-P Actin US 10 , 392 ,438 B2

BISPECIFIC ANTIBODIES related to the therapeutic indication can enhance specificity and reduce unwanted interaction , thereby widening the CROSS -REFERENCE TO RELATED therapeutic index . APPLICATIONS Although bispecific antibodies posses certain advantages 5 over canonical bivalent monospecific classical antibodies , This application is the U . S . National Phase of Interna use of bispecific antibodies has been hindered by the tional Application No . PCT/ 32015 /053537 , filed May 13, expense in obtaining BsAbs in sufficient quantity and purity . 2015 and published in English , which claims the benefit of To produce multispecific proteins, e . g . bispecific antibod U . S . provisional application No. 61/ 994 ,720 , filed May 16 , ies and other heterodimers or heteromultimers, it is desirable 2014 , U .S . provisional application No . 62 /150 ,680 , filed " to use methods that favor formation of the desired hetero Apr. 21 , 2015 , and U . S . provisional application No . 62 / 159 , multimer over homomultimer( s ) . One method for obtaining 201, filed May 8 , 2015 . The complete content of all of the Fc -containing BsAbs remains the hybrid hybridoma tech above - referenced patent applications are hereby incorpo nique , in which two antibodies are co -expressed . However, rated by reference for all purposes . 15 this approach is inefficient with respect to yield and purity , the desired heteromultimer often being difficult to further REFERENCE TO SEQUENCE LISTING purify from a relatively large level of contaminant compris ing improperly paired polypeptide chains . This application contains a Sequence Listing which has Other techniques to favor heteromultimer formation and been submitted electronically in ASCII format and is hereby 20 reduce improper matching involve engineering sterically incorporated by reference in its entirety . Said ASCII copy, complementary mutations in multimerization domains at the created on Nov . 3rd , 2016 , is named PC71995A _ CH3 domain interface, referred to as a “ knobs- into -holes ” Seq _ Listing _ ST25. txt and is 269, 724 bytes in size . strategy as described by Ridgway et al . ( U . S . Pat. No. 5 ,731 , 168 ) and Merchant et al. ( U . S . Pat. No. 7 , 183 , 076 ) . 25 Techniques that replace one or more residues that make up FIELD the CH3 -CH3 interface in both CH3 domains with a charged amino acid for promoting the heterodimer formation have The present invention relates to engineered bispecific also been described by Strop et al. (WO2011 / 143545 ) . antibodies and related polypeptides, multimeric forms A recent review also discusses various approaches for thereof, and methods of making such proteins . 30 overcoming chain association issues when generating bispe cific antibodies (Klein et al. , mAbs 4 (6 ): 653 -663 ( 2012 ) ). BACKGROUND However, most of these techniques are directed to ensur ing proper pairing of the heavy chain polypeptides and do Antibodies having binding specificities for at least two 25 not address the further matching of each light chain poly different antigens, called bispecific antibodies (BsAbs ), have peptide with its corresponding heavy chain polypeptide to been engineered . Unlike classical antibodies which com provide a functional antigen -binding site . Thus, production prise two identical heterodimer ( i. e . a light chain portion and of desired bispecific antibodies remains a technically diffi a heavy chain portion ) " arms” wherein each arm comprises cult and costly process not commercially feasible due to the an antigen binding site ( e . g . a Fab region ), bispecificic anti - 40 high cost of goods . bodies have different sequences in each of the two arms ( e . g . Therefore , there is a long - felt need in the art for methods Fab regions) so that each arm of the Y -shaped molecule for engineering bispecific antibody fragments and /or full binds to a different antigen or different epitope of the same length BsAbs which enable the BsAbs to be expressed and antigen . recovered directly and / or efficiently from recombinant cell By binding two different antigenic molecules or different 45 culture and / or which may be produced with efficient vields epitopes of the same antigen , BsAbs offer a wide variety of clinical applications as targeting agents for in vitro and in and purities at commercially reasonable costs . vivo diagnostics and immunotherapies . Bispecific antibod ies are also advantageous for in vitro or in vivo diagnoses of SUMMARY OF THE INVENTION various disease states , including cancer . For example , one 50 E1. According to a first embodiment of the invention , there arm of the BsAb can be engineered to bind a tumor is provided a heterodimeric protein , comprisinging : associated antigen and the other arm to bind a detectable (i ) a first CÆC , domain (CHCL ) , comprising a first Chl marker. domain (Chl ) and a first C? domain (CL ) , wherein the BsAbs can be used to direct a patient ' s cellular immune first Chl and the first C , interact together at a first defense mechanisms to a tumor cell or an infectious agent 55 Cyl , interface ; ( e . g . virally infected cells such as HIV or influenza virus ; ( ii ) a second CgCl, comprising a second Chl and a protozoa such as Toxoplasma gondii) . In particular , one can second Cz, wherein the second Chl and the second Cu redirect immune modulated cytotoxicity by engineering one interacting together at a second CÆC? interface ; arm of the BsAb to bind to a desired target ( e . g . a tumor cell wherein the first Cyl differs from the second Cyl by at least or pathogen ) and the other arm of the BsAb to bind to a 60 one Cyl mutant residue in the first Cyl ; and the first C cytotoxic trigger molecule , such as the T - cell receptor or a differs from the second C , by at least one C , mutant residue Fc gamma receptor, thereby activating downstream immune in the first Cz; effector pathways. Using this strategy, BsAbs which bind to such that the Cylmutant residue and the C? mutant residue the Fc gamma RIII have been shown to mediate tumor cell of the first C C , interact with each other in preference to the killing by natural killer (NK ) cell /large granular lymphocyte 65 corresponding residue positions on the second C C?, the ( LGL ) cells in vitro and to prevent tumor growth in vivo . interacting mutant residues of the first Cyl and first C Alternatively , targeting two separate antigens or targets thereby forming a first complementary residue set . US 10 , 392 , 438 B2 Described below are a number of further embodiments first complementary residue set is less than 10 Å ? as (E ) of this first embodiment of the invention , where for measured using a 2 . 5 Å probe . convenience E1 is identical thereto . E12 . The heterodimeric protein according to any one of E2 . The heterodimeric protein according to E1, wherein the E1 -E11 , wherein the solvent accessible surface area of the second Cyl differs from the first Cyl by at least one Cyl 5 first complementary residue set is less than 5 A as mutant residue in the second Cyl; and the second C , measured using a 2 . 5 À probe . differs from the first C , by at least one C , mutant residue E12 . The heterodimeric protein according to any one of in the second C?; such that the Chl mutant residue and the E1 -E12 , wherein the solvent accessible surface area of the C , mutant residue of the second CyC , interact with each first complementary residue set is less than 1 Å2 as other in preference to the corresponding residue positions 10 measured using a 2 . 5 Å probe .

on the first C A, C , , the interacting mutant residues of the E13 . The heterodimeric protein according to any one of second Chl and second C? thereby forming a second E1 - E12 ,wherein the solvent accessible surface area of the complementary residue set . second complementary residue set is less than 225 Å ? as E3. A heterodimeric protein , comprising measured using a 2 . 5 À probe. ( i) a first Chl domain (Chl ) and a first C , domain (CL ), 15 E14 . The heterodimeric protein according to any one of the first Cyl and the first C , interacting together at a E1- E13 , wherein the solvent accessible surface area of the first CÆC? interface to form a first CÆC domain second complementary residue set is less than 220 Å ? as (CHC ), measured using a 2 . 5 Å probe . ( ii ) a second Chl domain (Chl ) and a second C , domain E15 . The heterodimeric protein according to any one of (CL ) , the second Chl and the second C? interacting 20 E1 - E14 , wherein the solvent accessible surface area of the together at a second CÆC , interface to form a second second complementary residue set is less than 150 Å2 as CHC? domain ( C # CL) ; measured using a 2 . 5 A probe . wherein the first Chl is engineered to differ from the second E16 . The heterodimeric protein according to any one of Chl by at least one Cyl mutant residue in the first Cyl ; and E1 -E15 , wherein the solvent accessible surface area of the the first C , is engineered to differ from the second C? by at 25 second complementary residue set is less than 100 Å2 as least one C? mutant residue in the first C? ; measured using a 2 . 5 À probe . such that the Cyl mutant residue and the C , mutant residue E17. The heterodimeric protein according to any one of of the first CÆC , interact with each other in preference to the E1- E16 , wherein the solvent accessible surface area of the corresponding residue positions on the second CyC , the second complementary residue set is less than 50 Å2 as interacting mutant residues of the first Cyl and first C , 30 measured using a 2 . 5 Å probe . thereby forming a first complementary residue set . E18. The heterodimeric protein according to any one of E4 . The heterodimeric protein according to E3, wherein the E1- E17 , wherein the solvent accessible surface area of the second Chl is engineered to differ from the first Chl by second complementary residue set is less than 20 Å ? as at least one Cyl mutant residue in the second Chl; and measured using a 2 . 5 À probe . the second C , is engineered to differ from the first C , by 35 E19 . The heterodimeric protein according to any one of at least one C , mutant residue in the second C , ; such that E1- E18 , wherein the solvent accessible surface area of the the Cyl mutant residue and the C? mutant residue of the second complementary residue set is less than 10 Å ? as second ChC? preferentially interact with each other over measured using a 2 . 5 Å probe . the corresponding residue positions on the first CÆC?, the E20 . The heterodimeric protein according to any one of interacting mutant residues of the second Cyl and second 40 E1- E19 , wherein the solvent accessible surface area of the C , thereby forming a second complementary residue set . second complementary residue set is less than 5 Å ? as E5 . The heterodimeric protein according to any one of measured using a 2 . 5 Å probe . E1 - E4 , wherein the solvent accessible surface area of the E21. The heterodimeric protein according to any one of first complementary residue set is less than 225 Å ? as E1 - E20 , wherein the solvent accessible surface area of the measured using a 2 . 5 À probe . 45 second complementary residue set is less than 1 Å as E6 . The heterodimeric protein according to any one of measured using a 2 . 5 À probe . E1- E5 , wherein the solvent accessible surface area of the E21 . The heterodimeric protein according to any one of first complementary residue set is less than 220 Å ? as E1 -E20 , wherein the solventaccessible surface area of the measured using a 2 .5 Å probe . first or the second complementary residue set is about 0 E7 . The heterodimeric protein according to any one of 50 Å ? as measured using a 2 . 5 Å probe. E1 - E6 , wherein the solvent accessible surface area of the E22 . The heterodimeric protein according to any one of first complementary residue set is less than 150 Å2 as E1 - E21, wherein the mutant residues of the first comple measured using a 2 .5 Å probe . mentary residue set are different to the mutant residues of E8 . The heterodimeric protein according to any one of the second complementary residue set. E1 - E7, wherein the solvent accessible surface area of the 55 E23 . The heterodimeric protein according to any one of first complementary residue set is less than 100 Å2 as E1- E22 , wherein formation of the first CÆC , and second measured using a 2 . 5 Å probe . CÆC , preferentially occurs over formation of a CÆCL E9 . The heterodimeric protein according to any one of comprised of either the first Chl and second Cz, or second E1- E8 , wherein the solvent accessible surface area of the Cyl and first C . first complementary residue set is less than 50 Å2 as 60 E24 . The heterodimeric protein according to any one of measured using a 2 . 5 À probe . E1- E23 , wherein formation of the first C?C and second E10 . The heterodimeric protein according to any one of CÆC , preferentially occurs over formation of a CÆC E1- E9, wherein the solvent accessible surface area of the comprised of either the first Chl and second C? , or second first complementary residue set is less than 20 Å ? as Cyl and first C , by at least about 4 - fold . measured using a 2 . 5 À probe . 65 E25 . The heterodimeric protein according to any one of E11 . The heterodimeric protein according to any one of E1 -E24 , wherein the first Cyl is attached to a first E1 - E10 , wherein the solvent accessible surface area of the variable heavy domain (VH ), and the first C , is attached US 10 , 392 , 438 B2 to a first variable light domain ( V . ) , and the second Chi E39. The heterodimeric protein according to any one of is attached to a second Vh , and the second C , is attached E1 - E38 , wherein formation of the first CÆC , and second to a second V , CyC , preferentially occurs over formation of a C C E26 . The heterodimeric protein according to any one of comprised of either the first Chl and second C? , or second E1- E25 , wherein the preferential formation of first CyC , 5 Cyl and first C , by at least about 90 -fold . and second CÆC? does not rely on complementary pairing E40 . The heterodimeric protein according to any one of of the variable domains . E1 - E39 , wherein formation of the first C4C , and second E27 . The heterodimeric protein according to any one of C?C? preferentially occurs over formation of a CÆC E1- E26 , wherein formation of the first CÆC , and second comprised of either the first Chl and second C?, or second CÆC , preferentially occurs over formation of a CÆC 10 Chl and first C? , by at least about 100 - fold . comprised of either the first Cyl and second C , , or second E41 . The heterodimeric protein according to any one of Chl and first C?, by at least about 5 - fold . E25 - E39, wherein the preferential formation of the first E28 . The heterodimeric protein according to any one of CHC , and second CÆC? occurs in the absence of any E1 -E27 , wherein formation of the first C # C , and second complementary pairing in the variable domains . C?C , preferentially occurs over formation of a CyC , 15 E42 . The heterodimeric protein according to any one of comprised of either the first Cyl and second C? , or second E25 -E41 , wherein the first Vh , first V?, first Ch and first Chl and first C? , by at least about 6 - fold . C , combined together form a first Fab , and the second VH , E29. The heterodimeric protein according to any one of second V? , second Chl, and second C? combined E1 - E28 , wherein formation of the first CyC , and second together form a second Fab . CAC , preferentially occurs over formation of a C C 20 E43 . The heterodimeric protein according to E42, wherein comprised of either the first Cyl and second C , , or second the preferential formation of first Fab and second Fab Cyl and first C , , by at least about 8 - fold . does not rely on complementary pairing of the variable E30 . The heterodimeric protein according to any one of domains. E1- E29 , wherein formation of the first CyC , and second E44 . The heterodimeric protein according to any one of CyC , preferentially occurs over formation of a Cyl , 25 E42 - E43 , wherein the preferential formation of first Fab comprised of either the first Chl and second C? , or second and second Fab occurs in the absence of any complemen Cyl and first C , by at least about 10 - fold . tary pairing in the variable domains. E31 . The heterodimeric protein according to any one of E44 . The heterodimeric protein according to any one of E1 - E30 , wherein formation of the first CyC , and second E1 - E43 , wherein the preferential formation of first CÆC CHC , preferentially occurs over formation of a CAC , 30 and second ChC relies on complementary pairing of the comprised of either the first Chl and second C?, or second complementary residue sets . Chl and first C? , by at least about 15 - fold . E45 . The heterodimeric protein according to any one of E32. The heterodimeric protein according to any one of E1- E46 , wherein at least one of the Cz domains is a kappa E1 - E31, wherein formation of the first CÆC , and second domain . CHCl preferentially occurs over formation of a C C? 35 E46 . The heterodimeric protein according to any one of comprised of either the first Chl and second Cz, or second E1 - E45, wherein both the first C , and the second C , is a Cyl and first C? , by at least about 20 - fold . kappa domain . E33. The heterodimeric protein according to any one of E47 . The heterodimeric protein according to any one of E1 - E32 , wherein formation of the first CÆC , and second E1 -E46 , wherein the complementary residue sets com CHC , preferentially occurs over formation of a CÆC , 40 prise a positively or negatively charged residue in one comprised of either the first Cyl and second Cz, or second domain , and either a polar residue , or an oppositely Cyl and first C? , by at least about 25 - fold . charged residue in the other domain . E34 . The heterodimeric protein according to any one of E48. The heterodimeric protein according to any one of E1- E33 , wherein formation of the first CÆC , and second E1 -E47 , wherein the locations of the complementary CyC , preferentially occurs over formation of a CyC , 45 residue sets are selected from the group consisting of: comprised of either the first Chl and second C? , or second Cyl - 124 and C - 176 ; ( ii ) Cyl- 188 and C - 178 ; ( iii ) Cyl and first C? , by at least about 30 - fold . Cyl - 143 and C - 178 ; (iv ) Cy1- 143 and C - 131 ; ( v ) E35 . The heterodimeric protein according to any one of Chl- 221 and C 123 ; ( vi) Ch1 - 145 and C _ _ 131 ; (vii ) E1- E34 , wherein formation of the first CyC , and second Cyl- 179 and C , 131; ( viii ) Cyl- 186 and C , 131 ; and ( ix ) CAC , preferentially occurs over formation of a CAC , 50 Ch1 - 188 and C - 133 , according to Kabat numbering . comprised of either the first Cyl and second C , , or second E49 . The heterodimeric protein according to E48 , wherein Chl and first C? , by at least about 40 - fold . the mutation at the Cyl position is selected from the E36 . The heterodimeric protein according to any one of group consisting of W , H , K , R , S and T, and the mutation E1- E35 , wherein formation of the first C4C , and second at the C , position is selected from the group consisting of CÆC , preferentially occurs over formation of a CÆC 55 S , M , D and E . comprised of either the first Chl and second C?, or second E50 . The heterodimeric protein according to E49, wherein Cyl and first C , by at least about 50 - fold . the mutation at the Cyl position is selected from the E37 . The heterodimeric protein according to any one of group consisting of E , and D , and the mutation at the C , E1- E36 , wherein formation of the first CyC , and second position is selected from the group consisting of H , K , and C?C , preferentially occurs over formation of a C C 60 R . comprised of either the first Cyl and second C? , or second E51 . The heterodimeric protein according to any one of Cyl and first C , , by at least about 60 - fold . E49 -E50 , wherein the complementary residue sets further E38 . The heterodimeric protein according to any one of comprise one or more mutations selected from the group E1 - E37 , wherein formation of the first CAC , and second consisting of: Chl - 143D , Chl- 145S , Chl- 186A , Chl CAC preferentially occurs over formation of a C C 65 186E , Cy1 - 188G , Chl- 143S , Chl- 190S , Chl- 1901 , comprised of either the first Chl and second Cz , or second CZ - 133S , C2 - 1351, C2- 176G , C4 - 176M , and Cr- 178G , Chl and first C? , by at least about 80 - fold . C? -1785 . US 10 , 392 ,438 B2 E52 . The heterodimeric protein according to any one of 188W , Cyl- 122C , Cyl - 139C , Cyl - 174C , Cyl - 230S , E1 - E51 , wherein the first and second complementary Cz -133S , C2- 178S , C4 -131D , C4 -116C , C1- 123C , residue sets are selected from two of the following C4- 176C , C [ -214S . groups: Chl- 124K , C4 - 176D , CH1 - 190S , Cz - 133S ; ( ii ) E58 . The heterodimeric protein according to E57, wherein Chl - 124K , Cl - 176D , C? - 133S ; ( iii ) Chl- 124E , 5 the second CÆC , comprises residues from one of groups C4 - 176K ; ( iv ) Chl - 124E , C4 - 176K , Ch1 - 188G ; ( v ) Chl i - xxvii, provided the first and second CÆC , do not both 188E , C2 - 178K , C 1 - 143E ; ( vi) Chl- 188K , C7- 178D , comprises residues from the same group . Cyl - 143D ; ( vii ) Cyl - 143K , C - 178D ; (viii ) Cyl - 143D , E59. The heterodimeric protein according to any one of C4 - 178R ; ( ix ) Chl - 143K , C7- 178D ; (x ) Chl - 143D , E1- E58 , wherein the first Chl is connected to a first Cu2 C? -178K ; (xi ) Chl- 143D , C4 - 178K , C4 - 176M ; (xii ) Chl- domain (CH2 ), which is connected to a first CH3 domain 143E , CZ - 131R ; (xiii ) Chl- 143R , CL - 131E ; (xiv ) Chl (CH3 ) , and the second Chl is connected to second Ch2 , 143R , C2- 131E , CH1- 186A ; ( xv ) Chl- 221D , C2 - 123K ; which is connected to a second Ch3. (xvi ) Chl- 221D , C? - 123K , Cyl - 1901, C2- 1351; ( xvii ) E60 . The heterodimeric protein according to E59, wherein Chl- 145E , C2- 131H ; ( xviii ) Chl- 143H , CH1- 179D , Chl - 15 the first CH3 and second Cp3 comprises a first C3 186E , C7- 131H ; (xix ) Chl- 145E , C2- 131H ; (xx ) Chl mutant residue and second Cy3 mutant residue respec 186E , C2 - 131H , Chl- 145S ; (xxi ) Chl -143S , C4 - 131D , tively, the first CH3 mutant residue and second CH3 CH1 - 188W ,CZ - 133S , 178S ; (xxii ) Chl - 143S , CH1- 188W , mutant residues being engineered to differ from each Cz- 133M , C7- 176G , C4 - 178G ; (xxiii ) Ch1 - 143H , Chl other, and preferentially interact with each other and 179D , C71 - 186E , C2- 131H , CH - 1901, C2 - 1351, ( xxiv ) 20 thereby form Cy3 heterodimers over the formation of Ch3 CH - 186E , C2 -131H , CH - 145S ; (XXV ) Chl - 143S , homodimers . C2- 131D , CH1- 188W , Cz- 133S , C4- 176C ; (xxvi ) Chl - E61. The heterodimeric protein according to any one of 143S , C41 -188W , 133M , C?- 178G , C7- 176G ; ( xxvii ) E1- E60 , wherein the first Cyl is attached to a first CH1 - 143S , CH1- 188W , C2- 131D . variable heavy domain ( V ) , and the first C , is attached E53 . The heterodimeric protein according to any one of 25 to a first variable light domain (V1 ) , and the second Chl E1 - E52 , comprising an engineered disulfide bond is attached to a second Vy , and the second C , is attached between the first Chl and the first C?, and or the second to a second V?, and wherein the first Vy comprises Cyl and the second Cz : E54 . The heterodimeric protein according to E53 , wherein E62VvQ39. The heterodimeric and VH - Q105 . the engineered disulfide bond is located at one or more of 30 E62. The heterodimeric protein according to any one of the following positions (i ) Chl- 122 and C _ _ 123 ; ( ii ) E1- E61, wherein the first Chl is attached to a first Chl - 139 and C - 116 ; and ( iii ) Chl - 174 and C _ _ 176 . variable heavy domain (VH ), and the first C? is attached E55 . The heterodimeric protein according to any one of to a first variable light domain (V .) , and the second Chl E53 - E54 , wherein a wild type disulfide bond has been is attached to a second Vh , and the second C , is attached removed , by mutating one or both of Chl -C230 and 35 to a second V? , and wherein the second Vy comprises C2 -214 to any residue except C , on the first CÆC , and /or Vy -Q39 and V - 2105 . second C , Cr E63 . The heterodimeric protein according to any one of E56 . The heterodimeric protein according to E55 , wherein E1 - E62, wherein the first Cyl is attached to a first the first and /or second Chl - C230 and first , and /or second variable heavy domain (Vy ), and the first C , is attached C , -C214 are mutated to S . 40 to a first variable light domain (VL ) , and the second Chl E57 . The heterodimeric protein according to any one of is attached to a second Vy, and the second C , is attached E1 - E56 , wherein the first C / C , comprises residues from to a second V?, and wherein the first V , comprises: (i ) one of the following groups : ( i) Chl- 124K , C2 - 176D , V -238 ; and ( ii ) one of V2- Q1 ; V - S1, V -D1 , V -E1 , Chl - 190S , C4 - 133S ; ( ii ) Chl - 124K , C?- 176D , C2- 133S ; V4 - A1, or V2- N1 ; and ( iii ) one of V2- T42 , V2- Q42 , or ( iii ) Chl - 124E , C4 - 176K , C4 - 133S ; ( iv ) Chl- 124E , 45 V -K42 . C2 - 176K , CH1 - 188G , C2 - 133S ; ( v ) Cyl- 188E , C7- 178K , E64 . The heterodimeric protein according to any one of Chl - 143E ; ( v ) Chl- 188K , C7- 178D , Chl - 143D ; ( vi ) E1- E63 , wherein the first Cyl is attached to a first Chl - 143K , C? -178D ; ( vii ) Chl - 143D , C7 - 178R ; ( viii ) variable heavy domain ( VH ) , and the first C? is attached CH1 - 143K , C -178D ; ( ix ) Cy1- 143D , C7 - 178K ; ( x ) Chl to a first variable light domain ( V1) , and the second Cyl 143D , C4 - 178K , CL - 176M ; (xi ) Chl- 143E , C?- 131R ; 50 is attached to a second VH , and the second C is attached (xii ) Cyl -143R , C2 - 131E ; (xiii ) Cyl - 143R , C2 - 131E , to a second V?, and wherein the second V , comprises: (i ) Chl - 186A ; ( xiv ) Chl- 221D , C7- 123K ; (xv ) Chl - 221D , V2 - Q38 ; and ( ii ) one of V2 - Q1 ; V2 - S1, V2 - D1, V2- E1, C4 - 123K , Chl- 1901 , C2 - 1351, Chl - 174C , C71- 230S , Vi - A1, or V2- N1 ; and ( iii ) one of V2- T42 , V . - Q42, or CZ - 176C , C7- 214S ; (xvi ) Chl- 145E , CZ - 131H ; ( xvii ) V -K42 . Chl - 143H , Cyl- 179D , CH1- 186E , C7 - 131H ; ( xviii ) Chl- 55 E65. The heterodimeric protein according to any one of 122C , Cyl -145E , Cy1 - 230S , Cz - 123C , C1 -131H , E1 -E64 , wherein the first CHC , comprises Chl - 124K , CL - 214S ; (xix ) Chl- 186E , C?- 131H , Chl - 145S ; (xx ) C4 - 176D , CH1 - 190S , and C2- 133S . Chl - 143S , C2 - 131D , CH1- 188W , C4 - 133S , C4 - 178S ; E66 . The heterodimeric protein according to any one of ( xxi) Chl -143S , Chl- 188W , C7- 133M , C - 176G , E1 - E65, wherein the second CÆC , comprises Cyl- 124E , C? - 178G ; (xxii ) Chl- 143H , Cyl- 179D , Cyl- 186E , 60 C4 - 176K , C71 - 1886 , and C? - 133S . Cz -131H , CH - 1901, C7 - 1351, Cyl - 174C , Cyl - 230S , E67 . The heterodimeric protein according to any one of C4 - 176C , C -214S ; (xxiii ) Cyr- 186E , C4 -131H , CH - 145S , E1 -E66 , wherein the first CC, comprises Chl -124K , Chl- 139C , CH1 - 230S , C7- 116C , C2- 214S ; (xxiv ) Chl C - 176D , Chl - 190S , and C - 133S , and the second CHCL 143S , C? -131D , CH1 -188W , C7 - 133S , C7 - 178S , Cyl comprises Chl -124E , C4 -176K , Chl- 188G , and 174C , CH1 - 230S , C7 - 176C , C7- 214S ; ( XXV ) Cy1- 143S , 65 C2- 133S . CH1- 188W , CH1 - 122C , Ch1 - 230S , C? - 133M , C4 - 1786 , E68 . A bispecific antibody comprising a heterodimeric pro C2- 176G , C2 - 123C , C2- 214S ; (xxvi ) Chl- 143S , Chl- tein as in any one of E1- E67 . US 10 , 392 , 438 B2 10 E69. The bispecific antibody as set forth in E66 , wherein the Kabat numbering scheme. A dash (“ _” ) indicates an amino first CÆC , comprises Cyl- 124K , C4 - 176D , CH1- 190S , acid position that is occupied only in a different type of and C4 - 133S . antibody domain or in a different species. E70 . The bispecific antibody as set forth in any one of FIG . 3 depicts non - reducing and reducing SDS -PAGE E68- E69 ,wherein the second CÆC , comprises Chl - 124E , 5 analysis of wild - type 29D7 monoclonal IgG1 antibody with C4 -176K , CH1 - 1886 , and Cz- 133S . native disulphide bridge ( A ), no disulphide bridge ( B ) or E71. The bispecific antibody as set forth in any one of with disulphide bridges in novel positions (C - F ) as detailed E68 -E70 , wherein the first CC, comprises Cyl- 124K , in Table 1 for clones Cys1, Cys3a , Cys3b and Cys6 respec C4 - 176D , Cyl - 190S , and C , - 133S , and the second Chi tively . M : molecular weight marker. comprises Chl - 124E , C2 - 176K , Chl- 188G , and 10 FIG C , - 133S . FIG . 4 depicts mass spectrometric analysis of constructs E72 . A nucleic acid encoding the heterodimeric protein with engineered disulfides . Panel A : clone Cys _ Beta , con according to any one of E1 - E65, or a bispecific antibody struct with wild type disulfides removed ; Panel B : clone according to any one of E68 - E71 . Cys _ 1 ; Panel C : clone Cys _ 3a ; Panel D : clone Cys _ 3 b ; E73. A vector comprising the nucleic acid according to E72. 15 PanelFan E : clone Cys _ 6 . E74 . A cell comprising the nucleic acid according to E72 , or FIG . 5 depicts x - ray crystal structures of the interface comprising the vector according to E71 . region involved in design S1. Within each panel , the Chl E75 . A method ofmaking the heterodimeric protein , accord 60mdomain is shown on top in dark gray with key residues ing to any one of E1 -E67 , comprising: (i ) cotransfecting rendered in ball and stick form . The C , domain is shown on a cell line with one or more vectors to express the first 20 the bottom in light gray with key resides rendered as tubes. Chl , the first C , of the first C Cz; and the second Cyl , Key interactions are indicated by dotted lines with distances and the second C , of the second C , C , ; ( ii ) culturing the in Angstroms. Panel A : the orientation of key residues cell line under conditions to express the one or more Chl - 124 and C , - S176 , as well as the supporting residues vectors and that allow the first C4C , and second C _ C , to C - V133 , Cyl -S188 , and Cy1 - V190 in a native Fab arm assemble ; and (iii ) purifying the heterodimeric protein 25 comprising IgGi Chl and kappa CL . Panel B : Design to be from the cell culture. used in one Fab arm of a standard two -arm antibody , with E76 . The method of E75 , wherein the cell line is cotrans mutations Chl- L124K , C2 - S176D , C , -V133S , and Chl fected with vectors that express the first Cyl, first C , V190S . Panel C : Design to be used in the other Fab arm of second Cyl , and second C , in a 1 : 1: 1 : 1 ratio . a standard two -arm antibody, with mutations Cul -L124E , 30 Cz- S176K , CZ -V133S , and Cyl - S1886 . Without wishing to BRIEF DESCRIPTION OF THE DRAWINGS be bound by any particular theory , when the mutations shown in B and C are introduced into each of the two Fab FIG . 1 depicts potential products that may result from arms of an antibody, heavy /light mispairing will be disfa attempting to generate bispecific antibody via coexpression vored by Lys /Lys or Asp /Glu charge repulsion , and /or cor of 2 different antibody light chains and 2 different antibody 35 rect pairing will be encouraged by Lys /Asp or Lys /Glu heavy chains, where the C , 3 interface is engineered using charge attraction . Panel A depicts PDB entry 3QQ9, while established technology to favor heterodimer formation , but panels B and C are unpublished crystal structures . the heavy / light chain interface is devoid of the mutations of FIG . 