USOO82363.08B2

(12) United States Patent (10) Patent No.: US 8.236,308 B2 Kischel et al. (45) Date of Patent: Aug. 7, 2012

(54) COMPOSITION COMPRISING McLaughlin et al., Cancer Immunol. Immunother, 1999.48, 303 CROSS-SPECIES-SPECIFIC ANTIBODES 3.11. AND USES THEREOF The U.S. Department of Health and Human Services Food and Drug Administration, Center for Biologics Evaluation and Research, “Points to Consider in the Manufacture and Testing of Monoclonal (75) Inventors: Roman Kischel, Karlsfeld (DE); Tobias Antibody Products for Human Use.” pp. 1-50 Feb. 28, 1997.* Raum, München (DE); Bernd Hexham et al., Molecular Immunology 38 (2001) 397-408.* Schlereth, Germering (DE); Doris Rau, Gallart et al., Blood, vol.90, No. 4 Aug. 15, 1997: pp. 1576-1587.* Unterhaching (DE); Ronny Cierpka, Vajdos et al., J Mol Biol. Jul. 5, 2002:320(2):415-28.* München (DE); Peter Kufer, Moosburg Rudikoff et al., Proc. Natl. Acad. Sci. USA, 79: 1979-1983, Mar. (DE) 1982.* Colman P. M., Research in Immunology, 145:33-36, 1994.* (73) Assignee: Micromet AG, Munich (DE) International Search Report for PCT International Application No. PCT/EP2006/009782, mailed Nov. 7, 2007 (6 pgs.). *) Notice: Subject to anyy disclaimer, the term of this Bortoletto Nicola et al., “Optimizing Anti-CD3Affinity for Effective patent is extended or adjusted under 35 T Cell Targeting Against Tumor Cells'. European Journal of Immu U.S.C. 154(b) by 491 days. nology, Nov. 2002, vol. 32 (11), pp. 3102-3107. (XPO02436763). Fleiger, D. et al., “A Bispecific Single-Chain Antibody Directed Against EpCAM/CD3 in Combination with the Cytokines Interferon (21) Appl. No.: 12/083,351 Alpha and Interleukin-2 Efficiently Retargets T and CD3+CD56+ Natural-Killer-Like T Lymphocytes to EpCAM-Expressing Tumor (22) PCT Filed: Oct. 10, 2006 Cells', Cancer Immunology and Immunotherapy, Berlin, DE, Oct. 2000, vol. 49 (8), pp. 441-448. (XP002263709). (86). PCT No.: PCT/EP2006/0097.82 Jacobs, et al., “Efficiency of T Cell Triggering by Anti-CD3 Monoclonal Antibodies (mAb) with Potential Usefulness in S371 (c)(1), Bispecific mAb Generation.” Cancer Immunology and (2), (4) Date: Jun. 3, 2008 Immunotherapy, Jul. 1997, vol. 44(5), pp. 257-264. (XP002453511). Knechtle, S.J. et al., “FN18-CRM9 Immunotoxin Promotes Toler (87) PCT Pub. No.: WO2007/042261 ance in Primate Renal Allografts.”. Transplantation, Jan. 1997, vol. 63 (1), pp. 1-6. (XP002453509). PCT Pub. Date: Apr. 19, 2007 Nevell, et al., “In Vivo T-Cell Ablation by a Holo-Immunotoxin Directed at Human CD3”. Proceedings of the National Academy of (65) Prior Publication Data Sciences of USA, National Academy of Science, Washington D.C., US 2009/O252683 A1 Oct. 8, 2009 Apr. 1992, vol. 89, pp. 2585-2589. (XP002083390). Rogers, Kenneth et al., Identification and Characterization of Macaque CD89 (immunoglobulin A Fc receptor), Immunology, Oct. Related U.S. Application Data 2004, vol. 113 (2), pp. 178-186. (XP002453510). (60) Provisional application No. 60/724,781, filed on Oct. Salmeron A. et al., “A Confirmational Epitope Expressed Upon Asso ciate of CD3-Epsilon with Either CD3-Delta or CD3-gamma is the 11, 2005. Main Target for Recognition by Anti-CD3 Monoclonal Antibodies'. (51) Int. Cl. Journal of Immunology, Nov. 2001, vol. 147 (9), pp. 3047-3052. A 6LX39/395 (2006.01) (XPO02453512). (52) U.S. Cl...... 424/136.1; 424/144.1; 424/156.1 (Continued) (58) Field of Classification Search ...... None See application file for complete search history. Primary Examiner — Zachary Skelding (74) Attorney, Agent, or Firm — Foley & Lardner LLP (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS The present invention relates to uses of bispecific antibodies 2002/0142000 A1 10/2002 Digan et al. exhibiting cross-species specificity for evaluating the in vivo 2002/0147312 A1 10, 2002 O'Keefe et al. 2005/020 1994 A1 9, 2005 Korman et al. safety and/or activity and/or pharmacokinetic profile of the 2006.0034835 A1 2, 2006 Adams et al. same in non-human species and humans. The present inven tion moreover relates to methods for evaluating the in vivo FOREIGN PATENT DOCUMENTS safety and/or activity and/or pharmacokinetic profile of said WO WO 2004/106380 12, 2004 bispecific anti-bodies exhibiting cross-species specificity. WO WO 2005/061547 7/2005 The present invention also relates to methods of measuring WO WO 2006/084264 8, 2006 the biological activity and/or efficacy of such bispecific anti WO WO 2007OO8943 A2 * 1, 2007 bodies exhibiting cross-species specificity. In addition, the WO WO 2010.042904 A2 * 4, 2010 present invention relates to pharmaceutical compositions OTHER PUBLICATIONS comprising bispecific single chain antibodies exhibiting Macket al., The Journal of Immunology, 1997, 158: 3965-3970.* cross-species specificity and to methods for the preparation of Yoshino et al., Exp Anim. Apr. 2000:49(2):97-110.* pharmaceutical compositions comprising said bispecific Harlow etal. Antibodies, A Laboratory Manual, Cold Spring Harbor single chain antibodies exhibiting cross-species specificity Laboratory, 1988, pp. 567-569.* for the treatment of diseases. Lammers et al., Experimental Hematology 30 (2002), pp. 537-545.* Offner et al., Cancer Immunol Immunother (2005) 54: 431-445.* 9 Claims, 41 Drawing Sheets US 8.236,308 B2 Page 2

OTHER PUBLICATIONS Office Action Received in the corresponding European Patent Appli Schlereth, Bernd et al., “T-Cell Activation and B-Cell Depletion in cation No. EP 06806 155.5, dated Mar. 24, 2010. Chimpanzees Treated with a Bispecific Anti-CD19/Anti-CD3 Davis, et al., "Affinity improvement of single antibodyVH domains: Single-Chain Antibody Construct”. Cancer Immunology and residues in all three hypervariable regions affect antigen binding'. Immunotherapy, Springer-Verlag, BE, May 2006, vol. 55(5), pp. Immunotechnology, vol. 2, 1996, pp. 169-179. 503-5 14. (XPO19333227). Holt, et al., “Domain antibodies: proteins for therapy”. TRENDs in Wolf, et al. “Bites: Bispecific Antibody Constructs with Unique Biotechnolgy, vol. 21, No. 11, pp. 484-490, 2003. Anti-Timor Activity”. DDT-Drug Discovery Today, Elsevier Science Ltd, GB, vol. 10 (18), pp. 1237-1244. (XP005103829), 2005. * cited by examiner U.S. Patent Aug. 7, 2012 Sheet 1 of 41 US 8,236,308 B2

FIGURE 1

Anti-CD3

OKT-3

g Comprising SEO D NOS. 6 and 8

g comprising SEO D NOS. 2 and 4

UCHT-1 U.S. Patent Aug. 7, 2012 Sheet 2 of 41 US 8,236,308 B2

FIGURE 2

Cynomolgus PBMC HPB-ALL lg comprising SEQID NOS. 2 and 4

g Comprising SEO D NOS. 6 and 8

FN-18 U.S. Patent Aug. 7, 2012 Sheet 3 of 41 US 8,236,308 B2

FIGURE 3

Kato ill HPB-ALL 8 5-1 OLHXSEOD 2 NO.16 d5 1 00 101 102 103 104 F 2-H y 3. 5-1 OLHXSEOD NO.14 3 so of 2 3 4 FL2-H human EpCAM transfected CHO HPB-ALL S 8 t 5-10LHxSEQID 8 NO.12 8 currences10 Oto 10 m 102 103 to F2H

8 8 t 5-1 OLHXSEO D gto 8 NO.10 8 00 O00 U.S. Patent Aug. 7, 2012 Sheet 4 of 41 US 8,236,308 B2

FIGURE 4

9 O A 5-1 OLHXSEOD NO.14 - v 5-1 OLHXSEQ D NO.12 O5 A. Y. ) 0 5-1 OLHXSEQD NO.10 1 5

- 10 105 10 103 102 1 0 1 OP 10 1 0 10° 10' concentration in ng/ml of bispecific antibody U.S. Patent Aug. 7, 2012 Sheet 5 of 41 US 8,236,308 B2

FIGURE 5

65 5-1OLHXSECR D NO.14 40 5-1 OLHXSECR D NO.12 8 5-1 OLHXSECR D NO.10 s 5-1 OLHXdi-anti CD3 O 15 SS

- 10 10-5 10-4 10-3 10-2 10-1 100 101 102 103 104 concentration in ng/ml of bispecific antibody

U.S. Patent Aug. 7, 2012 Sheet 7 of 41 US 8,236,308 B2

FIGURE 7

M79 3B10 2G8 Cd Cd Cs O C Co E ce a C C

d O O d C O A O O . . . 'N M c) C) A 100 of 102 103 104 100 0 102 103 o' 100 0 1 102 0304 U.S. Patent Aug. 7, 2012 Sheet 8 of 41 US 8,236,308 B2

FIGURE 8 FIGURE 8(A) Kato HPB-ALL d O to 2 tle . 2G8LHXSEQID A Oo NO.12 Oo MY 100 to 102 103 10?

O O 2 E. 2. 2G8LHXSEOD Oo 3. NO.1O Oo 100 to 102 103 to 100 to 102 103 to F 2-H F2H O O in 2 29 2G8HLXSEOD O O NO.12 () O 100 to 102 103 10? 00 F2H

O O in 2 29 2C8HLXSEOD Oo g 2. NO.1 O Oo 100 to 102 103 10? 1 U.S. Patent Aug. 7, 2012 Sheet 9 of 41 US 8,236,308 B2

FIGURE 8(B)

cynomolgus EpCAM transfected CHO HPB-ALL

2G8LHXSEOD NO.16 OO 10 O2 103 04 FL2-H

2G8LHXSEOD NO.14

2G8HLXSEO ED NO.16

2G8HLXSEOD NO.14

5-1 OLHXSEO D NO.10 OO 101 102 103 101 F 2-- U.S. Patent Aug. 7, 2012 Sheet 10 of 41 US 8,236,308 B2

FIGURE 9

60 A 2G8LHXSEOD NO.1O 50 k () () v 2G8HLXSEOD NO.12 40 o 5-1 OLHXdi-anti CD3 30

20

10

O 10-4 10-3 10-2 10-1 10 0 1 0 1 102 103 104 concentration in ng/ml of bispecific antibody U.S. Patent Aug. 7, 2012 Sheet 11 of 41 US 8,236,308 B2

FIGURE 10

A 2G8LHXSEOD NO.1 O v 2G8HLXSECR D NO.12 O 5-1 OLHXCi-anti CD3

1 O O 10-4 10-3 10-2 10-1 100 101 102 103 104 concentration in ng/ml of bispecific antibody U.S. Patent Aug. 7, 2012 Sheet 12 of 41 US 8,236,308 B2

FIGURE 11

SEO ID NO. 2 EVKLEESGGGLVQPKGSLKLSCAASGFTFN h3-73 EVQLSESGGGLVOPGGSLKLSCAASGFTFS

SEO ID NO. 2 TYAMNWVROAPGKGLEWV RIRSKYNNYAT hu3-73 GSAMHWVROASGKGLEW RIRSKANSYAT

SEQ ID NO. 2 YYADSVKRFTISRDDSQSILYLOMNNLKT: hu3-73 AYAASVKGRETISRDDSKNTAYLOMNSLKT

SEO ID NO. 2 EDTAMYYCVRHGNFGNSYVSWFA WGQGTL i hu3-73/ ER4hu J1 EDTASYYCT WGOGTL

SEO ID NO. 2 WTVSA hu3-73 WTVSS

U.S. Patent Aug. 7, 2012 Sheet 14 of 41 US 8,236,308 B2

FIGURE 13

Counts

SEQID NO.10

SEQID NO.146

CD3 Positivity U.S. Patent Aug. 7, 2012 Sheet 15 of 41 US 8.236,308 B2

FIGURE 14

cynoPEFITC.008 e cynoPEFITC.002 e

y y a yaan l v- t v

0 ya> wn . S. se O A- 2 e 100 101 102 103 104 A FL1-H

O cynoPEFITC006

CD2 Positivity U.S. Patent Aug. 7, 2012 Sheet 16 of 41 US 8,236,308 B2

FIGURE 15

l 4 O human CD38 cyno CD38 QDGNEEMGSITOTPYQVSISGTTVILTCSOLGSEAQWOH 41 46 47 71 abcdefghk human CD38 NDKNIGGDE DDKNIGSDEDHLSLKEFSEEOSGYYvcYPR cyno CD3E NGKNKG------DSGDQLFLPEFSEMEQSGYYVCYPR E-F-loop

* ... - c. 96 human CD3E cyno CD3& GSNPEDASHELYLEARVCENCMEMD U.S. Patent Aug. 7, 2012 Sheet 17 of 41 US 8,236,308 B2 FIGURE 16 1. ODGNEEMGSITOT 2 GNEEMGSITOTPY 3 EEMGSITOTPYOV 4 MGSITOTPYOVSI 5 SITOTPYOVSISG 6 TOTPYOVSISGTT 7 TPYOVSISGTTVI 8 YOVSISGTTVILT 9 VSISGTTVILTSS 1 O ISGTTVILTSSOH 11 GTTVILTSSOHLG 12 TVILTSSOHLGSE 13 ELTSSOHLGSEAO 14 TSSOHLGSEAOWO 15 SOHLGSEAQWOHN 16 HLGSEAOWOHNGK 17 GSEAOWOHNGKNK 18 EAQWOHNGKNKGD 19 QWOHNGKNKGDSG 20 OHNGKNKGDSGDO 21 NGKNKGDSGDOLF 22 KNKGDSGDOLFLP 23 KGDSGDOLFLPEF 24 DSGDOLFLPEFSE 25 GDOLFLPEFSEME 26 OLFLPEFSEMEQS 27 FLPEESEMEOSGY 28 PEESEMEOSGYYV 29 ESEMEOSGYYVSY 3O EMEOSGYYVSYPR 31 EOSGYYVSY PRGS 32 SGYYVSYPRGSNP 33 YYWSYPRGSNPED 34 VSYPRGSNPEDAS 35 YPRGSNPEDASHH 36 RGSNPEDASHHILY 37 SNPEDASHHILYLK 38 PEDASHHLYLKAR 39 DASHHILYKARVS 4 O SHHILYLKARVSEN 41 HLYILKARVSENSM 42 YLKARVSENSMEM 43 KARVSENSMEMD U.S. Patent Aug. 7, 2012 Sheet 18 of 41 US 8,236,308 B2 FIGURE 17 1. DGNEEMGGITOTP 2 NEEMGGITOTPYK 3 EMGGITOTPYKVS 4 GGITOTPYKVSIS 5 ITOTPYKVSISGT 6 OTPYKVSISGTTV 7 PYKVSSGTTWIL 8 KVSISGTTWILTS 9 SISGTTVILTSPO 1 O SGTTVILTSPOYP 11 TTVILTSPOYPGS 12 VILTSPOYPGSEI 1.3 LTSPOYPGSEILW 14 SPOYPGSEILWOH 15 OYPGSEILWOHND 16 PGSEILWOHNDKN 17 SEILWOHNDKNIG 18 ILWOHNDKNIGGD 19 WOHNDKNIGGDED 2O HNDKNIGGDEDDK 21 DKNIGGDEDDKNI 22 NIGGDEDDKNIGS 23 GGDEDDKNIGS DE 24 DEDDKNIGSDEDH 25 DDKNIGS DEDHS 26 KNIGSDEDHLSLK 27 IGSDEDHLSLKEF 28 SDEDHLSLKEFSE 29 EDHLSL, KEESELE 3 O HLSLKEFSELEOS 31 SLKEFSELEOSGY 32 FSEEEOSGYYV 33 FSEEEOSGYYVSY 34 ELEOSGYYVSYPR 35 EOSGYYVSYPRGS 36 SGYYVSYPRGSKP 37 YYVSYPRGSKPED 38 WSYPRGSKPEDAN 39 YPRGSKPEDANFY 4 O RGSKIPEDANFYLY 41 SKIPEDANFYLYILR 42 PEDANFYLYLRAR 43 DANEYLYLRARVS 44 NFYLYLRARVSEN 45 YLYLRARVSENSM 46 YLRARVSENSMEM 47 LRARVSENSMEMD U.S. Patent Aug. 7, 2012 Sheet 19 of 41 US 8,236,308 B2

FIGURE 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617 18 19 20

(43 spots)

human CD3e (47 spots)

I-23 5 9 29 AR 59 PA cyno CD3e (43 spots)

human CD3& (47 spots) U.S. Patent Aug. 7, 2012 Sheet 20 of 41 US 8,236,308 B2

FIGURE 19

1 2 3 4 56789 10 cyno CD3s (43 spots)

human CD38 (47 spots) U.S. Patent Aug. 7, 2012 Sheet 21 of 41 US 8,236,308 B2

U.S. Patent Aug. 7, 2012 Sheet 22 of 41 US 8,236,308 B2

XI5)95), III

ZGT

(tz"ONOIÒBS) U.S. Patent Aug. 7, 2012 Sheet 23 of 41 US 8,236,308 B2

-?CIIÒ?S)-~~~~–·

AJOS9XIII-uetun? CIIÒGIS) 0Iç01?0I001(OLI’ON AHOSQu?Inuu (OI’ON U.S. Patent Aug. 7, 2012 Sheet 24 of 41 US 8,236,308 B2

e 2 C t 9 Z. S S is O r M

e s 9 ed N r O Z S Cya r V

É U.S. Patent Aug. 7, 2012 Sheet 25 of 41 US 8,236,308 B2

FIGURE 24

(A)

cyno CD3e (43 spots)

human CD3E (47 spots)

cyno CD3e (43 spots)

human CD3e (47 spots) U.S. Patent Aug. 7, 2012 Sheet 26 of 41 US 8,236,308 B2

FIGURE 25

U.S. Patent Aug. 7, 2012 Sheet 27 of 41 US 8.236,308 B2

O U.S. Patent Aug. 7, 2012 Sheet 28 of 41 US 8.236,308 B2

Wiz}(anbeoeuu)G-ug

e al

U.S. Patent Aug. 7, 2012 Sheet 30 of 41 US 8.236,308 B2

H-19)6ZEHT|5)|–}0/.?."ONCIIDESX(TH

O 8 (9 O sunoo U.S. Patent Aug. 7, 2012 Sheet 31 of 41 US 8,236,308 B2

OINEdSn6?ouuouÁo

0. E. 0/L"ONCIIDESXHTHH9B sho [ (ueuun?)TTV-gdH

s lee S. OO 8 9 O O 08 Og O' surhoo sino) U.S. Patent Aug. 7, 2012 Sheet 32 of 41 US 8.236,308 B2

O O7 surhoo U.S. Patent Aug. 7, 2012 Sheet 33 of 41 US 8.236,308 B2

(0. Og O' O. t76||"ONCIIDESXHTHH9B Stahoo

OO 8 Og O O2 O CO O sinoo suno U.S. Patent Aug. 7, 2012 Sheet 34 of 41 US 8.236,308 B2