6 depicts the results ofmass spectrometric analysis the present invention . LC1 paired with HC1 provides a Fab of heterodimeric bispecific antibody Abi / Ab2 comprising arm binding one epitope , and LC2 paired with HC2 provides 40 engineered favorable electrostatic interactions at the Chil a Fab arm binding a distinct epitope, possibly on a different Cl- Kappa interface of both Fab arms as described in antigen . LC1 paired with HC2, or LC2 paired with HC1, Example 4 (panel A ) and a control Ab1/ Ab2 construct with results in a Fab with reduced or no binding to those epitopes . native Cyl/ Cl - Kappa interfaces (panel B ) . The novel elec A : correct pairing of a representative bispecific antibody, trostatic interaction mutations led to a significant reduction showing on the left arm , a combination of first Vy and first 45 of incorrectly paired light chain in the isolated Fab frag V ( represented by heavy vertical stripes and light vertical ments . Key : * potential incomplete leader sequence process dashes respectively ) through preferential formation of first ing ; ^ Correctly paired (both H and L chains) Ab2 Fab arm CÆC ( facilitated by the interaction of a first complementary with a post - translational modification in the light chain . residue set, depicted by filled and unfilled circles ), and on FIG . 7 depicts mass spectrometric analysis of bispecific the right arm , a combination of second Vh and second V , 50 antibody Ab1/ Ab2 Fc domain ( panel A ) and of a control ( represented by heavy diagonal hatching and light check Abi /Ab2 containing HC heterodimerizing mutations but no pattern respectively ) through preferential formation of the Fab arm C C , interface mutations. Expected molecular second Cyc? ( facilitated by the interaction of a second weight of Fc (consisting ofheavy chain from Ab 1 & 2 ) were complementary residue set, depicted by filled and unfilled detected in both cases whilst no heavy chain homodimers triangles ). B : incorrect pairing of a bispecific antibody, 55 were detected . showing on the left arm , a combination of first Vh and FIG . 8 depicts a graph showing the results from separation second V? ( represented by heavy vertical stripes and light of bispecific antibody Abi/ Ab2 using anion -exchange chro check pattern respectively ) through formation of a third matography. Anion - exchange chromatography was used to C C? , and on the right arm , a combination of second V and assess protein heterogeneity within the bispecific Ab1/ Ab2 first V . ( represented by heavy diagonal hatching and light 60 antibody preparation after protein A and preparative SEC vertical dashes respectively ) through formation of a fourth chromatography. Analysis of the parental antibodies Abi C C . C and D each show semi- functional bispecific anti - and Ab2 are shown in panel A ( i ) and panel A ( ii ) respec bodies , where either the left or right arm has paired correctly , tively . Parental Abl displays an apparent single peak . Paren and the other arm has paired incorrectly . tal Ab2 shows a population of acidic and basic charge FIG . 2 depicts the sequence of wild type human IgG1Cyl 65 species which elute before and after the main peak , respec (panel A ), Kappa C , (panel B ) , and Lambda C , domains. tively . The heterodimeric bispecific Abi/ Ab2 antibody is The amino acid residues are numbered according to the shown in panel B . Fractions from Peak 1 , Peak 2A and Peak US 10 , 392 , 438 B2 11 2B from the bispecific Ab1 /Ab2 antibody ( panel B ) were chain backbone , shown using a light gray ribbon , is in the analyzed by mass spectrometry . front of the view , with the dark gray heavy chain backbone FIG . 9 depicts a graph showing a mass spectrometric ribbon more to the back . Key water molecules defining this analysis of Fab components from ion exchange fractionated pocket are shown as spheres. heterodimeric bispecific antibody Ab1/ Ab2 (derived in FIG . 5 FIG . 17 depicts graphs showing mass spectrometric 8B ). Panel 9A shows that peak 2B from FIG . 8B contains analysis of dual arm Fab fragments designed to show the enriched bispecific Abi/ Ab2 with correctly paired light impact of subsets of the S1 and S1 _ rev mutations . Panels A chains in each Fab arm , but with a post translational modi - and B show originalmonospecific antibodies with no Chl/ fication in the Ab2 Fab arm . Panel 9B shows Peak 2A from C , mutations. A bispecific combining the two parent anti FIG . 8B to has enriched incorrect light chain pairing ( anti - 10 bodies has significant mispairing in the absence of the Si body 1 heavy chain combined antibody 2 light chain ) , Panel and S1 _ rev mutations (Panel C ) but nearly eliminated mis 9C shows peak 1 from FIG . 8B . This peak represents only pairing when S1 and S1 _ rev are used ( Panel D ). Using correctly paired bispecific Ab1/ Ab2 Fab arms with no post - various subsets of the S1 and S1 _ rev mutations results in translational modifications. Key : * potential incomplete antibodies (Panels E and F ) with reduced mispairing relative leader sequence processing ; ^ Ab2 Fab with a post -transla - 15 to Panel C , but still lower fidelity than the full S1 and S1 _ rev tionalmodification . design used in Panel D . The peaks corresponding to mis FIG . 10 depicts a graph showing mass spectrometric paired Fabs are labled as “ Ab3H C5L ” and “ C5H Ab3L ” analysis of dual arm Fab fragment of constructs C5XAb3 - while the correct pairings are labeled as “ C5 Fab ” and “ Ab3 Mi & C5XAb3 -M1 - NEGATIVE . Significant reduction of Fab ” . incorrectly paired light chain between C5 & Ab3 was 20 FIG . 18 depicts a graph showing separation of bispecific observed in construct C5XAb3 -M1 compared to C5XAb3 - antibodies using hydrophobic chromatography . In Panel A , M1- NEGATIVE . Key : * potential incomplete leader the C5 and Ab3 antibodies are combined into a bispecific sequence processing. incorporating only CH3 mutations for heavy chain hetereo FIG . 11 depicts a graph showing mass spectrometric dimerization , but no bispecific - favoring mutations in the analysis of dual arm Fab fragment of constructs C5XAb3 - 25 heavy / light interface . There are three major peaks , indicat M2 (panel 11B ) and C5XAb3 -M2 - NEGATIVE (panel 11A ). ing a heterogenous sample . In Panel B , the S1 and S1 rev Significant reduction of incorrectly paired light chain designs are added to the heavy / light interface to disfavor between C5 & Ab3 was observed in construct C5XAb3- M2 mispairing of the wrong heavy and light chains ; the sample compared to C5XAb3 -M2 - NEGATIVE . Key : * potential homogeneity is greatly improved . If some of the secondary incomplete leader sequence processing . 30 supporting mutations of the S1 and S1 _ rev designs are not FIG . 12 depicts a graph showing separation of bispecific utilized (Panels C and D ) , the sample has an intermediate antibodies using hydrophobic interaction chromatography. level of heterogeneity . Panels E and F are controls showing The parental antibodies Ab3 and C5 shown in panel 12A ( 1 ) the level of homogeneity observed with the monospecific and panel 12A ( 2 ) , respectively, each display an apparent versions of the two antibodies used to assemble the bispe single peak . The heterodimerization approach M1 is shown 35 cific antibodies of Panels A - D . in panel 12C and heterodimerization approach M2 is shown FIG . 19 depicts Differential Scanning calorimetry ( DSC ) in panel 12B . The chromatograms on the left for both panel curves for bispecific Fabs with various combinations of 12B ( 1 ) and 12C ( 1 ) show the incorporation of the heavy - mutations in each Fab arm , as described in Table 23 . The chain heterodimerization mutations alone . The chromato solid thick lines indicate raw data , while the thin dotted lines grams on the right (panels 12B ( 2 ) and 12C ( 2 ) ) show bispe - 40 indicate the results of fitting the raw data to a two transition cific antibodies that contain both the heavy - chain and light- or three transition model, as appropriate . As summarized in chain mutations described in Example 5 . These results Table 24 , all Fabs showed good stability with their lowest demonstrate a reduction in the heterogeneity of antibody transition above 65° C . produced with the incorporation of both Cyl and Cl- Kappa FIG . 20 depicts mass spectrographic analysis of dual arm mutations for correct light chain pairing and Cy3 mutations 45 Fab fragments with various combinations of designs in each compared with a bispecific ( e . g . , “ NEGATIVE ” ) comprising Fab arm , as enumerated in Table 23 . Bispecific antibodies only the Cy3 /Cy3 mutations . with S1 in the Ab3 Fab arm and any of T1 , T2 , T3 , T4 , or FIG . 13 depicts the interface region between a Chi T9 in the C5 Fab arm displayed high fidelity of heavy / light domain and a C , domain ( from PDB entry 3009) . The view chain pairing ( Panels A - E ) . A minor amount of mispairing is along the interaction edge between the domains , with Cyl 50 ( ~ 3 % ) was detected in the sample combining S1 in one Fab in dark gray on the left, and C , in light gray on the right. arm with S1_ rev in the other Fab arm ( Panel F , mispair FIG . 14 depicts the interface region between a Cyl labeled as “ C5 H Ab3 L ” ). If one Fab arm (Panels G - H ) or domain and a C , domain ( from PDB entry 3QQ9) with a both Fab arms (Panel I) did not contain a bispecific - favoring drawing style similar to FIG . 13 . This view highlights the design , larger amounts of mispaired Fab ( 19 % or higher ) regions of Chl that interact with C? (backbone atoms of 55 were detected . interacting residues shown with ball and stick rendering ) . FIG . 21 depicts separation of bispecific antibodies using The primary Ig - fold B -strand regions are numbered 1 to 7 hydrophobic interaction chromatography . The antibodies are from N terminus to C terminus. enumerated in Table 23 . Bispecific antibodies with S1 in the FIG . 15 depicts the interface region between a Cyl Ab3 Fab arm and any of T1 , T2 , T3 , T4 , or T9 in the C5 Fab domain and a C , domains (from PDB entry 3QQ9 ) with a 60 arm displayed high fidelity of heavy / light chain pairing drawing style similar to FIG . 13 . This view highlights the (Panels A - E ). A minor amount of mispairing is apparent as regions of C? that interact with Chl (backbone atoms of a small tail on the left side of the main peak . This tail on the interacting residues shown with ball and stick rendering) . peak is slightly larger for Sl on Ab3 paired with S1_ rev on The primary Ig - fold B - strand regions are numbered 1 to 7 C5 (Panel F , see arrow ) . These results are consistent with the from N terminus to C terminus. 65 mass spectrographic analysis of Example 41 and FIG . 20 . If FIG . 16 depicts a mostly buried , solvated pocket between one Fab arm (Panels G - H ) or both Fab arms (Panel I ) did not the Cyl and C , domains of PDB entry 3QQ9 . The light contain a bispecific - favoring design , larger amounts of mis US 10 , 392 , 438 B2 13 14 paired Fab were detected , as indicated by the presence of FIG . 40 . TAM - 163 activates hTrkB , but not hTrkA or additional peaks . For reference , Panels J - K show the corre hTrkC in the SHC1 recruitment assay sponding profile of the monospecific Ab3 and C5 antibodies FIG . 41. FIG . 1 . TAM -163 activates TrkB -dependent on which these bispecific designs were based . puphosphorylation events in hTrkB - Cre cells FIG . 22 depicts a bispecific antibody according to the 5 FIG . 42 TAM - 163 does not activate Trk -dependent phos invention . The domains are labelled as follows: 1 - V , first phorylation events in hTrkA - Cre or hTrkC -Cre cells variable light domain ; 1 - V v : first variable light domain . FIG . 43 . TAM - 163 activates Trk - dependent phosphory 1 -Cz : first constant light domain . 1 -Chl : first constant heavy lation events in human neuroblastoma SH - SY5Y cells 1 domain . 1 - C 2 : first constant heavy 2 domain . 1 -C73 : first FIG . 44 . TAM - 163 induces internalization of TrkB in constant heavy 3 domain . 2 - V , second variable light 10 h TrkB - Cre and in human neuroblastoma SH - SY5Y cells domain ; 2 - Vu : second variable light domain . 2 -Cz : second FIG . 45. TAM - 163 induces degradation of TrkB in constant light domain . 2 -Chl : second constant heavy 1 h TrkB - Cre and in human neuroblastoma SH - SY5Y cells domain . 2 -C42 : second constant heavy 2 domain . 2 -CH3 : FIG . 46 . TAM - 163 does not bind to human p75NTR second constantheavy 3 domain . The first CÆC , and second FACS analysis C?C , domains are indicated between the braces ( 1 -CHCl , 15 FIG . 47. TAM - 163 does not bind human p75NTR - cell and 2 -CAC , respectively ) and encompass the respective C based ELISA and Ch domains . The dotted oval lines capture the four FIG . 48 . TAM - 163 binds to mouse , dog and cat TrkB with domains (V2 , VH , Cz, Chl) that make up the first and second high affinity Fab ( 1- Fab and 2 -Fab respectively ) . The first CaC , interface FIG . 49. TAM - 163 activates TrkB -dependent signaling in and second CyC , interface are patterned in brickwork . 20 cells transfected with mouse TrkB Mutant residues in the C? and Chl domains are represented FIG . 50 . TAM - 163 activates TrkB -dependent signaling in by filled and unfilled circles and triangles ( the set of filled cells transfected with dog TrkB and unfilled circles represent the complementary residue set of the first Fab and the set of filled and unfilled triangles DETAILED DESCRIPTION represent the complementary residue set of the second Fab ) . 25 The ‘ knobs and holes ' pairing of the first CH3 and second In some aspects , the invention relates to a heterodimeric Cy3 domains is represented by an arrow and ring . protein comprising ( i ) a first Cyl domain (Cyl ) and a first This figure further illustrates the two semi- functional and Cy domain (CL ) , the first Chl and the first C , interacting one non - functional permutations that are avoided by the together at a first CyC , interface to form a first C , C , domain present invention . That is , the present inventions reduce the 30 (CHCL ); ( ii ) a second Chl domain (Chl ) and a second C probability that that a first Chl ( 1 -Chl ) and a second Ch domain (CL ) , the second Chl and the second C interacting ( 2 - CL) will associate to form a third C C (FIG . 1B , left together at a second CÆC , interface to form a second CÆCz ; arm ) compared with the favored pairings shown herein and wherein the first Cyl is engineered to differ from the second in FIG . 1A . Similarly , the present invention reduces the Chl by at least one Cul mutant residue in the first Chl ; and likelihood of formation of a fourth CyC , (comprising a 35 the first C , is engineered to differ from the second C , by at second 2 -Cyl and a 1 - C , ) as illustrated in FIG . 1C ( right least one C , mutant residue in the first Cz; such that the Cyl arm ) . Likewise , the present invention reduces the formation mutant residue and the C mutant residue of the first C?C of a non - functional antibody ( e . g . , FIG . 1B ) comprising a interact with each other in preference to the corresponding third CAC , in one arm and a fourth CÆC , in the other arm . residue positions on the second CÆC?, the interacting mutant FIG . 23 : Chimeric TOA - 1 antibody binds human TrkB 40 residues of the first Chl and first C , thereby forming a first FIG . 24 : Chimeric TOA - 1 antibody binds mouse TrkB complementary residue set . FIG . 25 : Humanized TOA - 1 variants compete with bioti - In some aspects , the second Cyl is engineered to differ nylated chimeric TOA - 1 for binding to human TrkB from the first Chl by at least one Chl mutant residue in the FIG . 26 : Humanized TOA - 1 variants compete with bioti - second Cyl ; and the second C , is engineered to differ from nylated chimeric TOA - 1 for binding to human TrkB 45 the first C , by at least one C , mutant residue in the second FIG . 27 : Humanized TOA - 1 variants compete with bioti - Cz; such that the Cyl mutant residue and the C? mutant nylated chimeric TOA - 1 for binding to human TrkB residue of the second CÆC , preferentially interact with each FIG . 28 : Humanized TOA - 1 version 1 . 0 / 1 . 4 fully retains other over the corresponding residue positions on the first human TrkB binding properties relative to parental TOA - 1 CyCH , , the interacting mutant residues of the second Cyl and antibody 50 second C , thereby forming a second complementary residue FIG . 29 : Agonist activity of Anti - TrkB TOA - 1 antibodies set . FIG . 30 : Humanized TOA -1 activates the TrkB signalling The first Chl may be engineered to differ from wild type cascade Ch1. The second Chl may be engineered to differ from wild FIG . 31: The TOA - 1 and BDNF binding sites on hTrkB type C 1. The first C? may be engineered to differ from wild overlap 55 type C . The second C , may be engineered to differ from FIG . 32 : TOA - 1 binding to chimeric TrkB - TrkA receptors wild type C , FIG . 33 : Anti - TrkB antibodies bind to mouse , cat and dog The first Chl may comprise at least one Chl mutant TrkB residue engineered to differ from the corresponding position FIG . 34 : TOA - 1 antibodies do not bind to TrkA or TrkC on the second Cyl. The first C , may comprise at least one FIG . 35 : Humanized TOA - 1 does not bind to p75 60 C , mutant residue engineered to differ from the correspond FIG . 36 : Humanized TOA - 1 does not bind to p75 ing position on the second C?. The second Chl may com FIG . 37 : TOA - 1 does not activate the TrkA or TrkC prise at least one Cyl mutant residue engineered to differ signaling cascades from the corresponding position on the first Cyl. The second FIG . 38 . TAM - 163 activates the Cre - luciferase reporter C? may comprise at least one C , mutant residue engineered gene in hTrkB cells 65 to differ from the corresponding position on the first Cl . FIG . 39 . TAM - 163 does not activate the Cre - luciferase In some aspects of the invention , the identity of the reporter gene in hTrkA - Cre and hTrkC - Cre cells mutant residues of the first complementary residue set are US 10 , 392 , 438 B2 15 16 different from the identity of the mutant residues of the formation of the first and second CÆC , preferentially occurs second complementary residue set . In some aspects , the over formation of the third and fourth C?C by at least about location of the mutant residues of the first complementary 40 - fold . In some aspects , formation of the first and second residue set are different from the location of the mutant CÆC? preferentially occurs over formation of the third and residues of the second complementary residue set (locations 5 fourth CyC , by at least about 50 - fold . In some aspects , according to Kabat numbering as described herein ) . In some formation of the first and second CyC , preferentially occurs aspects of the invention , the identity and location of the over formation of the third and fourth CyC , by at least about mutant residues of the first complementary residue set are 60 -fold . In some aspects , formation of the first and second different to the identity and location of the mutant residues CyC , preferentially occurs over formation of the third and of the second complementary residue set . 10 fourth CyC , by at least about 70 - fold . In some aspects , Preferential Formation of Heterodimers formation of the first and second CyCz preferentially occurs Providing a second complementary residue set in the over formation of the third and fourth CyC , by at least about second CyC further decreases the risk ofmis - pairing of the 75 - fold . In some aspects , formation of the first and second domains. This strategy may be more effective when there is CÆC , preferentially occurs over formation of the third and little overlap between the engineered mutations of the dif - 15 fourth CyC , by at least about 80 - fold . In some aspects , ferent domains . In some aspects , the first complementary formation of the first and second C , Cl preferentially occurs residue set of the first CÆC , are located at different positions over formation of the third and fourth C , C , by at least about relative to the location of the second complementary residue 85 - fold . In some aspects , formation of the first and second set of the second CyC? CHCL preferentially occurs over formation of the third and Accordingly , in some aspects of the invention , formation 20 fourth CÆC , by at least about 90 - fold . In some aspects , of the first CyC , and second C C , preferentially occurs over formation of the first and second C4C , preferentially occurs formation of a CÆC , comprised of either the first Chl and over formation of the third and fourth CHC? by at least about second C , (hereinafter referred to as a third CXCL) , or 95 - fold . In some aspects , formation of the first and second second Cyl and first C? (hereinafter referred to as a fourth CÆC? preferentially occurs over formation of the third and C C ) . 25 fourth CyC , by at least about 99 - fold . In some aspects , FIG . 1A and FIG . 22 illustrate a correctly paired antibody formation of the first and second CÆC? preferentially occurs (comprising first CÆC , and second CÆC?. Improperly paired over formation of the third and fourth CÆC? by at least about domains are also depicted : a third C C ( comprising a first 100 - fold . In some aspects , formation of the first and second Cyl and a second C , ) is shown as the left arm of FIGS . 1B , CC, preferentially occurs over formation of the third and and 1D , and a fourth CyC , (comprising a second Cyl and 30 fourth CyC , by at least about 200 - fold . In some aspects , a first C ) is shown as the right arm of FIGS . 1B , and 1C . formation of the first and second CyC ,1 preferentially occurs Similarly , switching the right hand and left hand light chains over formation of the third and fourth CyC , by at least about of FIG . 22 would result in a non - functional antibody com 500 - fold . In some aspects , formation of the first and second prising third CHC? and fourth CyC? CyC , preferentially occurs over formation of the third and Favorably , formation of the first and second CÆC? pref - 35 fourth CÆC , by at least about 1000 - fold . erentially occurs over formation of the third and fourth In some aspects , formation of the first and second C2C , C?C , by at least about 4 - fold . In some aspects , formation of preferentially occurs over formation of the third and fourth the first and second CÆCl preferentially occurs over forma- CÆC? at a ratio of at least about 4 to about 1 . In some tion of the third and fourth CyC , by at least about 5 - fold . In aspects , formation of the first and second CyC , preferen some aspects , formation of the first and second ChC , 40 tially occurs over formation of the third and fourth CÆC? at preferentially occurs over formation of the third and fourth a ratio of at least about 5 to about 1 . In some aspects , CyC , by at least about 6 - fold . In some aspects , formation of formation of the first and second CyC , preferentially occurs the first and second CÆC? preferentially occurs over forma- over formation of the third and fourth CHC , at a ratio of at tion of the third and fourth CC , by at least about 7 - fold . In least about 6 to about 1 . In some aspects, formation of the some aspects , formation of the first and second CyC , 45 first and second Cyc , preferentially occurs over formation preferentially occurs over formation of the third and fourth of the third and fourth CC, at a ratio of at least about 7 to CyC , by at least about 8 - fold . In some aspects , formation of about 1 . In some aspects , formation of the first and second the first and second CÆCl preferentially occurs over forma- CÆCl preferentially occurs over formation of the third and tion of the third and fourth CÆC , by at least about 9 - fold . In fourth CyC , at a ratio of at least about 8 to about 1 . In some some aspects , formation of the first and second CÆC 50 aspects , formation of the first and second CÆC? preferen preferentially occurs over formation of the third and fourth tially occurs over formation of the third and fourth C / C , at CyC , by at least about 10 - fold . In some aspects, formation a ratio of at least about 9 to about 1. In some aspects , of the first and second CÆC? preferentially occurs over formation of the first and second C # C? preferentially occurs formation of the third and fourth CyC , by at least about over formation of the third and fourth CyC , at a ratio of at 12 - fold . In some aspects , formation of the first and second 55 least about 10 to about 1 . In some aspects, formation of the CAC preferentially occurs over formation of the third and first and second CÆC , preferentially occurs over formation fourth CÆC , by at least about 15 - fold . In some aspects , of the third and fourth CÆC? at a ratio of at least about 12 formation of the first and second CÆC , preferentially occurs to about 1 . In some aspects, formation of the first and second over formation of the third and fourth CyC , by at least about CC, preferentially occurs over formation of the third and 20 - fold . In some aspects , formation of the first and second 60 fourth CyC , at a ratio of at least about 15 to about 1 . In some C?C , preferentially occurs over formation of the third and aspects , formation of the first and second CÆC , preferen fourth CyC , by at least about 25 - fold . In some aspects, tially occurs over formation of the third and fourth CyC , at formation of the first and second CÆC , preferentially occurs a ratio of at least about 20 to about 1 . In some aspects , over formation of the third and fourth CÆC , by at least about formation of the first and second C?C , preferentially occurs 30 - fold . In some aspects , formation of the first and second 65 over formation of the third and fourth CAC , at a ratio of at CyC , preferentially occurs over formation of the third and least about 25 to about 1 . In some aspects , formation of the fourth CÆC , by at least about 35 - fold . In some aspects , first and second CÆC? preferentially occurs over formation US 10 , 392 , 438 B2 17 18 of the third and fourth CyC , at a ratio of at least about 30 empiral mass of each Fab arm and the Fc and values to about 1 . In some aspects, formation of the first and second obtained are compared to the theoretical mass of the two C , C , preferentially occurs over formation of the third and possible correct Fab arms and the two possible incorrect Fab fourth C , C , at a ratio of at least about 35 to about 1 . In some arms and for the Fc a comparison to theoretical mass of aspects, formation of the first and second Cyc , preferen - 5 homodimer vs heterodimer Fc is made. The signal intensity tially occurs over formation of the third and fourth CC , at for each fragment can be converted to a % of total intensity a ratio of at least about 40 to about 1 . In some aspects, of all fragments detected above background noise allowing formation of the first and second CyC , preferentially occurs for a ratio comparison of correct Fab product to incorrect over formation of the third and fourth CÆC , at a ratio of at Fab product. In a separate approach , post protein A bispe least about 45 to about 1 . In some aspects , formation of the 10 cific antibody preparation elute can be fractionated using ion first and second CyC , preferentially occurs over formation exchange or HIC chromatography and eluted fractions iden of the third and fourth CyC , at a ratio of at least about 50 tified using LCMS . Identified peaks are then assigned % to about 1 . In some aspects , formation of the first and second UC from A280 measurements associated with the chro CÆC? preferentially occurs over formation of the third and matography step . Ion exchange chromatography or hydro fourth CHCL at a ratio of at least about 55 to about 1. In some 15 phobic interaction chromatography fractionate bispecific aspects , formation of the first and second CÆC , preferen IgG containing correct and incorrect light chain pairings tially occurs over formation of the third and fourth CyC , at based on differential charge or hydrophobicity properties . a ratio of at least about 60 to about 1 . In some aspects , The % area under curve from the resulting A280 chromato formation of the first and second ChC? preferentially occurs grams can be used to quantitate the amount of correct over formation of the third and fourth CÆC? at a ratio of at 20 product . least about 65 to about 1 . In some aspects , formation of the Solvent Accessible Surface Area first and second CÆC , preferentially occurs over formation When introducing non -wild type human residues (such as of the third and fourth C / C at a ratio of at least about 70 the complementary residue sets herein ; see below ) into to about 1 . In some aspects, formation of the first and second antibodies intended for administration to human patients , Cyl , preferentially occurs over formation of the third and 25 there is a risk that the human immune system will recognize fourth CyC , at a ratio of at least about 75 to about 1 . In some the modified residues as foreign and generate antibodies aspects , formation of the first and second CyC , preferen - against the therapeutic (an anti - drug antibody or ADA tially occurs over formation of the third and fourth CyC , at response , which may result in faster clearance , reduced a ratio of at least about 80 to about 1. In some aspects, activity of circulating therapeutic , or both ) . In order to be formation of the first and second CyC , preferentially occurs 30 recognized by the ADA , the non -human residues of the over formation of the third and fourth CyC , at a ratio of at therapeutic antibody must be accessible to the ADA . Mini least about 85 to about 1 . In some aspects , formation of the mizing the surface area accessible to the ADA would be first and second CyC , preferentially occurs over formation expected to reduce the ability of the ADA to interact with the of the third and fourth CHC? at a ratio of at least about 90 therapeutic antibody. to about 1 . In some aspects, formation of the first and second 35 In some aspects , the solvent accessible surface area of the CC , preferentially occurs over formation of the third and first complementary residue set is less than 225 Å2 as fourth C , C , at a ratio of at least about 95 to about 1 . In some measured using a 2 . 5 Å probe . In some aspects, the solvent aspects , formation of the first and second Cyc , preferen - accessible surface area of the second complementary residue tially occurs over formation of the third and fourth CyC , at s et is less than 225 Å ? as measured using a 2 . 5 Å probe. In a ratio of at least about 99 to about 1 . In some aspects , 40 some aspects , the solvent accessible surface area of the first formation of the first and second C , C preferentially occurs complementary residue set is less than 220 Å as measured over formation of the third and fourth CyC , at a ratio of at using a 2 . 5 À probe . In some aspects , the solvent accessible least about 100 to about 1 . In some aspects , formation of the surface area of the second complementary residue set is less first and second CyC , preferentially occurs over formation than 220 Å ? as measured using a 2 . 5 À probe . In some of the third and fourth CyC , at a ratio of at least about 200 45 aspects , the solvent accessible surface area of the first to about 1 . In some aspects , formation of the first and second complementary residue set is less than 150 Å ? as measured CyC , preferentially occurs over formation of the third and using a 2 . 5 À probe . In some aspects , the solvent accessible fourth CyC , at a ratio of at least about 500 to about 1 . In surface area of the second complementary residue set is less some aspects , formation of the first and second CC, than 150 XP as measured using a 2 . 5 À probe . In some preferentially occurs over formation of the third and fourth 50 aspects , the solvent accessible surface area of the first CC , at a ratio of at least about 1000 to about 1 . In some complementary residue set is less than 120 Å ? as measured aspects , formation of the first and second CyC , preferen - using a 2 . 5 À probe . In some aspects , the solvent accessible tially occurs over formation of the third and fourth CyC , at surface area of the second complementary residue set is less a ratio of at least about 2000 to about 1 . In some aspects, than 120 Å ? as measured using a 2 . 5 À probe . In some formation of the first and second C , C preferentially occurs 55 aspects , the solvent accessible surface area of the first over formation of the third and fourth CC , at a ratio of at complementary residue set is less than 100 Å ? as measured least about 5000 to about 1 . using a 2 . 5 À probe . In some aspects, the solvent accessible The level of correct heterodimer light chain pairing ( i. e . surface area of the second complementary residue set is less first and second C , C , formed ) relative to “ incorrect light than 100 Å ? as measured using a 2 . 5 À probe . In some chain pairing ( i . e . third and fourth CyC , formed ) may be 60 aspects , the solvent accessible surface area of the first measured by Liquid Chromatography Mass Spectrometry complementary residue set is less than 80 Å as measured (LCMS ) . A bispecific antibody preparation may be purified using a 2 . 5 À probe . In some aspects , the solvent accessible by protein A chromatography and preparative size exclusion surface area of the second complementary residue set is less chromatography to remove any aggregates or lower molecu - than 80 Å2 as measured using a 2 . 5 Å probe . In some lar weight components is digested with LysC enzyme to 65 aspects , the solvent accessible surface area of the first release each Fab arm and the Fc as independent fragments complementary residue set is less than 50 Å2 as measured ( 3 fragments total) . LCMS may then be used to measure the using a 2 . 5 À probe . In some aspects, the solvent accessible US 10 , 392 , 438 B2 19 20 surface area of the second complementary residue set is less sphere radius is 1 .4 Å , as this corresponds to the approxi than 50 Å2 as measured using a 2 . 5 Å probe . In some mate radius of a water molecule . However, a larger value aspects , the solvent accessible surface area of the first ( such as 2 . 5 Å , as used herein ) may be appropriate , when complementary residue set is less than 40 Å ? as measured taking into account the experimental uncertainties in atom using a 2 . 5 À probe . In some aspects, the solvent accessible 5 positions inherent in a crystal structure , or if the molecular surface area of the second complementary residue set is less entity , whose access to biomolecule' s surface is in question , than 40 Å2 as measured using a 2. 5 Å probe . In some aspects , the solvent accessible surface area of the first is larger than a water molecule ( for example , the biomol complementary residue set is less than 30 Å as measured ecules of the potential host 's immune system ). using a 2 .5 Å probe. In some aspects , the solvent accessible 10 One aspect of the present invention is to provide a means surface area of the second complementary residue set is less of generating and maintaining bispecific heterogeneous anti than 30 Å2 as measured using a 2 . 5 Å probe . In some bodies or Fab fragments thereof through the use of engi aspects , the solvent accessible surface area of the first neered mutations in the Cyl and C , domains . However, complementary residue set is less than 20 Å ? as measured introducing non - canonical residues into antibodies for in using a 2 . 5 À probe . In some aspects , the solvent accessible 15 Vivo use risks triggering a host immune response . It is surface area of the second complementary residue set is less therefore advantageous to minimize the extent to which than 20 Å ? as measured using a 2 . 5 Å probe . In some introduced or engineered residues to an antibody or Fab aspects, the solvent accessible surface area of the first fragment thereof can potentially trigger a host immune complementary residue set is less than 10 Å2 as measured response . Accordingly , the solvent accessible surface area of using a 2 . 5 Å probe. In some aspects, the solvent accessible 20 a complementary residue set of some aspects of the inven surface area of the second complementary residue set is less tion is less than 50 Å ? as measured using a 2 . 5 À probe . than 10 Å ? as measured using a 2 . 5 À probe . In some In some aspects , the solvent accessible surface area of a aspects , the solvent accessible surface area of the first complementary residue set of the invention is less than 45 complementary residue set is less than 5 X2 as measured Å ? as measured using a 2 . 5 Å probe . In some aspects , the using a 2 . 5 A probe . In some aspects , the solvent accessible 25 solvent accessible surface area of a complementary residue surface area of the second complementary residue set is less set of the invention is less than 40 A ’ as measured using a than 5 Å ? as measured using a2 .5 À probe . In some aspects, 2 . 5 À probe . In some aspects , the solvent accessible surface the solvent accessible surface area of the first complemen area of a complementary residue set of the invention is less tary residue set is less than 2 Å ? as measured using a 2 . 5 Å than 35 Å ? as measured using a 2 . 5 À probe . In some probe . In some aspects , the solvent accessible surface area of 30 aspects , the solvent accessible surface area of a complemen the second complementary residue set is less than 2 A as tary residue set of the invention is less than 30 X as measured using a 2 . 5 À probe . In some aspects, the solvent measured using a 2 . 5 Å probe . In some aspects, the solvent accessible surface area of the first complementary residue accessible surface area of a complementary residue set of the set is less than 1 Å ? as measured using a 2 .5 Å probe . In invention is less than 25 Å ? as measured using a 2 .5 Å probe . some aspects , the solvent accessible surface area of the 35 In some aspects , the solvent accessible surface area of a second complementary residue set is less than 1 X2 as complementary residue set of the invention is less than 20 measured using a 2 .5 À probe . Å ? as measured using a 2 . 5 Å probe . In some aspects , the In some aspects , the solvent accessible surface area is solvent accessible surface area of a complementary residue measured using the surface area algorithm in Maestro 9 .6 , set of the invention is less than 15 Å ? as measured using a 9 . 7 , or 9 . 9 ( Schrodinger, LLC . ) . The resolution may be 0 . 3 . 40 2 . 5 Å probe . In some aspects , the solvent accessible surface Preferably , the solvent accessible surface area of the first area of a complementary residue set of the invention is less complementary residue set is less than 50 Å2 as measured than 10 Å2 as measured using a 2 . 5 À probe . In some using a 2 . 5 Å probe at high resolution ( for example, a aspects , the solvent accessible surface area of a complemen resolution of 0 . 3 ), using the surface area algorithm in tary residue set of the invention is less than 9 Å ? as measured Maestro 9 .6 , 9. 7 , or 9 .9 (Schrodinger , LLC . ). 45 using a 2 . 5 A probe . In some aspects , the solvent accessible It is well known in the art that mutation of a single side surface area of a complementary residue set of the invention chain can improve antibody binding potency by an order of is less than 8 Å ? as measured using a 2 . 5 À probe . In some magnitude or more . For example , a His / Tyr substitution with aspects , the solvent accessible surface area of a complemen an accessible surface area of - 90 Å2 is known to cause t ary residue set of the invention is less than 7 Å ? as measured a > 10 - fold binding improvement of bevacizumab ( J . Chem . 50 using a 2 . 5 A probe . In some aspects , the solvent accessible Inf. Model. 53 ( 11 ) , 2937 - 50 ( 2013 ) ) . However, it is well surface area of a complementary residue set of the invention known in the art that even smaller surface changes can have is less than 6 Å2 as measured using a 2 .5 Å probe . In some similar effects . An alanine side chain has an accessible aspects , the solvent accessible surface area of a complemen surface area of - 20 Å2. A mutation to alanine can be tary residue set of the invention is less than 5 Å ? as measured sufficient to change binding affinity between two proteins by 55 using a 2 . 5 Å probe . In some aspects , the solvent accessible greater than an order ofmagnitude . For example , see Mabs surface area of a complementary residue set of the invention 3 ( 5 ) , 479 -486 ( 2011 ) . Thus, a small mutated surface area is less than 4 Å2 as measured using a 2 . 5 Å probe . In some may be sufficient to allow the immune system to produce an aspects , the solvent accessible surface area of a complemen anti -drug antibody (ADA ) which recognizes an engineered tary residue set of the invention is less than 3 Å ’ as measured biotherapeutic antibody , while having significant selectivity 60 using a 2 . 5 A probe. In some aspects , the solvent accessible against binding native human antibodies. surface area of a complementary residue set of the invention The solvent accessible surface area (SASA ) is the surface is less than 2 Å ? as measured using a 2 .5 Å probe. In some of a biomolecule accessible to a solvent (typically water) . aspects , the solvent accessible surface area of a complemen SASA can be calculated by using the rolling ball' algorithm tary residue set of the invention is less than 1 Å ? as measured developed by Shrake & Rupley in 1973 , which models a 65 using a 2 . 5 À probe. In some aspects, the solvent accessible sphere approximating the size of the solvent molecule to surface area of a complementary residue set of the invention ‘probe ’ the surface of the molecule . A typical value for the is less than 0 .5 Å ? as measured using a 2 . 5 Å probe . In some US 10 , 392 , 438 B2 21 aspects , the solvent accessible surface area of a complemen - increase complementary pairing above that of the non tary residue set of the invention is about 0 Å ? as measured engineered or wild type V NH framework sequences . using a 2 . 5 À probe . There are multiple advantages realized by avoiding insert Variable Domain ing mutant residues of complementary residue sets into In some aspects , the first Cyl is attached to a first variable 5 variable domains. For example , it is sometimes advanta heavy domain (Vy ) , and the first C , is attached to a first geous to use different germline frameworks for the variable variable light domain (VI ) , and the second Cyl is attached region . Sequence variations in each germline present differ to a second Vh , and the second C , is attached to a second V . ing local environments for any mutations made in the When combined , the first Vy, first V , , first Cyl, and first C , variable domain ; mutations which work in some frameworks form a first Fab . When combined , the second V . , second V?, 10 may not work in other frameworks ( for example , problems second Cyl , and second C , form a second Fab . with expression , aggregation , stability, or other physical In some aspects, the first Vy is connected to the first Cyl , properties could occur ) . Also , mutations in the V ,Ny inter which in turn is connected to the first Ch2, which in turn is face ( the area most likely to affect pairing specificity ) are connected to the first Cy3 , thereby forming a first heavy near CDRs and may affect the relative orientation of V , and chain . In some aspects , the second Vy is connected to the 15 Vy in subtle ways that differ from antibody to antibody , and second Cyl, which in turn is connected to the secondC2 , between frameworks . A subtle variation in V ,Ny orienta which in turn is connected to the second Cy3 , thereby tion may be tolerated by some antibodies, but not others . In forming a second heavy chain . addition , mutating multiple regions of the protein surface In some aspects , the first V , is connected to the first C , (both variable and constant domains) provides additional thereby forming a first light chain . In some aspects , the 20 opportunities for the patient ' s immune system to recognize second V , is connected to the second C , , thereby forming a the antibody as foreign , and reject it via an anti- drug second light chain . antibody response (ADA ). Two possible results of an ADA In some aspects , the invention provides for preferential response are a faster rate of clearance of the therapeutic from formation of a first Fab and second Fab that does not rely on the patient, and neutralization of the drug ' s ability to bind its complementary pairing of the variable domains. 25 intended target (Jawa et . al , Clin . Immunol. 149( 3 ) , 534 - 55 Where the preferential formation of heterodimeric protein (2013 ) ) . In the development of bispecific antibodies , it is domain interactions is discussed as not relying on comple desirable to take steps to minimize the probability of the mentary residue pairing of the variable domains , this means patient ' s immune system mounting an ADA response . While that the complementary pairing of, for example, a first Cyl there are some computational models for predicting T cell and a first C , domain is sufficient to effect preferential 30 ADA response , accurate tools for conformational epitopes formation of a first CyC , ( or a first Fab ) . Additonal engi - are lacking. Therefore , given the limited accuracy of in silico neered residues in one or more of the variable or constant predictions, it is preferable to limit modifications of high domains may provide additive effects to increase the fidelity fidelity bispecific IgG molecules to the Cyl and C , domains of the preferential formation of the desired domain pairing rather than mutating multiple domains as required by the In some aspects , the first complementary residue set is 35 Lewis et al. method ( see below ) . necessary for preferential formation of the first C Cl. In In some aspects , the first Vy comprises Vr - Q39 (as in some aspects , the first complementary residue set is neces - DP54 or DP75 ) or Vy - Q105 (as in human J segments other sary for preferential formation of the first Fab . In some than JH2 ) . In some aspects , the second Vy comprises aspects , the second complementary residue set is necessary VH - Q39 ( as in DP54 or DP75 ) or VvQ105 ( as in human J for preferential formation of the second CyC , . In some 40 segments other than JH2 ) . aspects , the second complementary residue set is necessary In some aspects , the first V , comprises one or more of : (i ) for preferential formation of the second Fab . V -238 (as in DPK9 or DPL16 ) ; and ( ii) one of V , - Q1 ( as In some aspects , the first complementary residue set is in DPL7 ) , V -S1 ( as in DPL16 ) , V2 - D1 ( as in DPK9) , V2- E1 sufficent for preferential formation of the first CyCl . In (as in DPK23 ), V? -A1 (as in DPK3) , or V2 -N1 (as in some aspects, the first complementary residue set is suffi - 45 DPK2) ; and ( iii ) one of V - 142 (as in DPL7 ) , V - Q42 (as cient for preferential formation of the first Fab . In some in DPL16 ) , or V -K42 ( as in DPKI) . aspects , the second complementary residue set is sufficent In some aspects , the second V , comprises one or more of: for preferential formation of the second CÆCl . In some ( i ) V - 238 ( as in DPK9 or DPL16 ) ; and ( ii ) one of V - Q1 aspects , the second complementary residue set is sufficient ( as in DPL7) , V -S1 (as in DPL16 ), V2 -D1 ( as in DPK9 ) , for preferential formation of the second Fab . 50 V2 - E1 ( as in DPK23 ) , V? -A1 (as in DPK3) , or V -N1 (as in In some aspects , the invention provides for preferential DPK2) ; and ( iii ) one of V - 142 (as in DPL7 ) , V - Q42 ( as formation of a first Fab and second Fab that does not rely on in DPL16 ) , or V -K42 ( as in DPKI) . complementary pairing of the variable domains such that In some aspects , the first Vh comprises VH -Q39 (as in formation of the first and second CÆC preferentially occurs DP54 or DP75 ) and V - 2105 ( as in human J segments other over formation of the third and fourth C C? at a ratio of at 55 than JH2) . In some aspects , the second Vy comprises least about 4 to about 1 , and may occur at a ratio of at least Vy - Q39 (as in DP54 or DP75 ) and Vy -Q105 ( as in human a value selected from the group 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 20 , J segments other than JH2 ) . In some aspects , both the first 25 , 30 , 35 , 40 , 45 , 50 , 55 , 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 100 , and second Vy comprise these residues . 200 , 500 , 1000 , 2000 , and 5000 to 1 . In some aspects , the first V , comprises: ( i) Vz- Q38 (as in There are some instances of known V ,Ny pairs that have 60 DPK9 or DPL16 ) ; and ( ii ) one of V - Q1 ( as in DPL7) , a natural affinity with each other. Accordingly , in some V2 - S1 ( as in DPL16 ), V2- D1 (as in DPK9 ), V2 -E1 (as in aspects the invention provides for preferential formation of DPK23 ) , V . - A1 (as in DPK3 ) , or V , -N1 (as in DPK2 ) ; and a first Fab and second Fab that does not rely on the any of ( iii ) one of V [ - T42 (as in DPL7 ) , V2 - Q42 ( as in DPL16 ) , or the variable domains comprising engineered mutant residues V2- K42 ( as in DPK9) . that form complementary residue sets . In some aspects , the 65 In some aspects , the second V , comprises: (i ) V _ - 238 ( as multimeric proteins of the invention do not comprise muta in DPK9 or DPL16 ) ; and ( ii ) one of V? - Q1 ( as in DPL7) , tions in any of the variable domains that are engineered to V - S1 ( as in DPL16 ), V?- D1 (as in DPK9 ), V2 -E1 (as in US 10 , 392 , 438 B2 23 24 DPK23 ) , VL -A1 ( as in DPK3) , or V - N1 (as in DPK2) ; and In principle , heterodimer - favoring mutations could be ( iii ) one of V - T42 ( as in DPL7 ) , V2 - 042 ( as in DPL16 ), or included in either of the major interface regions between the V2 -K42 (as in DPK9) . heavy and light chain , which are the interface between the In some aspects , both the first V , and second V , comprise Chl and C , domains, and the interface between the variable the above residues . 5 heavy and variable light domains . However, as noted in part Lewis et al . (Nat . Biotechnol . 32 , 191- 98 (2014 ) , or above, mutations in the Ch1/ C , interface are highly pre “ Lewis publication ” hereafter ) reported mutations in the ferred for development of a robust bispecific platform . Mutations in the variable domain interface may affect the CH1, C _ , V2, and Vh domains which attempted to address conformation of the CDR loops : because the CDR loops the issue of pairing light chains with the proper heavy 10 form part of the variable domain interface, they may interact chains. In a related patent application , WO2014150973 , ( either directly or indirectly through nearby residues ) with bispecific antibodies are disclosed which all involve at least mutations made in the variable domains . If such interactions one mutation of a variable domain . The Lewis publication with heterodimer - enhancing mutations alter the CDR loop states : “ Our method requires the introduction of multiple conformations in ways which affect antibody affinity , these mutations into conserved framework regions of both vari- 15 heterodimer mutations will prove to be poor candidates for able and constant domains. ” The authors further noted that reliable use across a broad range of antibodies . in their experience , “ variable domains dominated the spe - In addition , it is known that the relative orientation of the cific assembly of heavy chains and light chains” . They two variable domains is not constant among all antibodies; hypothesized that during the protein folding pathway, the the angle between the two domains can vary by at least 30 variable domains may “ recognize one another first and drive 20 degrees between antibodies (Abhinandan and Martin , Pro the C , domain to interact with unfolded Cyl” , such that the tein Eng Des Sel. 23 (9 ) , 689 - 97 , (2010 ) ) . These changes heavy / light chain pairing is largely determined by interac necessarily alter the detailed pattern of contacts between tions of Vy and V? , before Chl and C , interact. That residues in the variable domains, and correspondingly alter hypothesis would explain their observation thatmutations in the range of amino acid substitutions that would be tolerated the variable region were required . 25 in the interface . In contrast , the present invention provides heterodimeric Given these facts , if variable domain mutations were used proteins ( e . g . bispecific antibodies ) which require no muta - in a heterodimer -favoring platform design , it would be tions of the CDRs or even the remainder of the variable difficult to demonstrate robust and reliable applicability region , and yet achieve high fidelity of chain pairing . Thus, without testing a large number of examples covering the relative to recent art in the field , specifically Lewis and 30 various CDR conformations and variable domain orientation WO2014150973 , the heterodimeric proteins and bispecific angles encountered in common practice in known antibody antibodies of the present invention are unexpected and structures. Accordingly , one of the advantages in generating provide significant beneficial advantages. heterodimers and bispecific anitbodies according to the As is known in the art , interactions between an antibody present invention is to rely solely on modifying the Chl /CL and its antigen are driven primarily by the CDR loops . While 35 interface . None of the embodiments of the present invention not all CDR loops participate in antigen binding for all require pairing with modifications of the variable domains as antigens, when designing a method of antibody engineering an essential feature to achieve useful levels of pairing to try and achieve high fidelity bispecific chain pairing , fidelity . mutation of positions within the CDRs and variable regionis The present invention advantageously provides that the a disadvantage due to the risk of negatively affecting anti - 40 preferential formation of first Fab and second Fab relies on body binding affinity . For cases involving the simultaneous complementary pairing of the complementary residue sets . production ofmultiple Fab sequences ( or a bispecific IgG ) In some aspects, preferential formation refers to the rather than a single Fab , the various embodiments of formation of a first Fab ( or first CÆCL ) comprising the first WO2014150973 all envision mutating the CDR2 region of Chl and first C? to a greater extent than the formation of a the heavy chain as defined by Kabat (“ the residue which is 45 Fab ( or CyC ) comprising the first Cyl with a second Cz , four amino acids upstream of the first residue of HFR3 or a second Cyl with a first C , according to Kabat” is mutated to glutamate , where HFR3 In some aspects , preferential formation refers to the refers to framework 3 of the heavy chain ) . The heterodi- formation of a second Fab (or second CÆC ) comprising the meric proteins and bispecific antibodies of the present second Cyl and second C , to a greater extent than the invention do not involve modification of the CDRs, and thus 50 formation of a Fab ( or CyC ) comprising the first Cyl with avoid this risk . In addition , position 1 of the light chain a second C , or a second Cyl with a first C , variable region ( which is mutated to Arg during production In some aspects , at least one of the C , domains is a kappa of four- chain mixtures according to the claims of WO2014 / domain . In some aspects , at least one of the C? domains is 150973A1) is near the CDR1 and CDR3 loops , which means a lambda domain . In some aspects , both of the C , domains that mutations at this position may also affect binding 55 are kappa domains . In some aspects, both of the C , domains affinity to some antigens . In PDB entry 4LLY, a crystal are lambda domains . In some aspects , one of the C , domains structure described in the Lewis publication , the side chain is a kappa domain , and the other C , domain is a lambda of position 1 is disordered beyond CB , but the backbone domain . atoms are within 5 Å of CDR L1 and within 6 Å of CDR L3 , In some aspects , the present invention provides for het and CB is oriented towards the face of the Fab containing the 60 erodimeric proteins and bispecific antibodies wherein for majority of the CDR residues ( ie , where antigen is expected mation of the first CÆC , and second CÆC , preferentially to bind ). In contrast , the heterodimeric proteins and bispe- occurs over formation of a C C , comprised of either the first cific antibodies of the present invention do not involve Cyl and second C? , or second Cal and first C? , by at least mutation of this position , or of any other position in the about an amount selected from the group consisting of variable domain , thus avoiding the risk of disturbing CDR 65 4 - fold , 5 - fold , 6 - fold , 8 - fold , 10 - fold , 15 - fold , 20 - fold , positioning and/ or antigen binding which exists when mutat 25 - fold , 30 - fold , 40 -fold , 50 - fold , 60 - fold , 80 - fold , 90 - fold , ing nearby framework residues. 100 - fold , 150 - fold , and 200 - fold . US 10 , 392 ,438 B2 25 26 The determination of correct C , CL pairing may be made Chl- 145 and C - 131. In some aspects , the complementary by mass spectrometry analysis . residue set comprises Chl - 179 and C - 131 . In some aspects , Complementary Residue Sets the complementary residue set comprises Cyl - 145 , Cyl In someaspects , the complementary residue sets comprise 179 , Chl - 186 and C _ _ 131 . In some aspects , the complemen a positively or negatively charged residue in one domain , 5 tary residue set comprises Chl - 143 , Chl - 179 , CH1- 186 , and and an oppositely charged residue in the other domain . In C - 131 . In some aspects , the complementary residue set some aspects , the complementary residue sets comprise a comprises Cyl - 186 and C , 131. In some aspects , the positively charged residue in one domain , and negatively complementary residue set comprises Chl- 143 and C - 133 . charged residue in the other domain . In some aspects , the In some aspects , the mutation at the Cyl position is complementary residue sets comprise a positively or nega - 10 selected from the group consisting of W , H , K , R , S and T , tively charged residue in one domain , and either a polar and the mutation at the C position is selected from the residue, or oppositely charged residue , in the other domain . group consisting of S , M , D and E . Positively charged residues may be selected from the group In some aspects , the mutation at the Chl position is consisting of H , K and R . Negatively charged residues may selected from the group consisting of E , and D , and the be selected form the group consisting of E and D . For the 15 mutation at the C , position is selected from the group avoidance of doubt, negatively charged residues are said to consisting of H , K , and R . be oppositely charged to positively charged residues, and In some aspects , one or more of the complementary vice versa . Polar residues may be selected from the group residue sets further comprise one or more further mutations. consisting of S , T , M , Q , N , W , and Y . Polar residues may be In some aspects , one or more of the complementary selected from the group consisting of S , T , M , Q , N , and W . 20 residue sets comprise one or more further mutations selected Polar residues may be selected from the group consisting of from the group consisting of: Cyl - 143D , C41 - 145S , Cyl S , T , M , Q , N , and Y . Polar residues may be selected from 186A , Cy1 - 186E , Cy1 - 188G , Cy1 - 188W , Cyl- 190S , Cul the group consisting of S , T, M , W , and Y . Polar residues may 1901, C - 133S , C?- 1351, C _ - 176G , C4 - 176M , and C4 - 178S . be selected from the group consisting of S , T, M , W , and Y . In some aspects , one or more of the complementary Polar residues may be selected from the group consisting of 25 residue sets comprise further mutations located at one or S , T , M , and W . Polar residues may be selected from the more positions selected from the group consisting of: Chl group consisting of S , M , W , and Y . Polar residues may be 143 , Cy1 - 145 , Cy1 - 186 , Cyl - 188 , Cy1 - 188 , Cul - 190 , selected from the group consisting of S , M , and W . Polar Cyl- 190 , C? - 133 , C2 - 135 , C?- 176 , C - 176 , and C? - 178 , residues may be selected from the group consisting of S and according to Kabat numbering as described herein . T . In some aspects , Mis not considered to be a polar residue. 30 In some aspects , one or more of the complementary For example , the Cyl mutant residue may comprise a residue sets comprise a further Cyl mutant residue at positively or negatively charged residue , and the C , mutant Chl- 143 . The mutant residue at Chl - 143 may be selected residue may comprise either a polar residue , or an oppositely from the group consisting of H , K , R , E , and D . The mutant charged residue . The C? mutant residue may comprise a residue at Chl- 143 may be selected from the group con positively or negatively charged residue , and the Cylmutant 35 sisting of E , and D . The mutant residue at Cyl- 143 may be residue may comprise either a polar residue , or an oppositely E . The mutant residue at Chl - 143 may be D . charged residue . The C? mutant residue may comprise a In some aspects , one or more of the complementary positively charged residue , and the Cyl mutant residue may residue sets comprise a further Cyl mutant residue at comprise a negatively charged residue . The Chl mutant Chl- 145 . The mutant residue at Chl - 145 may be selected residue may comprise a positively charged residue , and the 40 from the group consisting of S , T , M , Q , N , E , D , W , or Y . C? mutant residuemay comprise a negatively charged resi - The mutant residue at Chl -145 may be selected from the due . group consisting of S , T , M , Q , N , E , or D . The mutant In some aspects of the invention , the complementary residue at Cy1- 145 may be selected from the group con residue sets may comprise a Chl mutant residue and a C sisting of S , T, M , Q , or N . The mutant residue at Chl - 145 mutant residue whose oppositely charged side chains pro - 45 may be selected from the group consisting of S , T , or M . The mote electrostatic interaction . Favorably, the altered charge mutant residue at Cyl - 145 may be S . The mutant residue at polarity of the respective Cyl and C , domains resulting Cyl - 145 may be T . from the engineered mutant residues supports the formation In some aspects , one or more of the complementary of the first or second Fab , and similarly, a repulsive charge residue sets comprise a further Cyl mutant residue at interaction resulting from one or more of the engineered 50 Chl- 186 . The mutant residue at Cyl - 186 may be selected mutant residues suppresses the formation of the third or from the group consisting of G , A , L , V , I, W , F , or Y . The fourth Fab . mutant residue at Cyl- 186 may be selected from the group In some aspects , the locations of the complementary consisting of G , A , L , V , I, or W . The mutant residue at residue sets are selected from the group consisting of: ( i) Chl - 186 may be selected from the group consisting of G , A , CH1 - 124 and C?. 176 ; ( ii ) Chl - 188 and Cz- 178 ; ( iii) Chl- 55 L , V , or I . The mutant residue at Chl- 186 may be selected 143 and C _ _ 178 ; (iv ) Chl- 143 and C2_ 131; ( v ) Ch1 -221 and from the group consisting of G , A , V , or L . The mutant C __ 123 ; ( vi) Ch1 - 145 and C __ 131 ; (vi ) Chl- 179 and C , 131 ; residue at Chl- 186 may be selected from the group con ( vii ) Chl - 186 and C _ _ 131 ; and ( viii ) Chl- 143 and C?- 133 , sisting of G , A , or V . The mutant residue at Chl - 186 may be according to Kabat numbering as defined herein . selected from the group consisting of G , or A . The mutant In some aspects , the complementary residue set comprises 60 residue at Cyl- 186 may be selected from the group con Cyl - 124 and CX - 176 . In some aspects , the complementary sisting of A , or W . The mutant residue at Cyl- 186 may be residue set comprises Cyl - 188 and C , 178 . In some aspects, selected from the group consisting of F , Y , or W . The mutant the complementary residue set comprises Chl- 143 and residue at Ch1 - 186 may W . The mutant residue at Chl -186 CL -178 . In some aspects , the complementary residue setm ay A . comprises Ch1 - 143 and C _- 131. In some aspects , the 65 In some aspects , one or more of the complementary complementary residue set comprises Cyl - 221 and C , 123 . residue sets comprise a further Cyl mutant residue at In some aspects , the complementary residue set comprises Chl - 188 . The mutant residue at Chl- 188 may be selected US 10 , 392 , 438 B2 27 28 from the group consisting of G , A , L , V , I, W , F , or Y . The Chl - 143D , C7- 178K ; ( xii ) Chl- 143D , C7- 178K , C4 - 176M ; mutant residue at Cyl - 188 may be selected from the group (xiii ) Cyl - 143E , C7- 131R ; (xiv ) Chl - 143R , C7 -131E ; (XV ) consisting of G , A , L , V , I, or W . The mutant residue at Cyl- 143R , C2 - 131E , Chl- 186A ; (xvi ) Ch1 - 221D , Chl - 188 may be selected from the group consisting of G , A , C2vizi- 123K ,; (xvii vincle ) Chl- 221D Sv , C1.