OINEdSn6?ouuouÁo

09 2 O THH-150B.X0/1"ONCIIDES

(ueuun?)TTV-EdH

U.S. Patent Aug. 7, 2012 Sheet 38 of 41 US 8,236,308 B2

4º

O O o O O co % sisk oiloeds

-0010/|‘ONC]]OESXHTXIV/O 0/|;'ONCIIOESXTHXIV/OA

3 %) SSA opeds U.S. Patent US 8,236,308 B2

%) SSA Olyoeds

US 8,236,308 B2 1. 2 COMPOSITION COMPRISING clonal antibodies (e.g. against human antigens) by design CROSS-SPECIES-SPECIFIC ANTIBODES tend to have limited reactivity to phylogenetically distant AND USES THEREOF species such as rodents, except in the very rare cases in which the antigen is highly conserved. Even among the group of This application is a National Stage of PCT/EP2006/ monoclonal antibodies with reactivity to human and primate 009782, filed Oct. 10, 2006, and claims priority from U.S. antigens, there are numerous examples of antibodies which Provisional Application 60/724,781, filed Oct. 11, 2005, react only with the human and chimpanzee antigenhomologs. incorporated herein by reference in its entirety. This has also been observed for anti-CD3 monoclonal anti The present invention relates to uses of bispecific antibod bodies. One of the most widely used and best characterized ies exhibiting cross-species specificity for evaluating the in 10 monoclonal antibodies specific for the CD3 complex is vivo safety and/or activity and/or pharmacokinetic profile of OKT-3 which reacts with chimpanzee CD3 but not with the the same in non-human species and humans. The present CD3 homolog of other primates, such as macaques, or with invention moreover relates to methods for evaluating the in dog CD3 (Sandusky et al., J. Med. Primatol. 15 (1986), 441 vivo safety and/or activity and/or pharmacokinetic profile of 451). The anti-CD3 monoclonal antibody UCHT-1 is also said bispecific antibodies exhibiting cross-species specificity. 15 reactive with CD3 from chimpanzee but not with CD3 from The present invention also relates to methods of measuring macaques (own data; see the following Examples). On the the biological activity and/or efficacy of such bispecific anti other hand, there are also examples of monoclonal antibodies bodies exhibiting cross-species specificity. In addition, the which recognize macaque antigens, but not their human present invention relates to pharmaceutical compositions counterparts. One example of this group is monoclonal anti comprising bispecific single chain antibodies exhibiting body FN-18 directed to CD3 from macaques (Uda et al., J. cross-species specificity and to methods for the preparation of Med. Primatol.30 (2001), 141-147). Interestingly, it has been pharmaceutical compositions comprising said bispecific found that peripheral lymphocytes from about 12% of cyno single chain antibodies exhibiting cross-species specificity molgus monkeys lacked reactivity with anti-rhesus monkey for the treatment of diseases. CD3 monoclonal antibody (FN-18) due to a polymorphism of In order to be marketed, any new candidate medication 25 the CD3 antigen in macaques. Uda et al. described a Substi must pass through rigorous testing. Roughly, this testing can tution of two amino acids in the CD3 sequence of cynomolgus be subdivided into preclinical and clinical phases: Whereas monkeys which are not reactive with FN-18 antibodies, as the latter further subdivided into the generally known clini compared to CD3 derived from animals which are reactive cal phases I, II and III—is performed in human patients, the with FN-18 antibodies (Uda et al., J Med. Primatol. 32 former is performed in animals. Generally, the aim of pre 30 (2003), 105-10; Uda et al., J Med. Primatol. 33 (2004), 34-7). clinical testing is to prove that the drug candidate works and Similar difficulties with the high specificity of monoclonal is efficacious and safe. Specifically, the purpose of these antibodies in preclinical animal testing are observed with animal studies is to prove that the drug is not carcinogenic, bispecific antibodies, for example a recombinant bispecific mutagenic or teratogenic, as well as to understand the phar single chain antibody of the general type disclosed in, for macokinetic of the drug. Only when the safety in animals and 35 example, U.S. Pat. No. 5,260,203. This added difficulty is due possible effectiveness of the drug candidate has been estab to the fact that a bispecific antibody, for example a bispecific lished in preclinical testing will this drug candidate be single chain antibody, comprises two distinct binding approved for clinical testing in humans. domains, either one of which—or both—may fail to recog The behavior of a small molecule drug candidate, e.g. a nize the non-human counterpart of its human target molecule. new anthracycline-based antineoplastic agent, in animals will 40 Effectively, the risk that e.g. a bispecific single chain anti in many cases be indicative of the expected behavior of this body, will fail to recognize its intended respective target mol drug candidate upon administration to humans. As a result, ecules in an animal is twice as high as with a monospecific the data obtained from such preclinical testing will therefore antibody or fragment thereof. generally have a high predictive power for the human case. There exist several known Strategies for countering Such However, such compatibility is not to be expected with all 45 problems. types of drug candidates; certain molecular formats would be One known approach is to perform preclinical testing of the expected to behave one way in animals and another way in (bispecific) antibody drug candidate or fragment thereof in a humans. In such cases, the predictive power of preclinical chimpanzee model. The chimpanzee is the closest genetic tests—and hence the likelihood of approval of the drug can relative to human, identical to the latter in over 99% of its didate for clinical testing is greatly reduced. 50 genome, so the chimpanzee variant of a molecule specifically One format of drug candidate which often acts differently bound by a (bispecific) antibody drug candidate or fragment in animals than in humans is an antibody. Generally, antibod thereof is very likely to be identical to the human variant of ies function by way of highly specific recognition of usu this molecule. The danger of non-recognition of this molecule ally proteinaceous—target molecules. Most antibody drug by the (bispecific) antibody drug candidate or fragment candidates are monoclonal antibodies; they recognize only a 55 thereof in chimpanzee is therefore minimized. However, test single site, or epitope, on their target molecule. However, ing in chimpanzees is very expensive and fraught with ethical while this discriminatory ability inherent to monoclonal anti problems. Furthermore, chimpanzees are endangered ani bodies and fragments thereof makes these compounds very mals so that the number of animals which can be used in interesting candidates for drug development, it also compli experimentation is very limited. For most developers of cates their preclinical testing. This is because of species 60 (bispecific) antibody therapeutics, such preclinical testing in dependent variations in the sequence of the target molecule chimpanzees is therefore precluded. bound by Such antibodies. A monoclonal antibody or frag The above approach is described e.g. in Schlereth et al. ment thereof which specifically recognizes and binds to, say, (Cancer Immunol. Immunother. 20 (2005), 1-12). In this molecule Y via epitope X in humans, will often fail to spe study, the biological activity of a clinical drug candidate, cifically recognize and bind to the corresponding molecule Y 65 bispecific single chain antibody CD19xCD3, has been tested in a non-human species since the corresponding epitope X" in chimpanzee. CD19xCD3 antibody, previously described in may be different from its human counterpart X. Thus, mono WO99/54440 for therapeutic administration in humans, is a US 8,236,308 B2 3 4 bispecific single chain antibody specifically binding to mice allow the injection and growth of non-syngeneic cells, human B cell antigen CD19 and human T cell antigen CD3. for instance in Xenograft tumor models, the applicability of The authors of this paper found that this bispecific single the data obtained for the drug candidate in Such studies is chain antibody bound to both human and chimpanzee variants limited due to the phylogenetic distance between rodents and of the CD3 and CD19 molecule. However, no reactivity of 5 humans. In addition, multiple blood extractions are problem said bispecific single chain antibody to B and T cells from atic in lower animals, say a mouse. However, such multiple other species, i.e. mouse, beagle dog, and non-chimpanzee blood extractions are essential for the determination of phar primates (cynomolgus, rhesus and baboon), could be found, macokinetic parameters and the continuous testing of blood again confirming the extreme species sensitivity of mono parameters for evaluating the biological effects of a drug clonal antibodies. 10 Another approach adapts the molecule used in preclinical candidate in preclinical animal testing. testing to the animal used for this testing. According to this In Summary, there are two main approaches of obtaining approach, the requisite safety information is obtained in pre preclinical data on safety and toxicity of a drug candidate for clinical studies by constructing so-called “surrogate' anti administration in humans. One way is the application of the bodies for administration to test animals. Generally, such a 15 clinical drug candidate to transgenic animal models, mostly Surrogate antibody is an antibody which has been modified so mouse models. However, preclinical data are of limited as to specifically recognize and bind to the test animal coun explanatory power due to the fact that rodents are less related terpart of the target molecule bound by the non-surrogate to humans compared to primates. Another way is the testing antibody, i.e. the actual drug candidate in humans. Thus, in of Surrogate molecules in a relevant animal species. These approaches using Such 'surrogate' antibodies, two different Surrogate molecules are specific for the animals used and are molecules have to be separately developed and investigated: therefore different from the clinical drug candidate developed the clinical drug candidate and a candidate for preclinical for administration in humans. The problem is that the clinical testing in an animal species corresponding to the target speci drug candidate cannot directly be applied in an animal other ficity of the clinical candidate. The major drawback of such than chimpanzees which is closely related to humans and has Surrogate approaches is that the Surrogate antibody for pre 25 highly predictive power when used in preclinical testing. clinical testing has been modified vis-a-vis the actual drug Existing methods for obtaining meaningful preclinical data candidate antibody. Therefore, the data obtained in preclini regarding a (bispecific) antibody or fragment thereof under cal testing using a Surrogate antibody are often not directly going testing as a drug candidate either match this antibody to applicable to the human case. As explained above, this the test animal, in which case the data obtained are often of reduced applicability ultimately reduces the predictive power 30 only limited applicability for the drug candidate or, con of any preclinical study using these approaches. versely, match the test animal to the antibody, in which case While the above approach adapts the drug candidate to ethical and/or cost difficulty arise/sand, in the worst case, the match the animal used for testing, other known approaches do applicability of the data obtained for the drug candidate may exactly the converse; according to these other known still be limited. approaches, the animal used for testing is adapted to the drug 35 candidate intended for administration to humans. It is therefore an aim of the invention to provide a solution One example of the adaptation of the test animal to the drug to the problems outlined above. candidate intended for administration to humans, is the cre The solution to these problems is the provision of bispecific ation of a transgenic animal expressing the human molecule single chain antibodies exhibiting cross-species specificity specifically bound by the (bispecific) antibody or fragment 40 which bind to human and non-chimpanzee primate target thereof instead of the non-human molecule which is endog molecules and therefore can be used both for preclinical enous to its own species. In this way, the (bispecific) antibody evaluation of safety, activity and/or pharmacokinetic profile or fragment thereof administered in preclinical trials will of said bispecific antibody in primates and in the identical encounter and bind to the human antigen in the transgenic test form—as drugs in humans. animal. For example, in a study designed by Bugelski et al. 45 Accordingly, one aspect of the invention relates to the use (Bugelski et al., Hum Exp Toxicol. 19 (2000), 230-243), of a bispecific single chain antibody comprising a first bind preclinical safety assessment of monoclonal antibody Kelix ing domain binding to a non-chimpanzee primate CD3, and a imab has been carried out in a human CD4 transgenic mouse second binding domain binding to a cell Surface antigen, in order to support chronic treatment of rheumatoid arthritis wherein said first binding domain binds to human and non in human patients. Keliximab is a monoclonal antibody with 50 chimpanzee primate CD3, specificity for human and chimpanzee CD4. The authors con for evaluating the (in vivo) Safety and/or activity and/or phar clude that the use of transgenic mice expressing human pro macokinetic profile of said bispecific single chain antibody in teins provides a useful alternative to studies in chimpanzees humans, comprising (i) administering said bispecific single with biopharmaceutical agents having limited cross-species chain antibody to a non-chimpanzee primate, (ii) measuring specificity (Bugelski et al., Hum Exp Toxicol. 19 (2000), 55 said (in vivo) Safety and/or activity and/or pharmacokinetic 230-243). However, creation of transgenic animals for test profile of said bispecific single chain antibody in said non purposes is very labor- and therefore cost-intensive. chimpanzee primate, and (iii) evaluating the (in vivo) Safety In the same vein, an alternative approach often employed is and/or activity and/or pharmacokinetic profile of said bispe to inject a non-transgenic test animal with human cells cific single chain antibody in humans. expressing the molecule to be specifically bound by the 60 In another aspect, the invention relates to a method for (bispecific) antibody or fragment thereof being tested. How evaluating the biological activity/safety/toxicity of a bispe ever, while avoiding the costs and time associated with con cific single chain antibody as defined above, comprising structing transgenic animal species, this approach presents (i) administering said bispecific single chain antibody to a other problems. For one, in approaches using e.g. immuno non-chimpanzee primate, competent mice, foreign cells introduced into the animal are 65 (ii) measuring the in Vivo safety and/or activity and/or phar often recognized by the immune system of the test animal and macokinetic profile of said bispecific single chain antibody are systematically eliminated. Although immunodeficient in said non-chimpanzee primate, US 8,236,308 B2 5 6 (iii) evaluating the in vivo safety and/or activity and/or phar chimpanzee primates is well known to the person skilled in macokinetic profile of said bispecific single chain antibody the art and can be carried out e.g. by sequence alignments. in the non-chimpanzee primate, and The term “cross-species specificity' or “interspecies speci (iv) determining an effective and non-toxic dose of said bispe ficity” as used herein means binding of at least one of the two cific single chain antibody and administering said dose to binding domains, preferably of both binding domains, of the humans. bispecific single chain antibody described herein to the same In particular, it is an aim of the invention to provide means target molecule in humans and non-chimpanzee primates. and methods which improve the predictive value of data Thus, “cross-species specificity' or “interspecies specificity' obtained in preclinical animal testing for the administration is to be understood as an interspecies reactivity to the same 10 moleculeX, but not to a molecule other than X. Cross-species of the drug candidate to humans. specificity of a monoclonal antibody recognizing e.g. human As used herein, a “bispecific single chain antibody' CD3, to a non-chimpanzee primate CD3, e.g. macaque CD3. denotes a single polypeptide chain comprising two binding can be determined, for instance, by FACS analysis. The FACS domains. Each binding domain comprises one variable region analysis is carried out in a way that the respective monoclonal from an antibody heavy chain (“VH region'), wherein the VH 15 antibody is tested for binding to human and non-chimpanzee region of the first binding domain specifically binds to said primate cells, e.g. macaque cells, expressing said human and first molecule, i.e. the CD3 molecule, and the VH region of non-chimpanzee primate CD3 antigens, respectively. An the second binding domain specifically binds to a cell Surface appropriate assay is shown in the following examples. For the antigen, as defined in more detail below. The two binding generation of the first binding domain of the bispecific single domains are optionally linked to one another by a short chain antibodies as defined herein, e.g. monoclonal antibod polypeptide spacer generally comprising on the order of 5 ies binding to both the human and non-chimpanzee CD3 (e.g. amino acids. Each binding domain may additionally com macaque CD3) can be used. Similarly, for the generation of prise one variable region from an antibody light chain (“VL the second binding domain of the bispecific single chain region'), the VH region and VL region within each of the first antibodies as defined herein, monoclonal antibodies binding and second binding domains being linked to one another via 25 to both of the respective human and non-chimpanzee primate a polypeptide linker, for example of the type disclosed and cell Surface antigens can be utilized. Appropriate binding claimed in EP 623679 B1, but in any case long enough to domains for the bispecific single chain antibodies as defined allow the VH region and VL region of the first binding domain herein can be derived from cross-species specific monoclonal and the VH region and VL region of the second binding antibodies by recombinant methods described in the art. A domain to pair with one another such that, together, they are 30 monoclonal antibody binding to a human cell Surface antigen able to specifically bind to the respective first and second and to the homolog of said cell Surface antigen in a non molecules. chimpanzee primate can be tested by FACS assays as set forth As used herein, the term “binds” or related expressions above. It is evident to those skilled in the art that cross-species such as “binding or “reactivity with/to” etc. refer to the specific monoclonal antibodies can also be generated by ability of the first and/or second binding domains of the 35 hybridoma techniques described in the literature (Milstein bispecific single chain antibody as defined hereinto discrimi and Köhler, Nature 256 (1975), 495-7). For example, mice nate between the respective first and/or second molecule to may be alternately immunized with human and non-chimpan such an extent that, from a pool of a plurality of different Zee primate CD3. From these mice, cross-species specific molecules as potential binding partners, only said respective antibody-producing hybridoma cells are isolated via hybri first and/or second molecule is/are bound, or is/are signifi 40 doma technology and analysed by FACS as set forth above. cantly bound. Such binding measurements can be routinely The generation and analysis of bispecific single chain anti performed e.g. on a Biacore apparatus. bodies exhibiting cross-species specificity as described More specifically, the first binding domain of the bispecific herein is shown in the following examples. The advantages of single chain antibody as defined herein binds to human CD3 the bispecific single chain antibodies exhibiting cross-species and to non-chimpanzee primate CD3. The term “non-chim 45 specificity include the points enumerated below. panzee primate” is explained in more detail below. As evident As used herein, “human” and “man” refers to the species to the person skilled in the art, it is not excluded from the Homo sapiens. A “human molecule is therefore the variant scope of the invention that the first binding domain of the of that molecule as it is naturally expressed in Homo sapiens. bispecific single chain antibodies exhibiting cross-species As far as the medical uses of the constructs described herein specificity as defined herein may also bind, e.g., to chimpan 50 are concerned, human patients are to be treated with the same. Zee CD3. On the other hand, it is apparent that binding As used herein, a “non-chimpanzee primate' or “non domains which only bind to human CD3, but not to non chimp primate' or grammatical variants thereof refers to any chimpanzee primate CD3, are excluded from the scope of the primate other than chimpanzee, i.e. other than an animal of invention. This applies mutatis mutandis to binding domains belonging to the genus Pan, and including the species Pan which only bind to non-chimpanzee primate CD3, but not to 55 paniscus and Pan troglodytes, also known as Anthropopith human CD3. Such as e.g. those of monoclonal antibody ecus troglodytes or Simia satyrus. A "primate”, “primate spe FN-18. cies”, “primates' or grammatical variants thereof denote/s an The second binding domain of the bispecific single chain order of eutherian mammals divided into the two suborders of antibodies as defined herein binds to a cell Surface antigen, prosimians and anthropoids and comprising man, apes, mon preferably a tumor antigen, as set forth below. Preferably, 60 keys and lemurs. Specifically, “primates' as used herein com both binding molecules of the bispecific single chain antibod prises the Suborder Strepsirrhini (non-tarsier prosimians), ies as defined herein are binding to their respective human and including the infraorder Lemuriformes (itself including the non-chimpanzee primate target molecules. The second bind Superfamilies Chemogaleoidea and Lemuroidea), the ing domain, thus, binds to a human cell Surface antigen and to infraorder Chiromyiformes (itself including the family the corresponding homolog of the cell Surface antigen in a 65 Daubentoniidae) and the infraorder Lorisiformes (itself non-chimpanzee primate. The identification and determina including the families Lorisidae and Galagidae). “Primates' tion of homologs of human cell Surface antigens in non as used herein also comprises the Suborder Haplorrhini, US 8,236,308 B2 7 8 including the infraorder Tarsiiformes (itself including the Barbary Macaque, Macaca sylvanmus; Assam Macaque, family Tarsiidae), the infraorder Simiiformes (itself including Macaca assamensis; Tibetan Macaque or Milne-Edwards the Platyrrhini, or New World monkeys, and the Catarrhini, Macaque, Macaca thibetana; Arunachal Macaque or Mun including the Cercopithecidea, or Old-World Monkeys). Zala, Macaca munzala); within the genus Lophocebus (Gray The non-chimpanzee primate species may be understood 5 cheeked Mangabey, Lophocebus albigena, Lophocebus albi within the meaning of the invention to be a lemur, a tarsier, a gena albigena, Lophocebus albigena Osmani Lophocebus gibbon, a marmoset (belonging to New World Monkeys of the albigena johnstoni; Black Crested Mangabey, Lophocebus family Cebidae) or an Old-World Monkey (belonging to the aterrimus; Opdenbosch's Mangabey, Lophocebus opdenbos Superfamily Cercopithecoidea). chi; Highland Mangabey, Lophocebus kipunji); within the As used herein, an “Old-World Monkey' comprises any 10 genus Papio (Hamadryas Baboon, Papio hamadryas; Guinea monkey falling in the Superfamily Cercopithecoidea, itself Baboon, Papio papio; Olive Baboon, Papio anubis; Yellow subdivided into the families: the Cercopithecinae, which are Baboon, Papio cynocephalus; Chacma Baboon, Papio ursi mainly African but include the diverse genus of macaques nus); within the genus Theropithecus (Gelada, Theropithecus which are Asian and North African; and the Colobinae, which gelada); within the genus Cercocebus (Sooty Mangabey, include most of the Asian genera but also the African colobus 15 Cercocebus atys, Cercocebus atys atys, Cercocebus atys monkeys. lunulatus; Collared Mangabey, Cercocebus torquatus; Agile Specifically, within the subfamily Cercopithecinae, an Mangabey, Cercocebus agilis; Golden-bellied Mangabey, advantageous non-chimpanzee primate may be from the Cercocebus chrysogaster, Tana River Mangabey, Cercocebus Tribe Cercopithecini, within the genus Allenopithecus galeritus; Sanje Mangabey, Cercocebus sanjei); or within the (Allen's Swamp Monkey, Allenopithecus nigroviridis); genus Mandrillus (Mandrill, Mandrillus sphinx: Drill, Man within the genus Miopithecus (Angolan Talapoin, Miopith drillus leucophaeus). ecus talapoin; Gabon Talapoin, Miopithecus Ogouensis); Most preferred is Macaca fascicularis (also known as within the genus Erythrocebus (Patas Monkey, Erythrocebus Cynomolgus monkey and, therefore, in the Examples named patas); within the genus Chlorocebus (Green Monkey, Chlo “Cynomolgus') and Macaca mulatta (rhesus monkey, named rocebus sabaceus; Grivet, Chlorocebus aethiops; Bale Moun 25 “rhesus'). tains Vervet, Chlorocebus diamdiamensis; Tantalus Monkey, Within the subfamily Colobinae, an advantageous non Chlorocebus tantalus; Vervet Monkey, Chlorocebus chimpanzee primate may be from the African group, within pygerythrus; Malbrouck, Chlorocebus cynosuros); or within the genus Colobus (Black Colobus, Colobus satanas; Angola the genus Cercopithecus (Dryas Monkey or Salongo Monkey, Colobus, Colobus angolensis; King Colobus, Colobus Cercopithecus dryas; Diana Monkey, Cercopithecus diana; 30 polykomos; Ursine Colobus, Colobus vellerosus; Mantled Roloway Monkey, Cercopithecus rolloway; Greater Spot Guereza, Colobus guereza); within the genus Piliocolobus nosed Monkey, Cercopithecus nictitans; Blue Monkey, Cer (Western Red Colobus, Piliocolobus badius, Piliocolobus copithecus mitis; Silver Monkey, Cercopithecus doggetti; badius badius, Piliocolobus badius temminckii. Piliocolobus Golden Monkey, Cercopithecus kanditi: Sykes's Monkey, badius waldronae; Pennant's Colobus, Piliocolobus pennan Cercopithecus albogularis; Mona Monkey, Cercopithecus 35 tii, Piliocolobus pennantipennantii, Piliocolobus pennantii mona; Campbell's Mona Monkey, Cercopithecus Campbelli: epieni, Piliocolobus pennantibouvieri; Preuss's Red Colo Lowe's Mona Monkey, Cercopithecus lowei: Crested Mona bus, Piliocolobus preussi: Thollon's Red Colobus, Piliocolo Monkey, Cercopithecus pogonias; Wolfs Mona Monkey, bus tholloni; Central African Red Colobus, Piliocolobus foai; Cercopithecus wolfi: Dent's Mona Monkey, Cercopithecus Piliocolobus foai foai; Piliocolobus foai ellioti, Piliocolobus denti; Lesser Spot-nosed Monkey, Cercopithecus petaurista; 40 foai oustaleti; Piliocolobus foai Semlikiensis, Piliocolobus White-throated Guenon, Cercopithecus erythrogaster, Sclat foai partmentierorum; Ugandan Red Colobus, Piliocolobus er's Guenon, Cercopithecus Sclateri; Red-eared Guenon, tephrosceles; Uzyngwa Red Colobus, Piliocolobus gordo Cercopithecus erythrotis; Moustached Guenon, Cercopith norum; Zanzibar Red Colobus, Piliocolobus kirkii; Tana ecus cephus; Red-tailed Monkey, Cercopithecus ascanius; River Red Colobus, Piliocolobus rufomitratus); or within the L'Hoest's Monkey, Cercopithecus Ihoesti: Preuss's Monkey, 45 genus Procolobus (Olive Colobus, Procolobus verus). Cercopithecus preussi: Sun-tailed Monkey, Cercopithecus Within the subfamily Colobinae, an advantageous non solatus; Hamlyn's Monkey or Owl-faced Monkey, Cercop chimpanzee primate may alternatively be from the Langur ithecus hamlyni; De Brazza's Monkey, Cercopithecus (leaf monkey) group, within the genus Semnopithecus (Nepal neglectus). Gray Langur, Semnopithecus Schistaceus; Kashmir Gray Alternatively, an advantageous non-chimpanzee primate, 50 Langur, Semnopithecus ajax, Tarai Gray Langur, Semnopith also within the subfamily Cercopithecinae but within the ecus hector, Northern Plains Gray Langur, Semnopithecus Tribe Papionini, may be from within the genus Macaca (Bar entellus; Black-footed Gray Langur, Semnopithecus hypoleu bary Macaque, Macaca sylvanus, Lion-tailed Macaque, cos; Southern Plains Gray Langur, Semnopithecus dussum Macaca silenus; Southern Pig-tailed Macaque or Beruk, ieri; Tufted Gray Langur, Semnopithecus priam); within the Macaca nemestrina; Northern Pig-tailed Macaque, Macaca 55 T vetulus group or the genus Trachypithecus (Purple-faced leonina; Pagai Island Macaque or Bokkoi, Macaca pagensis; Langur, Trachypithecus vetulus; Nilgiri Langur, Trachypith Siberut Macaque, Macaca Siberu; Moor Macaque, Macaca ecus johnii); within the T. Cristatus group of the genus Trac maura; Booted Macaque, Macaca ochreata; Tonkean hypithecus (Javan Lutung, Trachypithecus auratus; Silvery Macaque, Macaca tonkeana; Heck's Macaque, Macaca Leaf Monkey or Silvery Lutung, Trachypithecus Cristatus; hecki; Gorontalo Macaque, Macaca nigriscens; Celebes 60 Indochinese Lutung, Trachypithecus germaini; Tenasserim Crested Macaque or Black “Ape', Macaca nigra; Cynomol Lutung, Trachypithecus barbei); within the T obscurus group gus monkey or Crab-eating Macaque or Long-tailed of the genus Trachypithecus (Dusky Leaf Monkey or Spec Macaque or Kera, Macaca fascicularis; Stump-tailed tacled Leaf Monkey, Trachypithecus obscurus; Phayre's Leaf Macaque or Bear Macaque, Macaca arctoides; Rhesus Monkey, Trachypithecus phayrei); within the T. pileatus Macaque, Macaca mulatta. Formosan Rock Macaque, 65 group of the genus Trachypithecus (Capped Langur, Trachyp Macaca cyclopis; Japanese Macaque, Macaca fiscata; Toque ithecus pileatus; Shortridge's Langur, Trachypithecus short Macaque, Macaca sinica; Bonnet Macaque, Macaca radiata; ridgei; Gee's Golden Langur, Trachypithecus geei); within US 8,236,308 B2 10 the T francoisi group of the genus Trachypithecus (Francois 142. The human CD3 delta is indicated in GenBank Acces Langur, Trachypithecus francoisi; Hatinh Langur, Trachyp sion No. NM 000732 and comprises SEQID NO. 143. ithecus hatinhensis; White-headed Langur, Trachypithecus The CD3 epsilon"FN-18 negative of Macaca fascicularis poliocephalus; Laotian Langur, Trachypithecus laotum; (i.e. CD3 epsilon not recognized by monoclonal antibody Delacours Langur, Trachypithecus delacouri; Indochinese FN-18 due to a polymorphism as set forth above) is indicated Black Langur, Trachypithecus ebenus); or within the genus in GenBank Accession No. AB073994 and comprises SEQ Presbytis (Sumatran Surili, Presbytis melalophos; Banded ID NO. 136. Surili, Presbytis femoralis; Sarawak Surili, Presbytis chry The CD3 epsilon “FN-18 positive' of Macaca fascicularis somelas; White-thighed Surili, Presbytis Siamensis; White (i.e. CD3 epsilon recognized by monoclonal antibody FN-18) fronted Surili, Presbytis frontata; Javan Surili, Presbytis 10 is indicated in GenBank Accession No. AB073993 and com comata; Thomas’s Langur, Presbytis thomasi. Hose's Lan prises SEQID NO. 135. The CD3 gamma of Macaca fascicu gur, Presbytis hosei: Maroon Leaf Monkey, Presbytis rubi laris is indicated in GenBank Accession No. AB073992 and cunda; Mentawai Langur or Joja, Presbytis potenziani; comprises SEQID NO. 144. The CD3 delta of Macaca fas Natuna Island Surili, Presbytis natunae). cicularis is indicated in GenBank Accession No. AB073991 Within the subfamily Colobinae, an advantageous non 15 and comprises SEQID NO. 145. chimpanzee primate may alternatively be from the Odd The nucleic acid sequences and amino acid sequences of Nosed group, within the genus Pygathrix (Red-shanked the respective CD3 epsilon, gamma and delta homologs of Douc, Pygathrix nemaeus; Black-shanked Douc, Pygathrix Macaca mulatta can be identified and isolated by recombi nigripes; Gray-shanked Douc, Pygathrix cinerea); within the nant techniques described in the art (Sambrook et al. Molecu genus Rhinopithecus (Golden Snub-nosed Monkey, Rhinop lar Cloning: A Laboratory Manual; Cold Spring Harbor ithecus roxellana; Black Snub-nosed Monkey, Rhinopithecus Laboratory Press, 3' edition 2001). This applies mutatis bieti; Gray Snub-nosed Monkey, Rhinopithecus brelichi: mutandis to the CD3 epsilon, gamma and delta homologs of Tonkin Snub-nosed Langur, Rhinopithecus avunculus); other non-chimpanzee primates as defined herein. within the genus Nasalis (Proboscis Monkey, Nasalis larva As pointed out above and as disclosed herein, it is envis tus); or within the genus Simias (Pig-tailed Langur, Simias 25 aged that the first binding domain of the bispecific single concolor). chain antibody comprised in the inventive pharmaceutical As used herein, the term “marmoset' denotes any New composition leads to an epitope of human and non-chimpan World Monkeys of the genus Callithrix, for example belong Zee primate CD3 which comprises the amino acid sequence ing to the Atlantic marmosets of Subgenus Callithrix (sic!) “phenylalanine (F)-serine (S)-glutamic acid (E)”. The person (Common Marmoset, Callithrix (Callithrix) jacchus; Black 30 skilled in the art is readily in the position to deduce an epitope tufted Marmoset, Callithrix (Callithrix) penicillata; Wied’s detected by a given antibody/binding molecule and/or (as in Marmoset, Callithrix (Callithrix) kuhlii; White-headed Mar the present invention) a given "binding domain” of a single moset, Callithrix (Callithrix) geoffroyi; Buffy-headed Mar chain construct by methods known in the art, said methods are moset, Callithrix (Callithrix) flaviceps; Buffy-tufted Marmo also illustrated in the appended examples and may comprise set, Callithrix (Callithrix) aurita); belonging to the 35 Western blot analysis, epitope mapping or pepspot analysis Amazonian marmosets of subgenus Mico (Rio Acari Marmo and the like. set, Callithrix (Mico) acariensis; Manicore Marmoset, Cal The epitope to be detected by said first binding domain is lithrix (Mico) manicorensis; Silvery Marmoset, Callithrix preferably in the range of 15 amino acids +/-3 amino acids. (Mico) argentata: White Marmoset, Callithrix (Mico) Envisaged are (but not limiting) 18, 17, 16, 15, 14, 13, 12, 11, leucippe: Emilia’s Marmoset, Callithrix (Mico) emiliae: 40 10,9,8,7,6, 5, 4 or 3 amino acids in said epitope comprising Black-headed Marmoset, Callithrix (Mico) nigriceps; Mar the “F-S-E stretch/"F-S-E core epitope. ca's Marmoset, Callithrix (Mico)marcai: Black-tailed Mar As shown in the following Examples, the minimum core moset, Callithrix (Mico) melanura: Santarem Marmoset, epitope of human and non-chimpanzee primate CD3 bound Callithrix (Mico) humerallifera; Maués Marmoset, Callithrix by the first binding domain of the bispecific single chain (Mico) mauesi; Gold-and-white Marmoset, Callithrix (Mico) 45 antibody as defined herein is an epitope comprising the amino chrysoleuca; Hershkovitz's Marmoset, Callithrix (Mico) acid residues “FSE. More specifically, the minimum epitope intermedia; Satéré Marmoset, Callithrix (Mico) saterei); comprises the amino acid residues “FSEXE' (SEQID NOs. Roosmallens' Dwarf Marmoset belonging to the Subgenus 202 and 204), wherein the substitution of methionine to leu Callibella (Callithrix (Callibella) humilis); or the Pygmy cine is a conserved amino acid substitution between two Marmoset belonging to the subgenus Cebuella (Callithrix 50 neutral, non-polar amino acid residues. The minimum (Cebuella) pygmaea). epitope may be part of a discontinuous epitope. As used As used herein, CD3 denotes a molecule expressed as part herein, the term “discontinuous epitope' is to be understood of the T cell receptor and has the meaning as typically as at least two non-adjacent amino acid sequence stretches ascribed to it in the prior art. In human, it encompasses in within a given polypeptide chain, here e.g. CD3 (preferably individual or independently combined form all known CD3 55 CD3 epsilon), which are simultaneously bound by an anti subunits, for example CD3 epsilon, CD3 delta, CD3 gamma, body. These amino acid stretches might be of different length CD3 Zeta, CD3 alpha and CD3 beta. The non-chimpanzee and may also be involved in the interaction of antibody and primate CD3 antigens as referred to herein are, for example, antigen. Accordingly, in addition to the minimum (core) Macaca fascicularis CD3 and Macaca mulatta CD3. In epitope as defined above, the bispecific single chain antibody Macaca fascicularis, it encompasses CD3 epsilon FN-18 60 may simultaneously bind to one, two or even more non negative and CD3 epsilon FN-18 positive. CD3 gamma and adjacent epitopes. This (these) non-adjacent epitope(s) in CD3 delta. In Macaca mulatta, it encompasses CD3 epsilon, combination with the minimal (core) epitope could represent CD3 gamma and CD3 delta. Preferably, said CD3 as used the contact site between antigen and antibody. According to herein is CD3 epsilon. The human CD3 epsilon is indicated in this definition, such simultaneous binding may be of the GenBank Accession No. NM 000733 and comprises SEQ 65 polypeptide in linear form. Here, one may imagine the IDNO. 134. The human CD3 gamma is indicated in GenBank polypeptide forming an extended loop, in one region of which Accession No. NM 000073 and comprises SEQ ID NO. the two sequences for example are more or less in parallel and US 8,236,308 B2 11 12 in proximity of one another. Non-adjacent epitopes in the The corresponding sequences of the human and non-chim linear sequence could form a three dimensional structure panzee primate nucleic acid and amino acid sequences can be leading to a close proximity of these epitopes. In this state found e.g. in NCBI databases. they are simultaneously bound by the bispecific single chain A cross-species-specific monoclonal antibody binding to a antibody as defined herein. According to this definition, 5 human cell Surface antigen (preferably a tumor antigen) and simultaneous binding of the at least two sequence stretches of to the homolog of said cell Surface antigen (preferably a the polypeptide indicated above (including the minimum tumorantigen) in a non-chimpanzee primate can be generated (core) epitope) may also take the form of antibody binding to as set out above. "Homologs' as used herein refer to genes a conformational epitope. Here, the mature polypeptide has (encoding e.g. CD3, CD3 epsilon, cell Surface antigens or 10 tumor antigens) which encode gene products with similar or already formed its tertiary conformation as it normally exists identical biological function in different species and which in vivo. In this tertiary conformation, the polypeptide chain is genes can be attributed to a common precursor gene. Cross folded in Such a manner as to bring the at least two sequence species specificity of said monoclonal antibody to the human stretches indicated above into spatial proximity, for example, and non-chimpanzee primate tumor antigen can be tested by on the outer Surface of a particular region of mature, folded 15 FACS assays as set forth above. Alternatively, immunohis polypeptide, where they are then recognized by virtue of their tochemistry, radioimmunoassay, or ELISA assays may be three-dimensional conformation in the context of the Sur used as known to the person skilled in the art. The second rounding polypeptide sequences. binding domain of the bispecific single chain antibody exhib The term "cell surface antigen” as used herein denotes a iting cross-species specificity as described herein can for molecule which is displayed on the surface of a cell. In most example be derived from Such cross-species specific mono cases, this molecule will be located in or on the plasma clonal antibodies by recombinant techniques described in the membrane of the cell such that at least part of this molecule following examples. remains accessible from outside the cell in tertiary form. A The term “evaluating the in vivo safety and/or activity non-limiting example of a cell Surface molecule which is and/or pharmacokinetic profile' of the bispecific single chain located in the plasma membrane is a transmembrane protein 25 antibody as used herein may be understood as set forth below. comprising, in its tertiary conformation, regions of hydrophi Before a new candidate medication can be marketed it must licity and hydrophobicity. Here, at least one hydrophobic pass through rigorous testing, which may be roughly Subdi region allows the cell surface molecule to be embedded, or vided into preclinical testing in animals and clinical phases in inserted in the hydrophobic plasma membrane of the cell human patients. The aim of preclinical testing in animals is to while the hydrophilic regions extend on either side of the 30 prove that the drug candidate is safe and efficacious (see e.g. plasma membrane into the cytoplasm and extracellular space, the Preclinical safety evaluation of biotechnology-derived respectively. Non-limiting examples of a cell surface mol pharmaceuticals; ICH Harmonised Tripartite Guideline: ICH ecules which are located on the plasma membrane are pro Steering Committee meeting on Jul. 16, 1997). teins which have been modified at a cysteine residue to bear a The term “drug”, “drug candidate' or “pharmaceutical palmitoyl group, proteins modified at a C-terminal cysteine 35 composition' as used herein refers to bispecific single chain residue to bear a farnesyl group or proteins which have been antibodies defined herein. modified at the C-terminus to bear a glycosyl phosphatidyl The biological activity of the bispecific single chain anti inositol (“GPI) anchor. These groups allow covalent attach body as defined herein can be determined for instance by ment of proteins to the outer Surface of the plasma membrane, cytotoxicity assays, as described in the following examples, where they remain accessible for recognition by extracellular 40 in WO99/54440 or by Schlereth et al. (Cancer Immunol. molecules such as antibodies. Immunother. 