- 123Kcom - 145R7EK, Chl - 1901: 4, :C 1764_ - 1351, ; L , V, or I. The mutant residue at Chl- 188 may be selected 5 (xviii ) Chl - 145E , CZ - 131H ; (xvix ) Chl- 143H , Chl - 179D , from the group consisting of G , A , V , or L . The mutant Cy1 - 186E , C2 - 131H ; ( xix ) Chl- 145E , C2 - 131H ; ( xx ) Chl residue at Chl- 188 may be selected from the group con 186E , C7 -131H , Cyl- 145S ; (xxi ) Cy1- 143S , C? - 131D , sisting of G , or A . The mutant residue at CH1 - 188 may be Ch1 - 188W , C? - 133S , C4 - 178S ; (xxii ) Chl- 143S , Chl selected from the group consisting ofG , A , or W . The mutant 188W , C7 - 133M , C2 - 176G , C7 - 178G ; (xxiii ) Chl - 143H , residue at Chl- 188 may be selected from the group con - 10 Chl - 179D , CH1- 186E , C4 - 131H , CH - 1901 , C2 - 1351, ( xxiv ) sisting of G , or W . The mutant residue at Chl- 188 may be CH- 186E, C2- 131H , CH - 145S ; ( XXV ) Chl- 143S , C7 - 131D , selected from the group consisting of F , Y , or W . The mutant Chl- 188W , C? - 133S , CL - 176C ; ( xxvi) Chl - 143S , Chl residue at Ch1 - 188 may be W . The mutant residue at 188W , C7 - 133M , C _ -178G , C? - 176G ; ( xxvii) Chl- 143S , Cyl- 188 may be A . The mutant residue at Cyl - 188 may be Cy1 - 188W , C , - 131D . G . 15 Novel Disulfide Linkage In some aspects , one or more of the complementary In some aspects , the invention provides for a novel residue sets comprise a further Chl mutant residue at disulfide bond between the first Chl and the first C? , and /or Cyl- 190 . The mutant residue at Chl - 190 may be selected the second Chl and the second C?. The novel disulfide bond from the group consisting of S , T , I , L . The mutant residue may be located at one or more of the following positions (i ) at Chl- 190 may be selected from the group consisting of I 20 Chl- 122 and C - 123 ; ( ii) Chl - 139 and CZ - 116 ; and ( iii ) or L . The mutant residue at Cyl - 190 may be selected from Cyl - 174 and C , 176 . the group consisting of S or T . The mutant residue at The wild type disulfide bond may be removed , by mutat Cyl- 190 may be selected from the group consisting of Sor ing one or both of Chl - C230 and C4 -214 to any residue I. The mutant residue at Cyl- 190 may be T . The mutant except C , on either or both of the first CC , and / or second residue at Cyl - 190 may be L . The mutant residue at 25 C Cl. In some aspects , the Cz- C214 is deleted in either or Cyl - 190 may be I . The mutant residue at Cyl - 190 may be both of the first and / or second C , C . In some aspects , the Chl -C230 is deleted in either or both of the first and /or In some aspects , one or more of the complementary second C , C , residue sets comprise a further C , mutant residue at C , - 133 . In some aspects , the first and/ or second Cyl -C230 and The mutant residue at C2- 133 may be selected from the 30 first , and / or second C2 -C214 are mutated to S . In some group consisting of S , T, Q or M . The mutant residue at aspects , the first CyC , comprises Cyl -C230S and C4 - 133 may be S . The mutant residue at Cz- 133 may be T . C2 -C214S , and further comprises one or more of the fol Themutant residue at C4- 133 may be M . The mutant residue lowing residue pairs : Cyl- 122C and C _ _ 123C ; Cyl- 139C at C - 133 may be Q . and C _ - 116C ; and Chl - 174C and C _ 176C . In some aspects , In some aspects , one or more of the complementary 35 the second CÆC , comprises Cyl -C230S and C2 - C214S , and residue sets comprise a further C , mutant residue at C2 - 135 . further comprises one or more of the following residue pairs : The mutant residue at C2- 135 may be selected from the CH1- 122C and C -123C ; Cyl - 139C and C _ _ 116C ; and Chl group consisting of I , T , or M . The mutant residue at C4- 135 174C and C2 _ 176C . Favorably , the first ChC? and second may be I . C?C do not comprise novel cytokine mutations located at In some aspects , one or more of the complementary 40 the same corresponding positions. residue sets comprise a further C , mutant residue at C , - 176 . In some aspects , wherein a given CC, comprises The mutant residue at C2 - 135 may be selected from the CH - 174C and C4 - 176C , the given CHC further comprises group consisting of G , A , V , I , L , M , N . or T. The mutant CH - 1901 and C4 - 1351. residue at C7- 176 may be selected from the group consisting CwC Mutations of G , A , V, I, L , or M . The mutant residue at C4 - 176 may be 45 In some aspects , the first CaC , and /or second CÆC , selected from the group consisting of G , A , V , L , or M . The comprises residues from one of the following groups: (i ) mutant residue at C?- 176 may be selected from the group Cyl - 124K , C , - 176D , Cyl- 190S , C7- 133S ; (ii ) Cy1 - 124E , consisting of G , A , V , or M . The mutant residue at C? - 176 C - 176K , CH1 -188G , C -133S ; ( iii ) Chl - 124K , C7 - 176D , may be selected from the group consisting of G , A , or M . C - 133S ; ( iv ) Chl- 124E , C2 - 176K , C - 133S ; ( v ) Chl The mutant residue at C4- 176 may be selected from the 50 188E , C2- 178K , C71 - 143E ; ( vi ) Chl - 188K , C2 - 178D , Chl group consisting of G , or M . The mutant residue at C4 - 176 143D ; (vii ) Cyl - 143K , C?- 178D ; (viii ) Cyl- 143D , may be G . The mutant residue at C2- 176 may be A . The Cz- 178R ; ( ix ) Chl- 143K , C7 - 178D ; ( x ) Chl- 143D , mutant residue at C?- 176 may be M . The mutant residue at C - 178K ; ( xi ) Cy1 - 143D , C - 178K , C7 - 176M ; ( xii ) Chl C , - 176 may be N . 143E , C7- 131R ; ( xiii ) Chl- 143R , C2 - 131E ; (xiv ) Chl In some aspects , one or more of the complementary 55 143R , C2 - 131E , Chl- 186A ; ( xv ) CH1- 221D , C4 - 123K ; residue sets comprise a further C , mutant residue at C4 - 178 . (xvi ) Cyl -221K , CZ - 123K , Cyl- 1901, C2 - 1351, Cy1 - 174C , The mutant residue at C? - 135 may be selected from the Ch1 - 230S , C? - 176C , C7- 214S ; (xvii ) Chl - 145E , C2 - 131H ; group consisting of G , S , V , or A . The mutant residue at (xviii ) Chl- 143H , Chl- 179D , CH1- 186E , C1- 131H ; (xix ) CL - 135 may be S . Chl -122C , Chl- 145E , Ch1- 230S , C4- 123C , C4 -131H , In some aspects , wherein the first and second comple - 60 C2- 214S ; (xx ) Chl- 186E, C2- 131H , CH1- 145S ; ( xxi) Chl mentary residue sets are selected from two of the following 143S , C7- 131D , CH1 - 188W , C? - 133S , C7 - 178S ; (xxii ) Cyl groups : (i ) Chl- 124K , C4 -176D ; ( ii ) Chl- 124K , C7- 176D , 143S , Chl- 188W , C - 133M , C4 - 176G , C2- 178G ; (xxiii ) Chl- 190S , C? - 133S ; (iii ) Chl- 124K , C7 - 176D , C - 133S ; Cyl - 143H , Chl- 179D , Chl- 186E, C / - 131H , CH - 1901 , ( iv ) Chl - 124E , C4 - 176K ; (v ) Chl - 124E , C - 176K , Chl - C - 1351, Chl- 174C , C?1- 230S , C4 - 176C , CZ - 214S ; (xxiv ) 188G ; ( vi) CH1 - 188E , C4 - 178K , Chl- 143E ; (vii ) Chl - 65 C - 186E , CZ - 131H , CH - 145S , Chl- 139C , Ch1 - 230S , 188K , C? - 178D , CH1 - 143D ; ( viii ) Chl- 143K , C - 178D ; C - 116C , C2 - 214S ; (xxv ) Chl - 143S , C? - 131D , CH1- 188W , (ix ) Chl- 143D , CZ- 178R ; ( x) C21- 143K , CZ- 178D ; ( xi) Cz- 1335, C4 -178S , Chl- 174C , Ch1- 230S , Cz- 176C , US 10 , 392 ,438 B2 29 30 C1 -214S ; (xxvi ) Cyl- 221D , 123K , Cyl - 1901, C? -1351 , In some aspects , the first Cyl , and /or second Cycy Chl -174C , Ch1 -230S , C4- 176C , C4 -214S ; (xxvii ) Chl- comprise the residues Ch1 -143R , C2- 131E , and Chl- 186A . 143S , C71- 188W , Chl- 122C , CH1- 139C , C71 - 174C , Chl- In some aspects , the first C C? and/ or second C / C , com 230S , Cz- 133S , C4- 178S , C2- 131D , C2- 116C , C4 -123C , prise the residues Chl- 143R , and C - 131E . In some aspects , C4- 176C , CZ -214S . 5 the first CÆC , and / or second CHC , comprise the residues Favorably , the first and second Fab do not both comprise C , 1- 143K . and C .- 131E . In some aspects , the first CC, residues from the same group . In some aspects , the first CÆC , and / or second Chc and /or second ChC , comprise the residues Chl - 143R , and comprises the residues Chl - 124K , C4- 176D , CH1 - 190S , and Cz - 131D . In some aspects , the first CÆC , and /or second CL - 133S . In some aspects , the first CaC , and /or second 10 CÆC comprise the residues Chl- 143K , and C2- 131D . CAC , comprise the residues Chl- 124K , and C4 - 176D . In In some aspects , the first CaC , and /or second CaCl some aspects , the first C C? and /or second ChC , comprises comprise the residues C71- 221D , C7- 123K , Chl- 1901 , the residues CH1 - 124K , C2 - 176E . In some aspects , the first C2- 1351, Chl- 174C , CH1 - 230S , C2- 176C , and CL - 214S . In C?C , and / or second CÆC , comprise the residues Cyl- 124R , some aspects, the first CC , and / or second CAC , comprise C , - 176D . In some aspects . the first C , C , and /or second 15 the residues Chl - 221D , and C1- 123K . In some aspects, the CHC , comprise the residues Cyl - 124R , C4 - 176E . first CÆC , and / or second CÆC , comprise the residues Chl In some aspects , the first CaC , and /or second Call 221E , and C? - 123K . In some aspects , the first C4C _ and / or comprise the residues Chl- 124E , C4 - 176K , Ch1- 188G , and Sendsecondespeciale CuCy, and orcomprisesecond che the aspects residues, the first Chl Czens- 221D, , con and C1 - 133S . In some aspects , the first ChC , and/ or second C - 123R . In some aspects , the first CÆC , and /or second CoC , comprise the residues Cyl - 124E , and C? - 176K . In 20 ChC , comprise the residues Cyl - 221E , and C4 - 123R . some aspects , the first CAC , and / or second CHC , comprise In some aspects , the first ChC , and /or second CÆC , the residues Cyl - 124E , and C4 - 176R . In some aspects , the comprise the residues Chl- 145E , and C4 - 131H . In some first CyC , and/ or second CÆC , comprise the residues Chl - aspects , the first CÆC , and /or second CÆC , comprise the 124D , and C? -176K . In some aspects, the first Chl , and /or residues Cyl - 145D , and C2- 131H . In some aspects , the first second C C comprise the residues Chl- 124D , and 25 CyC , and /or second CyC , comprise the residues Cyl - 145E , C4 - 176R . and CL - 131K . In some aspects , the first CÆC , and / or second In some aspects , the first CÆC , and /or second CAC , CC, comprise the residues Chl - 145E , and C2- 131R . In comprise the residues Chl - 188E , C4 - 178K , and Ch1- 143E . In some aspects , the first C C? and / or second CÆC , com some aspects , the first CÆC? and / or second CÆC , comprise prise the residues CH1- 188E , and C4 - 178K . In some aspects , 30 the residues Chl- 145D , and C - 131K . In some aspects , the the first CÆC , and /or second CÆC , comprise the residues 30 first CÆC , and / or second CÆC , comprise the residues Cyl CH1- 188D , and C2- 178K . In some aspects , the first CÆC 145E , and C4- 131H . and / or second C C comprise the residues Ch1 - 188E , In some aspects , the first CÆC , and /or second CÆC , Cl - 178R . In some aspects , the first CÆC , and /or second comprise the residues CH1- 143H , Cyl - 179D , C71- 186E , C?C , comprise the residues Chl- 188D , C7 - 178R . 35 and C4- 131H . In some aspects , the first CÆC , and /or second In some aspects , the first Chl? and/ or second CCL CÆC , comprise the residues CH1- 143E , C71- 179D , Chl comprise the residues C „ 1 - 188K , C , - 178D , and C , 1 - 143D . 186E , and Cr - 131H . In some aspects, the first Chºi and / or In some aspects , the first CyC , and /or second C / C , com - second C C comprise the residues Chl- 143D , Chl - 179D , prise the residues Cyl - 188K , C , - 178D . In some aspects , the Chl- 186D , and Cq - 131H . In some aspects , the first ChC , first CyC , and /or second C , C , comprise the residues Cyl - 40 and /or second CaC , comprise the residues Chl - 143H , Chl 188R , C4 - 178D . In some aspects , the first CÆC , and / or 179D , CH1- 186D , and CL - 131H . In some aspects , the first second ChC , comprise the residues Ch1- 188K , C4- 178E . In CÆC , and / or second ChC , comprise the residues Chl- 143H , some aspects , the first CC, and / or second CÆC , comprise Chl - 179E , Chl- 186D , and C4 - 131H . In some aspects , the the residues Cyl - 188R , C4 - 178E . first CÆC and / or second CÆC , comprise the residues Chl In some aspects , the first CÆC , and /or second CÆC , 45 143H , Chl- 179E , Chl- 186E , and C? - 131H . In some comprise the residues Ch1 - 143K , and C?- 178D . In some aspects , the first CAC , and / or second CÆC , comprise the aspects , the first CÆC , and / or second CÆC , comprise the residues Chl- 143H , Chl - 179E , Ch1 - 186D , and CL - 131H . residues Chl - 143K , and C? - 178E . In some aspects , the first In some aspects , the first CÆC , and /or second CÆC , com CÆC , and /or second CÆC comprise the residues Cyl - 143R , prise the residues Ch1- 143H , Chl- 179E , CH1- 186E , and and C4 - 178D . In some aspects , the first C C and / or second 50 C2- 131H . CAC , comprise the residues Cyl- 143R , and C4 - 178E . In some aspects , the first CAC , and /or second CÆC In some aspects , the first CaC and / or second C C comprise the residues Chl- 145E , C2- 131H , Chl- 122C , comprise the residues CH1 -143D , and C - 178R . In some CH1- 230S , C? - 123C , and CL - 214S . aspects , the first CHC , and /or second CÆC , comprise the In some aspects , the first CÆC , and /or second CÆC residues Chl- 143E , and C4 - 178R . In some aspects , the first 55 comprise the residues Ch1 - 186E , C4 - 131H , and Ch1 - 145S . CÆC , and / or second ChC , comprise the residues Chl- 143D , In some aspects , the first CÆC , and /or second C C com and C4- 178K . In some aspects , the first CÆC , and /or second prise the residues Chl - 186E , and C? - 131H . In some aspects , C?C , comprise the residues Chl - 143E , and Cl - 178K . the first C / C , and /or second CÆC , comprise the residues In some aspects , the first CÆC , and /or second CÆCz C hl - 186D , and C2 -131H . comprise the residues Ch1 -143D , C4 - 178K , and C4- 176M . 60 In some aspects , the first CaC , and /or second CÆC , In some aspects , the first ChC and / or second CACL comprise the residues Ch1 - 143S , C2- 131D , CH1 - 188W , comprise the residues Chl - 143E , and C - 131R . In some C? - 133S , and C4 - 178S . In some aspects , the first CXCL aspects , the first ChC , and / or second CaC , comprise the and / or second CÆC , comprise the residues Ch1 - 143S , Chl residues Cyl- 143D , and C . - 131R . In some aspects , the first 188W , and C? - 131D . In some aspects , the first CÆC , and /or C?C and /or second CÆC , comprise the residues Ch1- 143E , 65 second CÆC , comprise the residues Chl - 143T , CH1 - 188W , and C - 131K . In some aspects , the first CÆC , and /or second and C4 - 131D . In some aspects , the first CÆC , and/ or second C?C , comprise the residues Chl- 143D , and C? - 131K . CÆC comprise the residues Chl - 143S , Chl- 188W , and US 10 , 392 , 438 B2 31 32 C1- 131E . In some aspects , the first CÆC , and /or second 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and C?C , comprise the residues Chl- 143T, CH1- 188W , and 99 % . In some aspects , the invention comprises a Cyl CL - 131E. domain comprising a sequence identical to SEQ ID NO : 8 , In some aspects , the first CÆC? and /or second CÆC by at least an amount selected from the group consisting of comprise the residues Chl- 143S , Chl- 188W , C4 - 133M , 5 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , C?- 176G , and C4 - 1786 . In some aspects , the first CÆC 95 % , 96 % , 97 % , 98 % , and 99 % . and / or second CÆC , comprise the residues Chl - 143S , Chl In some aspects , the invention comprises a Cyl domain 188W , and Cz- 133M . In some aspects , the first CÆC? and /or comprising a sequence identical to SEQ ID NO : 33 , by at second CÆC , comprise the residues Ch1- 143T, CH1 - 188W , least an amount selected from the group consisting of 85 % , and C4- 133M . 10 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % 94 % , 95 % , In some aspects , the first Cycy and /or second Cyc? 96 % , 97 % , 98 % , and 99 % . In some aspects , the invention comprise the residues Chl- 143H , Chl - 179D , CH1- 186E , comprises a Chl domain comprising a sequence identical to Cr- 131H , CH - 1901 , C2 - 1351, Chl - 174C , CH1 -230S , 176C , SEQ ID NO :34 , by at least an amount selected from the and C -214S . In some aspects , the first CÆC , and /or second group consisting of 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , CAC , comprise the residues Cy1 - 143H , C71- 179D , Chl- 15 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 99 % . In some 186E , and C4- 131H . In some aspects, the first C4C , and /or aspects , the invention comprises a Cyl domain comprising second CÆC , comprise the residues Cyl- 143H , CH1- 179E , a sequence identical to SEQ ID NO :35 , by at least an amount Cyl- 186E , and Cl - 131H . In some aspects , the first C CL selected from the group consisting of 85 % , 86 , 87 % , 88 % , and / or second ChC , comprise the residues Chl- 143H , Chl 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 179D , Cyl- 186D , and CL - 131H . In some aspects , the first 20 and 99 % . In some aspects, the invention comprises a Cyl CÆC , and / or second ChC , comprise the residues Chl- 143H , domain comprising a sequence identical to SEQ ID NO : 36 , Chl - 179E , Chl- 186D , and C4- 131H . by at least an amount selected from the group consisting of In some aspects , the first CÆC , and /or second C C 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , comprise the residues Cy - 186E , C7 - 131H , C7- 145S , Cyl - 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects, the 139C , CH1 -230S , C4 - 116C , and CL - 214S . 25 invention comprises a Cyl domain comprising a sequence In some aspects , the first CuCz and /or second ChC identical to SEQ ID NO : 37 , by at least an amount selected comprise the residues Chl- 143S , C - 131D , CH1- 188W , from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , C2- 133S , C4- 178S , Chl- 174C , Ch1- 230S , C4 - 176C , and 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and C [ - 214S . 99 % . In some aspects , the invention comprises a Cyl In some aspects , the first CÆC , and /or second CyCZ 30 domain comprising a sequence identical to SEQ ID NO :38 , comprise the residues Cyl- 143S , Chl- 188W , C4 - 133M , by at least an amount selected from the group consisting of C4 - 178G , C -176G , CH1 -122C , CH1- 230S , C - 123C , and 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , CL - 214S . 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the In some aspects , the first CÆC? and /or second CÆC invention comprises a Cal domain comprising a sequence comprise the residues Chl- 143S , C7 - 131D , Chl- 188W , 35 identical to SEQ ID NO :39 , by at least an amount selected C1 - 133S , Cz -178S , Cyl - 122C , CH - 139C , CH - 174C , Chl - from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , 230S , C7- 116C , C7- 123C , C2 - 176C , and C -214S . 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and In some aspects , the invention comprises a Chl domain 99 % . In some aspects , the invention comprises a Chl comprising a sequence identical to SEQ ID NO : 1 , by at least domain comprising a sequence identical to SEQ ID NO :40 , an amount selected from the group consisting of 85 % , 86 , 40 by at least an amount selected from the group consisting of 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , 97 % , 98 % , and 99 % . In some aspects , the invention com - 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the prises a Cyl domain comprising a sequence identical to invention comprises a Cyl domain comprising a sequence SEQ ID NO :2 , by at least an amount selected from the group identical to SEQ ID NO :41 , by at least an amount selected consisting of 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % 92 % , 45 from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 99 % . In some 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and aspects , the invention comprises a Cyl domain comprising 99 % . a sequence identical to SEQ ID NO : 3 , by at least an amount In some aspects , the invention comprises a C , domain selected from the group consisting of 85 % , 86 , 87 % , 88 % , comprising a sequence identical to SEQ ID NO : 9 , by at least 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 50 an amount selected from the group consisting of 85 % , 86 , and 99 % . In some aspects , the invention comprises a Cyl 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , domain comprising a sequence identical to SEQ ID NO : 4 , 97 % , 98 % , and 99 % . In some aspects , the invention com by at least an amount selected from the group consisting of prises a C , domain comprising a sequence identical to SEQ 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % 92 % , 93 % , 94 % , ID NO : 10 , by at least an amount selected from the group 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the 55 consisting of 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % 92 % , invention comprises a Chl domain comprising a sequence 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 99 % . In some identical to SEQ ID NO : 5 , by at least an amount selected aspects , the invention comprises a C? domain comprising a from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , sequence identical to SEQ ID NO : 11 , by at least an amount 90 % , 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and selected from the group consisting of 85 % , 86 , 87 % , 88 % , 99 % . In some aspects , the invention comprises a Cyl 60 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , domain comprising a sequence identical to SEQ ID NO : 6 , and 99 % . In some aspects , the invention comprises a C , by at least an amount selected from the group consisting of domain comprising a sequence identical to SEQ ID NO : 12 , 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , by at least an amount selected from the group consisting of 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , invention comprises a Cyl domain comprising a sequence 65 95 % , 96 % , 97 % , 98 % , and 99 % . identical to SEQ ID NO : 7 , by at least an amount selected In some aspects , the invention comprises a C , domain from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , comprising a sequence identical to SEQ ID NO :24 , by at US 10 , 392 , 438 B2 33 34 least an amount selected from the group consisting of 85 % , charged amino acid for promoting the heterodimer forma 86 , 87 % , 88 % , 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 95 % , tion have also been described in WO2009 /089004 . 96 % , 97 % , 98 % , and 99 % . In some aspects , the invention In some aspects , the heterodimeric protein of the inven comprises a C? domain comprising a sequence identical to tion further comprises a first CH3 region and a second CH3 SEQ ID NO :25 , by at least an amount selected from the 5 region , which interact together to form a Ch3 interface , group consisting of 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , wherein one or more amino acids within the Cy3 interface 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 99 % . In some destabilizes homodimer formation and are not electrostati aspects , the invention comprises a C , domain comprising a cally unfavorable to homodimer formation . In some embodi sequence identical to SEQ ID NO : 26 , by at least an amount ments , the engineered Cy3 interface sterically favors het selected from the group consisting of 85 % , 86 , 87 % , 88 % , 10 erodimer formation over homodimer formation . In some 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , embodiments , the engineered Cy3 interface electrostatically and 99 % . In some aspects, the invention comprises a C favors heterodimer formation over homodimer formation . domain comprising a sequence identical to SEQ ID NO : 27 , In some embodiments , the amino acid modification in the by at least an amount selected from the group consisting of first Cy3 polypeptide is an amino acid substitution at C , 3 85 % , 86 , 87 % 88 % , 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 15 391, and the amino acid modification in the second Cy3 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the polypeptide is an amino acid substitution at C , 3 -441 (ac invention comprises a C , domain comprising a sequence cording to the numbering of SEQ ID NO : 18 ) . In some identical to SEQ ID NO :28 , by at least an amount selected embodiments , the amino acid modification in the first CH3 from the group consisting of 85 % , 86 , 87 % 88 % , 89 % , polypeptide is Cy3 -441R and the amino acid modification in 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 20 the second C3polypeptide is C73 - 391E or C73 -391D ( for 99 % . In some aspects , the invention comprises a C , domain greater detail, see WO2011 / 143545 ) . In some embodiments , comprising a sequence identical to SEQ ID NO : 29, by at the bispecific antibodies further comprise amino acid modi least an amount selected from the group consisting of 85 % , fication in the first hinge region at positions CH2- D232 and 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , CH2- P241 of SEQ ID NO : 42 ( hinge IgG1) , or CH2- C233 , 96 % , 97 % , 98 % , and 99 % . In some aspects , the invention 25 CH2- E237, and Ch2 -P241 of SEQ ID NO : 79 ( IgG2 hinge comprises a C , domain comprising a sequence identical to sequence ) in one arm , and the substituted /replaced amino SEO ID NO : 30 , by at least an amount selected from the acid in the first hinge region has an opposite charge to the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , corresponding amino acid in the second hinge region in 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 99 % . In some another arm ( for greater detail, see WO2011/ 143545 ). For aspects , the invention comprises a C , domain comprising a 30 example , the amino acid modification in the hinge region sequence identical to SEQ ID NO :31 , by at least an amount can be CH2- D232R , CH2 -D232E , CH2 -P241R , and/ or CH2 selected from the group consisting of 85 % , 86 , 87 % , 88 % , P241E . In another example , the amino acid modification in 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , the hinge region can be C42 -C233D , CH2 - C233E , CH2 and 99 % . In some aspects , the invention comprises a C C233K , CH2 -C223R , CH2 - E237E , CH2 - E237K , CH2 domain comprising a sequence identical to SEQ ID NO : 32 , 35 E237R , CH2 -P241D , CH2 - P241E , CH2 -P241K , and /or Ch2 by at least an amount selected from the group consisting of P228R . In some aspects , the Cy3 domain is selected from 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % the group consisting of SEQ ID NO :82 , 83 , 84 , and 85 . 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the invention comprises a Ch2 domain Modification to constant domains of antibodies to pro - comprising a sequence identical to SEQ ID NO : 13 , by at duce heterodimers are disclosed in U . S . Pat. No. 5 ,731 , 168 , 40 least an amount selected from the group consisting of 85 % , WO2009089004 , and WO2011143545 , each of whose con 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , tents is herein incorporated in its entirety . 96 % , 97 % , 98 % , and 99 % . In some aspects , the invention CH2 and Ch3 Domains comprises a Cu2 domain comprising a sequence identical to In some aspects , the first Cyl is connected to a first CH2 SEQ ID NO : 14 , by at least an amount selected from the domain (Cy2 ), and the second Cyl is connected to second 45 group consisting of 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , C42 . The first and second Cu2 may each comprise a first and 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 99 % . In some second Cy2 mutant residue respectively , the first and second aspects , the invention comprises a Cy2 domain comprising Ch2 mutant residues being engineered to differ from each a sequence identical to SEQ ID NO : 15 , by at least an amount other, and preferentially interact with each other and thereby selected from the group consisting of 85 % , 86 , 87 % , 88 % , form Ch2 heterodimers preferentially over the formation of 50 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , CH2 homodimers . and 99 % . In some aspects , the invention comprises a C 2 In some aspects , the heterodimeric protein of the inven - domain comprising a sequence identical to SEQ ID NO : 16 , tion further comprises a first Cy2 region and second Cy2 by at least an amount selected from the group consisting of region , which interact together to form a C , 2 interface , 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , wherein one or more amino acids within the Cy2 interface 55 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the destabilize homodimer formation and are not electrostati - invention comprises a Cy2 domain comprising a sequence cally unfavorable to homodimer formation . identical to SEQ ID NO : 17 , by at least an amount selected In some aspects , the first Chl or Ch2 is connected to a from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , first Cy3 domain (CH3 ), and the second Chl or CH2 is 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and connected to second Cy3 . The first and second Cy3 may 60 99 % . In some aspects , the invention comprises a Cy2 each comprise a first and second Cy3 mutant residue respec - domain comprising a sequence identical to SEQ ID NO :45 , tively , the first and second Cy3 mutant residues being by at least an amount selected from the group consisting of engineered to differ from each other, and preferentially 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , interact with each other and thereby form Cy3 heterodimers 95 % , 96 % , 97 % , 98 % , and 99 % . preferentially over the formation of Cy3 homodimers. Tech - 65 In some aspects , the invention comprises a Cp3 domain niques involving replacing one or more residues that make comprising a sequence identical to SEQ ID NO : 18 , by at up the CH3 -CH3 interface in both CH3 domains with a least an amount selected from the group consisting of 85 % , US 10 , 392 , 438 B2 35 36 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , selected from the group consisting of IgG1, IgG2, IgGz, 96 % , 97 % , 98 % , and 99 % . In some aspects, the invention IgG4, IgA , or IgA ,) . For example , the antibody of the comprises a Ch3 domain comprising a sequence identical to invention may comprise a first Fab from one antibody SEO ID NO : 19 , by at least an amount selected from the sub - class ( for example , selected from the group consisting group consisting of 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 5 of IgG , IgG , IgG , IgG , IgA , or IgA2) , and a second Fab 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and 99 % . In some from a different sub - class ( for example , selected from the aspects , the invention comprises a Cy3 domain comprising group consisting of IgG , IgG , IgGz, IgG4, IgA , or IgA2, a sequence identical to SEQ ID NO : 20 , by at least an amount provided the second Fab is of a different subclass to the first selected from the group consisting of 85 % , 86 , 87 % , 88 % , Fab ) , and first and second Cu2 domains and first and second 89 % , 90 % , 91 % 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 10 CH3 domains from a single antibody class ( for example , and 99 % . In some aspects , the invention comprises a Ch3 selected from the group consisting of IgG1, IgG2, IgGz, domain comprising a sequence identical to SEQ ID NO : 21 , IgG4, IgA , or IgA ) . by at least an amount selected from the group consisting of In another aspect of the invention , the antibody or Fab 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , region thereof (e . g ., bispecific antibody ) as described herein 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the 15 comprises a full - length human antibody , wherein a first invention comprises a CH3 domain comprising a sequence antibody variable domain of the antibody or Fab thereof is identical to SEQ ID NO : 22 , by at least an amount selected capable of recruiting the activity of a human immune from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , effector cell by specifically binding to an effector antigen 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and located on the human immune effector cell , wherein a 99 % . In some aspects , the invention comprises a Cy3 20 second antibody variable domain of the heterodimeric pro domain comprising a sequence identical to SEQ ID NO :23 , tein is capable of specifically binding to a target antigen . In by at least an amount selected from the group consisting of some embodiments , the human antibody has an IgG1, IgG2, 85 % , 86 , 87 % 88 % , 89 % , 90 % , 91 % 92 % 93 % 94 % , IgGz, or IgG , isotype . 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the Except where indicated otherwise by context, the terms invention comprises a Cy3 domain comprising a sequence 25 “ first” and “ second ” , and variations thereof, are merely identical to SEQ ID NO : 46 , by at least an amount selected generic identifiers, and are not to be taken as identifying a from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , specific or a particular Chl , C?, V , V1, CH2 , CH3, or Fab . 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and In another aspect of the invention , a heterodimeric protein 99 % . In some aspects , the invention comprises a Cy3 disclosed herein may be deimmunized to reduce immuno domain comprising a sequence identical to SEQ ID NO : 47 , 30 genicity upon administration to a subject using known by at least an amount selected from the group consisting of techniques such as those described , e . g . in PCT Publication 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , WO98 /52976 and W000 /34317 . 95 % , 96 % , 97 % , 98 % , and 99 % . In some aspects , the In other embodiments , a heterodimeric protein may be invention comprises a Ch3 domain comprising a sequence modified or derivatized , such as by making a fusion antibody identical to SEQ ID NO : 48 , by at least an amount selected 35 or immunoadhesin that comprises all or a portion of the from the group consisting of 85 % , 86 , 87 % , 88 % , 89 % , heterodimeric polypeptide , e . g . bispecific antibody dis 90 % , 91 % , 92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , and closed herein , linked to another polypeptide or molecular 99 % . In some aspects , the invention comprises a Cy3 agent. Heteromultimeric , e . g . heterodimeric polypeptides domain comprising a sequence identical to SEQ ID NO :49 , disclosed herein ( e . g . , bispecific antibodies ) may be modi by at least an amount selected from the group consisting of 40 fied or derivatized , for example , to extend in vivo half- lives, 85 % , 86 , 87 % , 88 % , 89 % , 90 % , 91 % , 92 % , 93 % , 94 % , by producing more stable fusion molecules and/ or by treat 95 % , 96 % , 97 % , 98 % , and 99 % . ment with biocompatible polymers such as polyethylene In some aspects , the invention further comprises a IgG glycol (PEG ) , commonly referred to as “ pegylation , ” or by hinge region between the Chl and Ch2 region . The IgG any of a number of other engineering methods well known hinge region may comprise SEQ ID NO : 42 . The IgG hinge 45 in the art . region may comprise SEQ ID NO :43 . The IgG hinge region A heterodimeric protein may be derivatized with a chemi may comprise SEQ ID NO : 44 . The IgG hinge region may be cal group , including but not limited to polyethylene glycol a IgG2 hinge region , and may comprise SEQ ID NO : 79 . (PEG ) , a methyl or ethyl group , an ester, a carbohydrate Ig Isotype and Subclass group and the like , using well known techniques . These In some embodiments , the heterodimeric protein may 50 chemical groups ( and others like them which have been used comprise one or more IgA domains . In some embodiments, to stability therapeutic compounds in vivo ) are useful to the heterodimeric protein may comprise one or more IgD improve the biological characteristics of the heterodimeric domains . In some embodiments , the heterodimeric protein polypeptide , e . g . , to increase serum half - life and bioactivity . may comprise one or more IgE domains . In some embodi- A heterodimeric protein may also be labeled using any of ments , the heterodimeric protein may comprise one or more 55 a multitude of methods known in the art . As used herein , the IgG domains. In some embodiments , the heterodimeric terms“ label” or “ labeled ” refers to incorporation of another protein may comprise one or more IgM domains . molecule in the antibody. In one embodiment, the label is a In some embodiments , at least one Fab is an IgA1, or detectable marker , e . g ., incorporation of a radio labeled IgA2 . In some embodiments , at least one Fab is an IgGi, amino acid or attachment to a polypeptide of biotinyl IgG2 , IgG3 , or IgG4 . In some embodiments , the IgG Fab 60 moieties that can be detected by marked avidin ( e . g ., strepta comprises a human IgG Fab ( e . g . IgG , IgG2, IgGz, or IgG4) . vidin containing a fluorescent marker or enzymatic activity In some embodiments , the first and second Fab are the same that can be detected by optical or colorimetric methods ) . In subclass ( i . e . both are IgG , or both are IgG , or both are another embodiment, the label or marker can be therapeutic , IgGz, or both are IgGa) . e . g ., a drug conjugate or toxin . Various methods of labeling In alternative embodiments , the first Fab is of a different 65 polypeptides and glycoproteins are known in the art and may subclass to the second Fab ( i. e . the first Fab and second Fab be used . Examples of labels for polypeptides include, but are may each be of a different subclass , and each may be not limited to : radioisotopes or radionuclides (e . g. , 3H , 14C , US 10 , 392 , 438 B2 37 38 15N , 35S , 90Y , 99Tc, 111 In , 1251, 1311) , fluorescent labels the expression of antibody chain genes in a host cell. It will ( e . g ., FITC , rhodamine , lanthanide phosphors ), enzymatic be appreciated by those skilled in the art that the design of labels ( e . g . , horseradish peroxidase , B - galactosidase, the expression vector, including the selection of regulatory luciferase , alkaline phosphatase ) , chemiluminescent mark sequences may depend on such factors as the choice of the ers , biotinyl groups , predetermined polypeptide epitopes 5 host cell to be transformed , the level of expression of protein recognized by a secondary reporter ( e . g ., leucine zipper pair desired , etc . Preferred regulatory sequences for mammalian sequences , binding sites for secondary antibodies , metal host cell expression include viral elements that direct high binding domains, epitope tags ) , magnetic agents , such as levels of protein expression in mammalian cells , such as gadolinium chelates , toxins such as pertussis toxin , taxol, promoters and / or enhancers derived from retroviral LTRS, cytochalasin B , gramicidin D , ethidium bromide , emetine , 10 cytomegalovirus (CMV ) ( such as the CMV promoterlen mitomycin , etoposide , tenoposide , vincristine, vinblastine , hancer ) , Simian Virus 40 (SV40 ) ( such as the SV40 pro colchicine, doxorubicin , daunorubicin , dihydroxy anthracin moter/ enhancer ) , adenovirus, (e . g . the adenovirus major late dione , mitoxantrone , mithramycin , actinomycin D , 1 - dehy - promoter (AdMLP ) ) , polyoma and strong mammalian pro drotestosterone , glucocorticoids, procaine, tetracaine , lido moters such as native immunoglobulin and actin promoters. caine , propranolol, and puromycin and analogs or homologs 15 In addition to the antibody chain genes and regulatory thereof. In some embodiments , labels are attached by spacer sequences , the recombinant expression vectors of the inven arms of various lengths to reduce potential steric hindrance . tion may carry additional sequences , such as sequences that Nucleic Acids and Methods of Producing Polypeptides and regulate replication of the vector in host cells (e . g . origins of Heterodimeric Proteins of the Invention replication ) and selectable marker genes. For example , typi In some embodiments , different nucleic acid molecules 20 cally the selectable marker gene confers resistance to drugs, encode one or more chains or portions of the heterodimeric such as G418 , hygromycin or methotrexate , on a host cell protein , e . g . bispecific antibody disclosed herein . In other into which the vector has been introduced . For example , embodiments , the same nucleic acid molecule encodes a selectable marker genes include the dihydrofolate reductase heterodimeric protein disclosed herein . (DHFR ) gene ( for use in dhfr host cells with methotrexate In one aspect, the present invention provides a nucleic 25 selection /amplification ) , the neo gene ( for G418 selection ) , acid sequence encoding one of the chains of a heterodimeric and the glutamate synthetase gene . protein disclosed herein , or portion thereof as described The term “ expression control sequence " as used herein above . Nucleic acid molecules of the invention include means polynucleotide sequences that are necessary to effect nucleic acids that hybridize under highly stringent condi the expression and processing of coding sequences to which tions, such as those at least about 70 % , 75 % , 80 % , 85 % , 30 they are ligated . Expression control sequences include 90 % , 95 % , 97 % , 98 % or 99 % or more identical to a nucleic appropriate transcription initiation , termination , promoter acid sequence of the invention . and enhancer sequences ; efficient RNA processing signals In some aspects, the nucleic acid is DNA . In some such as splicing and polyadenylation signals ; sequences that aspects , the nucleic acid is RNA . In some aspects , the stabilize cytoplasmic mRNA ; sequences that enhance trans nucleic acid is mRNA . In some aspects , the nucleic acid is 35 lation efficiency ( i . e . , Kozak consensus sequence ) ; a non -natural nucleic acid , such as PNA (peptide nucleic sequences that enhance protein stability ; and when desired , acid ), morpholino and locked nucleic acid , glycol nucleic sequences that enhance protein secretion . The nature of such acid , and threose nucleic acid . control sequences differs depending upon the host organism ; In a further aspect, the present invention provides a vector in prokaryotes , such control sequences generally include comprising a nucleic acid sequence encoding one or more of 40 promoter, ribosomal binding site , and transcription termina the chains or portions of the heteromultimeric or heterodi- tion sequence ; in eukaryotes , generally, such control meric protein disclosed herein , or portion thereof as sequences include promoters and transcription termination described herein . sequence . The term " control sequences ” is intended to In a further aspect, the present invention provides a vector include , at a minimum , all components whose presence is suitable for expressing one or more of the chains or portions 45 essential for expression and processing, and can also include of the heterodimeric protein disclosed herein , or portion additional components whose presence is advantageous , for thereof as described herein . In some aspects , the invention example , leader sequences and fusion partner sequences . provides for a vector that comprises a nucleic acid of the In some aspects, the invention comprises a nucleic acid invention . encoding at least one Cyl, or C , of the invention . The In another embodiment, a nucleic acid molecule of the 50 invention further provides for nucleic acids that encode for invention is used as a probe or PCR primer for a specific a Fab of the invention . In some aspects , the invention amino acid sequence , e . g . a specific antibody sequence such provides for a nucleic acid that encodes for a first Fab of the as in the hinge and constant heavy domain sequences . For invention . In some aspects , the invention provides for a instance , the nucleic acid can be used as a probe in diag - nucleic acid that encodes for a second Fab of the invention . nostic methods or as a PCR primer to amplify regions of 55 In some aspects , the invention provides for a nucleic acid DNA that could be used , inter alia , to isolate additional that encodes a first heavy chain of the invention . In some nucleic acid molecules encoding useful sequences . In some aspects , the invention provides for a nucleic acid that embodiments , the nucleic acid molecules are oligonucle - encodes a second heavy chain of the invention . In some otides. In some embodiments, the oligonucleotides are from aspects , the invention provides for a nucleic acid that hinge and constant domain regions of the heavy and light 60 encodes a first light chain of the invention . In some aspects , chains of an antibody of interest. In some embodiments , the the invention provides for a nucleic acid that encodes a oligonucleotides encode all or a part of one or more of the second light chain of the invention . modified Fab regions of the heterodimeric polypeptide, e. g . In some aspects , the invention provides for a cell that bispecific antibodies or fragments thereof of the invention as comprises a vector of the invention . In some aspects , the described herein . 65 invention provides for a cell that comprises a nucleic acid of Recombinant expression vectors of the invention may, in the invention . In some aspects , the invention provides for a some embodiments , carry regulatory sequences that control cell that expresses a nucleic acid of the invention . US 10 , 392 , 438 B2 39 40 Favorably, the invention provides for a cell that expresses transfer vehicle must consider penetration of, and retention a heterodimeric protein as herein described . Co -expressing within the blood brain barrier and / or the use of alternate the first CÆC , and second CÆC , in the same cell takes means of directly delivering such transfer vehicle to such advantage of the complementary residue sets that allow for target cell. In some aspects, the compositions of the present correct formation of the heteromultimeric protein . In some 5 invention may be combined with agents that facilitate the aspects , this permits a bispecific antibody to be expressed transfer of exogenous mRNA ( e . g . agents which disrupt or and generated in a fully assembled form , and requires little improve the permeability of the blood brain barrier and to no additional purification or processing steps over what thereby enhance the transfer of exogenous mRNA to the would be typically required for purification of a monoclonal target cells ) . antibody. 10 In some aspects , bispecific antibodies of the invention The use of liposomal transfer vehicles to facilitate the may be used in mRNA replacement therapy or RNA tran delivery of nucleic acids to target cells is contemplated by script therapy . Accordingly, in some aspects , the invention the present invention . In some aspects , the transfer vehicle comprises a cell, or vector, comprising one or more nucleic is formulated as a lipid nanoparticle . Examples of suitable acids encoding one or more polypeptide chains of the 15 lipids include , for example , the phosphatidyl compounds invention , such that expression of the polypeptide chains of ( e . g . , phosphatidylglycerol, phosphatidylcholine , phospha the invention in vivo result in the generation of a bispecific tidylserine , phosphatidylethanolamine , sphingolipids, cere antibody in vivo . Delivery mechanisms for such vectors brosides , and gangliosides ) . Also contemplated is the use of include lipid based systems and nanoparticles ( see for polymers as transfer vehicles , whether alone or in combi example , w02010053572 , WO2012170930 and 20 nation with other transfer vehicles . Suitable polymers may WO2011068810 , each of whose contents is incorporated include , for example , polyacrylates, polyalkycyanoacry entirely ) . lates, polylactide, polylactide -polyglycolide copolymers , In some aspects , the invention further comprises a transfer polycaprolactones , dextran , albumin , gelatin , alginate , col vehicle, defined herein as any of the standard pharmaceutical lagen , chitosan , cyclodextrins , dendrimers and polyethylen carriers , diluents , excipients and the like which can be used 25 imine . in connection with the administration ofbiologically active The invention contemplates the use of lipid nanoparticles agents , including nucleic acids. The compositions and in as transfer vehicles comprising a cationic lipid to encapsu particular the transfer vehicles described herein are capable of delivering nucleic acids of the invention to the target cell. late and /or enhance the delivery ofmRNA into the target cell In some embodiments , the transfer vehicle is a lipid nano - 30 thather will act as a depot for protein production . As used particles , suitable for transferring mRNA to a target cell . herein , the phrase " cationic lipid ” refers to any of a number In some aspects , the invention comprises an mRNA of lipid species that can a net positive charge at a selected encoding a bispecific antibody of the invention , a transfer pH , such as physiological pH . vehicle and , optionally , an agent to facilitate contact with , In one aspect, this invention provides a strategy for and subsequent transfection of a target cell . 355 enhancingcm the formation of a bispecific antibody , by altering In some embodiments the mRNA encoding one or more or engineering an interface between the light chain and the polypeptides of the invention can comprise one or more heavy chain of one or more Fab regions of the antibody. In modifications that confer stability to the mRNA ( e . g . , com - some embodiments , one or more residues that make up the pared to a wild - type or native version of the mRNA ) . For Chl / C , interface of the one more Fab regions are replaced example , the nucleic acids of the present invention may 40 with residues such that the modified residues favor pairing comprise modifications to one or both of the 5 ' and 3 of the specific heavy and light chain of the modified Fab untranslated regions . Such modifications may include , but region over mispairing with heavy chains or light chains of are not limited to , the inclusion of a partial sequence of a other Fab regions in the protein . In one embodiment , the cytomegalovirus (CMV ) immediate - early 1 ( IE1) gene , a modifications introduce novel disulfide bridges in the Fab poly A tail , a Capl structure or a sequence encoding human 45 region . In another embodiment, the modifications introduce growth hormone (hGH ) ) . In some embodiments , the mRNA disrupting mutations that disrupt the native interface is modified to decrease mRNA immunogenicity . between the Cyl and C , domains of a Fab region , as well as In some embodiments , the mRNA of the invention have restoring modifications that introduce non - native stable undergone a chemical or biologicalmodification to render interactions at the interface . In another embodiment, the them more stable . Exemplary modifications to an mRNA 50 disrupting mutations may introduce both novel disulfide include the depletion of a base ( e . g . by deletion or by the bridges and disrupting and restoring mutations . substitution of one nucleotide for another ) ormodification of In some embodiments , the formation of the heterodimeric a base , for example , the chemical modification of a base . In protein comprising one or more amino acid modifications in some aspects, a poly A tail can be added to an mRNA the Cyl / C , interface of one or more Fab regions disclosed molecule thus rendering the mRNA more stable . 55 herein is substantially increased in comparison to the wild In some aspects , the transfer vehicle in the compositions type heterodimeric protein without such modifications . In of the invention is a liposomal transfer vehicle , e . g . a lipid some embodiments , the formation of the heterodimeric nanoparticle . The transfer vehicle may be selected and/ or protein comprising one or more amino acid modifications in prepared to optimize delivery of the mRNA to a target cell. Cyl / C , interface of at least one Fab region is at least about For example, if the target cell is a hepatocyte the properties 60 any of 51 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , of the transfer vehicle ( e . g . , size , charge and / or pH ) may be 95 % , 97 % , 99 % , or 100 % in comparison to the wild - type optimized to effectively deliver such transfer vehicle to the heterodimeric protein without such modifications. target cell , reduce immune clearance and/ or promote reten - In another aspect, the present invention also provides tion in that target cell. Alternatively , if the target cell is the methods of producing a heteromultimeric protein , e . g . a central nervous system ( e . g . mRNA administered for the 65 heterodimeric protein of the invention , such as a bispecific treatment of neurodegenerative diseases may specifically antibody. In some embodiments , the method comprises the target brain or spinal tissue ), selection and preparation of the steps of: US 10 , 392 , 438 B2 42 a ) cotransfecting a cell line with vectors expressing each “ recombinant host cell” and “ host cell” mean not only the heavy chain and each light chain of each Fab region of the particular subject cell but also the progeny of such a cell. protein ; Because certain modifications may occur in succeeding b ) culturing the cell line under conditions to express each generations due to either mutation or environmental influ heavy chain and each light chain of each Fab region of the 5 ences , such progeny may not, in fact, be identical to the protein and that allow the heteromultimeric protein to parent cell, but are still included within the scope of the term assemble; and " host cell” as used herein . Such cell may comprise a vector c ) purifying the heteromultimeric protein from the cell according to the invention as described above . culture . In some embodiments , the cell line is cotransfected In another aspect, the present invention provides a method with vectors that express the heavy chain and the light chain 10 for making an antibody or portion thereof as described of each Fab region in a 1 : 1 : 1 : 1 ratio . above . According to one embodiment, said method com In some embodiments , the method comprises the steps of: prises culturing a cell transfected or transformed with a ( i ) cotransfecting a cell line with one or more vectors to vector as described above , and retrieving said antibody or express the first Cyl , the first C , of the first CyC , ; and the portion thereof. Nucleic acid molecules encoding antibodies second Cyl, and the second C , of the second C ,Ci 15 and vectors comprising these nucleic acid molecules can be ( i ) culturing the cell line under conditions to express the one used for transfection of a suitable mammalian , plant, bac or more vectors and that allow the first CyC , and second terial or yeast host cell . Transformation can be by any known CHC? to assemble; and method for introducing polynucleotides into a host cell . ( ii ) purifying the heteromultimeric protein from the cell Methods for introduction of heterologous polynucleotides culture . 20 into mammalian cells are well known in the art and include In some aspects , the cell line is cotransfected with vectors dextran -mediated transfection , calcium phosphate precipita that express the first Chl, first C?, second Chl , and second tion , polybrene- mediated transfection , protoplast fusion , C , in a 1 : 1 : 1 : 1 ratio . electroporation , encapsulation of the polynucleotide ( s ) in The skilled artisan can readily determine , using well - liposomes , and directmicroinjection of the DNA into nuclei. known techniques, the relative amounts of molecules or 25 In addition , nucleic acid molecules may be introduced into antibodies to use according to the methods disclosed herein . mammalian cells by viral vectors . Methods of transforming In the methods disclosed herein , incubations may be cells are well known in the art . See , e . g . , U . S . Pat . Nos . performed across a range of temperatures . Such tempera 4 ,399 ,216 , 4 ,912 ,040 , 4 ,740 , 461, and 4 , 959 , 455. Methods of tures will be recognized by those skilled in the art and will transforming plant cells are well known in the art, including , include , for example , incubation temperatures at which 30 e. g . , Agrobacterium -mediated transformation , biolistic deleterious physical changes such as denaturation or decom - transformation , direct injection , electroporation and viral position do not occur in the mixed molecules or antibodies. transformation . Methods of transforming bacterial and yeast In certain embodiments , the incubations are performed at cells are also well known in the art . about 37° C . Mammalian cell lines available as hosts for expression are Any of a number of host cells may be used in methods of 35 well known in the art and include many immortalized cell the invention . Such cells are known in the art ( some of which lines available from the American Type Culture Collection are described herein ) or can be determined empirically with (ATCC ) . These include , inter alia , Chinese hamster ovary respect to suitability for use in methods of the invention ( CHO ) cells , NSO cells , SP2 cells , HEK -293T cells , 293 using routine techniques known in the art . In certain embodi- Freestyle cells ( Invitrogen ) , NIH - 3T3 cells , Hela cells , ments, the host cell is prokaryotic . In some embodiments , a 40 baby hamster kidney (BHK ) cells , African green monkey host cell is a gram -negative bacteria cell. In other embodi - kidney cells (COS ) , human hepatocellular carcinoma cells ments , a host cell is E . coli. In some embodiments , the E . (e .g ., Hep G2) , A549 cells , and a number of other cell lines. coli is of a strain deficient in endogenous protease activities . Cell lines of particular preference are selected through In some embodiments , the genotype of an E . coli host cell determining which cell lines have high expression levels . lacks degP and prc genes and harbors a mutant spr gene . In 45 Other cell lines that may be used are insect cell lines , such other embodiments of the invention , the host cell is mam - as Sf9 or Sf21 cells. When recombinant expression vectors malian , for example , a Chinese Hamster Ovary (CHO ) cell. encoding antibody genes are introduced into mammalian In some embodiments , methods of the invention further host cells, the antibodies are produced by culturing the host comprise expressing in a host cell a polynucleotide or cells for a period of time sufficient to allow for expression recombinant vector encoding a molecule the expression of 50 of the antibody in the host cells or , more preferably , secre which in the host cell enhances yield of a bispecific antibody tion of the antibody into the culture medium in which the or a heterodimeric protein of the invention . For example , host cells are grown . Antibodies can be recovered from the such molecule can be a chaperone protein . In one embodi- culture medium using standard protein purification methods . ment, said molecule is a prokaryotic polypeptide selected Suitable plant host cells may include , e. g ., Nicotiana, Ara from the group consisting of DsbA , DsbC , DsbG and FkpA . 55 bidopsis , duckweed , corn , wheat, potato , etc . Suitable bac In some embodiments of these methods , the polynucleotide terial host cells may include, e . g . , E . coli and Streptomyces encodes both DsbA and DsbC . species . Suitable yeast host cells may include , e . g ., Schi In one aspect , the present invention provides recombinant zosaccharomyces pombe , Saccharomyces cerevisiae and host cells allowing the recombinant expression of the anti - Pichia pastoris . bodies of the invention or portions thereof . Antibodies 60 Expression of polypeptides of the invention or portions produced by such recombinant expression in such recom - thereof from production cell lines can be enhanced using a binant host cells are referred to herein as “ recombinant number of known techniques. For example , the glutamine antibodies” . The present invention also provides progeny synthetase gene expression system ( the GS system ) is a cells of such host cells, and antibodies produced by same. common approach for enhancing expression under certain The term “ recombinant host cell ” (or simply " host cell” ) , as 65 conditions. The GS system is discussed in whole or part in used herein , means a cell into which a recombinant expres - connection with EP0216846 , EP0256055 , EP0323997 , and sion vector has been introduced . It should be understood that EP0338841 . US 10 , 392 ,438 B2 43 44 It is likely that polypeptides comprising Fc polypeptides encoding proteins, domains and antibodies of the present or Fc regions and immunoglobulin -like hinge polypeptides, invention , and vectors comprising said polynucleotides. In such as antibodies , as expressed by different cell lines or in some aspects , the invention provides for a method of pro transgenic animals , will differ from each other in their ducing an antibody , immunoglobulin domain , or protein , glycosylation patterns . All such " glycoforms” of polypep - 5 comprising culturing a host cell under conditions that result tides of the invention , including all heterodimers of poly in production of the antibody , immunoglobulin domain , or peptides comprising immunoglobulin - like hinge sequences , protein , and isolating the antibody , immunoglobulin domain , bispecific polypeptides , antibodies and the like, are consid ered to be part of the instant invention , regardless of their or protein , from the host cell or culture . glycosylation state, and more generally , regardless of the 10 The invention provides improved methods, compositions, presence or absence of any post - translational modifi kits and articles of manufacture for generating heteromulti cation ( s ) . meric complex molecules, more preferably , heterodimeric In some embodiments , heterodimeric protein is an anti proteins, such as , e . g . a bispecific antibody . The invention body, a maxibody, a monobody, a peptibody, an Fc fusion provides methods to make and to purify heteromultimeric protein , or Fab region of any of the foregoing . In some 15 complex molecules in yields and purities desirable for embodiments , the heterodimeric protein is a bispecific anti commercial manufacture of biotherapeutics . The invention body . makes possible efficient production of complex molecules The heterodimeric protein thereof may comprise one or that, in turn , can be used for diagnosing and / or treating more human domains . The heterodimeric protein may com various disorders or conditions where use of multispecific prise one ormore humanized Ig domains. The heterodimeric 20 antibodies is desirable and /or required . Details of methods, protein may comprise one or more murine Ig domains. The compositions , kits and articles ofmanufacture of the inven heterodimeric protein may comprise one or more lg domains tion are provided herein . originating from a species selected from the group consist - Methods of Using Proteins of the Invention ing of human , monkey , mouse , rat , hamster , guinea pig , The present invention also provides various therapeutic rabbit , dog , cat, donkey, goat, camel, cow , horse , pig , 25 applications for the proteins of the invention . In one aspect , chicken , and shark . the proteins of the invention can be used for treating various mammalianIn some , avianaspects , or, theSqualiform antibodies in origin of the (notwithstanding invention are diseases ( e .g . cancer , autoimmune diseases , or viral infec the method used to generate any artificially mutated or tions ) by binding the first protein ( e . g . first human antibody otherwise engineered versions ). The mammalian , avian , or 30 variable domain ) to an effector antigen and by binding the squaliform species may be human , mouse, rabbit, rat, second protein ( e .g . second human antibody variable rodent, pig , cow , sheep , goat, donkey , horse , camel, llama, domain ) to a target antigen . For example , the proteins of the primate , monkey, dog , cat, chicken , or spiny dogfish . The invention can be used for redirecting cytotoxicity, delivering antibodies of the invention may be humanized . thrombolytic agents to clots , for delivering immunotoxins to In some aspects , the invention comprises mutant antibodd -. 