20 (2005), 1-12). “Efficacy” or “in vivo effi The "tumor antigen” as used herein may be understood as cacy” as used herein refers to the response to therapy by the those antigens that are presented on tumor cells. These anti bispecific single chain antibody as defined herein, using e.g. gens can be presented on the cell Surface with an extracellular standardized NCI response criteria. The success or in vivo part which is often combined with a transmembrane and 45 efficacy of the therapy using a bispecific single chain anti cytoplasmic part of the molecule. These antigens can some body as defined herein refers to the effectiveness of the bispe times be presented only by tumor cells and never by the cific single chain antibody as defined herein for its intended normal ones. Tumorantigens can be exclusively expressed on purpose, i.e. the ability of the bispecific antibody to cause its tumor cells or might represent a tumor specific mutation desired effect, i.e. depletion of pathologic cells, e.g. tumor compared to normal cells. In this case; they are called tumor 50 cells. The in vivo efficacy may be monitored by established specific antigens. More common are antigens that are pre standard methods for the respective disease entities includ sented by tumor cells and normal cells, and they are called ing, but not limited to white blood cell counts, differentials, tumor-associated antigens. These tumor-associated antigens Fluorescence Activated Cell Sorting, bone marrow aspira can be overexpressed compared to normal cells or are acces tion. In addition, various disease specific clinical chemistry sible for antibody binding in tumor cells due to the less 55 parameters and other established standard methods may be compact structure of the tumor tissue compared to normal used. Furthermore, computer-aided tomography, X-ray, tissue. Non-limiting examples of tumor antigens as used nuclear magnetic resonance tomography (e.g. for National herein are EpCAM (Naundorf, Int. J. Cancer 100/1 (2002), Cancer Institute-criteria based response assessment Cheson 101-110), EGFR (Liu, Br. J. Cancer 82/12 (2000), 1991 BD, Horning SJ, Coifier B, Shipp MA, Fisher R I, Connors 1999: Bonner, Semin. Radiat. Oncol. 12 (2002), 11-20; 60 J M. Lister T A, Vose J. Grillo-Lopez A. Hagenbeek A, Kiyota, Oncology 63/1 (2002), 92-98; Kuan, Brain Tumor Cabanillas F. Klippensten D. Hiddemann W. Castellino R. Pathol. 17/2 (2000), 71-78), EGFRVIII (Kuan, Brain Tumor Harris NL, Armitage J. O. Carter W. Hoppe R, Canellos G. P. Pathol. 17/2 (2000), 71-78), or Carboanhydrase IX (MN/CA Report of an international workshop to standardize response IX) (Uemura, Br. J. Cancer 81/4 (1999), 741-746; Long criteria for non-Hodgkin’s lymphomas. NCI Sponsored caster, Cancer Res. 61/17 (2001), 6394-6399; Chia, J. Clin. 65 International Working Group. J Clin Oncol. 1999 April; Oncol. 19/16 (2001),3660-3668; Beasley, Cancer Res.61/13 17(4): 1244), positron-emission tomography scanning, white (2001), 5262-5267). blood cell counts, differentials, Fluorescence Activated Cell US 8,236,308 B2 13 14 Sorting, bone marrow aspiration, lymph node biopsies/his diac arrhythmia, coagulation and the like, as set forth e.g. in tologies, and various lymphoma specific clinical chemistry the Common Terminology Criteria for adverse events v3.0 parameters (e.g. lactate dehydrogenase) and otherestablished (CTCAE). Laboratory parameters which may be tested standard methods may be used. include for instance haematology, clinical chemistry, coagu Another major challenge in the development of drugs is the lation profile and urine analysis and examination of other predictable modulation of pharmacokinetic properties. To body fluids such as serum, plasma, lymphoid or spinal fluid, this end, a pharmacokinetic profile of the drug candidate, i.e. liquor and the like. Safety can thus be assessed e.g. by physi a profile of the pharmacokinetic parameters that effect the cal examination, imaging techniques (i.e. ultrasound, X-ray, ability of a particular drug to treat a given condition, is estab CT scans, Magnetic Resonance Imaging (MRI), other mea lished. Pharmacokinetic parameters of the drug influencing 10 Sures with technical devices (i.e. electrocardiogram), vital the ability of a drug for treating a certain disease entity signs, by measuring laboratory parameters and recording include, but are not limited to: half-life, Volume of distribu adverse events. For example, adverse events in non-chimpan tion, hepatic first-pass metabolism and the degree of blood Zee primates in the uses and methods according to the inven serum binding. The efficacy of a given drug agent can be tion may be examined by histopathological and/or his influenced by each of the parameters mentioned above. 15 tochemical methods. “Half-life” means the time where 50% of an administered The term “effective and non-toxic dose” as used herein drug are eliminated through biological processes, e.g. refers to a tolerable dose of the bispecific single chain anti metabolism, excretion, etc. body as defined herein which is high enough to cause deple By “hepatic first-pass metabolism' is meant the propensity tion of pathologic cells, tumor elimination, tumor shrinkage of a drug to be metabolized upon first contact with the liver, or stabilisation of disease without or essentially without i.e. during its first pass through the liver. major toxic effects. Such effective and non-toxic doses may “Volume of distribution” means the degree of retention of be determined e.g. by dose escalation studies described in the a drug throughout the various compartments of the body, like art and should be below the dose inducing severe adverse side e.g. intracellular and extracellular spaces, tissues and organs, events (dose limiting toxicity, DLT). etc. and the distribution of the drug within these compart 25 The above terms are also referred to e.g. in the Preclinical mentS. safety evaluation of biotechnology-derived pharmaceuticals “Degree of blood serum binding means the propensity of S6; ICH Harmonised Tripartite Guideline; ICH Steering a drug to interact with and bind to blood serum proteins. Such Committee meeting on Jul. 16, 1997. as albumin, leading to a reduction or loss of biological activity It has been Surprisingly found that it is possible to generate of the drug. 30 bispecific antibody-based therapeutics for humans wherein Pharmacokinetic parameters also include bioavailability, the identical molecule can also be used in preclinical animal lag time (Tlag), Tmax, absorption rates, more onset and/or testing. This is due to the unexpected identification of bispe Cmax for a given amount of drug administered. cific single chain antibodies which, in addition to binding to “Bioavailability” means the amount of a drug in the blood human antigens (and due to genetic similarity likely to chim compartment. 35 panzee counterparts), also bind to the homologs of said anti “Lagtime” means the time delay between the administra gens of non-chimpanzee primates, such as macaques. Thus, tion of the drug and its detection and measurability in blood or the need to construct a Surrogate bispecific single chain anti plasma. body for testing in a phylogenetic distant (from humans) “Tmax’ is the time after which maximal blood concentra species disappears. As a result, the very same bispecific single tion of the drug is reached, and “Cmax’ is the blood concen 40 chain antibody can be used in animal preclinical testing as is tration maximally obtained with a given drug. The time to intended to be administered to humans in clinical testing as reach a blood or tissue concentration of the drug which is well as following market approval. The ability to use the same required for its biological effect is influenced by all param molecule for preclinical animal testing as in later administra eters. Pharmacokinetik parameters of the bispecific single tion to humans virtually eliminates, or at least greatly chain antibodies exhibiting cross-species specificity which 45 reduces, the danger that the data obtained in preclinical ani may be determined in preclinical animal testing in non-chim mal testing are not applicable to the human case. In short, panzee primates as outlined above are also set forth e.g. in the obtaining preclinical safety data in animals using the same publication by Schlereth et al. (Cancer Immunol. Immu molecule as will actually be administered to humans does nother. 20 (2005), 1-12). much to ensure the applicability of the data to a human The term “toxicity' as used herein refers to the toxic effects 50 relevant scenario. In contrast, in conventional approaches of a drug manifested in adverse events or severe adverse using Surrogate molecules, said Surrogate antibodies have to events. These side events might refer to a lack of tolerability be molecularly adapted to the animal test system used for of the drug in general and/or a lack of local tolerance after preclinical safety assessment. Thus, the Surrogate antibody to administration. Toxicity could also include teratogenic or be used in human therapy in fact differs in sequence and also carcinogenic effects caused by the drug. 55 likely in structure from the one used in preclinical testing in The term “safety”, “in vivo safety” or “tolerability” as used pharmacokinetic parameters and/or biological activity, with herein defines the administration of a drug without inducing the consequence that data obtained in preclinical animal test severe adverse events directly after administration (local tol ing have limited applicability/transferability to the human erance) and during a longer period of application of the drug. case. The use of Surrogate molecules requires the construc “Safety”, “in vivo safety” or “tolerability” can be evaluated 60 tion, production, purification and characterization of a com e.g. at regular intervals during the treatment and follow-up pletely new antibody construct. This leads to additional period. Measurements include clinical evaluation, e.g. organ development costs and time necessary to obtain that mol manifestations, and Screening of laboratory abnormalities. ecule. In Sum, Surrogates have to be developed separately in Clinical evaluation may be carried out and deviating to nor addition to the actual drug to be used in humantherapy, so that mal findings recorded/coded according to NCI-CTC and/or 65 two lines of development for two bispecific single chain anti MedDRA standards. Organ manifestations may include cri body molecules have to be carried out. Therefore, a major teria Such as allergy/immunology, blood/bone marrow, car advantage of the bispecific antibody-based constructs exhib US 8,236,308 B2 15 16 iting cross-species specificity described herein is that the First, the bispecific single chain antibody exhibiting cross identical molecule can be used for therapeutics inhumans and species specificity used in preclinical testing is the same as the in preclinical animal testing. one used in human therapy. Thus, it is no longer necessary to On the otherhand, it is also no longer necessary to adapt the develop two independent molecules which may differ in their test animal to the bispecificantibody-drug candidate intended 5 pharmacokinetic properties and biological activity. This is for administration to humans. Such as e.g. the creation of highly advantageous in that e.g. the pharmacokinetic results transgenic animals producing the human molecules bound by are more directly transferable and applicable to the human the bispecific antibody. The bispecific single chain antibodies setting than e.g. in conventional Surrogate approaches. exhibiting cross-species specificity according to the uses and Second, the uses of the bispecific antibody exhibiting methods of invention can be directly used for preclinical 10 cross-species specificity and methods according to the inven testing in non-chimpanzee primates, without any genetic tion for the preparation of therapeutics in human is less cost manipulation of the animals. As well knownto those skilled in and labor-intensive than Surrogate approaches. the art, approaches in which the test animal is adapted to the Third, chimpanzee as a species for animal testing is drug candidate always bear the risk that the results obtained in avoided. the preclinical safety testing are less representative and pre 15 Fourth, multiple blood samples can be extracted for exten dictive for humans due to the modification of the animal. For sive pharmacokinetic studies. example, in transgenic animals, the proteins encoded by the A further aspect of the invention relates to a method of transgenes are often highly over-expressed. Thus, data determining the biological activity and/or efficacy of a bispe obtained for the biological activity of an antibody against this cific single chain antibody as defined above, wherein said protein antigen may be limited in their predictive value for bispecific single chain antibody is administered to a non humans in which the protein is expressed at much lower, more chimpanzee primate and the in vivo activity is measured. physiological levels. Preferably, said in vivo activity is T cell activation, tumor A further advantage of the uses of the bispecific single target cell depletion, cytotoxicity, toxicity, occurrence of chain antibody exhibiting cross-species specificity of the adverse side effects, and/or cytokine release. Methods for the invention lies in the avoidance of chimpanzee as a species for 25 determination of said in vivo activity are set forth e.g. in WO animal testing. Chimpanzees are the closest relatives to 99/54440. humans and were recently grouped into the family of homi The present invention in another aspect also provides for a nids based on the genome sequencing data (Wildman et al., pharmaceutical composition for the treatment of a human PNAS 100 (2003), 7181). Therefore, data obtained with patient, comprising a bispecific single chain antibody which chimpanzee is generally considered to be highly predictive 30 comprises for humans. However, due to their status as endangered spe (i) a first binding domain binding to a non-chimpanzee pri cies, the number of chimpanzees which can be used for medi mate CD3, and cal experiments is highly restricted. As stated above, mainte (ii) a second binding domain binding to a cell Surface antigen, nance of chimpanzees for animal testing is therefore both wherein said first binding domain binds to human and non costly and ethically problematic. The uses of the bispecific 35 chimpanzee primate CD3. single chain antibody of the invention avoids both financial In accordance with this invention, the term “pharmaceuti burden and ethical objection during preclinical testing with cal composition' relates to a composition for administration out prejudicing the quality, i.e. applicability, of the animal to a patient, preferably a human patient. Preferably, the phar testing data obtained. In light of this, the uses of bispecific maceutical composition comprises suitable formulations of single chain antibodies exhibiting cross-species specificity 40 carriers, stabilizers and/or excipients. In a preferred embodi and methods according to the invention for preclinical animal ment, the pharmaceutical composition comprises a composi testing in non-chimpanzee primates provides for a reasonable tion for parenteral, transdermal, intraluminal, intraarterial, alternative for studies in chimpanzees. intrathecal and/or intranasal administration or by direct injec A further advantage of the bispecific single chain antibody tion into tissue. It is in particular envisaged that said compo of the invention is the ability of extracting multiple blood 45 sition is administered to a patient via infusion or injection. samples when using it as part of animal preclinical testing, for Administration of the suitable compositions may be effected example in the course of pharmacokinetic animal studies. by different ways, e.g., by intravenous, intraperitoneal, Sub Multiple blood extractions can be much more readily cutaneous, intramuscular, topical or intradermal administra obtained with a non-chimpanzee primate than with lower tion. The composition of the present invention may further animals, say a mouse. The extraction of multiple blood 50 comprise a pharmaceutically acceptable carrier. Examples of samples allows continuous testing of blood parameters for the Suitable pharmaceutical carriers are well known in the art and determination of the biological effects induced by the bispe include phosphate buffered saline solutions, water, emul cific single chain antibody of the invention. Furthermore, the sions, such as oil/water emulsions, various types of wetting extraction of multiple blood samples enables the researcherto agents, Sterile solutions, liposomes, etc. Compositions com evaluate the pharmacokinetic profile of the bispecific single 55 prising Such carriers can be formulated by well known con chain antibody as defined herein. In addition, potential side ventional methods. These compositions can be administered effects which may be induced by said bispecific single chain to the Subject at a suitable dose which can be determined e.g. antibody reflected in blood parameters can be measured in by dose escalating studies by administration of increasing different blood samples extracted during the course of the doses of the bispecific single chain antibody exhibiting cross administration of said antibody. This allows the determina 60 species specificity described herein to non-chimpanzee pri tion of the potential toxicity profile of the bispecific single mates, for instance macaques. As set forth above, the bispe chain antibody as defined herein. cific single chain antibody exhibiting cross-species The advantages of the pharmaceutical compositions com specificity described herein can be advantageously used in prising bispecific single chain antibodies exhibiting cross identical form in preclinical testing in non-chimpanzee pri species specificity, uses of said bispecific antibodies and 65 mates and as drug in humans. These compositions can also be methods according to the invention may be briefly Summa administered in combination with other proteinaceous and rized as follows: non-proteinaceous drugs. These drugs may be administered US 8,236,308 B2 17 18 simultaneously with the composition comprising the bispe sequence “QYPGSEILWQHND” (SEQ ID NO. 203). Also cific single chain antibody as defined herein or separately additional or (further) epitopes of cynomolgus CD3 epsilon before or after administration of said bispecific antibody in contained in said chain may be detected by the binding mol timely defined intervals and doses. The dosage regimen will ecule or molecule comprising the binding domains as defined be determined by the attending physician and clinical factors. therein. These additional or further sequences may comprise As is well known in the medical arts, dosages for any one the amino acid sequence “QDGNEEMGSITQT' (SEQ ID patient depend upon many factors, including the patients NO.199) and “YYVSYPRGSNPED” (SEQ ID NO. 200). size, body Surface area, age, the particular compound to be Thus, the minimal epitope is most likely part of a discontinu administered, sex, time and route of administration, general ous epitope or a conformational epitope. As evident to a health, and other drugs being administered concurrently. 10 person skilled in the art, the scope of the present invention Preparations for parenteral administration include sterile includes bispecific single chain antibodies not only binding to aqueous or non-aqueous solutions, Suspensions, and emul this minimum (core) epitope, but also to one, two or even sions. Examples of non-aqueous solvents are propylene gly more non-adjacent amino acid sequence stretches within col, polyethylene glycol, vegetable oils such as olive oil, and CD3 (preferably CD3 epsilon). Based on the results shown in injectable organic esters such as ethyl oleate. Aqueous carri 15 the following Examples it is concluded that cross-species ers include water, alcoholic/aqueous solutions, emulsions or specific anti-CD3 antibodies contact CD3 epsilon in the area Suspensions, including saline and buffered media. Parenteral of amino acid residues 57-61 of both cynomolgus and human vehicles include sodium chloride solution, Ringer's dextrose, CD3 epsilon comprising the amino acid stretches FSEME dextrose and sodium chloride, lactated Ringers, or fixed oils. (SEQ ID NO. 206) and FSELE (SEQID NO. 205) of cyno Intravenous vehicles include fluid and nutrient replenishers, molgus and human CD3 epsilon, respectively, with the motif electrolyte replenishers (such as those based on Ringer's FSE forming the epitope core. This result—although plau dextrose), and the like. Preservatives and other additives may sible because of the accessibility of the E-F-loop (amino acids also be present Such as, for example, antimicrobials, anti 56-62) of human CD3 epsilon (Kjer-Nielsenet al., PNAS 101 oxidants, chelating agents, inert gases and the like. In addi (2004), p. 7675-80) comprising the amino acids FSELE(SEQ tion, the composition of the present invention might comprise 25 ID NO. 205) or FSEME (SEQ ID NO. 206) is surprising proteinaceous carriers, like, e.g., serum albuminor immuno since there is no overlap of this newly defined epitope with the globulin, preferably of human origin. It is envisaged that the known epitope on the CD3 epsilon-chain of anti-CD3 anti composition of the invention might comprise, in addition to bodies OKT-3 and UCHT-1 (Kjer-Nielsen et al., loc.cit: the bispecific single chain antibody as defined herein, further Arnett et al., PNAS 101 (2004), p. 16268-73) which have so biologically active agents, depending on the intended use of 30 far been regarded as representative of all anti-CD3 antibodies the composition. Such agents might be drugs acting on the thought to form a single family with the same or a very similar gastro-intestinal System, drugs acting as cytostatica, drugs epitope. In summary, the epitopes “FSE' and “FSEXE' (SEQ preventing hyperurikemia, drugs inhibiting immunoreactions ID NO. 204) are distinct from the epitopes recognized by (e.g. corticosteroids), drugs modulating the inflammatory UCHT-1 or OKT-3 (Kjer-Nielsen et al., PNAS 101 (2004), p. response, drugs acting on the circulatory system and/or 35 7675-80; Arnett et al., PNAS 101 (2004), p. 16268-73) and agents such as cytokines known in the art. are unique for cross-species specific anti-CD3 antibodies According to a preferred embodiment of the pharmaceuti binding to human and macaque CD3. Preferably, the mini cal composition of the invention, the first binding domain of mum epitope comprises the amino acid sequence “FSEXE the bispecific single chain antibody as defined herein binds to (SEQ ID NO. 204), wherein X represents L (Leucine) or M an epitope of human and non-chimpanzee primate CD3 com 40 (Methionine) and stands for a substitution of non-polar, neu prising the amino acid sequence “FSE. The minimum core tral amino acid residues. epitope comprising the amino acid residues “FSE, the mini It is envisaged that in the pharmaceutical composition of mum epitope comprising the amino acid sequence “FSEXE the invention, said first binding domain of the bispecific (SEQ ID NOS. 202 and 204; wherein “X” corresponds to a single chain antibody of the pharmaceutical composition of leucine (L) or to a methionine (M)) or non-adjacent epitopes 45 the invention is located C-terminally to the second binding as defined herein are preferably 18, 17, 16, 15, 14, 13, 12, 11, domain. However, also part of this invention is a bispecific 10,9,8,7,6, 5, 4 or 3 amino acid residues in length. Prefer construct, wherein the “first binding domain to a non-chim ably, said epitopes are 13 amino acid residues in length. Even panzee primate CD3” is located N-terminally to the herein more preferred, the epitope with the “FSEXE' (SEQID NOs. defined 'second binding domain to a cell Surface antigen'. 202 and 204; wherein “X” corresponds to a leucine (L) or to 50 As shown in the following examples, the advantages as a methionine (M))—motif comprises the amino acid described hereinabove are realizable not only when the first sequence “EFSELEQSGYYVC (SEQ ID NO. 195) of binding domain (binding to CD3) is located C-terminally to human CD3 epsilon. In cynomolgus CD3 epsilon, the corre the second binding domain, i.e. closer to the C-terminus of the sponding epitope reads “EFSEMEQSGYYVC'' (SEQ ID bispecific single chain antibody than the second binding NO. 201). The substitution of methionine to leucine is a 55 domain, but also when the first binding domain (binding to conserved amino acid Substitution between two neutral, non CD3) is located N-terminally to the second binding domain, polaramino acid residues. The corresponding sequence of the i.e. closer to the N-terminus of the bispecific single chain preferred epitope “EFSEXEQSGYYVC” wherein X repre antibody than the second binding domain. The arrangement sents L (Leucine) or M (Methionine) is depicted in SEQID of the binding domains in the bispecific single chain antibody NO. 207. As shown in the following Examples, the bispecific 60 defined herein may therefore be one in which the first binding single chain antibody as defined herein not only binds to this domain is located C-terminally to the second binding domain. epitope, but also to amino-acid stretches non-adjacent to said The arrangement of the V chains may be VH (cell surface minimal epitope. For example, the bispecific single chain antigen)-VL(cell surface antigen)-VL(CD3)-VH(CD3), antibody as defined herein in addition to the minimum core VH(cell surface antigen)-VL(cell surface antigen)-VH epitope may simultaneously bind to (an) epitope(s) of human 65 (CD3)-VL(CD3), VL(cell surface antigen)-VH(cell surface CD3 epsilon contained in said CD3 epsilon chain. Accord antigen)-VL(CD3)-VH(CD3) or VL(cell surface antigen)- ingly, said epitope may additionally comprise the amino acid VH(cell surface antigen)-VH(CD3)-VL(CD3). For an US 8,236,308 B2 19 20 arrangement, in which the first binding domain is located H1) comprising an amino acid sequence as set out in SEQID N-terminally to the second binding domain, the following NO. 115. The VH region of the first binding domain thus may orders are possible: VH (CD3)-VL(CD3)-VL(cell surface comprise one, two or all three of the mentioned CDRs. The antigen)-VH(cell surface antigen), VH(CD3)-VL(CD3)-VH mentioned CDRs are included for example in the VH regions (cell surface antigen)-VL(cell surface antigen), VL(CD3)- shown in SEQID NOS. 2 and 110. VH(CD3)-VL(cell surface antigen)-VH(cell surface antigen) Alternatively, it is envisaged that the VH region of the first or VL(CD3)-VH(CD3)-VH(cell surface antigen)-VL(cell binding domain comprises a third CDR (CDR-H3) compris surface antigen). As used herein, “N-terminally to” or “C-ter ing an amino acid sequence as set out in SEQID NO. 119. minally to” and grammatical variants thereof denote relative Preferably, the first binding domain additionally comprises a location within the primary amino acid sequence rather than 10 second CDR (CDR-H2) comprising an amino acid sequence placement at the absolute N- or C-terminus of the bispecific as set out in SEQID NO. 120. Especially preferred, the first single chain antibody. Hence, as a non-limiting example, a binding domain additionally comprises a first CDR (CDR first binding domain which is “located C-terminally to the H1) comprising an amino acid sequence as set out in SEQID second binding domain simply denotes that the first binding NO. 121. The VH region of the first binding domain thus may domain is located to the carboxyl side of the second binding 15 comprise one, two or all three of the mentioned CDRs. The domain within the bispecific single chain antibody, and does above-indicated CDRs are included for example in the VH not exclude the possibility that an additional sequence, for region shown in SEQID NO. 6. example a His-tag, or another proteinaceous or non-proteina In another preferred embodiment of the pharmaceutical ceous compound Such as a radioisotope, is located at the composition, the first binding domain comprises a VL region ultimate C-terminus of the bispecific single chain antibody. having an amino acid sequence as shown in any of SEQID In another preferred embodiment of the pharmaceutical NOs. 4, 148, 168 or 8. It is envisaged and preferred that the VL composition, the second binding domain binds to a cell Sur region of the first binding domain comprises at least a third face antigen and to the non-chimpanzee primate homolog of CDR (CDR-L3) comprising an amino acid sequence as set said cell Surface antigen. out in SEQID NO. 116. The VL region may further comprise According to this embodiment of the invention, both the 25 a second CDR(CDR-L2) comprising an amino acid sequence first and second binding domains of the bispecific single as set out in SEQID NO. 117. The VL region may in addition chain antibody described herein specifically bind to both comprise a first CDR (CDR-L1) comprising an amino acid human and non-chimpanzee primate variants of said first and sequence as set out in SEQID NO. 118. The VL region of the second molecules, respectively. In light of the above state first binding domain thus may comprise one, two or all three ments, this is particularly advantageous since Sufficient 30 of the mentioned CDRs. The above-indicated CDRs are (cross-species) specificity exists on both sides of the bispe included for example in the VL regions shown in SEQ ID cific single chain antibody, thus ensuring interspecies com NOS. 4, 148 and 168. patibility with respect to both first and second molecules and Alternatively, it is envisaged that the VL region of the first hence optimal extrapolability of the data obtained in preclini binding domain comprises a third CDR (CDR-L3) compris cal animal studies to the case of administration in humans. 35 ing an amino acid sequence as set out in SEQID NO. 164. Preferably, said cell Surface antigen is a tumor antigen. Preferably, the first binding domain additionally comprises a Even more preferred, said tumor antigen is EpCAM (Naun second CDR (CDR-L2) comprising an amino acid sequence dorf, Int. J. Cancer 100/1 (2002), 101-110), EGFR (Liu, Br. J. as set out in SEQID NO. 165. Especially preferred, the first Cancer 82/12 (2000), 1991-1999: Bonner, Semin. Radiat. binding domain additionally comprises a first CDR (CDR Oncol. 12 (2002), 11-20; Kiyota, Oncology 63/1 (2002), 40 L1) comprising an amino acid sequence as set out in SEQID 92-98; Kuan, Brain Tumor Pathol. 17/2 (2000), 71-78), NO. 166. The VL region of the first binding domain thus may EGFRVIII (Kuan, Brain Tumor Pathol. 1772 (2000), 71-78), comprise one, two or all three of the mentioned CDRs. The or Carboanhydrase IX (MN/CAIX) (Uemura, Br. J. Cancer above-indicated CDRs are included for example in the VL 81/4 (1999), 741-746; Longcaster, Cancer Res.61/17 (2001), region shown in SEQID NO. 8. 6394-6399; Chia, J. Clin. Oncol. 19/16 (2001), 3660-3668; 45 Preferably, the first binding domain comprises CDR-L1 Beasley, Cancer Res. 61/13 (2001), 5262-5267). (SEQ ID NO. 118), CDR-L2 (SEQID NO. 117), and CDR Particularly preferred as cell surface antigen and/or tumor L3 (SEQ ID NO. 116) and CDR-H1 (SEQ ID NO. 115), antigen is EpCAM. As shown in the following Examples, the CDR-H2 (SEQID NO. 114) and CDR-H3 (SEQID NO. 112) present application for the first time provides for the nucleic or CDR-H3* comprising the amino acid sequence acid and amino acid sequences of the extracellular domain of 50 “VSWFAY (SEQID NO. 113). cynomolgus EpCAM shown in SEQ ID NOS. 47 and 48, Alternatively, the first binding domain comprises CDR-L1 respectively. Said sequences are essential tools for the gen (SEQ ID NO. 166), CDR-L2 (SEQID NO. 165), and CDR eration and characterization of the bispecific single chain L3 (SEQ ID NO. 164) and CDR-H1 (SEQ ID NO. 121), antibodies as defined herein exhibiting cross-species speci CDR-H2 (SEQID NO. 120) and CDR H3 (SEQID NO. 119). ficity to human and cynomolgus EpCAM. 