3535 cmtumor cells , or for converting enzyme activated prodrugs at ies and portions thereof, wherein a mutant is defined as a target site (e .g . a tumor ). sequence that has been engineered or altered to a sequence In another aspect, the proteins of the invention can be other than its natural canonical sequence , such that certain used for increasing specificity of a therapeutic agent and / or embodiments of polypeptides of the invention specifically modulating synergistic or additive pathways ( e . g . metabolic excludes naturally occurring sequences that fall within the 40 or biochemical pathways ) . For example, the proteins of the scope of the definition . In some aspects , therefore , the invention can engage receptor/ receptor, receptor/ ligand , present invention relates to polypeptides of the invention ligand /ligand , cell /cell , ligand /payload , receptor/ payload , or comprising mutations to enable heterodimeric Ig -domain single receptor. pairing such that the Ig domain polypeptide sequence differs Pharmaceutical Compositions from its naturally occurring corresponding sequence . 45 The invention provides a pharmaceutical composition Antibody Cyl domains may be selected from the group comprising proteins of the invention of the invention and a consisting of Cyal, Cydi, Chel, Chy1, and Caul. pharmaceutically acceptable carrier . As used herein , “ phar In some aspects , the constant light chain ( C ) domain of maceutically acceptable carrier ” includes any and all sol the invention is connected to a variable light chain ( V ) vents , dispersion media , coatings , antibacterial and antifun domain . Together, these may comprise an antibody light 50 gal agents , isotonic and absorption delaying agents , and the chain . The C , domain may be a C , K (constant light chain like that are physiologically compatible . Examples of phar kappa) . The C? domain may be a Cà ( constant light chain maceutically acceptable carriers include one or more of lambda ). water, saline , phosphate buffered saline, dextrose , glycerol, In some aspects , the Cyl domain of the invention is ethanol and the like , as well as combinations thereof, and connected to a variable heavy chain (Vu ) domain . Together, 55 may include isotonic agents, for example , sugars, polyalco these may comprise the heavy chain portion of a Fab hols such as mannitol, sorbitol, or sodium chloride in the molecule . In some aspects , the Vh and Chl domains are composition , and may include pharmaceutically acceptable connected to the remainder of the Ch domains typical for substances such as wetting or minor amounts of auxiliary that particular Ig isotype ( i . e . Cjal may be connected to substances such as wetting or emulsifying agents , preserva Cya2, and Cya3; Cy81 may be connected to Cy82 and 60 tives or buffers , which enhance the shelf life or effectiveness C?d3 ; Cykl may be connected to Cy£2 , Cy£3 , and Cy€4 ; of the antibody or antibody portion . Chyl may be connected to ChY2 , and Chy3 ; Chul may be In certain embodiments , the proteins of the invention may connected to Chu2 , CH43, and Ch?4 ). be present in a neutral form ( including zwitter ionic forms ) In some aspects , the invention provides for an isolated or as a positively or negatively -charged species. In some host cell that recombinantly produces an antibody of the 65 embodiments , the polypeptides may be complexed with a present invention . The present invention provides for an counterion to form a " pharmaceutically acceptable salt, ” isolated polynucleotide comprising a nucleotide sequence which refers to a complex comprising one or more poly US 10 , 392 ,438 B2 45 46 peptides and one or more counterions, where the counterions PEG -derivatized phosphatidylethanolamine ( PEG -PE ) . are derived from pharmaceutically acceptable inorganic and Liposomes are extruded through filters of defined pore size organic acids and bases . to yield liposomes with the desired diameter . The compositions of this invention may be in a variety of The active ingredients may also be entrapped in micro forms. These include , for example , liquid , semi- solid and 5 capsules prepared , for example , by coacervation techniques solid dosage forms, such as liquid solutions ( e . g . injectable or by interfacial polymerization , for example , hydroxym and infusible solutions ), dispersions or suspensions , tablets , ethylcellulose or gelatin -microcapsules and poly - (methyl pills , powders , liposomes and suppositories . The preferred methacrylate ) microcapsules , respectively , in colloidal drug form depends on the intended mode of administration and delivery systems ( for example , liposomes, albumin micro therapeutic application . Typical preferred compositions are 10 spheres , microemulsions, nano - particles and nanocapsules ) in the form of injectable or infusible solutions , such as or in macroemulsions. Such techniques are disclosed in compositions similar to those used for passive immunization Remington , The Science and Practice of Pharmacy , 20th of humans with antibodies in general. The preferred mode of Ed ., Mack Publishing ( 2000 ) . administration is parenteral ( e . g . intravenous, subcutaneous , Sustained - release preparations may be prepared . Suitable intraperitoneal, intramuscular ) . In a preferred embodiment, 15 examples of sustained -release preparations include semi the protein of the invention is administered by intravenous permeable matrices of solid hydrophobic polymers contain infusion or injection . In another preferred embodiment, the ing the antibody, which matrices are in the form of shaped protein of the invention is administered by intramuscular or articles , e. g . films, or microcapsules. Examples of sustained subcutaneous injection . release matrices include polyesters , hydrogels (for example , The pharmaceutical composition may further comprise 20 poly ( 2 -hydroxyethyl - methacrylate ), or ‘ poly (vinylalcohol ) ) , another component , such as an anti - tumor agent or an polylactides ( U . S . Pat . No . 3 , 773 ,919 ) , copolymers of L - glu imaging reagent. Another aspect of the present invention tamic acid and 7 ethyl- L - glutamate , non - degradable ethyl provides kits comprising antibodies of the invention and ene- vinyl acetate , degradable lactic acid - glycolic acid copo pharmaceutical compositions comprising these antibodies . lymers such as the LUPRON DEPOTTM injectable A kit may include , in addition to the antibody or pharma- 25 microspheres composed of lactic acid - glycolic acid copoly ceutical composition , diagnostic or therapeutic agents . A kit m er and leuprolide acetate ) , sucrose acetate isobutyrate , and may also include instructions for use in a diagnostic or poly - D -( - )- 3 -hydroxybutyric acid . therapeutic method . In some embodiments , the kit includes The formulations to be used for in vivo administration the antibody or a pharmaceutical composition thereof and a must be sterile . This is readily accomplished by , for diagnostic agent. In other embodiments , the kit includes the 30 example , filtration through sterile filtration membranes . antibody or a pharmaceutical composition thereof and one or Therapeutic compounds of the invention are generally more therapeutic agents , such as an additional antineoplastic placed into a container having a sterile access port, for agent, anti- tumor agent or chemotherapeutic agent. example , an intravenous solution bag or vial having a These agents and compounds of the invention can be stopper pierceable by a hypodermic injection needle. combined with pharmaceutically acceptable vehicles such as 35 An exemplary , non - limiting pharmaceutical composition saline , Ringer ' s solution , dextrose solution , and the like . The of the invention is a formulation as a sterile aqueous solution particular dosage regimen , i . e . dose , timing and repetition , having a pH that ranges from about 5 . 0 to about 6 . 5 and will depend on the particular individual and that individual' s comprising from about 1 mg/mL to about 200 mg/ mL of a medical history . composition comprising a heterodimeric protein of the Acceptable carriers, excipients , or stabilizers are nontoxic 40 invention , from about 1 millimolar to about 100 millimolar to recipients at the dosages and concentrations employed , of histidine buffer, from about 0 .01 mg/mL to about 10 and may comprise buffers such as phosphate , citrate , and mg/ mL of polysorbate 80 , from about 100 millimolar to other organic acids ; salts such as sodium chloride; antioxi- about 400 millimolar of trehalose , and from about 0 . 01 dants including ascorbic acid and methionine ; preservatives millimolar to about 1 . 0 millimolar of disodium EDTA ( such as octadecyldimethylbenzyl ammonium chloride ; hex - 45 dihydrate . amethonium chloride; benzalkonium chloride , benzetho Suitable emulsions may be prepared using commercially nium chloride ; phenol, butyl or benzyl alcohol; alkyl para - available fat emulsions, such as IntralipidTM , LiposynTM , bens, such asmethyl or propyl paraben ; catechol; resorcinol; InfonutrolTM , LipofundinTM and LipiphysanTM The active cyclohexanol; 3 - pentanol; and m - cresol) ; low molecular ingredient may be either dissolved in a pre -mixed emulsion weight ( less than about 10 residues ) polypeptides; proteins , 50 composition or alternatively it may be dissolved in oil ( e . g . such as serum albumin , gelatin , or Igs ; hydrophilic polymers soybean oil , safflower oil, cottonseed oil, sesame oil , corn oil such as polyvinylpyrrolidone; amino acids such as glycine , or almond oil ) and an emulsion formed upon mixing with a glutamine , asparagine , histidine , arginine , or lysine ; mono - phospholipid ( e . g . egg phospholipids , soybean phospholip saccharides , disaccharides , and other carbohydrates includ - ids or soybean lecithin ) and water. It will be appreciated that ing glucose, mannose , or dextrins ; chelating agents such as 55 other ingredients may be added , for example glycerol or EDTA ; sugars such as sucrose , mannitol, trehalose or sor - glucose , to adjust the tonicity of the emulsion . Suitable bitol; salt -forming counterions such as sodium ; metal com emulsions will typically contain up to 20 % oil, for example , plexes ( e . g . Zn -protein complexes ) ; and / or non - ionic sur- between 5 and 20 % . The fat emulsion can comprise fat factants such as TWEENTM , PLURONICSTM or droplets between 0 . 1 and 1 .0 um , particularly 0 .1 and 0 . 5 polyethylene glycol (PEG ). 60 um , and have a pH in the range of 5 . 5 to 8 .0 . Liposomes containing compounds of the invention are The emulsion compositions can be those prepared by prepared by methods known in the art , such as described in mixing a compound of the invention with IntralipidTM or the U . S . Pat. Nos. 4 ,485 , 045 and 4 ,544 ,545 . Liposomes with components thereof ( soybean oil, egg phospholipids , glyc enhanced circulation time are disclosed in U . S . Pat . No . erol and water ) . 5 ,013 ,556 . Particularly useful liposomes can be generated 65 Compositions for inhalation or insufflation include solu by the reverse phase evaporation method with a lipid com - tions and suspensions in pharmaceutically acceptable , aque position comprising phosphatidylcholine , cholesterol and ous or organic solvents , or mixtures thereof, and powders . US 10 , 392 , 438 B2 47 48 The liquid or solid compositionsmay contain suitable phar - Administration of compounds of the invention in accor maceutically acceptable excipients as set out above . In some dance with the method in the present invention can be embodiments , the compositions are administered by the oral continuous or intermittent, depending, for example , upon the or nasal respiratory route for local or systemic effect. Com recipient ' s physiological condition , whether the purpose of positions in preferably sterile pharmaceutically acceptable 5 the administration is therapeutic or prophylactic , and other factors known to skilled practitioners . The administration of solvents may be nebulised by use of gases . Nebulised a compound of the invention may be essentially continuous solutions may be breathed directly from the nebulising over a preselected period of time or may be in a series of device or the nebulising device may be attached to a face spaced doses . mask , tent or intermittent positive pressure breathing 10 Antibodies machine. Solution , suspension or powder compositionsmay An “ Antibody ” is an immunoglobulin molecule capable be administered , preferably orally or nasally , from devices of specific binding to a target or antigen , such as a carbo which deliver the formulation in an appropriate manner . hydrate , polynucleotide, lipid , polypeptide, etc ., through at Compounds and compositions of the invention may be least one antigen -binding site , located in the variable region used in conjunction with established treatments for the 15 of the immunoglobulin molecule . relevant indication . As used herein , unless otherwise indicated by context, the Therapeutic Methods of the Invention term is intended to encompass not only intact polyclonal or Therapeutic methods are also provided by the invention . monoclonal antibodies comprising two identical full - length A therapeutic method comprises administering a compound heavy chain polypeptides and two identical light chain or composition of the invention to a subject in need thereof. 20 polypeptides , but also fragments thereof ( such as Fab , Fab ', As used herein , an " effective dosage ” or “ effective Fab ' ) , , Fv ) , single chain (ScFv ) and domain antibodies amount " of drug , compound , or pharmaceutical composition (dAbs ), including shark and camelid antibodies, and fusion is an amount sufficient to affect any one or more beneficial proteins comprising an antibody portion , multivalent anti or desired results . For prophylactic use , beneficial or desired bodies , multispecific antibodies ( e .g . bispecific antibodies so results include eliminating or reducing the risk , lessening the 25 long as they exhibit the desired biological activity ) and severity , or delaying the outset of the disease , including antibody fragments as described herein , and any other biochemical, histological and /or behavioural symptoms of modified configuration of the immunoglobulin molecule that the disease , its complications and intermediate pathological comprises an antigen recognition site , for example without phenotypes presenting during development of the disease . limitation , minibodies , maxibodies , monobodies , peptibod For therapeutic use , beneficial or desired results include 30 ies, intrabodies, diabodies, triabodies , tetrabodies , V -NAR clinical results such as reducing tumor size , spread , vascu - and bis - scFv . lature of tumors , or one ormore symptomsof cancer or other Antigen -binding portions may be produced by recombi diseases associated with increased angiogenesis , decreasing nant DNA techniques or by enzymatic or chemical cleavage the dose of other medications required to treat the disease , of intact antibodies . Antigen -binding portions include, inter enhancing the effect of another medication , and /or delaying 35 alia , Fab , Fab ', F ( ab ') 2 , Fv , dAb , and complementarity the progression of the disease of patients . An effective determining region (CDR ) fragments , single - chain antibod dosage can be administered in one or more administrations. ies ( scFv ) , chimeric antibodies , diabodies and polypeptides For purposes of this invention , an effective dosage of drug , that contain at least a portion of an Ig that is sufficient to compound , or pharmaceutical composition is an amount confer specific antigen binding to the polypeptide . sufficient to accomplish prophylactic or therapeutic treat- 40 The immunoglobulin ( Ig ) domain is a type of protein ment either directly or indirectly. As is understood in the domain that typically consists of a 2 - layer sandwich of clinical context, an effective dosage of a drug , compound, or between 7 and 9 B -strands arranged in two B -sheets ( al pharmaceutical composition may or may not be achieved in though variations on these arrangements are known ) . A conjunction with another drug , compound , or pharmaceuti - B - strand is a stretch of polypeptide chain typically 3 to 10 cal composition . Thus , an " effective dosage ” may be con - 45 amino acids long with backbone in an almost fully extended sidered in the context of administering one or more thera - conformation . B sheets consist of B - strands connected lat peutic agents , and a single agent may be considered to be erally by at least two or three backbone hydrogen bonds , given in an effective amount if, in conjunction with one or forming a generally twisted , pleated sheet. The backbone of more other agents , a desirable result may be or is achieved . a strand switches repeatedly between interacting with its two An “ individual” or a " subject" is a mammal , more pref - 50 opposite neighboring strands in the sheet, or between sheet erably , a human . Mammals also include , but are not limited and non - sheet interactions for strands at the sheet edge . to , farm animals , sport animals , pets , primates , and horses. Members of the Ig superfamily are found in hundreds of For administration to human subjects , the total monthly proteins of different functions . Examples include antibodies , dose of an antibody of the invention is typically in the range the giant muscle kinase titin and receptor tyrosine kinases . of about 0 . 5 to about 1200 mg per patient, depending on the 55 Ig - like domains may be involved in protein - protein and mode of administration . For example , an intravenous protein — ligand interactions . monthly dose may require about 1 to about 1000 mg/ patient. An immunoglobulin (Ig ) is a heteromultimeric molecule . The total monthly dose may be administered in single or In a naturally occurring Ig , each multimer is composed divided doses and may , at the physician 's discretion , fall primarily of identical pairs of polypeptide chains, each pair outside of the typical range given herein . 60 having one “ light” ( about 25 kDa ) and one “ heavy ” chain An exemplary , non - limiting range for a therapeutically or (about 50 -70 kDa) . prophylactically effective amount of a heterodimeric pro - The amino - terminal portion of each chain includes a tein , e . g . a bispecific antibody or portion thereof, disclosed variable region , of about 100 to 110 or more amino acids herein is about 1 to about 1000 mg/ patient/ month . In certain primarily responsible for antigen recognition . The carboxy embodiments , the heterodimeric protein may be adminis - 65 terminal portion of each chain defines a constant region tered at about 1 to about 200 or about 1 to about 150 primarily responsible for effector function . Human light mg/ patient/month . chains are classified as K and a light chains. Heavy chains US 10 , 392 , 438 B2 49 50 are classified as a , , , Y , and y , and define the antibody' s Exceptwhere indicated otherwise explicitly or by context, isotype as IgA , IgD , IgE , IgG , IgM , respectively . Several of all Cyl residue numbering positions herein described are these classesmay be further subdivided into isotypes: IgG1, according to the numbering of SEQ ID NO : 1 , and all CL IgG2, IgG3, IgG4, IgA1, and IgA2 . residue positions are herein described according to the Within light and heavy chains, the variable and constant 5 numbering of SEQ ID NO : 9 . This numbering is most closely regions are joined by a “ J ” region of about 12 or more amino related to the numbering of Kabat, which is used herein acids , with the heavy chain also including a “ D ” region of except ( a ) in cases such as IgM domain where certain about 10 more amino acids in the context of an entire antibody sequence , the D and J regions are sometimes experimental data has shown Kabat to be incorrect, ( b ) when considered as parts of the variable region after they have 10 Kabat ’s reference is internally inconsistent, or (c ) when been joined ) . The variable regions of each light/heavy chain U otherwise noted . In the original Kabat reference , position pair form the antibody binding site such that an intact Ig has 107A is the first residue of the Cz. Many light chain 2 binding sites . sequences do not have any residue assigned to position Each domain in an antibody molecule has a similar 107A and many also do not have a residue at position 108 . structure of two B - sheets packed tightly against each other in 1515 The first residue of C , is the first residue numbered greater a compressed antiparallel B -barrel . This conserved structure than 107 , whatever that may be . is termed the immunoglobulin ( To ) fold The Jo fold of Cyl domain is a region of protein sequence , preferably constant domains contains two ß sheets packed against each at least 80 residues in length , and having more than 85 % of other, with each strand separated by a contiguous polypep its residues in common with one or more of SEQ ID NO : 1 , tide string ; these contiguous polypeptide strings typically 20 SEQ ID NO : 2 , SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO :5 , comprise a -helices , loops , turns , and short , sharp turns SEQ ID NO : 6 , SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID between two B - sheets called B - hairpins . NO :33 , SEQ ID NO : 34 , SEQ ID NO : 35 , SEQ ID NO : 36 , Variable domains exhibit the same general structure of SEQ ID NO :37 , SEQ ID NO :38 , SEQ ID NO : 39 , SEQ ID relatively conserved framework regions (FR ) joined by 3 NO :40 , or SEQ ID NO :41 . In some aspects , a Cyl domain hypervariable regions , also called complementarity deter - 25 is protein sequence having more than 85 % of its residues in mining regions or CDRs . The CDRs from the 2 chains of common with SEQ ID NO : 1 . each pair are aligned by the framework regions, enabling A Cu2 domain is a is a region of protein sequence , binding to a specific epitope. From N - terminus to C - term preferably at least 80 residues in length , and having more inus , both light and heavy chains comprise the domains FR1, than 85 % of its residues in common with one or more of CDR1, FR2 , CDR2, FR3 , CDR3 and FR4 . 30 SEQ ID NO : 13 , SEQ ID NO : 14 , SEQ ID NO : 15 , SEQ ID The identity of the amino acid residues in a particular NO : 16 , SEQ ID NO :17 , or SEQ ID NO : 45 . In some aspects , antibody that make up a CDR can be determined using a Ch2 domain is protein sequence having more than 85 % of methods well known in the art . For example , antibody CDRs its residues in common with SEQ ID NO : 13 . may be identified as the hypervariable regions originally hinge region is a region of protein sequence having defined by Kabat et al (Kabat et al. , 1991 , Sequences of 35 more than 80 % identity with one or more of SEQ ID NO :42 , Proteins of Immunological Interest, 5th ed . , Public Health SEQ ID NO :43 , SEQ ID NO :44 , SEQ ID NO :79 , SEQ ID Service, NIH , Washington D . C ., NIH Publication No. NO : 80 , or SEQ ID NO :81 . In some aspects , a hinge region 91 - 3242 ) . The positions of the CDRs may also be identified is protein sequence having more than 80 % of its residues in as the structural loop structures described by Chothia and common with SEQ ID NO : 42 . others (Chothia et al ., 1989, Nature 342: 877 -883 ) . Other 40 ACH3 domain is a region ofprotein sequence , preferably approaches to CDR identification include the " AbM defini - at least 80 residues in length , and having more than 85 % of tion ,” which is a compromise between Kabat and Chothia its residues in common with one or more of SEQ ID NO : 18 , and is derived the Abysis program (www .abysis .org ), or the SEQ ID NO : 19 , SEQ ID NO : 20 , SEQ ID NO :21 , SEQ ID “ contact definition ” of CDRs based on observed antigen NO : 22 , SEQ ID NO : 23 , SEQ ID NO :46 , SEQ ID NO :47 , contacts , set forth in MacCallum et al. , 1996 , J . Mol. Biol. , 45 SEQ ID NO : 48 , or SEQ ID NO : 49. In some aspects , a Cy3 262: 732 -745 . North has identified canonical CDR confor- domain is protein sequence having more than 85 % of its mations using a different preferred set of CDR definitions residues in common with SEQ ID NO : 18 . ( North et al. , 2011, J. Mol. Biol, 406 : 228 -256 ). In another AC , domain is a region of protein sequence preferably at approach , referred to herein as the " conformational defini- least 80 residues in length , and having more than 85 % of its tion " of CDRs , the positions of the CDRs may be identified 50 residues in common with one ormore of SEQ ID NO : 9 , SEQ as the residues that make enthalpic contributions to antigen ID NO : 10 , SEQ ID NO : 11 , SEQ ID NO : 12 , SEQ ID NO :24 , binding (Makabe et al. , 2008 , Journal of Biological Chem - SEQ ID NO :25 , SEQ ID NO : 26 , SEQ ID NO : 27 , SEQ ID istry , 283 : 1156 - 1166 ). Still other CDR boundary definitions NO :28 , SEQ ID NO : 29 , SEQ ID NO :30 , SEQ ID NO :31 , or may not strictly follow one of the above approaches , but will SEQ ID NO : 32 . In some aspects , the C , domain is a C , nonetheless overlap with at least a portion of the Kabat 55 kappa domain , and shares at least 85 % identity with one or CDRs, although they may be shortened or lengthened in more of SEQ ID NO : 9 , SEQ ID NO : 10 , or SEQ ID NO : 11 . light of prediction or experimental findings that particular In some aspects , the C domain is a C , lambda domain , and residues or groups of residues or even entire CDRs do not shares at least 85 % identity with SEQ ID NO : 12 . In some significantly impact antigen binding . As used herein , a CDR aspects , a C? domain is protein sequence having more than may refer to CDRs defined by any approach known in the 60 85 % of its residues in common with SEQ ID NO : 9 . art, including combinations of approaches . The methods Mammalian light chains are of two types , K and I , and in used herein may utilize CDRs defined according to any of any given naturally occurring antibody molecule only one these approaches . For any given embodiment containing type occurs . Approximately twice as many Kas à molecules more than one CDR , the CDRs ( or other residue of the are produced in humans but in other mammals this ratio can antibody ) may be defined in accordance with any of Kabat, 65 vary . Each free light chain molecule contains approximately Chothia , North , extended , AbM , contact, and /or conforma - 220 amino acids in a single polypeptide chain that is folded tional definitions . to form the constant and variable region domains. US 10 , 392 , 438 B2 51 52 During B cell development, a recombination event at the “ bifunctional” antibody is a hybrid antibody having two DNA level joins a single variable ( V ) segment with a joining different antigen binding sites . Bispecific antibodies are a ( 1) segment; the constant (C ) segment is later joined by species ofmultispecific antibody and may be produced by a splicing at the RNA level. Recombination ofmany different variety of methods including , but not limited to , fusion of V segments with several J segments provides a wide range 5 hybridomas or linking of Fab ' fragments. See, e .g . Songsiv of antigen recognition . Additional diversity is attained by ilailai & Lachmann ( 19901900 ) , Clin . Exp . Immunol. 79 : 315 -321 ; junctional diversity , resulting from the random additional of and Kostelny et al. ( 1992 ) , J . Immunol. 148 : 1547 - 1553 . The nucleotides by terminal deoxynucleotidyltransferase , and by somatic hypermutation , which occurs during B cell matu two binding sites of a bispecific antibody will bind to two ration in the spleen and lymph nodes. Constant kappa (CLK ) 10 different epitopes, which may reside on the same or different regions are encoded by a single gene , whereas lambda protein targets . constant (CLN ) regions are encoded by multiple genes , and The phrase " antigen binding arm , ” “ target molecule bind undergo splicing. Several markers associated with particular ing arm , " and variations thereof, as used herein , refers to a polymorphic species of CLa are known : IgCL1 (Mcg component part of an antibody of the invention that has an marker ) ; IgLC2 - IgCL22 (Kern -Oz - marker ); IgCLà 3 (Kern - 15 ability to specifically bind a target molecule of interest Oz + marker ) , and IgCL27, for example . The skilled person Generally and preferably, the antigen binding arm is a can easily establish all of the polymorphisms so far identi - complex of immunoglobulin polypeptide sequences, e . g . fied in human CLà chains . The sequences of the present CDR and /or variable domain sequences of an immunoglobu invention encompass other known polymorphisms of the lin light and heavy chain . CLK and CLà , and antibodies in general. Two polymorphic 20 The term “monoclonal antibody” as used herein refers to loci have been identified in the CLK ; CLK - V /A153 and an antibody obtained from a population of substantially CLK - L / 191 . The three polymorphisms so far identified are : homogeneous antibodies , i. e . , the individual antibodies Km ( 1 ) : CLK - V153 /L 191; Km ( 1 , 2 ) : CLK - A153 / L 191; and comprising the population are identical except for possible Km (3 ): CLK - A153/ 191. naturally occurring mutations thatmay be present in minor The term “ Fc region ” as used herein generally refers to a 25 amounts . Monoclonal antibodies are highly specific , being dimer complex comprising the C - terminal polypeptide directed against a single antigen . Further, in contrast to sequences of an immunoglobulin heavy chain , wherein a polyclonal antibody preparations that typically include dif C - terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region mayble ferent antibodies directed against different determinants comprise native or variant Fc sequences . The Fc sequence of 30 ( epitopes) , each monoclonal antibody is directed against a an immunoglobulin generally comprises two constant su single determinant on the antigen . domains, a CH2 domain and a Cy3 domain , and optionally As used herein , the term “ immunoadhesin ” designates comprises a C A domain . The term “ Fc polypeptide” is used antibody - like or immunoglobulin - like molecules which herein to refer to one of the polypeptides that makes up an combine the “ binding domain ” of a heterologous protein ( an Fc region . In some embodiments , an Fc polypeptide may be 35 " adhesin ” , e . g . a receptor, ligand or enzyme ) with the obtained or derived from any suitable immunoglobulin , such effector component of immunoglobulin constant domains . as from at least one of the various IgG1, IgG2, IgG3 , or IgG4 Structurally , the immunoadhesins comprise a fusion of the subtypes, or from IgA , IgE , IgD or IgM . In some embodi adhesin amino acid sequence with the desired binding mentsments , an FcFcpolynentide polypeptide comprises part or allall ofof a wild -- type specificity which is other than the antigen recognition and hinge sequence (generally at its N terminus ). In some 40 binding site (antigen combining site ) of an antibody ( i. e . is embodiments , an Fc polypeptide does not comprise a wild “ heterologous ” ) and an immunoglobulin constant domain type hinge sequence . An Fc polypeptide may comprise sequence. The immunoglobulin constant domain sequence native or variant Fc sequences . in the immunoadhesin may be obtained from any immuno The “ immunoglobulin - like hinge region ,” “ immuno globulin , such as IgG1 , IgG2, IgG3, or IgG4 subtypes, IgA , globulin - like hinge sequence , " and variations thereof, as 45 IgE , IgD or IgM . used herein , refer to the hinge region and hinge sequence of A Fab fragment is a monovalent fragment consisting of an immunoglobulin - like or an antibody - like molecule ( e . g . the V , Vy , C , and Cyl domains ; a F (ab ' ) 2 fragment is a immunoadhesins) . In some embodiments , the immuno - bivalent fragment comprising two Fab fragments linked by globulin - like hinge region can be from or derived from any a disulfide bridge at the hinge region ; a Fd fragment consists IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA , IgE , IgD 50 of the Vh and Chl domains; an Fv fragment consists of the or IgM , including chimeric forms thereof, e . g . a chimeric V , and V domains of a single arm of an antibody ; and a IgG1/ 2 hinge region . dAb fragment consists of a Vy domain or a V , domain ( e . g . " Antibody fragments ” comprise only a portion of an human , camelid , or shark ) . intact antibody , wherein the portion preferably retains at A single - chain antibody ( scFv ) is an antibody in which a least one , preferably most or all , of the functions normally 55 V , and V , region are paired to form a monovalent molecule associated with that portion when present in an intact via a synthetic linker that enables them to be made as a antibody . single protein chain . Diabodies are bivalent, bispecific anti A “ bivalent antibody ” comprises two antigen binding bodies in which Vy and V , domains are expressed on a sites per molecule (e . g . IgG ). In some instances , the two single polypeptide chain , but using a linker that is too short binding sites have the same antigen specificities . However, 60 to allow for pairing between the 2 domains on the same bivalent antibodies may be bispecific (see below ). chain , thereby forcing the domains to pair with complemen A “ monovalent antibody ” comprises one antigen binding tary domains of another chain and creating 2 antigen binding site per molecule ( e . g . IgG ) . In some instances , a monova - sites . One or more CDRs may be incorporated into a lent antibody can have more than one antigen binding site , molecule either covalently or noncovalently to make it an but the binding sites are from different antigens. 