55 Even more preferred, the VH region of the first binding In a further preferred embodiment of the pharmaceutical domain comprises or consists of the amino acid sequence composition of the invention, the first binding domain com shown in SEQID NO. 2 and the VL region of the first binding prises a VH region having an amino acid sequence as shown domain comprises or consists of the amino acid sequence in any of SEQ ID NOS. 2, 110 or 6. It is envisaged and shown SEQID NO. 4; or the VH region of the first binding preferred that the VH region of the first binding domain 60 domain comprises or consists of the amino acid sequence comprises at least a third CDR(CDR-H3) comprising an shown in SEQ ID NO. 110 and the VL region of the first amino acid sequence as set out in SEQID NO. 112 or CDR binding domain comprises or consists of the amino acid H3* comprising an amino acid sequence as set out in SEQID sequence shown in SEQID NO. 148; or the VH region of the NO. 113. The first binding domain may additionally comprise first binding domain comprises or consists of the amino acid a second CDR (CDR-H2) comprising an amino acid 65 sequence shown in SEQID NO. 110 and the VL region of the sequence as set out in SEQ ID NO. 114. Further, the first first binding domain comprises or consists of the amino acid binding domain may in addition comprise a first CDR (CDR sequence shown in SEQID NO. 168, or the VH region of the US 8,236,308 B2 21 22 first binding domain comprises or consists of the amino acid (a) an amino acid sequence as depicted in any of SEQID sequence shown in SEQID NO. 6 and the VL region of the NOs. 66, 68,74, 76, 122, 70, 72, 78,80, 172, 174, 176, first binding domain comprises or consists of the amino acid 178, 180, 182, 184, 186, 188, 190, or 192: sequence shown in SEQID NO. 8. Or the VH region of the (b) an amino acid sequence encoded by a nucleic acid first binding domain comprises or consists of the amino acid sequence as shown in SEQID NOS. 65, 67, 73,75, 123, sequence shown in SEQID NO. 2 and the VL region of the 69, 71, 77, 79, 171, 173, 175, 177, 179, 181, 183,185, first binding domain comprises or consists of the amino acid 187, 189, or 191; sequence shown in SEQID NO. 148. Or the VH region of the (c) an amino acid sequence encoded by a nucleic acid first binding domain comprises or consists of the amino acid sequence hybridizing under Stringent conditions to the sequence shown in SEQID NO. 110 and the VL region of the 10 complementary nucleic acid sequence of (b): first binding domain comprises or consists of the amino acid (f) an amino acid sequence encoded by a nucleic acid sequence shown in SEQID NO. 4. Or the VH region of the sequence which is degenerate as a result of the genetic first binding domain comprises or consists of the amino acid code to a nucleotide sequence of (b); and sequence shown in SEQID NO. 2 and the VL region of the 15 (g) an amino acid sequence at least 85% identical, more first binding domain comprises or consists of the amino acid preferred at least 90% identical, most preferred at least sequence shown in SEQID NO. 168. 95% identical to the amino acid sequence of (a) or (b). As set forth above, the order of the variable regions of the In this above-indicated embodiment, both the first and first binding domain may be VH-VL or VL-VH. Both second binding domains exhibit cross-species specificity. arrangements are within the scope of the invention. For a first In another preferred embodiment of the pharmaceutical binding domain comprising the VH of SEQID NO. 2 and the composition, the non-chimpanzee primate is a baboon, mar VL of SEQ ID NO. 4, the VH-VL arrangement is shown in moset or an old world monkey. SEQ ID NOS. 9 and 10, whereas the VL-VHarrangement is In an even more preferred embodiment of the pharmaceu depicted in SEQID NOS. 11 and 12. tical composition, the old world monkey is a monkey of the For a first binding domain comprising the VH of SEQID 25 macaque genus. NO. 110 and the VL of SEQID NO. 148, the VH-VL arrange Most preferably, the monkey of the macaque genus is Assa ment is shown in SEQ ID NOS. 146 and 147. For a first mese macaque (Macaca assamensis), Barbary macaque binding domain comprising the VH of SEQID NO. 110 and (Macaca sylvanus), Bonnet macaque (Macaca radiata), the VL of SEQID NO. 168, the VH-VL arrangement is shown Booted or Sulawesi-Booted macaque (Macaca ochreata), in SEQID NOS. 169 and 170, whereas the VL-VHarrange 30 Sulawesi-crested macaque (Macaca nigra), Formosan rock ment is depicted in SEQ ID NOs. 193 and 194. For a first macaque (Macaca Cyclopsis), Japanese Snow macaque or binding domain comprising the VH of SEQID NO. 6 and the Japanese macaque (Macaca fiscata), Cynomologus monkey VL of SEQ ID NO. 8, the VH-VL arrangement is shown in or crab-eating macaque or long-tailed macaque or Java SEQID NOS. 13 and 14, whereas the VL-VHarrangement is macaque (Macaca fascicularis), Lion-tailed macaque depicted in SEQID NOS. 15 and 16. 35 (Macaca silenus), Pigtailed macaque (Macaca nemestrina), Similarly, the order of the variable regions of the second Rhesus macaque (Macaca mulatta), Tibetan macaque binding domain may be VH-VL or VL-VH. Both arrange (Macaca thibetana), Tonkean macaque (Macaca tonkeana), ments are within the scope of the invention. For example, the Toque macaque (Macaca sinica), Stump-tailed macaque or VH-VL arrangement of a second binding domain exhibiting 40 Red-faced macaque or Bear monkey (Macaca arctoides), or cross-species specificity to human and cynomolgus EpCAM Moor macaque (Macaca maurus). is shown in SEQ ID NOS. 53 and 54, whereas the VL-VH Preferably, the non-chimpanzee primate CD3 comprises or arrangement is depicted in SEQID NOS. 55 and 56. consists of an amino acid sequence shown in SEQ ID NOS. In a particularly preferred embodiment of the pharmaceu 135, 136, 144, or 145. tical composition of the invention, the bispecific single chain 45 According to a further embodiment of the pharmaceutical antibody as defined herein comprises an amino acid sequence composition of the invention, at least one of said first or selected from the group consisting of: second binding domains is human, humanized, CDR-grafted (a) an amino acid sequence as depicted in any of SEQID and/or deimmunized. NOs. 38, 40, 124, 42 or 44; The term “human' antibody as used herein is to be under (b) an amino acid sequence encoded by a nucleic acid 50 stood as meaning that the bispecific single chain antibody as sequence as shown in SEQID NOS. 37, 39, 125, 41 or defined herein, comprises (an) amino acid sequence(s) con 43; tained in the human germline antibody repertoire. For the (c) an amino acid sequence encoded by a nucleic acid purposes of definition herein, said bispecific single chain sequence hybridizing under Stringent conditions to the antibody may therefore be considered human if it consists of complementary nucleic acid sequence of (b): 55 Such (a) human germline amino acid sequence(s), i.e. if the (d) an amino acid sequence encoded by a nucleic acid amino acid sequence(s) of the bispecific single chain anti sequence which is degenerate as a result of the genetic body in question is (are) identical to (an) expressed human code to a nucleotide sequence of (b); and germline amino acid sequence(s). A bispecific single chain (e) an amino acid sequence at least 85% identical, more antibody as defined herein may also be regarded as human if preferred at least 90% identical, most preferred at least 60 it consists of (a) sequence(s) that deviate(s) from its (their) 95% identical to the amino acid sequence of (a) or (b). closest human germline sequence(s) by no more than would In the above-indicated preferred embodiment, only the first be expected due to the imprint of Somatic hypermutation. binding domain (binding to CD3) exhibits cross-species Additionally, the antibodies of many non-human mammals, specificity. for example rodents such as mice and rats, comprise VH Most preferably, the bispecific single chain antibody as 65 CDR3 amino acid sequences which one may expect to existin defined herein comprises an amino acid sequence selected the expressed human antibody repertoire as well. Any Such from the group consisting of sequence(s) of human or non-human origin which may be US 8,236,308 B2 23 24 expected to exist in the expressed human repertoire would give rise to an antibody response by direct stimulation of T also be considered “human for the purposes of the present cells to produce said antibodies. “Reduced propensity togen invention. erate T-cell epitopes” and/or “deimmunization' may be mea As used herein, the term "humanized”, “humanization', sured by techniques known in the art. Preferably, de-immu “human-like' or grammatically related variants thereof are nization of proteins may be tested in vitro by T cell used interchangeably to refer to a bispecific single chain proliferation assay. In this assay PBMCs from donors repre antibody comprising in at least one of its binding domains at senting >80% of HLA-DR alleles in the world are screened least one complementarity determining region (“CDR) from for proliferation in response to either wild type or de-immu a non-human antibody or fragment thereof. Humanization nized peptides. Ideally cell proliferation is only detected upon approaches are described for example in WO 91/09968 and 10 loading of the antigen-presenting cells with wild type pep U.S. Pat. No. 6,407.213. As non-limiting examples, the term tides. Alternatively, one may test deimmunization by express encompasses the case in which a variable region of at least ing HLA-DR tetramers representing all haplotypes. These one binding domain comprises a single CDR region, for tetramers may be tested for peptide binding or loaded with example the third CDR region of the VH, from another non peptides Substitute for antigen-presenting cells in prolifera human animal, for example a rodent, as well as the case in 15 tion assays. In order to test whether deimmunized peptides which a or both variable region/s comprise at each of their are presented on HLA-DR haplotypes, binding of e.g. fluo respective first, second and third CDRs the CDRs from said rescence-labeled peptides on PBMCs can be measured. Fur non-human animal. In the event that all CDRs of a binding thermore, deimmunization can be proven by determining domain of the bispecific single chain antibody have been whether antibodies against the deimmunized molecules have replaced by their corresponding equivalents from, for been formed after administration in patients. Preferably, anti example, a rodent, one typically speaks of “CDR-grafting. body derived molecules are deimmunized in the framework and this term is to be understood as being encompassed by the regions and most of the CDR regions are not modified in order term “humanized' or grammatically related variants thereof to generate reduced propensity to induce T cell epitope so that as used herein. The term “humanized' or grammatically the binding affinity of the CDR regions is not affected. Even related variants thereof also encompasses cases in which, in 25 elimination of one T cell epitope results in reduced immuno addition to replacement of one or more CDR regions within a genicity. VH and/or VL of the first and/or second binding domain The invention also provides for a pharmaceutical compo further mutation/s (e.g. Substitutions) of at least one single sition comprising a nucleic acid sequence encoding a bispe amino acid residue/s within the framework (“FR) regions cific single chain antibody as defined herein. between the CDRs has/have been effected such that the amino 30 The invention further relates to a pharmaceutical compo acids at that/those positions correspond/s to the amino acid/s sition comprising a vector which comprises a nucleic acid at that those position/s in the animal from which the CDR sequence as defined above. Preferably said vector further regions used for replacement is/are derived. As is known in comprises a regulatory sequence which is operably linked to the art, such individual mutations are often made in the frame said nucleic acid sequence defined above. More preferably, work regions following CDR-grafting in order to restore the 35 said vector is an expression vector. original binding affinity of the non-human antibody used as a In a further aspect, the invention relates to a pharmaceutical CDR-donor for its target molecule. The term “humanized' composition comprising a host transformed or transfected may further encompass (an) amino acid Substitution(s) in the with a vector defined above. CDR regions from a non-human animal to the amino acid(s) A further aspect of the invention relates to a pharmaceuti of a corresponding CDR region from a human antibody, in 40 cal composition as defined hereinabove, further comprising a addition to the amino acid substitutions in the framework proteinaceous compound capable of providing an activation regions as described above. signal for immune effector cells. As used herein, the term “deimmunized,” “deimmuniza Preferably, the pharmaceutical composition further com tion” or grammatically related variants thereof denotes modi prises suitable formulations of carriers, stabilizers and/or fication of the first and/or second binding domain vis-a-vis an 45 excipients. original wild type construct by rendering said wild type con In another aspect, the invention relates to a process for the struct non-immunogenic or less immunogenic in humans. production of a pharmaceutical composition as defined Deimmunization approaches are shown e.g. in WOO0/34317. above, said process comprising culturing a host as defined WO 98/52976, WO 02/0794.15 or WO92/10755. The term above under conditions allowing the expression of the bispe “deimmunized also relates to constructs, which show 50 cific single chain antibody as defined hereinabove and recov reduced propensity to generate T cell epitopes. In accordance ering the produced bispecific single chain antibody from the with this invention, the term “reduced propensity to generate culture. T cell epitopes' relates to the removal of T-cell epitopes A further aspect of the invention relates to a use of a leading to specific T-cell activation. Furthermore, “reduced bispecific single chain antibody as defined hereinabove or as propensity to generate T cell epitopes' means Substitution of 55 produced by the process as defined hereinabove, a nucleic amino acids contributing to the formation of T cell epitopes, acid molecule as defined hereinabove, a vector as defined i.e. Substitution of amino acids, which are essential for for hereinabove or a host as defined hereinabove for the prepa mation of a T cell epitope. In other words, “reduced propen ration of a pharmaceutical composition for the prevention, sity to generate T cell epitopes' relates to reduced immuno treatment or amelioration of a disease. Another aspect of the genicity or reduced capacity to induce antigen independent T 60 invention relates to a method for the prevention, treatment or cell proliferation. The term “T cell epitope' relates to short amelioration of a disease in a Subject in the need thereof, said peptide sequences which can be released during the degrada method comprising the step of administration of an effective tion of peptides, polypeptides or proteins within cells and amount of a pharmaceutical composition of the invention or Subsequently be presented by molecules of the major histo as produced according by the process set forth above. compatibility complex (MHC) in order to trigger the activa 65 Preferably, said disease is a proliferative disease, a tumor tion of T cells; see inter alia WO 02/066514. For peptides ous disease, or an immunological disorder. Even more pre presented by MHC class II such activation of T cells can then ferred, said tumorous disease is a malignant disease, prefer US 8,236,308 B2 25 26 ably cancer. Cross-species specific bispecific single chain contrast, the anti-CD3 antibody shown in SEQ ID NO.162, antibodies as defined herein with specificity for EpCAM, OKT-3 and UCHT-1 fail to bind to macaque CD3. EGFR or EGFRVIII can be used for the therapy of epithelial FIG. 2: FACS assay for binding of an Ig comprising SEQ cancers and tumors. Cross-species specific bispecific single ID NOS. 2 and 4, an Ig comprising SEQID NOS. 6 and 8 and chain antibody constructs as defined herein with specificity 5 monoclonal antibody (mAb) FN-18 to HPB-ALL cells and for CAIX can be used for the treatment of tumors with PBMC of Macaca fascicularis (cynomolgus). HPB-ALL hypoxical regions or areas. Moreover, said CAIX constructs cells express the human CD3 complex. Cells stained with the may be used for the treatment of renal or cervical carcinomas. respective antibodies are shown in comparison to unstained In another preferred embodiment of the uses or methods of cells. Strong antigen binding on human as well as on cyno the invention, said pharmaceutical composition as defined 10 molgus cells was detected for the Ig comprising SEQID NOS. hereinabove is suitable to be administered in combination 2 and 4. For the Ig comprising SEQID NOS. 6 and 8, strong with an additional drug, i.e. as part of a co-therapy. In said binding to human cells but weaker binding to cynomolgus co-therapy, an active agent may be optionally included in the cells was observed. For FN-18, strong binding to cynomolgus same pharmaceutical composition as the bispecific single cells could be observed, whereas no binding to human cells chain antibody, or may be included in a separate pharmaceu 15 could be detected. tical composition. In this latter case, said separate pharma FIG. 3: FACS assay for binding of 5-10LHXSEQ ID ceutical composition is Suitable for administration prior to, NO.12, 5-10LHXSEQ ID NO.10, 5-10LHXSEQ ID NO.16 simultaneously as or following administration of said phar and 5-10LHXSEQID NO.14 to human Kato III cells express maceutical composition comprising the bispecific single ing EpCAM or human EpCAM transfected CHO cells and to chain antibody. The additional drug or pharmaceutical com HPB-ALL cells. Cells bound by the respective constructs position may be a non-proteinaceous compound or a pro (depicted as non-filled curves) are shown in comparison to teinaceous compound. In the case that the additional drug is a cells incubated only with the detection antibodies (depicted proteinaceous compound, it is advantageous that the pro as filled curves). Antigen binding of all bispecific constructs teinaceous compound be capable of providing an activation was clearly detectable for the antihuman EpCAM specificity signal for immune effector cells. 25 as well as for the anti CD3 specificities on the HPB-ALL cell Preferably, said proteinaceous compound or non-proteina line positive for human CD3. ceous compound may be administered simultaneously or FIG. 4: Cytotoxicity assay for 5-10LHXSEQ ID NO.12, non-simultaneously with a bispecific single chain antibody as 5-10LHXSEQ ID NO.10 and 5-10LHXSEQID NO.14 with defined hereinabove, a nucleic acid molecule as defined here human Kato III cells as target cells and human PBMC as inabove, a vector as defined as defined hereinabove, or a host 30 effector cells. All constructs showed cytotoxic activity. as defined as defined hereinabove. Preferably, said subject to FIG. 5: Cytotoxicity assay for 5-10LHXSEQ ID NO.12, be treated is a human. 5-10LHXSEQID NO.10, and 5-10LHXSEQID NO.14 with In a further aspect, the invention relates to a kit comprising Kato III cells as target cells and cynomolgus PBMC as effec a bispecific single chain antibody as defined hereinabove, a tor cells. 5-10LHXSEQID NO.14, 5-10LHXSEQID NO.12 nucleic acid molecule as defined hereinabove, a vector as 35 and 5-10LHXSEQ ID NO.10 showed cytotoxic activity. defined hereinabove, or a host as defined hereinabove. 5-10LHxdi-anti CD3 (deimmunised anti-CD3 antibody These and other embodiments are disclosed and encom shown in SEQID NO.163) which fails to bind to cynomolgus passed by the description and Examples of the present inven CD3 was used as a negative control. tion. Recombinant techniques and methods in immunology FIG. 6: Amino acid sequencealignment of the extracellular are described e.g. in Sambrook et al. Molecular Cloning: A 40 portion of the cynomolgus EpCAM antigen (also shown in Laboratory Manual; Cold Spring Harbor Laboratory Press, SEQID NO. 48) and the human EpCAM antigen. 3" edition 2001: Lefkovits; Immunology Methods Manual: FIG. 7: FACS assay for the detection of the cynomolgus The Comprehensive Sourcebook of Techniques; Academic EpCAM antigen on transfected CHO cells. Supernatants of Press, 1997: Golemis; Protein-Protein Interactions: A three different antihuman EpCAM hybridomas (M79, 3B10, Molecular Cloning Manual: Cold Spring Laboratory Press, 45 2G8) were tested for binding. Transfectants (depicted as non 2002. Further literature concerning any one of the antibodies, filled curves) as compared to untransfected cells (depicted as methods, uses and compounds to be employed in accordance filled curves) showed binding only with the supernatant of the with the present invention may be retrieved from public 2G8 hybridoma which is therefore recognized as antibody libraries and databases, using for example electronic devices. cross-species specific for human and cynomolgus EpCAM. For example, the public database “Medline', available on the 50 FIG. 8: FACS assay for binding of 2G8LHXSEQ ID Internet, may be utilized, for example under http://ww NO.12, 2G8LHXSEQ ID NO.10, 2G8LHXSEQ ID NO.16, w.ncbi.nlm.nih.gov/PubMed/medline.html. Further data 2G8LHXSEQID NO.14, 2G8HLXSEQID NO.12, 2G8HLX bases and addresses, such as http://www.ncbi.nim.nih.gov/, SEQID NO.10, 2G8HLXSEQ ID NO.16 and 2G8HLXSEQ http://www.infobioaen.fr/. http://www.fini.ch/bioloqV/re ID NO.14 on Kato III (FIG.8A) cells or cynomolgus EpCAM search tools.html, http://www.tiqr. or Q/. are known to the 55 transfected CHO cells (FIG. 8B) and HPB-ALL cells. Anti person skilled in the art and can also be obtained using, e.g., genbinding was clearly detectable for the anti EpCAM speci http://www.lvcos.com ficities as well as for the anti CD3 specificities. As a negative The Figures show: control for binding to cynomolgus EpCAM, the 5-10LHx FIG. 1: Identification of cross-species specific antibodies SEQID NO.10 construct was included which shows binding to macaque CD3: Cross-species specificity of an anti-CD3 60 to human CD3 (on HPB-ALL cells) but no binding to cyno antibody shown in SEQ ID NO.162 described in WO molgus EpCAM (cynomolgus EpCAM transfected CHO 99/54440, OKT-3, an Ig comprising SEQID NOS. 6 and 8, an cells). Ig comprising SEQID NOS. 2 and 4 and UCHT-1 to macaque FIG.9: Cytotoxicity assay for 2G8LHXSEQID NO.10 and (cynomolgus) CD3 were tested with Flow Cytometry as 2G8HLXSEQ ID NO.12 with cynomolgus EpCAM trans described in Example 1. An immunoglobulin (Ig) comprising 65 fected CHO cells as target cells and human PBMC as effector SEQID NOS. 6 and 8 and an Ig comprising SEQID NOS. 2 cells. 2G8LHXSEQ ID NO.10 and 2G8HLXSEQ ID NO.12 and 4 show cross-species specificity to macaque CD3. In showed cytotoxic activity. 5-10LHxdi-anti CD3 (deimmu US 8,236,308 B2 27 28 nised anti-CD3 antibody shown in SEQ ID NO.163) was phatase conjugated goat-anti-mouse IgG (B) Pepspot with included as negative control. 5-10LH fails to bind to cyno cross-species specific anti-CD3 antibody comprising the molgus EpCAM. human-like VH shown in SEQ ID NO. 110 and the human FIG. 10: Cytotoxicity assay for 2G8LHXSEQ ID NO.10 like VL shown in SEQID NO. 168 as described in Example and 2G8HLXSEQ ID NO.12 with cynomolgus EpCAM 5 18. transfected CHO cells as target cells and cynomolgus PBMC FIG. 25: Dot Blot Assay with the cross-species specific as effector cells. 2G8LHXSEQ ID NO.10 and 2G8HLXSEQ anti-CD3 antibody comprising the human-like VH of SEQID ID NO.12 showed cytotoxic activity. 5-10LHxdi-anti CD3 NO. 110 and the human-like VL of SEQ ID NO. 168 as (deimmunised anti-CD3 antibody shown in SEQID NO.163) described in Example 19 in (A) and the anti-CD3 murine was included as negative control. This construct fails to bind 10 to cynomolgus CD3 and cynomolgus EpCAM. IgG1 antibody UCHT1 (B) binding to the blotted peptides FIG. 11: Amino acid comparison of SEQ ID NO. 2 and “biotin-linker-EFSELEQSGYYVC (1) and “EFSELEQS human VH segment (hu)3–73. GYYVC-biotin” (2) derived from human CD3 epsilon FIG. 12: Amino acid and nucleotide sequences of a cross FIG. 26: FACS binding analysis of cross-species specific species specific human-like VH region (also shown in SEQ 15 bispecific single chain construct CAIXHLXSEQID NO.194 ID NOS. 110 and 111, respectively). to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol FIG. 13: FACS analysis of a schv comprising the human gus CD3+), A549 (human CAIX+) and CYNOM-KI (cyno like VH chain shown in SEQID NO. 110 and the VL chain molgus CAIX--) cells, respectively. The FACS staining was shown in SEQ ID NO: 148. The complete schvamino acid performed as described in Example 23. The thick line repre sequence is shown in SEQID NO.146. The control scFv of sents cells incubated with 1 ug/ml purified monomeric pro SEQ ID NO.10 shows a clear shift on human CD3 positive tein that were subsequently incubated with the anti-his anti HPB-All cells and thus binds to human CD3. The ScFv body and the PE labeled detection antibody. The thin depicted in SEQID NO. 146 also shows clear binding to said histogram line reflects the negative control: cells only incu CD3 positive human cells. bated with the anti-his antibody and the detection antibody. FIG. 14: Binding analysis of the scEv of SEQID NO. 146. 25 FIG. 27: FACS binding analysis of cross-species specific The control scFv of SEQ ID NO. 10 shows a clear shift on bispecific single chain construct CAIXHLXSEQID NO. 170 cynomolgus CD3 positive T cells and thus binds to cynomol to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol gus CD3 positive cells. Also the scFv of SEQ ID NO: 146 gus CD3+), A549 (human CAIX--) and 4 MBr-5 (macaque shows clear binding to cynomolgus CD3 positive cells. CAIX--) cells, respectively. The FACS staining was per FIG. 15: Alignment of amino acid sequences of human and 30 formed as described in Example 23. The thick line represents cynomolgus CD3 epsilon. cells incubated with 1 g/ml purified monomeric protein that FIG. 16: Amino acid sequences of the 13mer peptides were subsequently incubated with the anti-his antibody and derived from cynomolgus CD3 epsilon (43 peptide-spots). the PE labeled detection antibody. The thin histogram line FIG. 17: Amino acid sequences of the 13mer peptides reflects the negative control: cells only incubated with the derived from human CD3 epsilon (47 peptide-spots). 35 anti-his antibody and the detection antibody. FIG. 18: Pepspots developed by enhanced chemilumines FIG. 28: FACS binding analysis of cross-species specific cence (A) Control pepspot with horseradish-peroxidase con bispecific single chain construct CAIX LHXSEQID NO. 170 jugated goat-anti-mouse IgG (B) Pepspot with cross-species to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol specific anti-CD3 antibody I corresponding to an immuno gus CD3+), A549 (human CAIX--) and 4MBr-5 (macaque globulin (Ig) comprising the VH chain shown in SEQID NO. 40 CAIX--) cells, respectively. The FACS staining was per 2 and the VL chain shown in SEQID NO. 4. formed as described in Example 23. The thick line represents FIG. 19: Pepspot with cross-species specific anti-CD3 cells incubated with 1 g/ml purified monomeric protein that antibody II corresponding to an immunoglobulin (Ig) com were subsequently incubated with the anti-his antibody and prising the VH chain shown in SEQ ID NO. 6 and the VL the PE labeled detection antibody. The thin histogram line chain shown in SEQID NO. 8. 45 reflects the negative control: cells only incubated with the FIG. 20: Contact residues of OKT-3 and UCHT-1 and anti-his antibody and the detection antibody. E-F-loop epitope of cross-species specific anti-CD3 antibod FIG. 29: FACS binding analysis of cross-species specific ies I and II referred to in FIGS. 18 and 19, respectively, on bispecific single chain construct EGFRHLXSEQID NO. 170 cynomolgus and human CD3 epsilon. to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol FIG. 21: Amino acid sequence comparison of the murine 50 gus CD3+), A431 (human EGFR+) and CHO cells trans VL shown in SEQID NO. 4 to the human germline lambda 7a fected with cynomolgus EGFR (cynomolgus EGFR+). Segment. respectively. The FACS staining was performed as described FIG.22: Binding of the murine sclv shown in SEQID NO. in Example 23. The thick line represents cells incubated with 10 and the human-like scFv shown in SEQ ID NO. 170 to 1 ug/ml purified monomeric protein that were Subsequently human CD3-positive HPB-ALL cells. 55 incubated with the anti-his antibody and the PE labeled detec FIG. 23: Upper Panel: Equal binding of the murine sclv tion antibody. The thin histogram line reflects the negative shown in SEQID NO. 10 and the human-like schv shown in control: cells only incubated with the anti-his antibody and SEQ ID NO. 170 to human and cynomolgus T cells in the detection antibody. PBMCs. Lower Panel When preincubated with 10 g/ml of FIG. 30: FACS binding analysis of cross-species specific the murine IgG antibody mAb I described in Example 1 60 bispecific single chain construct EGFRLHXSEQID NO. 170 having the same binding specificity as the scFVs (i.e. for CD3 to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol epsilon), the shifts of cells stained with the above-mentioned gus CD3+), A431 (human EGFR+) and CHO cells trans murine ScFv or the human-like ScFv decrease significantly, fected with cynomolgus EGFR (cynomolgus EGFR+). underlining the similar binding region of the scFVs and the respectively. The FACS staining was performed as described original murine antibody mAb I. 65 in Example 23. The thick line represents cells incubated with FIG. 24: Pepspots developed by the alkaline phosphatase 1 ug/ml purified monomeric protein that were Subsequently detection system (A) Control pepspot with alkaline phos incubated with the anti-his antibody and the PE labeled detec US 8,236,308 B2 29 30 tion antibody. The thin histogram line reflects the negative fected with cynomolgus EGFR (cynomolgus EGFR+). control: cells only incubated with the anti-his antibody and respectively. The FACS staining was performed as described the detection antibody. in Example 23. The thick line represents cells incubated with FIG. 31: FACS binding analysis of cross-species specific 1 ug/ml purified monomeric protein that were Subsequently bispecific single chain construct EGFRHLXSEQID NO.194 incubated with the anti-his antibody and the PE labeled detec to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol tion antibody. The thin histogram line reflects the negative gus CD3+), A431 (human EGFR+) and CHO cells trans control: cells only incubated with the anti-his antibody and fected with cynomolgus EGFR (cynomolgus EGFR+). the detection antibody. respectively. The FACS staining was performed as described FIG. 37: Cytotoxic activity induced by CAIX and CD3 in Example 23. The thick line represents cells incubated with 10 cross-species specific bispecific single chain antibody con 1 g/ml purified monomeric protein that were Subsequently structs redirected to indicated target cell lines. Stimulated incubated with the anti-his antibody and the PE labeled detec CD8 positive T cells from human and cynomolgus origin tion antibody. The thin histogram line reflects the negative were used as effector cells, respectively. The assay was per control: cells only incubated with the anti-his antibody and formed as described in Examples 24 and 25. In the left panel the detection antibody. 15 of FIG. 37, a bispecific single chain antibody with a variable FIG. 32: FACS binding analysis of cross-species specific domain reactive with CAIX and a de-immunized human bispecific single chain construct EGFRLHXSEQID NO.194 CD3-specific variable domain has been used as a positive to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol control. In the right panel, the same construct has been used as gus CD3+), A431 (human EGFR+) and CHO cells trans a negative control. fected with cynomolgus EGFR (cynomolgus EGFR+). FIG.38: Cytotoxic activity induced by the CAIX and CD3 respectively. The FACS staining was performed as described cross-species specific bispecific single chain antibody con in Example 23. The thick line represents cells incubated with struct CAIX HLXSEQ ID NO. 194 redirected to target cell 1 g/ml purified monomeric protein that were Subsequently line A549. Stimulated CD8 positive T cells from human and incubated with the anti-his antibody and the PE labeled detec cynomolgus origin were used as effector cells, respectively. tion antibody. The thin histogram line reflects the negative 25 The assay was performed as described in Examples 24 and control: cells only incubated with the anti-his antibody and 25. the detection antibody. FIG. 39: Cytotoxic activity induced by EGFR and CD3 FIG. 33: FACS binding analysis of cross-species specific cross-species specific bispecific single chain antibody con bispecific single chain construct SEQID NO. 170xEGFRHL structs redirected to CHO cells transfected with cynomolgus to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol 30 EGFR as target cell line. Stimulated CD8 positive T cells gus CD3+), A431 (human EGFR+) and CHO cells trans from cynomolgus origin were used as effector cells. The fected with cynomolgus EGFR (cynomolgus EGFR+), measurements shown in this figure were performed in a single respectively. The FACS staining was performed as described assay. The assay was performed as described in Example 24. in Example 23. The thick line represents cells incubated with A bispecific single chain antibody with a variable domain 1 g/ml purified monomeric protein that were Subsequently 35 reactive with EGFR and a de-immunized human CD3-spe incubated with the anti-his antibody and the PE labeled detec cific variable domain (EGFRLHxdi-anti CD3) has been used tion antibody. The thin histogram line reflects the negative as a negative control. control: cells only incubated with the anti-his antibody and FIG. 40: Cytotoxic activity induced by EGFR and CD3 the detection antibody. cross-species specific bispecific single chain antibody con FIG. 34: FACS binding analysis of cross-species specific 40 structs redirected to human A431 as target cell line. Stimu bispecific single chain construct SEQID NO. 170xEGFRLH lated CD8 positive T cells from human origin were used as to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol effector cells. The measurements shown in this figure were gus CD3+), A431 (human EGFR+) and CHO cells trans performed in a single assay. The assay was performed as fected with cynomolgus EGFR (cynomolgus EGFR+). described in Example 24. A bispecific single chain antibody respectively. The FACS staining was performed as described 45 with a variable domain reactive with EGFR and a de-immu in Example 23. The thick line represents cells incubated with nized human CD3-specific variable domain (EGFR LHxdi 1 g/ml purified monomeric protein that were Subsequently anti CD3) has been used as a positive control. As a negative incubated with the anti-his antibody and the PE labeled detec control, an irrelevant bispecific single chain antibody has tion antibody. The thin histogram line reflects the negative been used. control: cells only incubated with the anti-his antibody and 50 The following Examples illustrate the invention: the detection antibody. FIG. 35: FACS binding analysis of cross-species specific EXAMPLE 1. bispecific single chain construct SEQID NO. 194xEGFRHL to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol Flow Cytometric Analysis of Cross-Species Specific gus CD3+), A431 (human EGFR+) and CHO cells trans 55 Antibodies fected with cynomolgus EGFR (cynomolgus EGFR+). respectively. The FACS staining was performed as described Cross-species specificity of anti-human CD3 antibodies to in Example 23. The thick line represents cells incubated with macaque CD3 (CD3 of Macaca fascicularis, in the following 1 g/ml purified monomeric protein that were Subsequently also named “Cynomolgus') was tested by flow cytometric incubated with the anti-his antibody and the PE labeled detec 60 analysis. Antibodies tested were an anti-CD3 antibody as tion antibody. The thin histogram line reflects the negative described in WO99/54440 (as shown in SEQID NO. 162 of control: cells only incubated with the anti-his antibody and the present application), monoclonal antibody (mAb) OKT-3 the detection antibody. (Jansen-Cillag). UCHT-1-PE (BD PharMingen, San Diego, FIG. 36: FACS binding analysis of cross-species specific Calif.), an immunoglobulin (Ig) comprising the VH and VL bispecific single chain construct SEQID NO. 194xEGFRLH 65 chains shown in SEQID NOS. 2 and 4, respectively, and an Ig to HPB-ALL (human CD3+), cynomolgus PBMC (cynomol comprising the VH and VL chains shown in SEQID NOS. 6 gus CD3+), A431 (human EGFR+) and CHO cells trans and 8, respectively. 2x10 cells (macaque T cell lines of US 8,236,308 B2 31 32 Macaca fascicularis and Macaca mulata, respectively, as body and there were two different sets of primers for the 5' kindly provided by H. Fickenscher, Heidelberg, Germany) region, one for the light chain variable region and the other for per sample were stained for 30 minutes at 4°C. in 25 ul of the heavy chain variable region. The primer combinations PBS/1% FCS/0.050% NaNs containing working dilutions of used in the PCR reactions are given below. monoclonal antibodies (as determined individually by titra- 5 Heavy Chain Variable Region: tion). Cells were washed two times in PBS/1% FCS/0.05% NaNs and a secondary antibody was added where necessary. After the addition of the secondary antibody, cells were 5' primer: washed again two times in the same solution and 10.000 (SEQ ID NO. 81) living cells were acquired. A FACS Calibur flow cytometer 10 5'-SAGGTGCAGCTCGAGGAGTCAGGACCT-3' and the CellOuest software from Becton Dickinson were used (SEQ ID NO. 82) to collect and analyze the data. Non viable cells were 5 - GAGGTCCAGCTCGAGCAGTCTGGACCT-3' excluded using forward and side scatter electronic gating. Isotype control or secondary antibody only were used as a (SEQ ID NO. 83) negative control. As can be seen from FIG. 1, only the Ig 15 5 - CAGGTCCAACTCGAGCAGCCTGGGGCT-3' comprising the VH and VL chains shown in SEQID NOS. 2 (SEQ ID NO. 84) and 4, respectively, and the Ig comprising the VH and VL 5 - GAGGTTCAGCTCGAGCAGTCTGGGGCA-3' chains shown in SEQID NOS. 6 and 8, respectively, showed (SEO ID NO. 85) cross-species specificity for a non-chimpanzee primate CD3. 5 " - GARGTGAAGCTCGAGGAGTCTGGAGGA-3' i.e. macaque CD3. 2O (SEQ ID NO. 86) EXAMPLE 2 5 - GAGGTGAAGCTTCTCGAGTCTGGAGGT-3'

- (SEO ID NO. 87) FACS Assay for Binding of an Ig Comprising SEQ 5 " - GAAGTGAAGCTCGAGGAGTCTGGGGGA-3' ID NOS. 2 and 4, an Ig Comprising SEQID NOS. 6 25 and 8 and mAb FN 18 to HPB-ALL Cells and (SEQ ID NO. 88) Cynomolgus PBMC 5 - GAGGTTCAGCTCGAGCAGTCTGGAGCT-3'

Binding- of an Ig comprising SEQID NOS. 2 and 4, an Ig 5 - GGGCTCGAGCACCATGGRATGSAGCTGKGTMATSCTCTT-3'(SEQ ID NO. 89) comprising SEQ ID NOS. 6 and 8 and mAb FN18 to the 30 cynomolgus CD3 antigen on cynomolgus PBMC and to the (SEQ ID NO. 90) human CD3 antigen on HPB-ALL cells (DSMZ No. ACC 5-GGGCTCGAGCACCATGRACTTCGGGYTGAGCTKGGTTTT-3 483) was tested using an FACS assay. For that purpose, 2.5.x 10 cells were incubated with the FITC-conjugated Ig com (SEQ ID NO. 91) prising SEQ ID NOS. 6 and 8 and the FITC-conjugated Ig 35 s' - GGGCTCGAGCACCATGGCTGTCTTGGGGCTGCTCTTCT-3' comprising SEQID NOS. 2 and 4 diluted 1:25 in 50 ul PBS 3' primer: with 2% FCS, respectively. The incubation with the FITC- (SEQ ID NO. 92) conjugated mAb FN18 antibody (Biosource International) 5 - GAGGAATTCGAACTGGACAGGGATCCAGAGTTCC-3' was performed in 50 ul of undiluted antibody. The samples (SEQ ID NO. 93) were measured on a FACSscan (BD biosciences, Heidelberg, 40 5 - CGGAATTCGAATGACATGGACATCTGGGTCATCC-3' FRG). The results for the assay are shown in FIG. 2. Strong antigenbinding on human as well as on cynomolgus cells was Light Chain Variable Region: detected for the Ig comprising SEQID NOS. 2 and 4. For the Ig comprising SEQID NOS. 6 and 8, strong binding to human cells but weaker binding to cynomolgus cells was observed. 45 5' primer: For FN 18, strong binding to cynomolgus cells could be (SEQ ID NO. 94) observed whereas no binding to human cells could be s' - CCAGTTCCGAGCTCGTTGTGACTCAGGAATCT-3' detected. (SEO ID NO. 95) s' - CCAGTTCCGAGCTCGTGTTGACGCAGCCGCCC-3' EXAMPLE 3 50 (SEQ ID NO. 96) Sequence Determination of the Variable Regions of s' - CCAGTTCCGAGCTCGTGCTCACCCAGTCTCCA-3' Two Anti-Human CD3 Antibodies Exhibiting (SEO ID NO. 97) Species Specificity for Non-Human Primates s' - CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3' 55 For the sequence determination of the variable regions of 5 - CCAGATGTGAGCTCGTGATGACCCAGACTCCA-3'(SEQ ID NO. 98) the cross-species specific anti-CD3 Igs of Examples 1 and 2. PCR (denaturation at 93°C. for 5 min, annealing at 58°C. for (SEO ID NO. 99) 1 min, elongation at 72° C. for 1 min for the first cycle: s' - CCAGATGTGAGCTCGTCATGACCCAGTCTCCA-3' denaturation at 93°C.O for 1 min, annealing at 58 O C. for 1 min, 60 (SEQ ID NO. 1 OO) elongation at 72°C. for 1 min for 30 cycles; terminal exten- s' - CCAGTTCCGAGCTCGTGATGACACAGTCTCCA-3' sion at 72° C. for 5 min) was used to amplify the coding sequences of the variable regions of the antibodies. As the (SEQ ID NO. 101) sequence of the 5' region- - of the variable regions is not known, 5 - GGGGAGCTCCACCATGGAGACAGACACACTCCTGCTAT-3' instead of a single primer a set of 5" primers was used in 65 (SEQ ID NO. 102) combination with a constant 3' primer whereby the 3' primer 5 - GGGGAGCTCCACCATGGATTTTCAAGTGCAGATTTTCAG-3 was chosen according to the isotype of the respective anti US 8,236,308 B2 33 34 - Continued required. The resulting schv antibody is flanked at the 5' end with a small Ser(Gly)Serlinker preceded by the restriction (SEQ ID NO. 103) enzyme recognition site for BspEI and at the 3' end with a 6 5 - GGGGAGCTCCACCATGGAGWCACAKWCTCAGGTCTTTRTA-3' histidine affinity tag followed by a stop codon and by the (SEO ID NO. 104) 5 restriction enzyme recognition site for SalI. The second s' - GGGGAGCTCCACCATGKCCCCWRCTCAGYTYCTKGT-3' single chain FV antibody was an anti human EpCAM speci 3' primer: ficity designated “5-10” which is described in SEQID NO. 33 (SEQ ID NO. 105) and 34 of the sequence listing included in the description. To 5 - GAGGAATTCGAACTGCTCACTGGATGGTGGG-3 accomplish the fusion of the single chain Fvantibodies and to 10 allow for eukaryotic expression, the coding sequence of the (SEQ ID NO. 106) single chain Fv antibodies was then cloned via BspEI (5' to 5 - CGGAATTCGAACAAACTCTTCTCCACAGTGTGACC-3' the Ser(Gly)Serlinker) and SalI into the pEFDHFR expres All PCR products with a length between 350 and 700 base sion vector (pEFDHFR was described in Mack et al. Proc. pairs were isolated, purified and sequenced with the respec Natl. Acad. Sci. USA 92 (1995) 7021-7025) containing the tive 3' primer according to standard protocols (Sambrook, 15 coding sequence for the human EpCAM specific single chain Molecular Cloning: A Laboratory Manual, Cold Spring Har Fv antibody 5-10 and the restriction enzyme recognition site bor Laboratory Press, Cold Spring Harbor, N.Y. for BspEI. The coding sequence of an murine immunoglobu (1989)(2001)). lin leaderpeptide is described in SEQID NO. 35 and 36 of the The obtained sequences were examined for functional vari sequence listing included in the description, preceded by a able region coding sequences and for the heavy chain and the 20 Kozak translation initiation consensus sequence and the light chain of each antibody a sequence coding for the vari restriction enzyme recognition site for EcoRI. Single clones able region was obtained. The nucleotide and amino acid of the constructs were isolated and sequenced with primers sequences of the heavy and light chain variable regions of the complementary to flanking regions in the vector according to cross-species specific anti-CD3 antibodies are described in standard protocols (Sambrook, Molecular Cloning: A Labo SEQID NOS. 1 through 8 in the sequence listing included in 25 ratory Manual, Cold Spring Harbor Laboratory Press, Cold the description, respectively. Spring Harbor, N.Y. (1989) (2001)). For further experiments a clone of each construct was selected. The nucleotide and EXAMPLE 4 amino acid sequences are described for 5-10LHXSEQ ID NO.12 in SEQ ID NOs. 37 and 38, for 5-10LHXSEQ ID Cloning of Anti Human EpCAM and CD3 30 NO.10 in SEQ ID NOs. 39 and 40, for 5-10LHXSEQ ID Cross-Species Specific Bispecific Single Chain NO.16 in SEQID NOS. 41 and 42 and for 5-10LHXSEQ ID Antibodies NOs.14 in SEQ ID NO. 43 and 44 of the sequence listing included in the description. To generate bispecific single chain antibodies comprising the aforementioned CD3 cross-species specificities, the 35 EXAMPLE 5 amplified variable regions had to be modified by PCR to obtain the corresponding single chain FV antibody fragments. Expression of the 5-10LHXSEQ ID NO.12, To determine Suitable arrangements of the light and heavy 5-10LHXSEQID NO.10, 5-10LHXSEQID NO.16 chain variable regions in the single chain FV antibody, two and 5-10LHXSEQID NO.14 Bispecific Single Chain different single chain Fv antibodies were generated for each 40 Antibodies in CHO Cells antibody. To this end, a two-step fusion PCR was used to amplify the sequence coding for the variable regions. A set of The plasmids with the sequences coding for the bispecific appropriate primers was designed to perform the PCR-based single chain antibodies were transfected into DHFR deficient cloning steps, finally resulting in a single chain antibody CHO cells for eukaryotic expression of the construct as connecting the two variable domains with a 15 amino acid 45 described in Kaufmann R. J. (1990) Methods Enzymol. 185, linker (Gly-Ser) in the order VH-Linker-VL and 537-566). Gene amplification of the construct was induced by VL-Linker-VH. The corresponding nucleotide and amino increasing concentrations of Methotrexat (MTX) to a final acid sequences are described in SEQID NO. 9through 12 and concentration of up to 500 nM MTX. The transfected cells in SEQID NO. 13 through 16 of the sequence listing included were then expanded and 1 liter of supernatant produced. The in the description. 50 construct was finally purified out of the culture Supernatant as In short the following primer combinations were used: described in Kuferetal. Cancer immunity Vol. 1, p. 10 (2001). For VL-VH scFv antibody shown in SEQID NOS. 11 and 12: SEQID NOs. 17 to 20. EXAMPLE 6 For VH-VL sclv antibody shown in SEQID NOS. 9 and 10: SEQID NOs. 21 to 24. 55 FACS Assay for Binding of 5-10LHXSEQ ID For VL-VH scFv antibody shown in SEQID NOS. 15 and 16: NO.12, 5-10LHXSEQ ID NO.10, 5-10LHXSEQ ID SEQID NOs. 25 to 28. NO.16 and 5-10LHXSEQID NO.14 to Kato III Cells For VH-VL scFv antibody shown in SEQID NOS. 13 and 14: or Human EpCAM Transfected CHO Cells and to SEQID NOs. 29 to 32. HPB-ALL Cells To generate the single chain antibody, two PCRs with the 60 respective primer combinations were performed. During this Binding of the bifunctional constructs to the EpCAM anti PCR overlapping complementary sequences were introduced gen on human Kato III cells expressing EpCAM (ATCC No. into the PCR-products stemming from the respective linker HTB-103) or on human EpCAM transfected CHO cells and primers that combined to form the coding sequence of the 15 to the human CD3 antigen on HPB-ALL cells was tested amino acid linker during the subsequent fusion PCR. The 65 using an FACS assay. For that purpose 2.5x10 cells were amplified VH and VL domains were fused in a next PCR in incubated with 50 ul of cell culture supernatant containing the which only the outer primers and both PCR-products were construct. The binding of the construct was detected with an US 8,236,308 B2 35 36 anti-His antibody (Penta-His Antibody, BSA free, obtained based in vitro cytotoxicity assays using the human EpCAM from Qiagen GmbH, Hilden, FRG) at 2 ug/ml in 50 lul PBS positive Kato III cells as target cells and cynomolgus PBMCs with 2% FCS. As a second step reagent a R-Phycoerythrin as effector cells. conjugated affinity purified F(ab')2 fragment, goat anti Target cells were washed twice with PBS and labeled with mouse IgG, Fc-gamma fragment specific antibody, diluted PKH26 dye (Sigma-Aldrich, Germany) according to the 1:100 in 50 ul PBS with 2% FCS (obtained from Dianova, manufacturers instructions. Labeled target cells were Hamburg, FRG) was used. The samples were measured on a washed twice with RPMI/10% FCS and mixed with freshly FACSscan (BD biosciences, Heidelberg, FRG). Antigen isolated effector cells at an E:T ratio of 10:1. Two times 10' binding was clearly detectable for the anti human EpCAM target and 2x10 effector cells in a volume of 50 ul RPMI/ specificity as well as for the anti CD3 specificities on the cell 10 10% FCS were added per well in a 96-well round bottom plate. Ten-fold serial dilutions of different Bispecific single line positive for human CD3 (see FIG. 3). chain antibodies were prepared in RPMI/10% FCS to obtain EXAMPLE 7 a starting concentration of 1000 ng/ml in the final reaction volume. 50 ul of the different solutions were added in tripli Cytotoxicity Assay for 5-10LHXSEQID NO. 12, 15 cates to the corresponding wells. Individual cytotoxicity mix tures were incubated for 24 to 48 hours at 37° C., 5% CO. 5-10LHXSEQ ID NO.10, and 5-10LHXSEQ ID Subsequently the measurement of cytotoxic activity was NO.14 with Kato III Cells as Target Cells and performed. To this end, propidium iodide (PI) was added to a Human PBMC as Effector Cells final concentration of 1 Lug/ml per well and plates were incu bated for 10 minutes at room temperature. The number of Bioactivity of 5-10LHXSEQID NO.12, 5-10LHXSEQID PKH and PI positive and negative target cells was determined NO.10, and 5LH-10xSEQID NO.14 was analyzed by FACS by FACS. Cytotoxicity was measured as the ratio of PKH based in vitro cytotoxicity assays using the human EpCAM positive and PI negative (living target cells) over the mean of positive Kato III cells as target cells and human PBMCs as living target cells (PKH-positive and PI negative) in the con effector cells. Target cells were washed twice with PBS and 25 trol containing no construct according to the formula: cyto labeled with PKH26 dye (Sigma-Aldrich, Germany) accord toxicity (%)=(PI-negative cells/mean of PI-negative cells in ing to the manufacturers instructions. Labeled target cells control)x100. Sigmoidal dose response killing curves were were washed twice with RPMI/10% FCS and mixed with analyzed by Prism Software (GraphPad Software Inc., San freshly isolated effector cells at an E:T ratio of 10:1. Two Diego, USA) and the bispecific single chain antibody con times 104 target and 2x10 effector cells in a volume of 50 ul 30 centration calculated that induced half maximal killing RPMI/10% FCS were added per well in a 96-well round (EC50 value). The results of this assay are shown below in bottom plate. Ten-fold serial dilutions of different bispecific FIG. 5.5-10LHXSEQ ID NO.14, 5-10LHXSEQ ID NO.12 single chain constructs were prepared in RPMI/10% FCS to and 5-10LHXSEQ ID NO.10 showed cytotoxic activity. The obtain a starting concentration of 1000 ng/ml in the final resulting EC50 values for 5-10LHXSEQID NO.14, 5-10LHx reaction volume. 50 ul of the different solutions were added in 35 SEQID NO.12 and 5-10LHXSEQID NO.10 were 87 pg/ml, triplicates to the corresponding wells. Individual cytotoxicity 69 pg/ml and 52 pg/ml respectively. 5-10LHxdi-anti CD3 (deimmunised anti-CD3 antibody as shown in SEQ ID mixtures were incubated for 24 to 48 hours at 37°C., 5% CO. NO.163) showed no activity. This is due to the fact that di-anti Subsequently the measurement of cytotoxic activity was CD3 antibody only binds to human CD3, but not to cynomol performed. To this end, Propidium iodide (PI) was added to a 40 gus CD3. final concentration of 1 Lug/ml per well and plates were incu bated for 10 minutes at room temperature. The number of EXAMPLE 9 PKH and PI positive and negative target cells was determined by FACS. Cytotoxicity was measured as the ratio of PKH Sequence Determination of the Cynomolgus positive and PI negative (living target cells) over the mean of 45 EpCAM Antigen and Generation of Cynomolgus living target cells (PKH-positive and PI negative) in the con EpCAM Transfected CHO Cells trol containing no construct according to the formula: cyto toxicity (%)=(PI-negative cells/mean of PI-negative cells in To obtain the cynomolgus EpCAM antigen for testing of control)x100. Sigmoidal dose response killing curves were cross-species specificity of anti human EpCAM antibodies, analyzed by Prism Software (GraphPad Software Inc., San 50 first the coding sequence of the cynomolgus EpCAM antigen Diego, USA) and the BiTE concentration calculated that had to be determined. To this end, colon tissue samples of 3 induced half maximal killing (EC50 value). The results of this animals were used in parallel for the isolation of total RNA assay are shown below in FIG. 4. All constructs showed and cDNA synthesis by random-primed reverse transcription, cytotoxic activity. The resulting EC50 values for 5-10LHx which were performed according to standard protocols (Sam SEQID NO.14, 5-10LHXSEQID NO.12 and 5-10LHXSEQ 55 brook, Molecular Cloning: A Laboratory Manual, Cold ID NO.10 were 1.3 pg/ml, 1.5 pg/ml and 5.8 pg/ml respec Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. tively. (1989) (2001)). A PCR (denaturation at 93° C. for 5 min, annealing at 58°C. for 1 min, elongation at 72°C. for 1 min EXAMPLE 8 for the first cycle; denaturation at 93°C. for 1 min, annealing 60 at 58° C. for 1 min, elongation at 72° C. for 1 min for 35 Cytotoxicity Assay for 5-10LHXSEQID NO.12, cycles; terminal extension at 72° C. for 5 min) was used to 5-10LHXSEQID NO. 10, and 5-10LHXSEQ ID amplify the coding sequence of the EpCAM antigen. As the NO.14 with Kato III Cells as Target Cells and coding sequence of the cynomolgus EpCAM antigen was not Cynomolgus PBMC as Effector Cells known, appropriate primers (5' primer described in SEQ ID 65 NO. 45, 3' primer described in SEQID NO. 46) for the PCR Bioactivity of 5-10LHXSEQID NO1.12, 5-10LHXSEQID reaction were designed according to the known coding NO.10, and 5-10LHXSEQID NO.14 was analyzed by FACS sequence of the human EpCAM antigen (Szala S. et al., Proc US 8,236,308 B2 37 38 Natl AcadSci USA. 87 (1990): p. 3542-6). Primers were also extension at 72°C. for 5 min) was used to amplify the coding designed as to allow for expression of the coding sequence of sequences of the variable regions of the antibody. As the the entire antigen. For the 3 samples, PCR of 960 base pairs sequence of the 5' region of the variable regions is not known were isolated, purified and subcloned via Xbal and SalI, into the aforementioned set of 5" primers was used in combination pEFDHFR. Multiple clones for each sample were sequenced with a constant 3' primer whereby the 3' primer was chosen according to standard protocols (Sambrook, Molecular Clon according to the isotype of the antibody. ing: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (2001)) using appro priate sequencing primers complementary to flanking Heavy chain variable region: sequences in the vector. 10 3' primer: The novel nucleotide and amino acid sequences of the (SEO ID NO. 107) cynomolgus EpCAM antigen are described in SEQID NOS. 5'-TATGCAACTAGTACAACCACAATCCCTGGG-3 47 and 48 in the sequence listing included in the description, Light chain variable region: respectively. 3' primer: The obtained sequences were examined by comparison 15 (SEQ ID NO. 108) with the coding sequence of the human EpCAM antigen. As s' - GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3' shown in FIG. 6, there is a high degree of sequence homology All PCR products with a length between 350 and 700 base between the coding sequence of the human EpCAM antigen pairs were isolated, purified and sequenced with the respec and the sequences obtained from the colon samples of the 3 tive 3' primer according to standard protocols (Molecular cynomolgus monkeys. Cloning: A Laboratory Manual, Cold Spring Harbor Labora To generate a cell line positive for cynomolgus EpCAM, a tory Press, Cold Spring Harbor, N.Y. (1989)(2001)). clone of the aforementioned coding sequence of the cynomol The obtained sequences were examined for functional vari gus EpCAM antigen subcloned into pEFDHFR with a veri able region coding sequences and for the heavy chain and the fied nucleotide sequence was transfected into DHFR deficient light chain of the antibody a sequence coding for the variable CHO cells for eukaryotic expression of the construct as 25 region was isolated. The nucleotide and amino acid described in Kaufmann R. J. (1990) Methods Enzymol. 185, sequences of the variable regions are described in SEQ ID 537-566). Gene amplification of the construct was induced by NOs. 49 through 52 in the sequence listing included in the increasing concentrations of MTX to a final concentration of description, respectively. up to 500nM MTX. The transfected cells were then tested for expression of cynomolgus EpCAM using an FACS assay. For 30 EXAMPLE 11 that purpose, a number of 2.5x10 cells was incubated with 50 ul supernatant three different mouse anti human EpCAM Cloning of EpCAM and CD3 Cross-Species Specific hybridomas (M79 Fogler et al., Cancer Res. 48 (1988); p. Bispecific Single Chain Antibodies 6303-8; 3B10 Passlick et al. Int. J. Cancer 87 (2000), p. 548-552; 2G8 Balzar et al., J. Mol. Med. 77 (1999), p. 35 To generate bispecific single chain antibody molecules 699-712). The binding of the antibodies was detected with a comprising the aforementioned CD3 cross-species specific R-Phycoerythrin-conjugated affinity purified F(ab')2 frag ity and the aforementioned EpCAM cross-species specificity, ment, goat anti-mouse IgG, Fc-gamma fragment specific the amplified variable regions of the 2G8 antibody had to be antibody, diluted 1:100 in 50 ul PBS with 2% FCS (obtained modified by PCR to obtain the corresponding single chain Fv from Dianova, Hamburg, FRG) was used. The samples were 40 antibody fragments. Two single chain Fv antibodies with measured on a FACSscan (BD biosciences, Heidelberg, different arrangements of the light and heavy chain variable FRG). The anti EpCAM antibody 2C8 was recognized as regions were generated. To this end, a two-step fusion PCR cross-species specific and the expression of cynomolgus was used to amplify the sequence coding for the variable EpCAM was confirmed (see FIG. 7). Transfectants (depicted regions. A set of appropriate primers was designed to perform as non-filled curves) as compared to untransfected cells (de 45 the PCR-based cloning steps, finally resulting in a 2G8 single picted as filled curves) showed binding only with the super chain antibody connecting the two variable domains with a 15 natant of the 2G8 hybridoma which is therefore recognized as amino acid linker (Gly-Ser) in the order VH-Linker-VL antibody species specific for human and cynomolgus and VL-Linker-VH. The nucleotide and amino acid EpCAM. sequences are described in SEQID NOS. 53 through 56 of the 50 sequence listing included in the description, respectively. EXAMPLE 10 In short the following primer combinations were used: For 2G8 VL-VH schv antibody (hereafter designated as Sequence Determination of the Variable Regions of 2G8LH shown in SEQID NOS. 55 and 56): SEQID NOs. an Anti Human EpCAM Antibody Cross-Species 57 to 60. Specific for Non-Human Primates 55 For 2G8 VH-VL schv antibody (hereafter designated as 2G8HL shown in SEQID NOS. 53 and 54): SEQID NOs. For the sequence determination of the variable regions of 61 to 64. the anti-EpCAM antibody 2G8, the respective hybridoma cell To generate the single chain antibody, two PCRs with the line was used for isolation of total RNA and cDNA synthesis respective primer combinations were performed. During this by random-primed reverse transcription, which were per 60 PCR, overlapping complementary sequences were intro formed according to standard protocols (Sambrook, Molecu duced into the PCR-products (stemming from the respective lar Cloning: A Laboratory Manual, Cold Spring Harbour linker primers that combined to form the coding sequence of Laboratory Press, Cold Spring Harbour, New York (1989) the 15 amino acid linker during the subsequent fusion PCR). (2001)). A PCR (denaturation at 93° C. for 5 min, annealing The amplified VH and VL domains were fused in this fusion at 58°C. for 1 min, elongation at 72°C. for 1 min for the first 65 PCR in which only the outer primers and both PCR-products cycle; denaturation at 93°C. for 1 min, annealing at 58°C. for were required. The resulting schv antibody is flanked at the 5' 1 min, elongation at 72°C. for 1 min for 30 cycles; terminal end with the restriction enzyme recognition site for BSrGI and US 8,236,308 B2 39 40 at the 3' end with the restriction enzyme recognition site for binding was clearly detectable for the anti EpCAM specifici BspEI. The coding sequence of the EpCAM specific single ties as well as for the anti CD3 specificities (see FIG. 8). As a chain Fvantibodies was then cloned via BsrGI and BspEI into negative control for binding to cynomolgus EpCAM, the the pEFDHFR expression vectors described above replacing 5-10LHXSEQID NO.10 construct was included which shows the 5-10LH scFv. Single clones of the constructs were iso binding on human CD3 (HPB-ALL cells) but no binding to lated and sequenced with primers complementary to flanking cynomolgus EpCAM (cynomolgus EpCAM transfected regions in the vector according to standard protocols (Sam CHO cells). The 5-10LH part only binds to human EpCAM. brook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. EXAMPLE 1.4 (1989) (2001)). For further experiments a clone of each con 10 struct was selected. The nucleotide and amino acid sequences Cytotoxicity Assay for 2G8LHXSEQID NO.10 and are described for 2G8LHXSEQID NO.12 in SEQID NOS. 65 2G8HLXSEQID NO.12 with Cynomolgus EpCAM and 66, for 2G8LHXSEQID NO.10 in SEQID NOS. 67 and Transfected CHO Cells as Target Cells and Human 68, for 2G8LHXSEQ ID NO.16 in SEQID NOs. 69 and 70, PBMC as Effector Cells for 2G8LHXSEQID NO.14 in SEQID NOS. 71 and 72, for 15 2G8HLXSEQ ID NO.12 in SEQ ID NOs. 73 and 74, for Bioactivity of selected bispecific single chain antibodies 2G8HLXSEQ ID NO.10 in SEQ ID NOS. 75 and 76, for was analyzed by FACS-based in vitro cytotoxicity assays 2G8HLXSEQID NO.16 in SEQID NOS. 77 and 78, and for using the cynomolgus EpCAM transfected CHO cells as tar 2G8HLXSEQID NO. 14 in SEQ ID NOS. 79 and 80 of the get cells and human PBMCs as effector cells. sequence listing included in the description. Target cells were washed twice with PBS and labeled with PKH26 dye (Sigma-Aldrich, Germany) according to the EXAMPLE 12 manufacturers instructions. Labeled target cells were washed twice with RPMI/10% FCS and mixed with freshly Expression of the 2G8LHXSEQID NO.12, isolated effector cells at an E:T ratio of 10:1.2x10 target and 2G8LHXSEQID NO.10, 2G8LHXSEQID NO.16, 25 2x10 effector cells in a volume of 50 ul RPMI/10% FCS 2G8LHXSEQID NO.14, 2G8HLXSEQID NO. 12, were added per well in a 96-well round bottom plate. Ten-fold 2G8HLXSEQID NO. 10, 2G8HLXSEQID NO. 16 serial dilutions of different bispecific single chain antibodies and 2G8HLXSEQ ID NO. 14 Bispecific Single were prepared in RPMI/10% FCS to obtain a starting concen Chain Antibodies in CHO Cells tration of 5000 ng/ml in the final reaction volume. 50 ul of the 30 different solutions were added in triplicates to the corre The plasmids with the sequences coding for the bispecific sponding wells and incubated for 24 to 48 hours at 37°C., 5% single chain antibodies were transfected into DHFR deficient CO. CHO cells for eukaryotic expression of the construct as Subsequently, the measurement of cytotoxic activity was described in Kaufmann R. J. (1990) Methods Enzymol. 185, performed. To this end propidium iodide (PI) was added to a 537-566). Gene amplification of the construct was induced by 35 final concentration of 1 Lug/ml per well and plates were incu increasing concentrations of MTX to a final concentration of bated for 10 minutes at room temperature. The number of up to 500 nMMTX. The transfected cells were then expanded PKH and PI positive target cells was determined by FACS. and 1 liter of Supernatant produced. The construct was finally Cytotoxicity was measured as the ratio of PI positive (dead purified out of the culture supernatant as described in Kufer et cells) over total number of target cells (PKH-positive) accord al. Cancer Immunity Vol. 1, p. 10 (2001). 