65 immunoadhesin . An immunoadhesin may incorporate the A “ multispecific antibody ” is one that targets more than CDR ( s ) as part of a larger polypeptide chain , may cova one antigen or epitope . A “ bispecific ,” “ dual- specific ” or lently link the CDR ( s ) to another polypeptide chain , or may US 10 , 392 ,438 B2 53 54 incorporate the CDR ( s ) noncovalently . The CDRs permit the molecule fusion partners may include any therapeutic agent immunoadhesin to specifically bind to a particular antigen of that directs the Fc fusion to a therapeutic target. Such targets interest . may be any molecule , for example without limitation , an An antibody may have one or more binding sites. If there extracellular receptor that is implicated in disease. is more than one binding site , the binding sites may be 5 One type of derivatized antibody is produced by cross identical to one another or may be different. For instance , a linking 2 or more antibodies ( of the same type or of different naturally - occurring antibody has 2 identical binding sites , a types ; e . g . to create bispecific antibodies ) . Suitable cross single - chain antibody or Fab fragment has one binding site , linkers include those that are heterobifunctional, having 2 while a “ bispecific ” or “ bifunctional” antibody has 2 differ - distinctly reactive groups separated by an appropriate spacer ent binding sites . 10 ( e . g ., m -maleimidobenzoyl - N - hydroxysuccinimide ester ) or An “ isolated antibody ” is an antibody that ( 1 ) is not homobifunctional ( e . g . disuccinimidyl suberate ) . associated with naturally -associated components , including Another type of derivatized antibody is a labelled anti other naturally -associated antibodies, that accompany it in body. Useful detection agents with which an antibody or its native state , ( 2 ) is free of other proteins from the same antibody portion of the invention may be derivatized include species , ( 3 ) is expressed by a cell that does not naturally 15 fluorescent compounds , including fluorescein , fluorescein express the antibody , or is expressed by a cell from a isothiocyanate , rhodamine , 5 - dimethylamine - 1 -napthalene different species , or ( 4 ) does not occur in nature . sulfonyl chloride , phycoerythrin , lanthanide phosphors and The term “ human antibody ” includes all antibodies that the like. An antibody may also be labelled with enzymes that have one or more variable and constant regions derived from are useful for detection , such as horseradish peroxidase , human Ig sequences. In some embodiments of the present 20 galactosidase, luciferase, alkaline phosphatase , glucose oxi invention , all of the variable and constant domains of the dase and the like . When an antibody is labelled with a antibody are derived from human Ig sequences ( a fully detectable enzyme, it is detected by adding additional human antibody ). reagents that the enzyme uses to produce a reaction product A humanized antibody is an antibody that is derived from that can be discerned . For example , when the agent horse a non -human species , in which certain amino acids have 25 radish peroxidase is present, the addition of hydrogen per been mutated so as to avoid or abrogate an immune response oxide and diaminobenzidine leads to a colored reaction in humans. Alternatively , a humanized antibody may be product, which is detectable . An antibody may also be produced by fusing the constant domains from a human labelled with biotin , and detected through indirect measure antibody to the variable domains of a non -human species . ment of avidin or streptavidin binding . An antibody may be The term " chimeric antibody ” refers to an antibody that 30 labelled with a magnetic agent, such as gadolinium . An contains one or more regions from one antibody and one or antibody may also be labelled with a predetermined poly more regions from one or more other antibodies . Each peptide epitope recognized by a secondary reporter ( e . g . antibody may originate from seperate species (such as leucine zipper pair sequences , binding sites for secondary human and mouse ) . antibodies , metal binding domains, epitope tags ) . In some The term " epitope ” includes any molecular determinant 35 embodiments , labels are attached by spacer arms of various capable of specific binding to an Ig or T - cell receptor. lengths to reduce potential steric hindrance . Epitopic determinants usually consist of surface groupings The antibody may also be derivatized with a chemical of atoms such as amino acids or sugar side chains and group such as polyethylene glycol (PEG ), a methyl or ethyl usually have specific 3 dimensional structural characteris - group , or a carbohydrate group . These groups may be useful tics, as well as specific charge characteristics . An antibody 40 to improve the biological characteristics of the antibody, e .g . is said to specifically bind an antigen when the dissociation to increase serum half - life or to increase tissue binding . constant is < 1 uM , preferably < 100 nM and more preferably : Antibody Specificity < 10 nM . In some embodiments comprising antigen binding Fully human antibodies are expected to minimize the domains, at least the antigen binding domain ( for example , immunogenic and allergic responses intrinsic to mouse or 45 but not limited to , an antibody variable region having all 6 mouse -derivatized monoclonal antibodies (Mabs ) and thus CDRs, or an equivalent region that is at least 90 percent to increase the efficacy and safety of the administered identical to an antibody variable region ) is chosen from that antibodies . The use of fully human antibodies can be found in : abagovomab , abatacept (ORENCIA® ) , abciximab expected to provide a substantial advantage in the treatment (REOPROB , c7E3 Fab ) , adalimumab (HUMIRA® ) , adeca of chronic and recurring human diseases , such as inflam - 50 tumumab , alemtuzumab (CAMPATH® , MabCampath or mation and cancer, which may require repeated antibody Campath - 1H ), altumomab , afelimomab , anatumomab administrations . mafenatox , anetumumab , anrukizumab , apolizumab , arcitu In addition , fusion antibodies can be created in which 2 momab , aselizumab , atlizumab , atorolimumab , bapineu ( or more ) single - chain antibodies are linked to one another. Zumab , basiliximab (SIMULECT® ) , bavituximab , bectu This is useful if one wants to create a divalent or polyvalent 55 momab ( LYMPHOSCAN® ) , belimumab (LYMPHO -STAT antibody on a single polypeptide chain , or if one wants to B® ), bertilimumab , besilesomab , Bcept (ENBREL® ) , create a bispecific antibody . bevacizumab (AVASTIN® ) , biciromab brallobarbital, biva By “ Fc fusion ” as used herein is meant a protein wherein tuzumab mertansine, brentuximab vedotin ( ADCETRIS® ) , one or more polypeptides is operably linked to an Fc canakinumab ( ACZ885 ) , cantuzumab mertansine, capromab polypeptide . An Fc fusion combines the Fc region of an 60 (PROSTASCINT® ) , catumaxomab (REMOV ABC ) ) , immunoglobulin with a fusion partner, which in general may cedelizumab (CIMZIA® ) , certolizumab pegol, cetuximab be any protein , polypeptide or small molecule . Virtually any (ERBITUX® ) , clenoliximab , dacetuzumab , dacliximab , protein or small molecule may be linked to Fc to generate an daclizumab (ZENAP AX ( ® ) , denosumab ( AMG 162) , detu Fc fusion . Protein fusion partners may include , but are not momab , dorlimomab aritox , dorlixizumab , duntumumab , limited to , the target -binding region of a receptor, an adhe - 65 durimulumab , durmulumab , ecromeximab , eculizumab sion molecule , a ligand , an enzyme, a cytokine , a ( SOLIRIS® ), edobacomab , edrecolomab (Mab17 -1A , PAN chemokine, or some other protein or protein domain . Small OREX® ) , efalizumab (RAPTIVA® ), efungumab (MY US 10 , 392 ,438 B2 55 56 COGRAB® ) , elsilimomab , enlimomab pegol, epitumomab s100A8, s100A9 , Nav1. 7 , GLPI, RSV , RSV F protein , cituxetan , efalizumab , epitumomab , epratuzumab , erli Influenza HA protein , Influenza NA protein , HMGBI, CD16 , zumab , ertumaxomab (REXOMUN® ) , etaracizumab ( et CD19 , CD20 , CD21, CD28 , CD32 , CD32b , CD64, CD79 , aratuzumab , VITAXIN® , ABEGRINTM ) , exbivirumab , CD22 , ICAM - I, FGFRI, FGFR2 , HDGF, EphB4, GITR , fanolesomab (NEUTROSPEC® ) , faralimomab , felvizumab , 5 B -amyloid , hMPV, PIV - I, PIV - 2 , OX40L , IGFBP3 , cMet, fontolizumab ( HUZAF® ), galiximab , gantenerumab , gavili PD - I , PLGF, Neprolysin , CTD , IL - 18 , IL - 6 , CXCL - 13 , momab ( ABX -CBL® ) , gemtuzumab ozogamicin (MYLO - IL - IRI, IL - 15 , IL - 4R , IgE , PAI- I , NGF, EphA2 , uPART, TARG® ), golimumab (CNTO 148 ), gomiliximab, ibali - DLL -4 , avß5 , avß6 , a561 , a3f1, interferon receptor type zumab ( TNX - 355 ) , ibritumomab tiuxetan (ZEVALIN? ), I and type II , CD 19 , ICOS, IL - 17 , Factor II , Hsp90 , IGF, igovomab , imciromab , infliximab (REMICAD E® ) , inoli - 10 IGF - I , IGF - II, CD 19 , GM -CSFR , PIV - 3 , CMV, IL - 13 , IL - 9 , momab , inotuzumab ozogamicin , ipilimumab (YERVOY® , and EBV . MDX - 010 ) , iratumumab , keliximab , labetuzumab , lemale - In some embodiments comprising antigen binding somab , lebrilizumab , lerdelimumab , lexatumumab (HGS - domains, at least the first antigen binding domain specifi ETR2, ETR2 -ST01 ) , lexitumumab , libivirumab , lintu - cally binds to a member (receptor or ligand ) of the TNF zumab , lucatumumab , lumiliximab , mapatumumab (HGS - 15 superfamily . Various molecules include , but are not limited ETRI, TRM - I) , maslimomab , matuzumab (EMD72000 ), to Tumor Necrosis Factor- a ( “ TNF - a ” ), Tumor Necrosis mepolizumab (BOSATRIA® ) , metelimumab , milatuzumab , Factor -R ( “ TNF - R ” ) , Lymphotoxin - a (“ LT - a ” ) , CD30 minretumomab , mitumomab , morolimumab , motavizumab ligand , CD27 ligand, CD40 ligand , 4 - 1 BB ligand , Apo - 1 (NUMAXTM ) , muromonab (OKT3 ) , nacolomab tafenatox , ligand (also referred to as Fas ligand or CD95 ligand ), Apo - 2 naptumomab estafenatox , natalizumab ( TYSABRI? , 20 ligand (also referred to as TRAIL ) , Apo - 3 ligand ( also ANTEGREN® ) , nebacumab , nerelimomab , nimotuzumab referred to as TWEAK ) , osteoprotegerin (OPG ) , APRIL , ( THERACIM hR3® , THERA -CIM -hR3? , THER - RANK ligand ( also referred to as TRANCE ) , TALL - I (also ALOC® ), nofetumomab merpentan (VERLUMA® ) , ocre referred to as BlyS , BAFF or THANK ) , DR4 , DR5 (also lizumab , odulimomab , ofatumumab , omalizumab (XO - known as Apo - 2 , TRAIL -R2 , TR6 , Tango - 63 , hAPOS , LAIR® ) , oregovomab (OVAREX® ) , otelixizumab , 25 TRICK2, or KILLER ), DR6 , DcRI, DcR2, DcR3 (also pagibaximab , palivizumab (SYNAGIS® ) , panitumumab known as TR6 or M68 ) , CARI, HVEM (also known as ( ABX - EGF , VECTIBIX® ) , pascolizumab , pemtumomab A TAR or TR2 ) , GITR , ZTNFR - 5 , NTR - I , TNFLI, CD30 , ( THERAGYN® ), pertuzumab (2C4 , OMNITARG® ) , pex - LTBr, 4 - 1BB receptor and TRI. elizumab , pintumomab , ponezumab , priliximab , pritu - In some embodiments comprising antigen binding mumab , ranibizumab (LUCENTIS? ), raxibacumab , rega - 30 domains , at least the first antigen binding domain is capable virumab , reslizumab , rituximab (RITUXAN® , of binding one or more targets chosen from 5T4 , ABL , MabTHERA® ), rovelizumab , ruplizumab , satumomab , ABCB5, ABCFI, ACVRI, ACVRIB , ACVR2 , ACVR2B , sevirumab , sibrotuzumab , siplizumab (MEDI - 507 ) , sontu ACVRLI, ADORA2A , Aggrecan , AGR2, AICDA , AIFI, zumab , stamulumab (Myo -029 ) , sulesomab (LEUKO - AIGI, AKAPI, AKAP2 , AMH , AMHR2, angiogenin (ANG ) , SCAN® ), tacatuzumab tetraxetan , tadocizumab , talizumab , 35 ANGPTI, ANGPT2 , ANGPTL3, ANGPTL4 , Annexin A2 , taplitumomab paptox , tefibazumab ( AUREXIS® ) , teli ANPEP , APC , APOCI, AR , aromatase , ATX , AXI, AZGPI momab aritox , teneliximab , teplizumab , ticilimumab , tocili ( zinc - a - glycoprotein ) , B7 . 1 , B7. 2 , B7 -H1 , BAD , BAFF , zumab (ACTEMRA® ) , toralizumab , tositumomab , trastu BAGI, BAII , BCR , BCL2 , BCL6 , BDNF , BLNK , BLRI zumab (HERCEPTIN® ) , tremelimumab ( CP- 675 ,206 ) , (MDR15 ) , BlyS , BMP1, BMP2 , BMP3B (GDFIO ) , BMP4 , tucotuzumab celmoleukin , tuvirumab , urtoxazumab , usteki- 40 BMP6 , BMP7 , BMP8 , BMP9, BMP11 , BMP12 , BMPR1A , numab (CNTO 1275 ) , vapaliximab , veltuzumab , vepali - BMPR1B , BMPR2, BPAGI (plectin ) , BRCAI, C19orfIO momab , visilizumab (NUVION® ) , volociximab (M200 ) , (IL27w ) , C3, C4A , C5 , C5R1, CANTI, CASPI, CASP4 , votumumab (HUMASPECT® ), zalutumumab , zanolim CAVI, CCBP2 (D6 / JAB61 ), CCLI ( 1 -309 ), CCLI 1 ( eo umab (HUMAX -CD4 ) , ziralimumab , or zolimomab aritox. taxin ), CCL13 (MCP - 4 ) , CCL15 (MIP -Id ) , CCL16 (HCC In some embodiments comprising antigen binding 45 4 ), CCL17 ( TARC ) , CCL18 (PARC ), CCL19 (MIP - 3b ) , domains, the antigen binding domain comprises a heavy and CCL2 (MCP - 1 ) , MCAF , CCL20 (MIP - 3a ), CCL21 (MEP light chain variable domain having six CDRs , and /or com - 2 ) , SLC , exodus - 2 , CCL22 (MDC /STC - I ) , CCL23 (MPIF - I ) , petes for binding with an antibody selected from the pre CCL24 (MPIF - 2 /eotaxin - 2 ) , CCL25 ( TECK ), CCL26 ( eo ceding list. In some embodiments comprising antigen bind - taxin - 3 ) , CCL27 (CTACK / ILC ) , CCL28 , CCL3 (MIP - Ia ) , ing domains, the antigen binding domain binds the same 50 CCL4 (MIP - Ib ) , CCL5 (RANTES ) , CCL7 (MCP - 3 ) , CCLS epitope as the antibodies in the preceding list. In some (mcp - 2 ), CCNAI, CCNA2 , CCNDI, CCNEI, CCNE2, CCRI embodiments comprising antigen binding domains, the anti (CKRI / HM145 ) , CCR2 (mcp - IRB /RA ), CCR3 (CKR3 / CM gen binding domain comprises a heavy and light chain KBR3 ), CCR4, CCR5 (CMKBR5 / ChemR13 ) , CCR6 (CM variable domain having six total CDRs, and binds to the KBR6 /CKR -L3 / STRL22 /DRYO ) , CCRT (CKRZ / EBI1 ) , same antigen as the antibodies in the preceding list. 55 CCR8 (CMKBR8 / TERI/CKR -LI ) , CCR9 (GPR -9 -6 ), In some embodiments comprising antigen binding CCRLI (VSHKI ) , CCRL2 ( L -CCR ), CD164 , CD19 , CDIC , domains , at least the first antigen binding domain comprises CD20 , CD200 , CD -22 , CD24 , CD28 , CD3 , CD33 , CD35 , a heavy and light chain variable domain having six ( 6 ) total CD37 , CD38 , CD3E , CD3G , CD3Z , CD4, CD40 , CD40L , CDRs, and specifically binds to an antigen selected from : CD44 , CD45RB , CD46 , CD52 , CD69, CD72 , CD74 , PDGFRa , PDGFRB , PDGF, VEGF, VEGF - A , VEGF- B , 60 CD79A , CD79B , CD8 , CD80 , CD81, CD83 , CD86 , CD105 , VEGF - C , VEGF - D , VEGF- E , VEGF- F , VEGFR1, CD137 , CDHI ( E - cadherin ), CDCP1CDH10 , CDH12 , VEGFR2, VEGFR3 , FGF, FGF2 , HGF, KDR , flt - 1 , FLK - 1 , CDH13 , CDH18 , CDH19 , CDH20 , CDH5 , CDH7, CDH8 , Ang - 2 , Ang - 1 , PLGF, CEA , CXCL13 , Baff , IL - 21 , CCL21 , CDH9, CDK2, CDK3, CDK4 , CDK5 , CDK6 , CDK7 , TNF- a , CXCL12 , SDF - I , bFGF, MAC - I, IL23p19 , FPR CDK9 , CDKNIA (p21 Wapl /Cipl ) , CDKNIB (p27Kipl ) , IGFBP4, CXCR3 , TLR4 , CXCR2 , EphA2 , EphA4, Eph - 65 CDKNIC , CDKN2A ( p16INK4a ), CDKN2B , CDKN2C , rinB2, EGFR ( ErbBl) , HER2( ErbB2 or p185neu ) , HER3 CDKN3 , CEBPB , CERI, CHGA , CHGB , Chitinase , ( ErbB3 ), HER4 ErbB4 or tyro2 ), SCI, LRP5, LRP6 , RAGE , CHSTIO , CKLFSF2 , CKLFSF3 , CKLFSF4 , CKLFSF5 , US 10 , 392 ,438 B2 57 58 CKLFSF6 , CKLFSF7, CKLFSF8 , CLDN3, CLDN7 ( clau - MS4A1, MSMB , MT3 (metallothionectin - Ui) , mTOR , din - 7 ) , CLN3, CLU ( clusterin ), CMKLRI, CMKORI MTSSI, MUCI (mucin ) , MYC , MYD88 , NCK2, neurocan , (RDCI ) , CNRI, COLI 8AI, COL1A1. COL4A3 , COL6A1 , neuregulin - 1 , neuropilin - 1 , NFKBI, NFKB2 , NGFB (NGF ) , CR2, Cripto , CRP , CSFI ( M - CSF ) , CSF2 (GM -CSF ) , CSF3 NGFR , NOR - Lingo , NgR -Nogo66 (Nogo ), NgR - p75 , NOR (GCSF ) , CTLA4 , CTL8, CTNNBI ( b - catenin ) , CTSB 5 Troy . NMEI (NM23A ) . NOTCH . NOTCHI. NOX5 . NPPB . (cathepsin B ), CX3CLI (SCYDI ) , CX3CR1 (V28 ) , CXCLI NROBI. NROB2. NRIDI. NR1D2. NR1H2. NR1H3 . (GROI ) , CXCLIO ( IP - 10 ) , CXCLII ( I - TAC /IP - 9 ) , CXCL12 NR1H4, NR112 , NR113 , NR2C1, NR2C2 , NR2E1 , NR2E3 , (SDFI ) , CXCL13 , CXCL 14 , CXCL 16 , CXCL2 (GRO2 ) , CXCL3 (GRO3 ), CXCL5 (ENA - 78 /LIX ), CXCL6 (GCP - 2 ), NR2F1, NR2F2 , NR2F6 , NR3C1, NR3C2 , NR4A1, CXCL9 (MIG ) , CXCR3 (GPRS / CKR - L2) , CXCR4 , 10 NR4A2 , NR4A3, NR5A1, NR5A2 , NR6A1, NRPI, NRP2, CXCR6 ( TYMSTR / STRL33 /Bonzo ) , CYB5, CYCI, Cyr61 , NT5E , NTN4 , OCT- 1, ODZI, OPN1, OPN2, OPRDI, CYSLTRI , C -Met . DAB2IP , DES , DKFZp451J0118 , P2RX7, PAP, PARTI, PATE , PAWR , PCA3 , PCDGF, PCNA , DNCLI, DPP4, E2F1, ECGFI5EDGI, EFNAI, EFNA3, PDGFA , PDGFB , PDGFRA , PDGFRB , PECAMI, peg EFNB2 , EGF, ELAC2 , ENG , endoglin , ENOI, ENO2 , EN03 , asparaginase , PF4 ( CXCL4 ) , Plexin B2 (PLXNB2 ) , PGF, EPHAI. EPHA2 . EPHA3 . EPHA4. EPHA5. EPHA6 . 15 PGR , phosphacan , PIAS2 , P13 Kinase , PIK3CG , PLAU EPHA7, EPHA8 , EPHAI , EPHAIO , EPHBI, EPHB2, (UPA ) , PLG5PLXDCI, PKC , PKC - B , PPBP (CXCL7 ), EPHB3, EPHB4 , EPHB5 , EPHB6 , EPHRIN - AI, EPHRIN PPID , PRI, PRKCQ , PRKDI, PRL , PROC , PROK2, pro A2, EPHRIN - A3 , EPHRIN - A4, EPHRIN - A5 , EPHRIN - A6 , NGF, prosaposin , PSAP, PSCA , PTAFR , PTEN , PTGS2 EPHRIN -BI . EPHRIN - B2 . EPHRTN -B3 , EPHB4 . EPG , (COX - 2 ) , PTN , RAC2 (P21Rac2 ), RANK , RANK ligand , ERBB2 (Her - 2 ), EREG , ERK8, Estrogen receptor, ESRI, 20 RARB , RGSI, RGS13 , RGS3 , RNFI10 (ZNF144 ), Ron , ESR2 , F3 ( TF ) , FADD , farnesyltransferase , FasL , FASNE, ROB02 , RXR , selectin , S100A2 , S100A8 , S100A9 , SCGB FCERIA , FCER2 , FCGR3A , FGF , FGFI (AFGF ) , FGFIO , 1D2 ( lipophilin B ) , SCGB2A1 (mammaglobin 2 ) , FGFI 1 , FGF12 , FGF12B , FGF13 , FGF14 , FGF16 , FGF17 , SCGB2A2 (mammaglobin 1 ) , SCYEI ( endothelial Mono FGF18 , FGF19 , FGF2 (bFGF ) , FGF20 , FGF21 ( such as cyte - activating cytokine ) , SDF2, SERPENA1, SERPINA3, mimAbl) , FGF22 , FGF23 , FGF3 ( int - 2 ) , FGF4 (HST ) , 25 SERPINB5 (maspin ) , SERPINEI ( PAI- I ) , SERPINFI, SHIP FGF5 , FGF6 (HST - 2 ) , FGF7 (KGF ) , FGF8 , FGF9 , FGFR3 , I , SHIP - 2 , SHBI, SHB2, SHBG , SfcAZ , SLC2A2, FIGF (VEGFD ) , FILI( EPSILON ) , FBLI ( ZETA ) , SLC33A1, SLC43A1, SLIT2 , SPPI, SPRRIB ( Spr . ) , FLJ12584 . FLJ25530 , FLRTI ( fibronectin ) , FLTI, FLT - 3 , STOGALI, STABI, STATE , STEAP, STEAP2 , SULF - 1 , FOS, FOSLI( FRA - 1) , FY (DARC ), GABRP (GABA ) , Sulf - 2 , TB4R2 , TBX21 , TCPIO , TDGFI, TEK , TGFA , GAGEBI, GAGECI, GALNAC4S -6ST , GATA3, GD2, 30 TGFBI, TGFBIII , TGFB2 , TGFB3 , TGFBI, TGFBRI, GD3 , GDF5, GDF8 , GFII, GGTI, GM -CSF , GNASI, TGFBR2 , TGFBR3, THIL , THBSI (thrombospondin - 1 ) , GNRHI, GPR2 (CCRIO ) , GPR31 , GPR44 . GPR81 THBS2 / THBS4 , THPO , TIE ( Tie - 1 ) , TIMP3 , tissue factor. ( FKSG80 ), GRCCIO (CIO ), gremlin ,GRP , GSN (Gelsolin ), TIKI2 , TLR10 , TLR2 , TLR3, TLR4, TLR5, TLR6JLR7 , GSTPI, HAVCR2 , HDAC , HDAC4 , HDAC5 , HDAC7A , TLR8 , TLRI, TM4SF1 , TNF, TNF - a , TNFAIP2 (B94 ) , HDACI, Hedgehog , HGF, HIFIA , HIPI, histamine and 35 TNFAIP3 , TNFRSFIIA , TNFRSFIA , TNFRSFIB , histamine receptors , HLA - A , HLA -DRA , HM74 , HMOXI, TNFRSF21, TNFRSF5 , TNFRSF6 ( Fas ), TNFRSF7 , HSP90 , HUMCYT2A , ICEBERG , ICOSL , ID2 , IFN - a , TNFRSF8 , TNFRSF9 , TNFSFIO ( TRAIL ) , TNFSFI 1 IFNAI, IFNA2 , IFNA4 , IFNA5 , EFNA6 , BFNA7, IFNBI, ( TRANCE ) , TNFSF12 (APO3L ), TNFSF13 ( April) , IFNgamma, IFNWI, IGBPI, IGFI, IGFIR , IGF2 , IGFBP2 , TNFSF13B , TNFSF14 (HVEM - L ) , TNFSF15 (VEGI ) , IGFBP3 , IGFBP6 , DL - I, ILIO , ILIORA , ILIORB , IL - 1 , 40 TNFSF 18 , TNFSF4 (OX40 ligand ) , TNFSF5 (CD40 ILIRI (CD121a ), ILIR2 (CD121b ) , IL - IRA , IL - 2 , IL2RA ligand ) , TNFSF6 (FasL ) , TNFSF7 (CD27 ligand ) , TNFSF8 (CD25 ) , IL2RB (CD122 ) , IL2RG (CD132 ) , IL - 4 , IL - 4R (CD30 ligand ) , TNFSF9 ( 4 - 1BB ligand ) , TOLLIP , Toll - like (CD123 ), IL - 5 , IL5RA (CD125 ), IL3RB ( CD131) , IL - 6 , receptors, TLR2, TLR4 , TLR9, TOP2A (topoisomerase lia ), ILORA (CD126 ) , IRORB (CD130 ), IL - 7 , IL RA (CD127 ) , TP53 , TPMI, TPM2, TRADD , TRAFI, TRAF2 , TRAF3 , IL - 8 , CXCRI ( ILSRA ) , CXCR2 ( ILSRB /CD128 ) , IL - 9 , 45 TRAF4 . TRAF5 , TRAF6 , TRKA , TREMI, TREM2, ILOR (CD129 ) , IL - 10 , IL10RA (CD210 ), IL10RB TRPC6 , TROY, TSLP, TWEAK , Tyrosinase , uPAR , VEGF, (CDW210B ) , IL - 11 , ILI IRA , IL - 12 , IL - 12A , IL - 12B , VEGFB , VEGFC , versican , VHL C5 , VLA - 4 , Wnt - 1 , XCLI IL - 12RB1, IL - 12RB2, IL - 13 , IL13RA1, IL13RA2 , IL14 , (lymphotactin ) , XCL2 (SCM - Ib ), XCRI (GPR5 / CCXCRI ) , IL15 , IL15RA , 1L16 , IL 17 , IL17A , IL17B , IL17C , IL17R , YYI, and ZFPM2. IL18 , IL18BP, IL18R1, IL18RAP, IL19 , ILIA , ILIB , ILI- 50 Definitions FIO , IL1F5 , IL1F6 , IL1F7 , IL1F8 , DL1F9 , ILIHYI, ILIRI, Generally, nomenclatures used in connection with , and IL1R2 , ILIRAP , ILIRAPLI, ILI RAPL2, ILIRLI, ILI RL2 , techniques of, biochemistry , analytical chemistry , synthetic ILIRN , IL2, IL20 , IL2ORA , IL21R , IL22 , IL22R , IL22RA2 , organic chemistry , medicinal and pharmaceutical chemistry , IL23 . DL24 , IL25 , IL26 , IL27 , IL28A , IL28B , IL29 , cell and tissue culture , molecular biology , immunology , IL2RA , IL2RB , IL2RG , IL3 , IL30 , IL3RA , IL4 , IL4R , 55 microbiology, genetics and protein and nucleic acid chem IL6ST ( glycoprotein 130 ), ?LK , INHA , INHBA , INSL3 , istry and hybridization described herein are those well INSL4 , IRAKI, IRAK2, ITGAI, ITGA2 , ITGA3, ITGA6 known and commonly used in the art . The methods and ( a6 integrin ) , ITGAV , ITGB3 , ITGB4 (84 integrin ) , JAKI, techniques of the present invention are generally performed JAK3, JTB , JUN , KOHF , KAII , KDR , KIM - 1 , KITLG , according to conventional methods well known in the art KLFS (GC Box BP ) , KLF6 , KLKIO , KLK12 , KLK13 , 60 and as described in various general and more specific KLK14 , KLK15 , KLK3, KLK4 , KLKS , KLK6 , KLKO, references that are cited and discussed throughout the pres KRTI, KRT19 (Keratin 19 ) , KRT2A , KRTHB6 (hair -spe - ent specification unless otherwise indicated . Reactions and cific type II keratin ), LAMA5 , LEP ( leptin ), Lingo -p75 , purification techniques are performed according to manu Lingo - Troy , LPS , LRPS, LRP6 , LTA ( TNF- b ) , LTB , LTBAR facturer ' s specifications, as commonly accomplished in the (GPR16 ) , LTB4R2 , LTBR , MACMARCKS , MAG or 65 art or as described herein . As used herein , the 20 natural, or Omgp , MAP2K7 ( c - Jun ) , MCP - I , MDK , MIBI, midkine , conventional, amino acids and their abbreviations follow MIF , MISRII ,MJP - 2 , MK , MK167 (Ki - 67 ) , MMP2, MMP9, IUPAC single letter and three letter codes . US 10 , 392 , 438 B2 59 60 A “ complementary residue set" , as used herein , refers to nition are , for example , polypeptides containing one or more at least one amino acid in a Cy - 1 domain , and at least one analogs of an amino acid ( including , for example , unnatural amino acid in the C , domain that are engineered to interact amino acids, etc . ) , as well as other modifications known in with each other. By interacting with each other, they drive the art. It is understood that the polypeptides can occur as their respective domains to heterodimerize and form an 5 single chains or associated chains . interface comprising at least some of the interaction between Unless indicated otherwise by a “ D ” prefix , e . g . D - Ala or the residues of the complementary residue set . The interac - N -Me - D - Ile , or written in lower case format, e . g . a , i, 1 , ( D tion may be characterized by a salt bridge , electrostatic versions of Ala , Ile , Leu ) , the stereochemistry of the a - car interaction , or van der Waals force . A complementary resi- bon of the amino acids and aminoacyl residues in peptides due set may comprise more than one engineered residue in 10 described in this specification and the appended claims is the each domain . natural or “ L ” configuration . Any given residue within a complementary residue set All peptide sequences are written according to the gen will be within 5 Å of at least one other residue of that erally accepted convention whereby the a - N - terminal amino complementary residue set . acid residue is on the left and the a - C - terminal amino acid In the context of complementary residue sets, two resi - 15 residue is on the right. As used herein , the term “ N - termi dues are said to interact if at least one atom of each residue nus” refers to the free a -amino group of an amino acid in a is within 5 Å of each other. Residue interaction may be peptide , and the term “ C - terminus” refers to the free a - car characterized as either a salt bridge , electrostatic interaction , boxylic acid terminus of an amino acid in a peptide . A or van der Waals force . For avoidance of doubt, in other peptide which is N -term inated with a group refers to a contexts it is recognized that interatomic forces may act over 20 peptide bearing a group on the a -amino nitrogen of the longer distances. N -terminal amino acid residue . An amino acid which is " Complementary pairing " between domains refers to the C -terminated with a group refers to an amino acid bearing a interaction of those two domains, at least in part , through a group on the carboxyl moiety , such as a methyl group complementary residue set. resulting in a methyl ester. “ Engineered ” , as used herein , refers to the deliberate 25 As used herein , “ biological activity ” refers to the in vivo mutation of residues that are not found in the predominant activities of a compound , composition , or other mixture , or wild type sequence , and may be an engineered insertion , physiological responses that result upon in vivo administra deletion or substitution mutation . tion of a compound , composition or other mixture . Biologi A “ heteromultimer” , “ heteromultimeric complex ” , or cal activity thus encompasses therapeutic effects , diagnostic " heteromultimeric polypeptide ” is a molecule comprising at 30 effects and pharmaceutical activity of such compounds , least a first polypeptide and a second polypeptide , wherein compositions, and mixtures. the second polypeptide differs in amino acid sequence from The term “ biologically compatible ” as used herein means the first polypeptide by at least one amino acid residue. The something that is biologically inert or non reactive with heteromultimer can comprise a " heterodimer ” formed by the intracellular and extra cellular biological molecules , and non first and second polypeptide or can form higher order 35 toxic . tertiary structures where polypeptides in addition to the first “ About” or “ approximately ,” when used in connection and second polypeptide are present. with a measurable numerical variable , refers to the indicated A “ heterodimer ," " heterodimeric protein ," " heterodimeric value of the variable and to all values of the variable that are complex ," or " heteromultimeric polypeptide” is a molecule within the experimental error of the indicated value (e . g . comprising a first polypeptide and a second polypeptide , 40 within the 95 % confidence interval for the mean ) or within wherein the second polypeptide differs in amino acid 10 percent of the indicated value , whichever is greater . sequence from the first polypeptide by at least one amino Numeric ranges are inclusive of the numbers defining the acid residue . 