40 ing to the formula: cytotoxicity (%)=(PI-positive cells/PKH positive cells)x100. Sigmoidal dose response killing curves EXAMPLE 13 were analyzed by Prism Software (GraphPad Software Inc., San Diego, USA) and the bispecific single chain antibody FACS Assay for Binding of 2G8LHXSEQID NO.12, concentration calculated that induced half maximal killing 2G8LHXSEQID NO.10, 2G8LHXSEQID NO.16, 45 (EC50 value). The results of this assay are shown below in 2G8LHXSEQID NO.14, 2G8HLXSEQID NO.12, FIG. 9. The resulting EC50 values for 2G8LHXSEQ ID 2G8HLXSEQID NO.10, 2G8HLXSEQID NO.16 NO.10 and 2G8HLXSEQ ID NO.12 were 1103 pg/ml and and 2G8HLXSEQID NO.14 on Kato III Cells or 3638 pg/ml, respectively. 5-10LHxdi-anti CD3 (deimmu Cynomolgus EpCAM Transfected CHO Cells and nised version of the anti-CD3 antibody as shown in SEQID HPB-ALL Cells 50 NO.163 binding to human CD3, but not to cynomolgus CD3) was included as negative control and showed no activity. This Binding of the bifunctional constructs from cell culture is due to the fact that 5-10LH only binds to human EpCAM Supernatants or binding of purified bifunctional constructs to but lacks cross-species specificity to cynomolgus EpCAM. the human EpCAM antigen on Kato III cells or cynomolgus EpCAM transfected CHO cells and to the CD3 antigen on 55 EXAMPLE 1.5 HPB-ALL cells was tested using an FACS assay. For that purpose 2.5x10 cells were incubated with 50 ul supernatant Cytotoxicity Assay for 2G8LHXSEQID NO.10 and or with 5 g/ml of the purified constructs in 50 lul PBS with 2G8HLXSEQID NO.12 with Cynomolgus EpCAM 2% FCS. The binding of the constructs was detected with an Transfected CHO Cells as Target Cells and anti-His antibody (Penta-His Antibody, BSA free, obtained 60 Cynomolgus PBMC as Effector Cells from Qiagen GmbH, Hilden, FRG) at 2 ug/ml in 50 lul PBS with 2% FCS. As a second step reagent a R-Phycoerythrin Bioactivity of selected bispecific single chain antibodies conjugated affinity purified F(ab')2 fragment, goat anti was analyzed by FACS-based in vitro cytotoxicity assays mouse IgG, Fc-gamma fragment specific antibody, diluted using the cynomolgus EpCAM transfected CHO cells as tar 1:100 in 50 ul PBS with 2% FCS (obtained from Dianova, 65 get cells and cynomolgus PBMCs as effector cells. Hamburg, FRG) was used. The samples were measured on a Target cells were washed twice with PBS and labeled with FACSscan (BD biosciences, Heidelberg, FRG). Antigen PKH26 dye (Sigma-Aldrich, Germany) according to the US 8,236,308 B2 41 42 manufacturers instructions. Labeled target cells were ID NO. 4 in Comparison to the Human-Like VHShown in washed twice with RPMI/10% FCS and mixed with freshly SEQID NO. 110/VL Shown in SEQID NO. 148 isolated effector cells at an E:T ratio of 10:1.2x10 target and Plasmid DNA encoding a) for the original murine VH 2x10 effector cells in a volume of 50 ul RPMI/10% FCS (SEQ ID NO. 2) and VL (SEQ ID NO. 4) and b) for the were added per well in a 96-well round bottom plate. Ten-fold 5 human-like VH (SEQ ID NO. 110) combined with the VL serial dilutions of different bispecific single chain antibodies (SEQ ID NO. 148) was each transformed into E. coli TG1 were prepared in RPMI/10% FCS to obtain a starting concen according to standard protocols. The nucleotide and amino tration of 5000 ng/ml in the final reaction volume. 50 ul of the acid sequences of the VH-VL Schv comprising the original different solutions were added in triplicates to the corre murine VH (SEQ ID NO. 2) and VL (SEQ ID NO. 4) are sponding wells. Individual cytotoxicity mixtures were incu- 10 shown in SEQID NOS.9 and 10, respectively. The nucleotide bated for 24 to 48 hours at 37° C., 5% CO. Subsequently the measurement of cytotoxic activity was and amino acid sequences of the VH-VL Schv comprising the performed as described in Example 14. The resulting EC50 human-like VH (SEQID NO. 110) and the VL (SEQID NO. values for 2G8LHXSEQ ID NO.10 and 2G8HLXSEQ ID 148) are shown in SEQID NOS. 147 and 146, respectively. NO.12 were 39810 pg/ml and 60350 pg/ml respectively. 15 Expression of different clones was performed in E. coli 5-10LHxdi-anti CD3 (deimmunised version of the anti-CD3 TG-1 in 96-well format. 100 ul LB/0.1% glucose were inocu antibody as shown in SEQ ID NO.163) was included as lated with 10 ul of an overnight culture of single clones and negative control and showed no activity. Di-anti CD3 only grown for 4 h at 37° C. After addition of IPTG to a final binds to human CD3, but fails to bind to macaque/cynomol concentration of 1 mM, the culture was grown at 30° C. for gus CD3.5-10LH only binds to human EpCAM but lacks 20 another 18-20h. Perwell, 40 ul of BEL-buffer (400 mMboric cross-species specificity to cynomolgus EpCAM. acid, 320 mM. NaCl, 4 mM EDTA pH 8.0+2.5 mg/ml lysozyme) were added and shaken at room temperature for 1 EXAMPLE 16 h. Cellular debris was eliminated by centrifugation and super natants were tested in flow cytometric experiments. Generation of a Human-Like CD3 Antibody 25 The human T cell line HPB-All and T cells in cynomolgus Fragment that Binds to Human and Cynomolgus peripheric blood mononuclear cells (PBMC) were used as CD3 human CD3 and cynomolgus CD3 positive cells, respec tively. Typically 100,000 cells were incubated with 50 ul of 1. Determination of a Correlating Human VH the ScFv containing bacterial Supernatants and incubated for The amino acid sequence of the murine VH chain shown in 30 30 min on ice. SEQ ID NO. 2 was aligned to the repertoire of human VH Afterwards the cells were washed three times with PBS and germline sequences (http://vbase.mrc-cpe.cam.ac.uk) using subsequently resuspended in 50 ul PBS containing anti-His the Vector NTI DNA analysis software. On the basis of this antibody (Pentahis, Roche) and further incubated on ice for analysis, the human VH segment 3-73 was chosen as a tem 30 min. Then the cells were washed three times with PBS and plate sequence (see FIG. 11). Definitions of CDRs and frame- 35 incubated with a PE labeled anti mouse IgG antibody for 30 works are according to the Kabat numbering scheme. more min. on ice (in this step cynomolgus PBMCs were The corresponding amino acid residues that differ between coincubated with anti-CD2 FITC to identify the T cells in the the VH chain shown in SEQ ID NO. 2 and the human VH PBMC mixture). After washing the cells for one time the cells segment 3-73 within the framework regions were mutated on were resuspended in a suitable buffer and positivity of cell the DNA level towards the human residues. However, the 40 bound antibody construct determined in a flow cytometer construct retained potentially crucial framework residues of (FACScalibur) and analyzed. The control sclv of SEQ ID the original murine VH sequence (according to the Kabat NO. 10 shows a clear shift on human CD3 positive cells as numbering scheme): H-30, H-41, H49, H82b, H-93 (see FIG. well as on cynomolgus CD3 positive cells indicative of bind 12). In this way, an amino acid sequence was designed that ing to both human and cynomolgus CD3. The sclv shown in was identical to the murine VH chain shown in SEQID NO. 45 SEQID NO. 146 containing the human-like VH also shows 2 sequence within its CDRS. The corresponding amino acid clear binding to CD3 positive human (see FIG. 13) and cyno sequence is shown in SEQID NO. 110, whereas the corre molgus cells (see FIG. 14). sponding nucleic acid sequence is shown in SEQID NO. 111; 4. Determination of a Correlating Human VL see also FIG. 12. The N-terminal VH sequence was changed The amino acid sequence of the murine VH chain shown in to “EVOLLE to generate a suitable N-terminal cloning site 50 SEQ ID NO. 2 was aligned to the repertoire of human VL (see FIG. 12). germline sequences (http://vbase.mrc-cpe.cam.ac.uk) using 2. Gene Synthesis and Cloning of the Human-Like VH the Vector NTI DNA analysis software. On the basis of this Region analysis, the human Vlambda segment 7a was chosen as a The afore-mentioned human-like VH region was gene syn template sequence (see FIG. 21). Definitions of CDRs and thesized (Entelechon, Germany) and subcloned via the 55 frameworks are according to the Kabat numbering scheme. restriction sites XhoI and BstEII into a suitable bacterial The corresponding amino acid residues that differ between expression vector. This vectoralready contained the sequence the murine VL chain shown in SEQID NO. 4 and the human coding foraVL chain (amino acid sequence shown in SEQID Vlambda segment 7a within the framework regions were NO. 148: N-terminus in comparison to the original VL shown mutated on the DNA level towards the human residues. How in SEQID NO. 4 slightly changed for cloning reasons) pair- 60 ever, the construct retained potentially crucial framework ing with the human like VH region followed by a Flag and a residues of the original murine Vlambda sequence (according His-6 Tag and preceded by a leader sequence that directs the to the Kabat numbering scheme): L 36, L 46, L 49, L 57 (see functional sclv into the periplasma of E. coli. The functional FIG. 21). In this way, an amino acid sequence was designed domain arrangement after cloning was Leader sequence-VH that was identical to the murine VL chain shown in SEQID (GS)-VL-Flag-His6. 65 NO. 4 sequence within its CDRs. The corresponding amino 3. Functional Analysis of scFv Constructs Having the Origi acid sequence of the generated human-like VL is shown in nal Murine VHShown in SEQID NO. 2/VL Shown in SEQ SEQ ID NO. 168, whereas the corresponding nucleic acid US 8,236,308 B2 43 44 sequence is shown in SEQID NO. 167. The N-terminal VL b) Human and cynomolgus PBMCs (containing T cells) were sequence was changed to “EL to generate a suitable N-ter washed three times with PBS and subsequently resus minal cloning site. pended in 50 ul PBS containing biotinylated anti-His anti 5. Gene Synthesis and Cloning of the Human-Like VL body (biotinylated Pentahis, Roche) and further incubated Region on ice for 30 min. Then the cells were washed three times The above-mentioned human-like VL region was gene with PBS and incubated with PE labeled Streptavidin for synthesized (Entelechon, Germany) and subcloned via the 30 more min. on ice. In this step, PBMCs were coincubated restriction sites SacI and BsiWI into a suitable bacterial with anti-CD2 FITC to identify the T cells in the PBMC expression vector. This vectoralready contained the sequence mixture. 10 After washing the cells from a) or b) for one time the cells coding for the above-mentioned human-like VH chain were resuspended in a suitable buffer and positivity of cell (amino acid sequence shown in SEQ ID NO. 110) pairing bound antibody construct determined in a flow cytometer with the human-like VL region (amino acid sequence shown (FACScalibur) and analyzed. in SEQID NO. 168) followed by a Flag and a His-6 Tag and The control scFv of SEQID NO. 10 (murine VH of SEQID preceded by a leader sequence that directs the functional schv 15 NO.4 murine VL of SEQID NO. 2) shows a clear shift on into the periplasma of E. coli. The functional domain arrange human CD3 positive cells as depicted in FIG.22. The shift on ment after cloning was Leader sequence-VH-(GS) linker human and cynomolgus T cells is less pronounced, most VL-Flag tag-His6 tag. probably due to the less sensitive detection system (FIG. 23). 6. Functional Analysis of scFv Constructs Having the The human-like sclv of SEQID NO. 170 (human-like VH Human-Like VHShown in SEQID NO. 110 Combined with of SEQ ID NO. 110 human-like VL of SEQ ID NO. 168) the Human-Like VL Shown in SEQID NO. 168 shows a positive shift on HPB-ALL cells (FIG.22) and clear Plasmid DNA encoding a) for the original murine VH shifts on human as well as cynomolgus T cells (FIG. 23, upper (SEQ ID NO. 2) and VL (SEQ ID NO. 4) and b) for the panel). human-like VH (SEQ ID NO. 110) combined with the When preincubated with 10 ug/ml of the murine IgG anti human-like VL (SEQID NO. 168) was each transformed into 25 body mab I described in Example 1 having the same speci E. coli TG1 according to standard protocols. The nucleotide ficity as the scFvs (i.e. for CD3 epsilon), the shifts of cells and amino acid sequences of the VH-VL Schv comprising the stained with the above-mentioned murine scEw or the human original murine VH (SEQID NO. 2) and VL (SEQID NO. 4) like Sclv decrease significantly, underlining the similar bind are shown in SEQID NOS. 9 and 10, respectively. The nucle ing region of the Sclvs and the original murine antibody; see otide and amino acid sequences of the VH-VL scFv compris 30 FIG. 23 lower panel. ing the human-likeVH (SEQID NO. 110) and the human-like VL (SEQ ID NO. 168) are shown in SEQ ID NOs. 169 and EXAMPLE 17 170, respectively. The nucleotide and amino acid sequences of the VL-VH schv comprising the human-like VL (SEQID Determination of an Epitope for Cross-Species NO. 168) and the human-like VH (SEQ ID NO. 110) are 35 Specific Anti-CD3 Antibodies Binding Both Human shown in SEQ ID NOs. 193 and 194, respectively. Due to and Cynomolgus CD3 Epsilon different cloning strategies, the amino acid sequence of the VL-VH scFv of SEQ ID NO. 194 shows three amino acid In order to determine the epitope of human and cynomol exchanges in comparison to the one of the VH-VL scFv of gus CD3 epsilon bound by cross-species specific anti-CD3 SEQ ID NO. 170, however, without affecting the binding 40 antibodies, epitope mapping was carried out with antibody I capacity and specificity of said ScFv. (Ig comprising the VH chain shown in SEQID NO. 2 and the Expression of different clones was performed in E. coli VL chain shown in SEQ ID NO. 4) and antibody II (Ig TG-1 in 96-well format. 100 ul LB/0.1% glucose were inocu comprising the VH chain shown in SEQID NO. 6 and the VL lated with 10 ul of an overnight culture of single clones and chain shown in SEQID NO. 8), both binding to human and grown for 4 h at 37° C. After addition of IPTG to a final 45 cynomolgus CD3 epsilon; see also FIG. 1. For the peptide concentration of 1 mM, the culture was grown at 30° C. for spotting (pepspot') analysis, overlapping 13mer peptides another 18-20h. Perwell, 40 ul of BEL-buffer (400 mMboric derived from the amino acid sequences of human and cyno acid, 320 mM. NaCl, 4 mM EDTA pH 8.0+2.5 mg/ml molgus CD3 epsilon (see FIG. 15) were covalently linked to lysozyme) were added and shaken at room temperature for 1 a Whatman 50 cellulose-B-alanine-membrane via the C-ter h. Cellular debris was eliminated by centrifugation and super 50 minus while the acetylated N-terminus remained free. In the natants were tested in flow cytometric experiments. peptides, the amino acid cystein wherever occurring in the The human T cell line HPB-ALL and human and cynomol corresponding CD3 epsilon sequence—was exchanged by gus T cells in peripheric blood mononuclear cells (PBMCs) the amino acid serin. The individual 13mer peptides gener were used as human CD3 and cynomolgus CD3 positive cells, ated (by JPT Peptide Technologies GmbH) are shown in respectively. 55 FIGS. 16 and 17. For cynomolgus CD3 epsilon, 43 spots have Typically 100,000 cells were incubated with 50 ul of the been tested, whereas for the human CD3 epsilon 47 spots ScFv containing bacterial Supernatants and incubated for 30 have been tested. The length of the overlapping sequence of min on ice. two adjacent peptides was set to be 11 amino acids. a) HPB-ALL cells were washed three times with PBS and The pepspot experiments were performed as follows. subsequently resuspended in 50 ul PBS containing anti-His 60 According to the manufacturer's protocol, the membrane was antibody (Pentahis, Roche) and further incubated on ice for rinsed with methanol for 1 min, washed with 1xTBS and 30 min. Then the cells were washed three times with PBS blocked with 1xTBS/1% (w/v) blocking reagent (BMChemi and incubated with a PE labeled anti mouse IgG antibody luminescence Blotting Substrate (POD) of Roche Diagnos for 30 more min. on ice. After washing the cells for one tics GmbH) for 3 h. All incubation and washing steps were time the cells were resuspended in a suitable buffer and 65 performed on an orbital shaker at room temperature, except positivity of cell bound antibody construct determined in a for the overnight incubation of the primary antibody. Directly flow cytometer (FACScalibur) and analyzed. after discarding the blocking Solution, the membranes were US 8,236,308 B2 45 46 incubated overnight with 5 or 3 g/ml of cross-species spe FIG. 15). The small stretch of spots 32 to 33 corresponds to cific anti-CD3 antibodies as set forth above in 1XTBS/0.5% amino acid residues 55-69 containing the minimal peptide (w/v) blocking reagent at 4°C. on an orbital shaker. After FSELE (amino acids 57-61). The spots 37 and 39 match washing 4 times with 1xTBS/0.05% Tween for 15 min, detec amino acid residues 65-77 (YYVSYPRGSKPED) and 69-81 tion of bound anti-CD3 antibody was accomplished by incu (YPRGSKPEDANFY) of human CD3 epsilon, respectively. bation for 2 h with a commercially available horseradish The correlations of spots 15 and 40 are already mentioned peroxidase-conjugated anti-IgG (F(ab) specific) antibody or above. an alkaline phosphatase-labeled anti-IgG antibody (diluted In Summary, both cross-species specific anti-CD3 antibod according to the manufacturer's recommendation in 1XTBS/ ies recognize discontinous epitopes on human and cynomol 0.5% blocking reagent, respectively). Subsequently, the 10 gus CD3 epsilon. Regarding cynomolgus CD3 epsilon both membranes were washed 6 times with 1xTBS/0.05% Tween cross-species specific anti-CD3 antibodies recognized a clear for 15 min. Horseradish-peroxidase was visualized by overlapping stretch of peptide-spots 27-29 (see FIG. 16). All enhanced chemiluminescence (luminescence Substrate Solu 13mer peptides covering this region contain one minimal tion A and starting solution B mixed 100:1: BM Chemilumi peptide FSEME (amino acid residues 57-61 of cynomolgus nescence Blotting Substrate (POD) of Roche Diagnostics 15 CD3 epsilon). The peptide-intersection on human CD3 epsi GmbH) and a BioMax Film (Kodak). Alkaline phosphatase lon bound by both antibodies can be determined for spots 32 was visualized using 5-bromo-4-chloro-indolyl phosphate/ and 33 (see FIG. 17). This section contains the minimal nitro blue tetrazolium liquid Substrate system (Sigma). peptide FSELE corresponding to residues 57-61 of human To exclude unspecific binding of horseradish-peroxidase CD3 epsilon. conjugated secondary antibody, the membrane was incubated Based on these results it is concluded that cross-species with secondary antibody only. All other steps were performed specific CD3 antibody fragments contact CD3 epsilon in the as in the experiment above. area of amino acid residues 57-61 of both cynomolgus and The control pepspot assay (see FIG. 18(A)) showed signals human CD3 epsilon comprising the amino acid stretches on spots 33 and 42 of cynomolgus CD3 epsilon and on spots FSEME and FSELE of cynomolgus and human CD3 epsilon, 37, 39 and 46 of human CD3 epsilon. These signals are 25 respectively, with the motif FSE forming the epitope core. regarded as unspecific and will not be mentioned further. This result—although plausible because of the accessibility 1. Anti-CD3 Antibody I (Ig Comprising the VHChain Shown of the E-F-loop (amino acids 56-62; see FIG. 15) of human in SEQID NO. 2 and the VL Chain Shown in SEQID NO. 4) CD3 epsilon (Kjer-Nielsen et al., PNAS 101 (2004), p. 7675 (i) Binding on Cynomolgus CD3 Epsilon 80) comprising the amino acids FSELE or FSEME is nev Strong binding signals of cross-species specific anti-CD3 30 ertheless Surprising since there is no overlap of this newly antibody I (Ig comprising the VH chain shown in SEQID NO. defined epitope with the known epitope on the CD3 epsilon 2 and the VL chain shown in SEQ ID NO. 4) to peptides chain of anti-CD3 antibodies OKT3 and UCHT1 (see FIG. derived from cynomolgus CD3 epsilon were detected on spot 17; Kjer-Nielsenet al., loc.cit; Arnettet al., PNAS 101 (2004), 1 as well as on the stretch of peptide-spots 24-29 (FIG. p. 16268-73) which have so far been regarded as representa 18(B)). The latter corresponds to amino acid residues 47-69 35 tive of all anti-CD3 antibodies thought to form a single family of cynomolgus CD3 epsilon (see FIG. 15). All 13mer peptides with the same or a very similar epitope. covering this region contain one minimal amino acid motif 56-59 (EFSE). Spot 1 corresponds to amino acid residues EXAMPLE 1.8 1-13 (QDGNEEMGSITQT) of cynomolgus CD3 epsilon. (ii) binding on Human CD3 Epsilon 40 Determination of the Epitope for the Human-Like Cross-species specific anti-CD3 antibody I bound to pep Cross-Species Specific Anti-CD3 Antibody Binding tide-spots 15, 28, 32, 33 and 40 derived from human CD3 Both to Human and Cynomolgus CD3 Epsilon epsilon (see FIG. 18(B)). The stretch of peptide-spots 28 to 33 corresponds to the amino acid residues 47-69 of human CD3 The epitope mapping of the human-like cross-species spe epsilon and comprises the minimal amino acid motif 57-59 45 cific anti-CD3 antibody fragment described in Example 16 (FSE). Spots 15 and 40 correspond to amino acid residues (SEQID NO. 170) was carried out by peptide-spotting (“pep 30-42 (QYPGSEILWQHND) and 71-83 (RGSKPEDAN spot') analysis as described in Example 17. For this purpose, FYLY), respectively. said single chain Fv fragment shown in SEQID NO. 170 was 2. Anti-CD3 Antibody II (Ig Comprising the VH Chain converted into a full IgG antibody with a murine gammal Shown in SEQID NO. 6 and the VL Chain Shown in SEQID 50 heavy chain comprising the VH region as shown in SEQID NO. 8) NO. 110 and a kappa light chain comprising the VL region as (i) binding on Cynomolgus CD3 Epsilon shown in SEQ ID NO. 168. The procedure of the pepspot The pepspot analysis with cross-species specific anti-CD3 experiment was identical to the protocol used in Example 17. antibody II (Ig comprising the VH chain shown in SEQ ID The pepspot membrane was incubated with 4 ug/ml of the NO. 6 and the VL chain shown in SEQ ID NO. 8) showed 55 mentioned IgG1 antibody, and an alkaline phosphatase-la strong signals to cynomolgus CD3 epsilon on the stretch of beled goat-anti-mouse IgG antibody detecting bound CD3 peptide-spots 27-29 as well as on spot 33 (see FIG. 19). The antibody. A second membrane was incubated with alkaline stretch spanning spots 27 and 29 corresponds to the amino phosphatase-labeled goat-anti-mouse IgG antibody alone to acid residues 53-69 of cynomolgus CD3 epsilon (see FIG. reveal unspecific binding of the detection antibody. 15), wherein the 13mer peptides have the minimal stretch of 60 The following signals detected in the control pepspot assay amino acids 57-61 (FSEME) in common. Spot 33 correlates (see FIG. 24(A)) have been regarded as unspecific and will with amino acid residues 65-77 (YYVSYPRGSNPED). not be mentioned further: the stained spot-stretches 10-13, (ii) Binding on Human CD3 Epsilon 15-19, 30-32, 35-41 of cynomolgus CD3 epsilon and 2-6, Cross-reactive anti-CD3 antibody II bound the peptide 14-19, 26, 34-39 and 46 of human CD3 epsilon. spots 15, 19, 32 and 33, 37.39 and 40 of human CD3 epsilon 65 (i) binding on Cynomolgus CD3 Epsilon (see FIG. 19). Spot 19 corresponds to amino acid residues The cross-species specific anti-CD3 antibody (murine 38-46d (WQHNDKNIGGDED) of human CD3 epsilon (see IgG1 comprising the VH chain shown in SEQID NO. 110 and US 8,236,308 B2 47 48 the VL chain shown in SEQID NO. 168) bound to the pep 4°C. on an orbital shaker. As a control, the anti-CD3 murine tide-spots 1 and 33 as well as to the amino acid stretch of IgG1 antibody UCHT1 (BD Biosciences) binding to human peptide-spots 24-29 (FIG. 24(B)) derived from cynomolgus CD3 epsilon was applied to a second membrane blotted with CD3 epsilon. The stretch spanning spots 24 and 29 corre the same amounts of the two peptides. After washing three sponds to the amino acid residues 47-69 of cynomolgus CD3 times with 1xTBS/0.05% Tween for 10 min, detection of epsilon (see FIGS. 15 and 16), wherein the 13mer peptides bound anti-CD3 antibody was accomplished by incubation have the minimal stretch of amino acids 56-59 (EFSE) in for 2 h with a commercially available alkaline phosphatase common. Spot 1 and spot 33 correspond to amino acid resi conjugated anti-IgG antibody (diluted according to the manu dues 1-13 (“QDGNEEMGSITQT: SEQ ID NO.199) and facturer's recommendation in 1XTBS/0.5% blocking 65-77 (“YYVSYPRGSNPED: SEQ ID NO. 200) of cyno 10 reagent). Subsequently, the membranes were washed three molgus CD3 epsilon, respectively. times with 1xTBS/0.05% Tween for 10 min. Alkaline phos (ii) Binding on Human CD3 Epsilon phatase was visualized using 5-bromo-4-chloro-indolyl Binding signals of the mentioned cross-species specific phosphate/nitro blue tetrazolium liquid Substrate system anti-CD3 IgG1 antibody to peptides derived from human (Sigma). CD3 epsilon (see FIG. 24(B)) were found on spots 28 and 33, 15 which correspond to the amino acid residues 47-59 and 57-69 The mentioned CD3 specific antibody comprising the VH of human CD3 epsilon (see FIG. 17), respectively. The two region shown in SEQID NO. 110 and the VL region shown in stained spots comprise the minimal amino acid motif 57-59 SEQ ID NO. 168 bound to both forms of the peptide (FSE). “EFSELEQSGYYVC (SEQ ID NO. 195) blotted to the The human-like cross-species specific anti-CD3 antibody membrane (see FIG. 25(A)(1) and (2)), whereas no binding recognizes the same discontinuous epitopes on human and could be obtained for the anti-CD3 murine IgG antibody cynomolgus CD3 epsilon as antibody I and II described in UCHT1 (see FIG.25 (B)(1) und (2)). The epitope recognized Examples 1 and 17. Binding signals of said human-like anti by anti-CD3 antibody UCHT1 is described e.g. in Kjer body on the peptide membrane reveal the peptide-intersec Nielsenet al., loc.cit; Arnettet al., PNAS (2204), p. 16268-73. tions corresponding to the amino acid sequence “FSEME 25 These results support the identification of the newly defined (amino acid residues 57-61) of cynomolgus CD3 epsilon and epitope of the herein-described anti-CD3 antibody (with the those corresponding to amino acid sequence “FSELE” VH region shown in SEQ ID NO. 110 and the VL region (amino acid residues 57-61) of human CD3 epsilon as core shown in SEQID NO. 168). Said epitope corresponds to the region. This is in line with the epitope determined for the amino acid residues “EFSELEQSGYYVC (SEQ ID NO. cross-species specific anti-CD3 antibodies I and II on both 30 195) on the human CD3 epsilon chain and comprises the cynomolgus and human CD3 epsilon (see Example 17). amino acid stretch “FSELE. EXAMPLE19 EXAMPLE 20

Verification of the Identified Epitope on Human CD3 35 Generation of CHO Cells Transfected with Epsilon for the Human-Like Cross-Species Specific Cynomolgus EGFR Anti-CD3 Antibody A shock frozen piece of EGFR positive cynomolgus colon To Verify the epitope of the human-like cross-species spe was used to obtain the total RNA that was isolated according cific anti-CD3 antibody fragment described in Example 16 on 40 to the instructions of the kit manual (Qiagen, RNeasy Mini human CD3 epsilon, the identified binding region as deter Kit). The obtained RNA was used for cDNA synthesis by mined in Experiment 18 was further analyzed by a dot-blot random-primed reverse transcription. For cloning of the full ting assay using a 13mer peptide covering the defined binding length sequence of the EGFR antigen the following oligo area of amino acid residues “FSELE' on human CD3 epsilon. nucleotides were used: This peptide comprises the amino acid sequence 45

“EFSELEQSGYYVC (SEQ ID NO. 195) of human CD3 5 EGFR. AG Xa epsilon. The peptide exists in two forms and is either bioti (SEO ID NO. 197) nylated N- or C-terminally. In case of the N-terminal label 5 - GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG-3 ling, a short linker connects the peptide with the biotin. As described in Example 18, the antibody fragment was con 50 3" EGFR. AG Sal Verted to a murine IgG format with a murine gammal heavy (SEQ ID NO. 198) chain comprising the VH region as shown in SEQID NO. 110 s' - TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT-3 '' . and a kappa light chain comprising the VL region as shown in A PCR (denaturation at 93° C. for 5 min, annealing at 58 SEQID NO.168. The dot blotting was performed as follows. C. for 1 min, elongation at 72°C. for 2 min for the first cycle: The Minifold I Spot Blot System from Schleicher & Schuell 55 denaturation at 93°C. for 1 min, annealing at 58°C. for 1 min, was used for immobilizing the peptides on a nitrocellulose elongation at 72°C. for 2 min for 30 cycles; terminal exten membrane (Protran BA85, 0.45um).75ug of each peptide in sion at 72° C. for 5 min) was used to amplify the coding 100 ul TBS were filtered through the membrane using sequence. The PCR product was subsequently digested with vacuum. After the filtration step the membrane was blocked Xbaland SalI, ligated into the appropriately digested expres with 1xTBS/1% (w/v) blocking reagent (BMChemilumines 60 sion vector pBF-DHFR, and transformed into E. coli. The cence Blotting Substrate (POD) of Roche Diagnostics afore-mentioned procedures were carried out according to GmbH) for 2 h. All incubation and washing steps were per standard protocols (Sambrook, Molecular Cloning: A Labo formed on an orbital shaker at room temperature, except for ratory Manual, 3rd edition, Cold Spring Harbour Laboratory the overnight incubation of the primary antibody. Directly Press, Cold Spring Harbour, New York (2001)). A clone with after discarding the blocking Solution, the membrane was 65 sequence-verified nucleotide sequence was transfected into incubated overnight with 3 g/ml of the above-mentioned DHFR deficient CHO cells for eukaryotic expression of the anti-CD3 antibody in 1xTBS/0.5% (w/v) blocking reagent at construct. Eukaryotic protein expression in DHFR deficient US 8,236,308 B2 49 50 CHO cells was performed as described in Kaufmann R. J. research grade and purchased from Sigma (Deisenhofen) or (1990) Methods Enzymol. 185, 537-566. Gene amplification Merck (Darmstadt). Immobilized metal affinity chromatog of the construct was induced by increasing concentrations of raphy (“IMAC) was performed using a Fractogel(R) column MTX to a final concentration of up to 20 nM MTX. (Merck) which was loaded with ZnCl2 according to the pro tocol provided by the manufacturer. The column was equili EXAMPLE 21 brated with buffer A2 (20 mM sodium phosphate buffer pH 7.5,0.4MNaCl) and the cell culture supernatant (500 ml) was Generation of EGFR and CD3 Cross-Species applied to the column (10 ml) at a flow rate of 3 ml/min. The Specific Bispecific Single Chain Antibodies column was washed with buffer A2 to remove unbound 10 sample. Bound protein was eluted using a two stepgradient of Generally, bispecific single chain antibody molecules, buffer B2 (20 mM sodium phosphate buffer pH 7.5, 0.4 M each comprising a domain with a binding specificity for the NaCl, 0.5 MImidazol) according to the following: human and the cynomolgus CD3 antigen as well as a domain Step 1: 20% buffer B2 in 6 column volumes: with a binding specificity for the human and the cynomolgus Step 2: 100% buffer B2 in 6 column volumes. 15 Eluted protein fractions from step 2 were pooled for further EGFR antigen, were designed as set out in the following purification. Table 1: Gel filtration chromatography was performed on a Sepha dex S200 HiPrep column (Pharmacia) equilibrated with PBS TABLE 1. (Gibco). Eluted protein samples (flow rate 1 ml/min) were Formats of anti-CD3 and anti-EGFR cross-species specific subjected to standard SDS-PAGE and Western Blot for detec bispecific single chain antibody molecules tion. Prior to purification, the column was calibrated for molecular weight determination (molecular weight marker SEQ ID Formats of protein constructs kit, Sigma MW GF-200). Protein concentrations were deter (nucliprot) (N -> C) mined using protein assay dye (MicroBCA, Pierce) and IgG 171 172 EGFRHLx SEQ ID NO. 170 25 (Biorad) as standard protein. 173,174 EGFRLHx SEQ ID NO. 170 175,176 EGFRHLx SEQ ID NO.194 The bispecific single chain antibodies were isolated in a 177,178 EGFRLHx SEQ ID NO.194 two step purification process of IMAC and gel filtration. The 179,180 SEQ ID NO. 170x EGFR HL main product had a molecular weight of about 52 kDa under 181,182 SEQ ID NO. 194x EGFR HL native conditions as determined by gel filtration in PBS. This 183,184 SEQ ID NO. 170x EGFR LH 30 molecular weight corresponds to the bispecific single chain 18S, 186 SEQ ID NO. 194x EGFR LH antibody. All constructs were purified according to this method. The afore-mentioned constructs containing the variable Purified bispecific single chain antibody protein was ana light-chain (L) and variable heavy-chain (H) domains reac lyzed in SDS PAGE under reducing conditions performed tive with the human and cynomolgus EGFR derived from 35 with pre-cast 4-12% Bis Tris gels (Invitrogen). Sample prepa murine hybridomas were obtained by gene synthesis and ration and application were performed according to the pro Subsequent cloning into an expression vector comprising the tocol provided by the manufacturer. The molecular weight CD3 specific VH and VL combinations reactive with the was determined with MultiMark protein standard (Invitro human and cynomolgus CD3. Herein, SEQID NO. 170 cor gen). The gel was stained with colloidal Coomassie (Invitro responds to amino acid sequence of the anti-CD3 VH-VL 40 gen protocol). The purity of the isolated protein was >95% as schv comprising the human-like VH (SEQID NO. 110) and determined by SDS-PAGE. the human-like VL (SEQ ID NO. 168). SEQ ID NO. 194 Western Blot was performed using an OptitranR) BA-S83 corresponds to the amino acid sequence of the anti-CD3 membrane and the Invitrogen Blot Module according to the VL-VH sclv comprising the human-like VL (SEQ ID NO. protocol provided by the manufacturer. The antibodies used 168) and the human-like VH (SEQ ID NO. 110). The con 45 were directed against the His Tag (Penta His, Qiagen) and structs were then transfected into DHFR-deficient CHO-cells Goat-anti-mouse Ig labeled with alkaline phosphatase (AP) by electroporation. (Sigma), and BCIP/NBT (Sigma). as substrate. The bispecific single chain antibody could be specifically detected by West EXAMPLE 22 ern Blot. A single band was detected at 52 kD corresponding 50 to the purified bispecific molecule. Expression and Purification of the EGFR and CD3 Cross-Species Specific Bispecific Single Chain EXAMPLE 23 Antibodies Flow Cytometric Binding Analysis of the EGFR and The bispecific single chain antibodies were expressed in 55 CD3 Cross-Species Specific Bispecific Antibodies chinese hamster ovary cells (CHO). Eukaryotic protein expression in DHFR deficient CHO cells was performed as In order to test the functionality of the cross-species spe described in Kaufmann R. J. (1990) Methods Enzymol. 185, cific bispecific antibody constructs with regard to binding 537-566. Gene amplification of the constructs were induced capability to human and cynomolgus EGFR and CD3, respec by increasing concentrations of MTX to a final concentration 60 tively, a FACS analysis was performed. For this purpose the of up to 20 nMMTX. After two passages of stationary culture EGFR positive epidermoid carcinoma A431 cells (ATCC, the cells were grown in roller bottles with CHO modified CRL-1555) and CD3 positive human T cell leukemia cell line MEM medium for 7 days before harvest. The cells were HPB-ALL (DSMZ. Braunschweig, ACC483) were used to removed by centrifugation and the Supernatant containing the check the binding to human antigens. The binding reactivity expressed protein was stored at -20°C. 65 to cynomolgus antigens was tested by using the generated Akta(RFPLC System (Pharmacia) and Unicorn R Software cynomolgus EGFR transfectants described in Example 20 were used for chromatography. All chemicals were of and cynomolgus PBMCs which were obtained by Ficollden US 8,236,308 B2 51 52 sity gradient centrifugation. 200,000 cells of the respective taneous lysis (without effector cells). All measurements were cell population were incubated for 30 minonice with 50 ul of done in quadruplicates. Measurement of chromium activity in the purified protein of the cross-species specific bispecific the Supernatants was performed with a Wizard 3 gamma antibody constructs (1 g/ml). The cells were washed twice in counter (Perkin Elmer Life Sciences GmbH, Köln, Ger PBS and binding of the construct was detected with an unla 5 many). Analysis of the experimental data was performed with beled murine Penta His antibody (diluted 1:20 in 50 ul PBS Prism 4 for Windows (version 4.02, GraphPad Software Inc., with 2% FCS; Qiagen; Order No. 34.660). After washing, San Diego, Calif., USA). Sigmoidal dose response curves bound anti His antibodies were detected with an Fc gamma typically had R values >0.90 as determined by the software. specific antibody (Dianova) conjugated to phycoerythrin, ECso values calculated by the analysis program were used for diluted 1:100 in 50 ul PBS with 2% FCS. Fresh culture 10 comparison of bioactivity. medium was used as a negative control. Cells were analyzed by flow cytometry on a FACS-Calibur As shown in FIGS. 39 and 40, all of the generated cross apparatus (Becton Dickinson, Heidelberg). FACS staining species specific bispecific single chain antibody constructs and measuring of the fluorescence intensity were performed revealed cytotoxic activity against human EGFR positive tar as described in Current Protocols in Immunology (Coligan, 15 get cells elicited by human CD8+ cells and cynomolgus Kruisbeek, Margulies. Shevach and Strober, Wiley-Inter EGFR positive target cells elicited by cynomolgus CD8+ science, 2002). cells. In FIG. 39, a bispecific single chain antibody with a The binding ability of several domain arrangements were variable domain reactive with EGFR and a de-immunized clearly detectable as shown in FIGS. 29 to 36. In FACS human CD3-specific variable domain (EGFR LHxdi-anti analysis, all constructs with different arrangement of VHand CD3) has been used as a negative control. In FIG. 40, the same VL domains specific for EGFR and CD3 showed binding to construct (EGFRLHxdi-anti CD3) has been used as a posi CD3 and EGFR compared to the negative control using cul tive control. As a negative control, an irrelevant bispecific ture medium and 1. and 2. detection antibody. In Summary, single chain antibody has been used. the cross-species specificity of the bispecific antibody to EXAMPLE 25 human and cynomolgus CD3 and EGFR antigens could 25 clearly be demonstrated. Generation and Characterization of Carboanhydrase IX (CAIX) and CD3 Cross-Species Specific EXAMPLE 24 Bispecific Single Chain Antibodies Bioactivity of EGFR and CD3 Cross-Species 30 Specific Bispecific Single Chain Antibodies TABLE 2 Bioactivity of the generated bispecific single chain anti Formats of CAIX and CD3 cross-species specific bodies was analyzed by chromium 51 release in vitro cyto bispecific single chain antibodies toxicity assays using the EGFR positive cell lines described in 35 Example 23; see also FIGS. 39 and 40. As effector cells SEQID Formats of protein constructs stimulated human CD8 positive T cells or stimulated cyno (nucliprot) (N-> C) molgus PBMCs were used, respectively. 189,190 CAIX HLx SEQID NO. 170 The generation of the stimulated CD8+ T cells was per 191,192 CAIX LHx SEQID NO. 170 formed as follows: 40 187,188 CADX HLx SEQID NO.194 A Petri dish (85 mm diameter, Nunc) was pre-coated with a commercially available anti-CD3 specificantibody in a final In analogy to the afore-mentioned Examples, Carboanhy concentration of 1 lug/ml for 1 hour at 37°C. Unbound protein drase IX (CAIX/MN) and CD3 cross-species specific bispe was removed by one washing step with PBS. The fresh cific single chain antibodies containing the variable light PBMC's were isolated from peripheral blood (30-50 ml 45 chain (L) and variable heavy-chain (H) domains reactive with human blood or 10 ml cynomolgus blood) by Ficoll gradient the human and cynomolgus CAIX antigen were created and centrifugation according to standard protocols. 3-5x107 Subsequently cloned into an expression vector comprising the PBMCs were added to the precoated petri dish in 50 ml of CD3 specific VH and VL combinations reactive with the RPMI 1640/10% FCS/IL-220U/ml (Proleukin, Chiron) and human and cynomolgus CD3. The experiments were carried stimulated for 2 days. At the third day the cells were collected, 50 out in essence as described in Examples 20 to 24, with the washed once with RPMI 1640. IL-2 was added to a final following exceptions: concentration of 20 U/ml and cultivated again for one day. The FACS binding experiments were performed with the The CD8+ CTLs were isolated by depleting CD4+ T cells an CAIX positive human lung carcinoma cell line A549 (ATCC, CD56+NK cells. CCL-185) to assess the binding capability to the human Target cells were washed twice with PBS and labeled with 55 CAIX antigen. The cross-species specificity to cynomolgus 11.1 MBq 'Cr in a final volume of 100 ul RPMI with 50% tissue was tested by deploying the cynomolgus skin cell line FCS for 45 minutes at 37° C. Subsequently the labeled target CYNOM-K1 (National Institute for Cancer Research (IST) cells were washed 3 times with 5 ml RPMI and then used in of Genova, Italy, ECACC 90071809) or the rhesus monkey the cytotoxicity assay. The assay was performed in a 96 well epithelial cell line 4 MBr-5 (ATCC, CCL-208). The same plate in a total volume of 250 ul supplemented RPMI (as 60 changes in cell lines apply to the cytotoxicity assays per above) with an E:T ratio of 10:1 corresponding to 1000 target formed with the CAIX and CD3 cross-species specific bispe cells and 10000 effector cells per well. 1 lug/ml of the cross cific single chain antibodies. species specific bispecific single chain antibody molecules As depicted in FIGS. 26 to 28, the generated CAIX and and 20 threefold dilutions thereof were applied. The assay CD3 cross-species specific bispecific single chain antibodies time was 18 hours and cytotoxicity was measured as relative 65 demonstrated binding to both the human and cynomolgus values of released chromium in the supernatant related to the antigens and proved to be fully cross-species specific. The difference of maximum lysis (addition of Triton-X) and spon cytolytic bioactivity of the analysed constructs is shown in