14range . In the context of the invention , the term heterodimeric is The term “ identity, ” as known in the art, refers to a used to indicate a heteromultimer comprising at least two 45 relationship between the sequences of two or more poly polypeptides with differing amino acid sequences ; but it will peptide molecules or two or more nucleic acid molecules , as be readily appreciated that in many embodiments , particu - determined by comparing the sequences. In the art , " iden larly those where the invention relates to IgG antibodies and tity ” also means the degree of sequence relatedness between similar molecules, heterodimeric proteins of the invention polypeptide or nucleic acid molecule sequences, as the case may equally be referred to as heteromultimeric proteins , as 50 may be , as determined by the match between strings of there will necessarily be four distinct polypeptides ( the first nucleotide or amino acid sequences . “ Identity ” measures the heavy and light chain , and the second heavy and light chain ) . percent of identicalmatches between two or more sequences “ Polypeptide , " " peptide, " and " protein ” are used inter - with gap alignments addressed by a particular mathematical changeably to refer to a polymer of amino acid residues. As model of computer programs ( i. e . “ algorithms” ). used herein , these terms apply to amino acid polymers in 55 The term " similarity ” is a related concept, but in contrast which one or more amino acid residues is an artificial to “ identity ” , refers to a measure of similarity which chemical analog of a corresponding naturally occurring includes both identical matches and conservative substitu amino acid . These terms also apply to naturally occurring tion matches. Since conservative substitutions apply to amino acid polymers . Amino acids can be in the L - form or polypeptides and not nucleic acid molecules, similarity only D - form as long as the binding and other desired character - 60 deals with polypeptide sequence comparisons. If two poly istics of the peptide are maintained . A polypeptide may be peptide sequences have , for example , 10 out of 20 identical monomeric or polymeric . The terms also encompass an amino acids , and the remainder are all nonconservative amino acid chain that has been modified naturally or by substitutions, then the percent identity and similarity would intervention ; for example , disulfide bond formation , glyco - both be 50 % . If in the same example , there are 5 more sylation , lipidation , acetylation , phosphorylation , or any 65 positions where there are conservative substitutions , then the other manipulation or modification , such as conjugation percent identity remains 50 % , but the percent similarity with a labeling component. Also included within the defi - would be 75 % ( 15 outof 20 ). Therefore , in cases where there US 10 , 392 , 438 B2 61 62 are conservative substitutions , the degree of similarity hydroxyl groups ordinarily present in the sugars may be between two polypeptide sequences will be higher than the replaced , for example , by phosphonate groups , phosphate percent identity between those two sequences. groups , protected by standard protecting groups , or activated The term “ conservative amino acid substitution ” refers to to prepare additional linkages to additional nucleotides, or a substitution of a native amino acid residue with a nonna - 5 may be conjugated to solid or semi- solid supports . The 5 ' tive residue such that there is little or no effect on the and 3 ' terminal OH can be phosphorylated or substituted polarity , charge, and approximate volume of the amino acid with amines or organic capping group moieties of from 1 to residue at that position . For example , a conservative substi tution results from the replacement of a non -polar residue in 20 carbon atoms. Other hydroxyls may also be derivatized a polypeptide with any other non -polar residue . The term 10 to standard protecting groups . Polynucleotides can also may also refer to a substitution identified as frequently contain analogous formsof ribose or deoxyribose sugars that occurring between highly similar proteins , as in the BLO are generally known in the art , including , for example , SUM62 matrix or related matrices (PNAS , USA 89 (22 ), 2 '- O -methyl - , 2 - 0 - allyl, 2 - fluoro - or 2 ' - azido - ribose , car 10915 - 9 , 1992 ). bocyclic sugar analogs, alpha -anomeric sugars , epimeric The term “ vector. ” as used herein , is intended to refer to 15 sugars such as arabinose , xyloses or lyxoses, pyranose a nucleic acid molecule capable of transporting another sugars , furanose sugars, sedoheptuloses , acyclic analogs and nucleic acid to which it has been linked . One type of vector abasic nucleoside analogs such as methyl riboside . One or is a " plasmid , ” which refers to a circular double stranded more phosphodiester linkages may be replaced by alterna DNA loop into which additional DNA segments may be tive linking groups . These alternative linking groups ligated . Another type of vector is a phage vector. Another 20 include, but are not limited to , embodiments wherein phos type of vector is a viral vector, wherein additional DNA phate is replaced by P ( O ) S (“ thioate ” ) , P ( S ) S ( “ dithioate ” ) , segments may be ligated into the viral genome. Certain “ O )NR2 (“ amidate ” ), P ( O ) R , P ( O )OR ', CO or CH2 ( “ for vectors are capable of autonomous replication in a host cell macetal ” ), in which each R or R ' is independently H or into which they are introduced ( e . g . bacterial vectors having substituted or unsubstituted alkyl ( 1 - 20 C .) optionally con a bacterial origin of replication and episomal mammalian 25 taining an ether ( 0 % ) linkage , aryl , alkenyl, cycloalkyl , vectors) . Other vectors ( e . g . non - episomal mammalian vec - cycloalkenyl or araldyl. Not all linkages in a polynucleotide tors ) can be integrated into the genome of a host cell upon need be identical. The preceding description applies to all introduction into the host cell, and thereby are replicated polynucleotides referred to herein , including RNA and along with the host genome. Moreover, certain vectors are DNA capable of directing the expression of genes to which they 30 are operatively linked . Such vectors are referred to herein as " Oligonucleotide, ” as used herein , generally refers to " recombinant expression vectors ” (or simply , “ recombinant short , generally single stranded , generally synthetic poly vectors” ) . In general, expression vectors of utility in recom nucleotides that are generally, but not necessarily , less than binant DNA techniques are often in the form of plasmids. In about 200 nucleotides in length . The terms " oligonucle the present specification , " plasmid ” and “ vector” may be 35s otideou ” and “ polynucleotide ” are notmutually exclusive. The used interchangeably as the plasmid is the most commonly description above for polynucleotides is equally and fully used form of vector. applicable to oligonucleotides. “ Polynucleotide ,” or “ nucleic acid molecule ,” which may A reference to a nucleotide sequence as used herein be used interchangeably herein , refers to a polymeric , pos encompasses its complement unless otherwise specified . sibly isolated , form of nucleosides or nucleotides of at least 40 Thus, a reference to a nucleic acid having a particular 10 bases in length . The term includes single and double sequence should be understood to encompass its comple stranded forms. The nucleotides can be deoxyribonucle - mentary strand , with its complementary sequence , unless otides , ribonucleotides, modified nucleotides or bases, and otherwise defined by context. or their analogs , or any substrate that can be incorporated “ Cell” or “ cell line ,” as used herein , includes various into a polymer by DNA or RNA polymerase , or by a 45 types of cells that can be used to express a heterodimeric synthetic reaction . protein , a polypeptide or a nucleic acid of the invention , e . g . A polynucleotide may comprise modified nucleotides , prokaryotic cells , eukaryotic cells , mammalian cells , rat such as methylated nucleotides and their analogs . If present, cells, human cells. modification to the nucleotide structure may be imparted The term “ purify ,” and grammatical variations thereof, is before or after assembly of the polymer. The sequence of 50 used to mean the removal , whether completely or partially , nucleotides may be interrupted by non - nucleotide compo - of at least one impurity from a mixture containing the nents. A polynucleotide may be further modified after syn - polypeptide and one or more impurities , which thereby thesis , such as by conjugation with a label . Other types of improves the level of purity of the polypeptide in the modifications include , for example , " caps” , substitution of composition ( i . e . by decreasing the amount (ppm ) of impu one or more of the naturally occurring nucleotides with an 55 rity ( ies ) in the composition ) . analog , internucleotide modifications such as , for example , The terms “ ion - exchange ” and “ ion - exchange chromatog those with uncharged linkages ( e . g . methyl phosphonates raphy” refer to a chromatographic process in which an phosphotriesters , phosphoamidates, carbamates , etc . ) and ionizable solute of interest ( e . g . a protein of interest in a with charged linkages (e .g . phosphorothioates, phosphoro - mixture ) interacts with an oppositely charged ligand linked dithioates , etc . ), those containing pendant moieties, such as, 60 ( e . g . by covalent attachment) to a solid phase ion exchange for example , proteins ( e .g . nucleases, toxins , antibodies, material under appropriate conditions of pH and conductiv signal peptides, ply - L - lysine , etc . ), those with intercalators ity, such that the solute of interest interacts non - specifically ( e . g . acridine , psoralen , etc . ) , those containing chelators ( e . g . with the charged compound more or less than the solute metals , radioactive metals , boron , oxidative metals , etc . ), impurities or contaminants in the mixture . The contaminat those containing alkylators, those with modified linkages 65 ing solutes in the mixture can be washed from a column of ( e . g . alpha anomeric nucleic acids, etc . ) , as well as unmodi- the ion exchange material or are bound to or excluded from fied forms of the polynucleotide ( s ). Further, any of the the resin , faster or slower than the solute of interest. “ Ion US 10 , 392 , 438 B2 63 64 exchange chromatography” specifically includes cation others known to the skilled person , such as those described exchange , anion exchange , and mixed mode chromatogra - herein ) , and is able to align sequences, particularly antibody phies. constant domain sequences according to known structural A “ blocking ” antibody or an “ antagonist ” antibody is one motifs , especially due to the large number of exemplary which inhibits or reduces biological activity of the antigen it 5 structural studies already existent for immunoglobulin binds. An " agonist antibody " , as used herein , is an antibody domains, antibodies and antibody constant domains in par which mimics at least one of the functional activities of a ticular, across subtype and species . polypeptide of interest . Computational approaches to sequence alignment gener The term " immune effector cell ” or “ effector cell ” as used ally fall into two categories : global alignments and local herein refers to a cell within the natural repertoire of cells in 10 alignments . Calculating a global alignment is a form of the human immune system which can be activated to affect global optimization that " forces” the alignment to span the the viability of a target cell. The viability of a target cell can entire length of all query sequences. By contrast, local include cell survival, proliferation , and /or ability to interact alignments identify regions of similarity within long with other cells . sequences that are often widely divergent overall . Local Salt bridges are a type of noncovalent interaction . A salt 15 alignments are often preferable , but can be more difficult to bridge involves close - range direct interactions between two calculate because of the additional challenge of identifying atoms with opposite formal charges . In the context of protein the regions of similarity . A variety of computational algo structure , salt bridges most often form between the anionic rithms have been applied to the sequence alignment prob carboxylate (RCOO - ) of either aspartic acid or glutamic lem . These include slow but formally correct methods like acid and the cationic ammonium (RNH3 - ) from lysine or the 20 dynamic programming and also efficient, heuristic algo guanidinium (RNHC (NH ) , + ) of arginine , with histidine rithms or probabilistic methods designed for large -scale another possibility. However, other amino acids may par - database search , that do not guarantee to find best matches . ticipate depending on changes to their pKa values and Global alignments, which attempt to align every residue locations in the polypeptide chain ( the N and C terminal in every sequence , are most useful when the sequences in the residues may be ionized , and thus capable of salt bridge 25 query set are similar and of roughly equal size . A general formation , regardless of amino acid type ). global alignment technique is the Needleman - Wunsch algo Electrostatic interactions are noncovalent interactions rithm , which is based on dynamic programming . Local between atoms having nonzero charge . They may have alignments are more useful for dissimilar sequences that are favorable , unfavorable , or neutral interaction energies and suspected to contain regions of similarity or similar may involve atomswhich have formal charges, or which are 30 sequence motifs within their larger sequence context. The polarized despite the lack of formal charge . Hydrogen Smith - Waterman algorithm is a general local alignment bonds, salt bridges, and pi- cation stacking are examples of method also based on dynamic programming . electrostatic interactions frequently observed in protein Pairwise sequence alignment methods are used to find the structures . best -matching piecewise ( local) or global alignments of two Structural alignments , which are usually specific to pro - 35 query sequences . The three primary methods of producing tein and sometimes RNA sequences , use information about pairwise alignments are dot- matrix methods , dynamic pro the secondary and tertiary structure of the protein or RNA gramming , and word methods ; however, multiple sequence molecule to aid in aligning the sequences . These methods alignment techniques can also align pairs of sequences . are used for two or more sequences and typically produce Although each method has its individual strengths and local alignments; however, because they depend on the 40 weaknesses , all three pairwise methods have difficulty with availability of structural information , they can only be used highly repetitive sequences of low information content for sequences whose corresponding structures are known especially where the number of repetitions differ in the two ( usually through X - ray crystallography or NMR spectros - sequences to be aligned . One way of quantifying the utility copy ) . Because both protein and RNA structure is more of a given pairwise alignment is the ‘maximum unique evolutionarily conserved than sequence , structural align - 45 match ' (MUM ) , or the longest subsequence that occurs in ments can be more reliable between sequences that are very both query sequences . Longer MUM sequences typically distantly related and that have diverged so extensively that reflect closer relatedness . Preferred methods to determine sequence comparison cannot reliably detect their similarity . identity and/ or similarity are designed to give the largest Where there is no available structural data on one of the match between the sequences tested . Methods to determine proteins , a comparison can still be made if structural data is 50 identity and similarity are codified in publicly available available on one or preferably more closely related proteins, computer programs. Preferred computer program methods such as immunoglobulins across species, and in particular to determine identity and similarity between two sequences antibody constant domains across species and subtype . include , but are not limited to , the GCG program package , Structural alignments are used as the “ gold standard ” including GAP (Devereux et al. , Nuc . Acids Res . 12 : 387 because they explicitly align regions of the protein sequence 55 (1984 ) ;Genetics Computer Group , University of Wisconsin , that are structurally similar rather than relying exclusively Madison , Wis . ) , BLASTP , BLASTN , and FASTA ( Atschul on sequence information . A commonly used algorithm for et al. , J. Mol . Biol . 215 : 403 - 10 ( 1990 )) . The BLAST X structural alignments is TM - ALIGN ( Zhang and Skolnick , program is publicly available from the National Center for Nucleic Acids Research , 33 : 2302 - 2309 ( 2005 ) ), which Biotechnology Information (NCBI ) and other sources (Alts assigns increased weight to the most similar regions of the 60 chul et al. , BLAST Manual (NCB NLM NIH , Bethesda, structure during superposition . Md. ); Altschul et al ., 1990 , supra ). The well -known Smith Sequence Alignment Waterman algorithm may also be used to determine identity . Where structural alignment with protein sequences of the By way of example , using the computer algorithm GAP invention is not possible , for example due to an absence of (Genetics Computer Group ) , two polypeptides for which the target sequence NMR or crystal structure data , sequence 65 percent sequence identity is to be determined are aligned for alignment may be used . The skilled person is familiar with optimal matching of their respective amino acids ( the sequence alignment tools (such as BLAST, CLUSTAL and "matched span ” , as determined by the algorithm ). A gap US 10 , 392, 438 B2 65 66 opening penalty (which is calculated as 3x the average release of Abysis (www . abysis . org ) is used herein to assign diagonal; the " average diagonal” is the average of the Kabat numbering to variable regions unless otherwise noted . diagonal of the comparison matrix being used ; the “ diago - The term “ percent sequence identity ” in the context of nucleic acid sequences means the residues in two sequences nal” is the score or number assigned to each perfect amino 5 that are the same when aligned for maximum correspon acid match by the particular comparison matrix ) and a gap 5 dence . The length of sequence identity comparison may be extension penalty (which is usually 0 . 1x the gap opening over a stretch of at least about nine nucleotides, usually at penalty ), as well as a comparison matrix such as PAM 250 least about 18 nucleotides , more usually at least about 24 or BLOSUM 62 are used in conjunction with the algorithm . nucleotides, typically at least about 28 nucleotides, more Preferred parameters for polypeptide sequence comparison 10 typically at least about 32 nucleotides , and preferably at least include the following : Algorithm : Needleman and Wunsch , 11 about 36 , 48 or more nucleotides. There are a number of J . Mol. Biol. 48 : 443 -53 ( 1970 ). Comparison matrix : BLO different algorithms known in the art which can be used to SUM 62 from Henikoff et al ., Proc . Natl . Acad . Sci. U .S . A measure nucleotide sequence identity . For instance , poly 89 : 10915 - 19 (1992 ) . nucleotide sequences can be compared using FASTA , Gap Other exemplary algorithms, gap opening penalties , gap 15 or Bestfit, which are programs in Wisconsin Package Ver extension penalties , comparison matrices, thresholds of sion10 . 0 , Genetics Computer Group (GCG ) , Madison , Wis . similarity , etc . may be used by those of skill in the art , FASTA , which includes , e . g . the programs FASTA2 and including those set forth in the Program Manual, Wiscon - FASTA3 , provides alignments and percent sequence identity sinPackage , Version 9 , September , 1997 . The particular of the regions of the best overlap between the query and choices to be made will depend on the specific comparison o search sequences ( Pearson , Methods Enzymol . 183 :63 - 98 to be made , such as DNA to DNA , protein to protein , protein (1990 ) ; Pearson , Methods Mol. Biol. 132: 185 - 219 (2000 ); to DNA ; and additionally , whether the comparison is Pearson , Methods Enzymol. 266 :227 - 258 ( 1996 ) ; Pearson , J . between given pairs of sequences ( in which case GAP or Mol. Biol . 276 :71 - 84 ( 1998 ); incorporated herein by refer BestFit are generally preferred ) or between one sequence ence ) . Unless otherwise specified , default parameters for a and a large database of sequences ( in which case FASTA or 35 particular program or algorithm are used . For instance , BLASTA are preferred ) . percent sequence identity between nucleic acid sequences For specific protein families with conserved structure , can be determined using FASTA with its default parameters other alignment algorithms are available . In the case of ( a word size of 6 and the NOPAM factor for the scoring antibodies , various algorithms for assigning Kabat number - matrix ) or using Gap with its default parameters as provided ing are available . The algorithm implemented in the 2012 in GCG Version 6 . 1 , incorporated herein by reference . TABLE 1 Sequence list . Dashes ( " - " ) indicate gaps introduced to align the various allotype and isotype domains with Kabat numbering . Alignments were adjusted so that boundaries ( spaces ) between blocks of 10 residues always fall between residue numbering positions ending in " O " and residue numbering positions ending in " 1 " . Where a domain does not begin with a residue whose numbering position ends with " 1 " , phonypadding gaps were inserted to adjust the alignment . For example , for Chi ( Seq ID 1 ) the Kabat domain begins at position 114 . Three gap characters were inserted to put the alignment in the correct frame ( starting with position 111 ) . These three gap residues ( corresponding to Kabat positions 111 - 113 ) are properly part of the VH domain and would generally be occupied by real amino acids belonging to the VH in a full antibody sequence . Similar padding gaps may be found at the beginning and end of some other sequences . The sequences herein described for 29D7 ( SEQ ID NOS : 51 and 53 , and other sequences that comprise those sequences ) are those of the antibody TAM - 163 . All examples herein described as using antibody 29D7 used the antibody TAM - 163 . SEQUENCE LIST SEQ Description Sequence IgG1 CH1 numbered - - - ASTKGPS VFPLAPSSKS - - TSGGTAAL GCLVKDYFPE PVTV - SW - - - from residue 111 - N - - - SGALT SG - VHTFPAV LOS - SGLYSL SSWTVPSSS LGT - - Q - TYI to 231 (with CNVNHKPSNT KVDKKV - - EP KSC insertions 162A , 162B , and 1620 after position 162 ) IgG1 CH1 G1m3 - - - ASTKGPS VFPLAPSSKS - - TSGGTAAL GCLVKDYFPE PVTV - SW - - - using the - N - - - SGALT SG - VHTFPAV LOS - SGLYSL SSWTVPSSS LGT - - Q - TYI numbering range of CNVNHKPSNT KVDKRV - - EP KSC Seq 1

??? IgG2 CH1 using the - - - ASTKGPS VFPLAPCSRS - - TSESTAAL GCLVKDYFPE PVTV - SW - - - numbering range of - N - - - SGALT SG - VHTFPAV LOS - SGLYSL SSWTVPSSN FGT - - Q - TYT Seq 1 CNVDHKPSNT KVDKTV - - ER K - - - IgG2 CH1 G2m23 - - - ASTKGPS VFPLAPCSRS - - TSESTAAL GCLVKDYFPE PVTV - SW - - - using the - N - - - SGALT SG - VHTFPAV LQS - SGLYSL SSWTVTSSN FGT - - Q - TYT numbering range of CNVDHKPSNT KVDKTV - - ER K - - - Seq 1 US 10 , 392 , 438 B2 67 68 TABLE 1 - continued Sequence list . Dashes ( " - " ) indicate gaps introduced to align the various allotype and isotype domains with Kabat numbering . Alignments were adjusted so that boundaries ( spaces ) between blocks of 10 residues always fall between residue numbering positions ending in " O " and residue numbering positions ending in " 1 " . Where a domain does not begin with a residue whose numbering position ends with " 1 " , phonypadding gaps were inserted to adjust the alignment . For example , for Cyl (Seq ID 1 ) the Kabat domain begins at position 114 . Three gap characters were inserted to put the alignment in the correct frame ( starting with position 111 ) . These three gap residues ( corresponding to Kabat positions 111 - 113 ) are properly part of the VH domain and would generally be occupied by real amino acids belonging to the VH in a full antibody sequence . Similar padding gaps may be found at the beginning and end of some other sequences . The sequences herein described for 29D7 ( SEQ ID NOS : 51 and 53 , and other sequences that comprise those sequences ) are those of the antibody TAM - 163 . All examples herein described as using antibody 29D7 used the antibody TAM - 163 . SEQUENCE LIST SEQ Description Sequence IgG3 CH1 using the - - - ASTKGPS VFPLAPCSRS - - TSGGTAAL GCLVKDYFPE PVTV - SW - - - numbering range of - N - - - SGALT SG - VHTFPAV LOS - SGLYSL SSWTVPSSS LGT - - Q - TYT Seq 1 CNVNHKPSNT KVDKRV - - EL KTP IgG4 CH1 using the - - - ASTKGPS VFPLAPCSRS - - TSESTAAL GCLVKDYFPE PVTV - SW - - - numbering range of - N - - - SGALT SG - VHTFPAV LQS - SGLYSL SSWTVPSSS LGT- - K - TYT Seq 1 CNVDHKPSNT KVDKRV - - ES KYG IgM CH1 seq1 - - - GSASAPT LFPLVSCENS P - SDTSSVAV GCLAQDFLPD SITL - SW - - - numbered from - KYKNNSDIS S - - TRGFPSV LRG - - GKYAA TSQVLLPSKD VMQGTDEHVV residue 111 to 226 CKVQH - PNGN - - KEKNVPLP ( with insertions 162A , 162B , and 162C after position 162 ) IgM CH1 seq2 using - - - GSASAPT LFPLVSCENS P - SDTSSVAV GCLAODFLPD SITF - SW - - - the numbering - KYKNNSDIS S - - TRGFPSV LRG - - GKYAA TSQVLLPSKD VMAGTDEHVV range of Seq 7 CKVQH - PNGN - - KEKNVPLP CL - Kappa - KM3 ------RTV AAPSVFIFPP SDEOLKSGTA SVVCLLNNFY PREAKVQWKV numbered from DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG position 101 to LSSPVTKSFN RGEC 215 10 CL - Kappa - KM1 using ------RTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV the numbering DNVLQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK LYACEVTHQG range of Seq 9 LSSPVTKSFN RGEC 11 CL - Kappa - KM1 , 2 ------RTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV using the DNALOSGNSO ESVTEQDSKD STYSLSSTLT LSKADYEKHK LYACEVTHOG numbering range of LSSPVTKSFN RGEC Seq 9 12 CL - Lambda using ------QPK AAPSVTLFPP SSEELQANKA TLVCLISDFY PGAVTVAWKA the numbering DSSPVKAGVE TTTPSKOS - N NKYAASSYLS LTPEQWKSHR SYSCOV THEG range of Seq 9 - - STVEKTVA PTECS 13 IgG1 CH2 numbered - - - APELLGG PSVFLFPPKP KDTLMI - SRT PEVTCVVVDV SHEDPEVKFN from 241 to 360B WYV - - DG - - V EVH -NAKTKP REEQYN - - - - STYRVVSVLT VLHQDWLNGK with insertions EYKCKVSNKA LPAPIEKTI - SKAK - - 266A , 302A , 316A , 316B , 360A , and 36 OB 14 IgG2 CH2 using the - - - APPVA - G PSVFLFPPKP KDTLMI - SRT PEVTCVVVDV SHEDPEVOFN numbering range of WYV - - DG - - V EVH - NAKTKP REEQFN - - - - STFRVVSVLT VVHQDWLNGK Seq 13 EYKCKVSNKG LPAPIEKTI - SKTK - - 15 IgG3 CH2 using the - - - APELLGG PSVFLFPPKP KDTLMI - SRT PEVTCVVVDV SHEDPEVOFK numbering range of WYV - - DG - - V EVH - NAKTKP REEOYN - - - - STFRVVSVLT VLHODWLNGK Seq 13 EYKCKVSNKA LPAPIEKTI - SKTK - - 16 ITG4 CH2 using the - - - APEFLGG PSVFLFPPKP KDTLMI - SRT PEVTCVVVDV SQEDPEVQFN numbering range of WYV - - DG - - V EVH - NAKTKP REEQFN - - - - STYRVSVLT VLHODWLNGK Seq 13 EYKCKVSNKG LPSSIEKTI - SKAK - - 17 IgM CH2 using the - - - - VIAELP PKVSVFVPPR DGFFGN - PRK SKLICQATGF S - - PRQIQVS numbering range of WLR - - EG - - K OVGSGVTTDO VOAEAKESGP TTYKVTSTLT IKESDWLGOS Seq 13 MFTCRVDHRG L - - TFQQNA - SSMCVP US 10 , 392 , 438 B2 69 70 TABLE 1 - continued Sequence list . Dashes ( " - " ) indicate gaps introduced to align the various allotype and isotype domains with Kabat numbering . Alignments were adjusted so that boundaries ( spaces ) between blocks of 10 residues always fall between residue numbering positions ending in " O " and residue numbering positions ending in " 1 " . Where a domain does not begin with a residue whose numbering position ends with " 1 " , phonypadding gaps were inserted to adjust the alignment . For example , for Cyl (Seq ID 1 ) the Kabat domain begins at position 114 . Three gap characters were inserted to put the alignment in the correct frame ( starting with position 111 ) . These three gap residues ( corresponding to Kabat positions 111 - 113 ) are properly part of the VH domain and would generally be occupied by real amino acids belonging to the VH in a full antibody sequence . Similar padding gaps may be found at the beginning and end of some other sequences . The sequences herein described for 29D7 ( SEQ ID NOS : 51 and 53 , and other sequences that comprise those sequences ) are those of the antibody TAM - 163 . All examples herein described as using antibody 29D7 used the antibody TAM - 163 . SEQUENCE LIST

SEQ Description Sequence 18 IqG1 CH3 numbered G - QPREPOVY TLPPSREE - - MTKNOVSLTC LVKGFYPS - D IAV - - EWES - from 361 to 478 NG - - QPENNY KTTPPVLDS - D - -GSFFLYS KLTVDKSRWO QGNVFSCSVM with insertion HEALHNHYTO KSLSLSPGK 398A 19 IgG1 CH3 alternate G - OPREPOVY TLPPSRDE - - LTKNOVSLTC LVKGFYPS -DIAV - - EWES - isotype using the NG - - QPENNY KTTPPVLDS - D - - GSFFLYS KLTVDKSRWQ QGNVFSCSVM numbering range of HEALHNHYTO KSLSLSPGK Seq 18 20 IgG2 CH3 using the G - QPREPOVY TLPPSREE - - MTKNQVSLTC LVKGFYPS - D IAV - - EWES numbering range of NG - - QPENNY KTTPPMLDS - D - -GSFFLYS KLTVDKSRWQ QGNVFSCSVM Seq 18 HEALHNHYTO KSLSLSPGK 21 IgG3 CH3 using the G - QPREPQVY TLPPSREE - - MTKNQVSLTC LVKGFYPS - D IAV - - EWES - numbering range of SG - - OPENNY NTTPPMLDS - D - -GSFFLYS KLTVDKSRWO OGNIFSCSVM Seq 18 HEALHNRFTQ KSLSLSPGK 22 IgG4 CH3 using the G - OPREPOVY TLPPSOEE - - MTKNOVSLTC LVKGFYPS - D IAV - - EWES - numbering range of NG - - QPENNY KTTPPVLDS - D - -GSFFLYS RLTVDKSRWO EGNVFSCSVM Seq 18 HEALHNHYTO KSLSLSLGK 23 IgM CH3 using the D - ODTAIRVF AIPPSFASI - FLTKSTKLTC LVTDLTTYDS VTI - - SWTRO numbering range of NG - - EAV - KT HTNISESHP . N - - ATFSAVG EASICEDDWN SGERFTCTVT Seq 18 HTDLPSP - LK QTISRPK 24 CL - V133S - 5176D ------RTV AAPSVFIFPP SDEOLKSGTA SVSCLLNNFY PREAKVOWKV using the DNALOSGNSO ESVTEODSKD STYSIPSTLT LSKADYEKHK VYACEVTHOG numbering range of LSSPVTKSFN RGEC Seq 9 25 CL -V133S - 5176K ------RTV AAPSVFIFPP SDEOLKSGTA SVSCLLNNFY PREAKVQWKV using the DNALQSGNSO ESVTEQDSKD STYSLKSTLT LSKADYEKHK VYACEVTHOG numbering range of LSSPVTKSFN RGEC Seq 9 26 CL - L1 . 1 using the ------RTV AAPSVFIFPP SDKOLKSGTA SVVCILNNFY PREAKVOWKV numbering range of DNALQSGNSQ ESVTEQDSKD STYSLCSTLT LSKADYEKHK VYACEVTHQG Seq 9 LSSPVTKSFN RGES 27 CL - L4 . 1 using the ------RTV AAPSVFIFPP SDCQLKSGTA HVVCLLNNFY PREAKVQWKV numbering range of DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHOG Seq 9 LSSPVTKSFN RGES 28 CL -L4 . 2 using the ------RTV AAPSVFIFPP SDEOLKSGTA HVVCILNNFY PREAKVQWKV numbering range of DNALQSGNSO ESVTEQDSKD STYSLCSTLT LSKADYEKHK VYACEVTHQG Seq 9 LSSPVTKSFN RGES 29 CL - L4 . 3 using the ------RTV AAPSVCIFPP SDEQLKSGTA HVVCLLNNFY PREAKVQWKV numbering range of DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG Seq 9 LSSPVTKSFN RGES 30 CL -H10 . 1 using the ------RTV AAPSVFIFPP SDEOLKSGTA DVSCLLNNFY PREAKVQWKV numbering range of DNALOSGNSO ESVTEQDSKD STYSLCSSLT LSKADYEKHK VYACEVTHOG Seq 9 LSSPVTKSFN RGES 31 CL - H10 . 4 using the ------RTV AAPSVFIFPP SDCOLKSGTA SVMCLLNNFY PREAKVQWKV numbering range of DNALQSGNSQ ESVTEQDSKD STYSLGSGLT LSKADYEKHK VYACEVTHOG Seq 9 LSSPVTKSFN RGES US 10 , 392 , 438 B2 72 TABLE 1 - continued Sequence list . Dashes ( " - " ) indicate gaps introduced to align the various allotype and isotype domains with Kabat numbering . Alignments were adjusted so that boundaries ( spaces ) between blocks of 10 residues always fall between residue numbering positions ending in " O " and residue numbering positions ending in " 1 " . Where a domain does not begin with a residue whose numbering position ends with " 1 " , phonypadding gaps were inserted to adjust the alignment . For example , for Cyl (Seq ID 1 ) the Kabat domain begins at position 114 . Three gap characters were inserted to put the alignment in the correct frame ( starting with position 111 ) . These three gap residues ( corresponding to Kabat positions 111 - 113 ) are properly part of the VH domain and would generally be occupied by real amino acids belonging to the VH in a full antibody sequence . Similar padding gaps may be found at the beginning and end of some other sequences . The sequences herein described for 29D7 ( SEQ ID NOS : 51 and 53 , and other sequences that comprise those sequences ) are those of the antibody TAM - 163 . All examples herein described as using antibody 29D7 used the antibody TAM - 163 . SEQUENCE LIST SEQ Description Sequence 32 CL - 5 . 6 using the ------RTV AAPSVCIFPP SDCOLKSGTA DVSCLLNNFY PREAKVQWKV numbering range of DNALQSGNSQ ESVTEQDSKD STYSLCSSLT LSKADYEKHK VYACEVTHOG Seq 9 LSSPVTKSFN RGES 33 CH1 - L124K - V190S - - - ASTKGPS VFPKAPSSKS - - TSGGTAAL GCLVKDYFPE PVTV - SW - - - numbered from - N - - - SGALT SG - VHTFPAV LOS - SGLYSL SSSVTVPSSS LGT - - Q - TYI residue 111 to 230 CNVNHKPSNT KVDKKV - - EP KSC ( with insertions 162A , 162B , and 162C after position 162 ) 34 CH1 - L124E - S188G - - - ASTKGPS VFPEAPSSKS - - TSGGTAAL GCLVKDYFPE PVTV - SW - - - numbered using the - N - - - SGALT SG - VHTFPAV LQS - SGLYSL GSWTVPSSS LGT - - Q - TYI range of Seq 33 CNVNHKPSNT KVDKKV - - EP KSC 35 CH1- L1 . 1 numbered - - - ASTKGPS VFPLAPSSKS - - TSGGTAAL GCLVKDYFPE PVTV - SW - - - using the range of - N - - - SGALT SG - VHTCPAV LOS - SGLYSL SSIVTVPSSS LGT - - Q - TYI Seq 33 CNVNHKPSNT KVDDKV - - EP KSS 36 CH1 - L4 . 1 numbered - - - ASTKGPS VCPLAPSSKS - - TSGGTAAL GCLVEDYFPE PVTV - SW - - - using the range of - N - - - SGALT SG - VHTFPAV LOS - SGLYSL SSWTVPSSS LGT - - Q - TYI Seq 33 CNVNHKPSNT KVDKKV - - EP KSS 37 CH1 - L4 . 2 numbered - - - ASTKGPS VFPLAPSSKS - - TSGGTAAL GCHVKDYFPE PVTV - SW - - - using the range of - N - - - SGALT SG - VHTCPAV LDS - SGLYEL SSIVTVPSSS LGT - - Q - TYI Seq 33 CNVNHKPSNT KVDKKV - - EP KSS 38 CH1 - L4 . 3 numbered - - - ASTKGPS VFPLAPSSKS - - TSGGTACL GCLVSDYFPE PVTV - SW - - - using the range of - N - - - SGALT SG - VHTFPAV LQS - SGLYEL SSVVTVPSSS LGT- - Q - TYI Seq 33 CNVNHKPSNT KVDKKV - - EP KSS 39 CH1 - H10 . 1 numbered - - - ASTKGPS VFPLAPSSKS - - TSGGTAAL GCSVKDYFPE PVTV - SW - - - using the range of - N - - - SGALT SG - VHTCPAV LOS - SGLYSL WSWTVPSSS LGT - - Q - TYI Seq 33 CNVNHKPSNT KVDKKV - - EP KSS 40 CH1 - H10 . 4 numbered - - - ASTKGPS VCPLAPSSKS - - TSGGTAAL GCSVKDYFPE PVTV - SW - - - using the range of - N - - - SGALT SG - VHTFPAV LOS - SGLYSL WSWTVPSSS LGT - - Q - TYI Seq 33 CNVNHKPSNT KVDKKV - - EP KSS 41 CH1 - 5 . 6 numbered - - - ASTKGPS VCPLAPSSKS - - TSGGTACL GCSVKDYFPE PVTV - SW - - - using the range of - N - - - SGALT SG - VHTCPAV LOS - SGLYSL WSVTVPSSS LGT - - O - TYI Seq 33 CNVNHKPSNT KVDKKV - - EP KSS DESSE EXT -* * TYI 42 IGG1 - HINGE - D - - KTHTCP PCP numbered from 231 to 243 43 IgG1 -HINGE - EE - E - - KTHTCP ECP numbered using the range of Seq 42 IGG1 - HINGE - RR - R - - KTHTCP RCP numbered using the range of Seq 42 45 CH2 - WINTER - - - APEAAGA PSVFLFPPKP KDTLMI - SRT PEVTCVVVDV SHEDPEVKFN numbered using the WYV - - DG - - V EVH - NAKTKP REEQYN - - - - STYRVSVLT VLHQDWLNGK range of Seq 13 EYKCKVSNKA LPAPIEKTI - SKAK - -