US 8,236,308 B2 107 108

SEQUENCE LISTING

< 16 Os NUMBER OF SEO ID NOS: 2O7

SEQ ID NO 1 LENGTH: 375 TYPE: DNA ORGANISM: mus musculus FEATURE: NAMEAKEY: misc feature LOCATION: (1) ... (375) OTHER INFORMATION: VH region <4 OOs, SEQUENCE: 1 gaggtgaagc titcticagtic tggaggagga ttggtgcagc Ctalaagggit c attgaaactic 6 O t catgtgcag cct ctoggatt Cacct tcaat accitacgc.ca tgaactgggt cc.gc.caggct 12 O cCaggaaagg gtttggaatg ggttgctcgc ataagaagta aatataataa. titatgcaa.ca 18O tattatgcc.g att cagtgaa agacaggttc acCatct CCa gagatgattic acaaagcatt 24 O cit citat citac aaatgaacaa cittgaaaact gagga cacag c catgtact a Ctgtgtgaga 3OO

Catgggaact tcggtaatag citacgttt co tggitttgctt actggggc.ca agggactictg 360 gtcactgtct ctgca 375

SEQ ID NO 2 LENGTH: 125 TYPE : PRT ORGANISM: mus musculus FEATURE: NAMEAKEY: misc feature LOCATION: (1) ... (125) OTHER INFORMATION: VH regi Col. <4 OOs, SEQUENCE: 2

Glu Val Lys Lieu. Lieu. Glu Ser Gly Gly Gly Lieu. Wall Glin Pro Lys Gly 1. 5 1O 15

Ser Luell Lys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Asn Thr Tyr 25

Ala Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Val 35 4 O 45

Ala Arg Ile Arg Ser Llys Tyr Asn Asn Tyr Ala Thir Ala Asp SO 55 6 O

Ser Wall Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Glin Ser Ile 65 70 7s 8O

Lell Tyr Luell Gln Met Asn. Asn Lieu Lys Thr Glu Asp Thir Ala Met Tyr 85 90 95

Wall Arg His Gly Asn. Phe Gly Asn. Ser Wall Ser Trp Phe 105 11 O

Ala Trp Gly Glin Gly Thr Lieu Wall. Thir Wall Ser Ala 115 12 O 125

SEQ ID NO 3 LENGTH: 327 TYPE: DNA ORGANISM: MUS MUSCULUS FEATURE: NAMEAKEY: misc feature LOCATION: (1) ... (327) OTHER INFORMATION: VL region SEQUENCE: 3 caggctgttg tdacticagga atctgcactic accacat cac ctdgtgaaac agt cacactic 6 O US 8,236,308 B2 109 110 - Continued acttgtc.gct Caagtactgg ggctgttaca act agta act atgccaactg. g.gt ccaagaa 12 O aalaccagatc atttatt cac tetctaata ggtgg tacca acaag.cgagc tcc aggtgtg 18O Cctgccagat t ct caggctic cct gattgga gacaaggctg C cct cac cat cacaggggca 24 O Cagactgagg atgaggcaat at atttctgt gct ct atggit acagcaacct Ctgggtgttc 3OO ggtggaggaa c caaactgac titccta 327

<210s, SEQ ID NO 4 &211s LENGTH: 109 212. TYPE: PRT <213s ORGANISM: mus musculus 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (109) <223> OTHER INFORMATION: VL region <4 OOs, SEQUENCE: 4 Glin Ala Val Val Thr Glin Glu Ser Ala Lieu. Thir Thr Ser Pro Gly Glu 1. 5 1O 15 Thr Val Thr Lieu. Thr Cys Arg Ser Ser Thr Gly Ala Val Thir Thr Ser 2O 25 3O Asn Tyr Ala Asn Trp Val Glin Glu Lys Pro Asp His Lieu. Phe Thr Gly 35 4 O 45 Lieu. Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe SO 55 6 O Ser Gly Ser Lieu. Ile Gly Asp Lys Ala Ala Lieu. Thir Ile Thr Gly Ala 65 70 7s 8O Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Lieu. Trp Val Phe Gly Gly Gly Thr Lys Lieu. Thr Val Lieu 1OO 105

<210s, SEQ ID NO 5 &211s LENGTH: 363 &212s. TYPE: DNA & 213s ORGANISM: MUS MUSCULUS 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (363) <223> OTHER INFORMATION: VH region <4 OOs, SEQUENCE: 5 Cagg to cagc tigcagcagtic tigggctgaa citggcaa.gac Ctgggggct C agtgaagatg 6 O t cctgcaagg Cttctggcta Cacct ttact agatctacga tigcactgggit aaaacagagg 12 O

Cctgga Cagg gtctggaatggattggatac attaatccta gcagtgctta tactaattac 18O aatcagaaat t caaggacaa gogo cacattg actgcagaca aatcc to cag tacagcc tac 24 O atgcaactgagtagcctgac atctgaggac totgcagt ct attactgtgc aagtcc.gcaa. 3OO gtcCactatg attacaacgg gtttic cttac tigggccaag ggactictggit cactgtctict 360 gca 363

<210s, SEQ ID NO 6 &211s LENGTH: 121 212. TYPE: PRT <213s ORGANISM: mus musculus 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (121) <223> OTHER INFORMATION: VH region

<4 OOs, SEQUENCE: 6 US 8,236,308 B2 111 112 - Continued

Glin Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Ala Arg Pro Gly Ala 1. 5 1O 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thir Phe Thir Arg Ser 2O 25

Thr Met His Trp Val Lys Glin Arg Pro Gly Glin Gly Lell Glu Trp Ile 35 4 O 45

Gly Tyr Ile Asin Pro Ser Ser Ala Tyr Thr Asn Tyr Asn Glin Llys Phe SO 55 6 O

Lys Asp Lys Ala Thr Lieu. Thir Ala Asp Llys Ser Ser Ser Thir Ala Tyr 65 70 7s 8O

Met Glin Leu Ser Ser Lieu. Thir Ser Glu Asp Ser Ala Wall Tyr Cys 85 90 95

Ala Ser Pro Glin Val His Tyr Asp Tyr Asn Gly Phe Pro Tyr Trp Gly 1OO 105 11 O Gln Gly Thr Lieu Val Thr Val Ser Ala 115 12 O

<210s, SEQ ID NO 7 &211s LENGTH: 318 &212s. TYPE: DNA & 213s ORGANISM: MUS MUSCULUS 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (318) <223> OTHER INFORMATION: VL region <4 OO > SEQUENCE: 7 caagttgttct cacccagtic ticcagcaatc atgtctgcat titcCagggga gaagg to acc 6 O atgacctgca gtgc.ca.gctic aagtgtaagt tacatgaact ggtaccagca gaagt caggc 12 O acct coccca aaagatggat titatgactica tocaaactgg Cttctggagt c cctdct cqc 18O ttcagtggca gtgggtctgg gacct ctitat it ct ct cacala t cagoag cat ggagactgaa 24 O gatgctgc.ca cittatt actg C cagcagtgg agt cqta acc cacccacgtt cggagggggg 3OO accaagctac aaattaca 3.18

<210s, SEQ ID NO 8 &211s LENGTH: 106 212. TYPE: PRT <213s ORGANISM: mus musculus 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (106) <223> OTHER INFORMATION: VL region <4 OOs, SEQUENCE: 8

Glin Wall Wall Lieu. Thir Glin Ser Pro Ala Ile Met Ser Ala Phe Pro Gly 1. 5 1O 15

Glu Lys Wall Thr Met Thr Cys ser Ala Ser Ser Ser Wall Ser Tyr Met 25

Asn Trp Tyr Glin Glin Llys Ser Gly Thir Ser Pro Arg Trp Ile Tyr 35 4 O 45

Asp Ser Ser Llys Lieu Ala Ser Gly Wall Pro Ala Arg Phe Ser Gly Ser SO 55 6 O

Gly Ser Gly Thir Ser Tyr Ser Leu Thir Ile Ser Ser Met Glu Thr Glu 65 70 7s

Asp Ala Ala Thr Tyr Tyr Cys Glin Gln Trp Ser Arg Asn Pro Pro Thir 85 90 95

Phe Gly Gly Gly. Thir Lys Lieu. Glin Ile Thr US 8,236,308 B2 113 114 - Continued

1OO 105

<210s, SEQ ID NO 9 &211s LENGTH: 747 &212s. TYPE: DNA <213> ORGANISM: ARTIFICIAL SEQUENCE 22 Os. FEATURE: <223> OTHER INFORMATION: VH-VL scFV single chain Fv <4 OOs, SEQUENCE: 9 gaggtgaagc titcticagtic tigaggagga ttggtgcago Ctaaagggit c attgaaactic 6 O t catgtgcag cct ctdgatt cacct tcaat accitacgc.ca togaactgggit cc.gc.caggct 12 O cCaggaaagg gtttggaatg ggttgctcgc ataagaagta aatataataa titatgcaa.ca 18O tattatgc.cg attcagtgaa agacaggttc accatct coa gagatgattic acaaagcatt 24 O ctictat ctac aaatgaacaa cittgaaaact gagga cacag ccatgtact a Ctgtgtgaga 3OO

Catgggaact tcggtaatag ctacgttt Co. ttittgctt actggggc.ca agggactictg 360 gtcactgtct Ctgcaggtgg ttggttct ggcggcggcg gct Coggtgg tggtggttct

Caggctgttg tacticagga atctgcactic accacat cac Ctggtgaaac agt cacactic acttgtc.gct caagtactgg ggctgttaca act agtaact atgccaactg ggtccaagaa 54 O aaaccagatc atttatt cac togg totaata ggtgg tacca acaag.cgagc tcc aggtgtg cctgccagat t ct caggctic cct gattgga gacaaggctg. ccct cac cat Cacaggggca 660

Cagactgagg atgaggcaat at atttctgt gct ct atggit acagcaacct Ctgggtgttc 72 O ggtggaggaa c caaactgac titcc ta 747

<210s, SEQ ID NO 10 &211s LENGTH: 249 212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: VH-VL scFv single chain Fv <4 OOs, SEQUENCE: 10 Glu Val Lys Lieu. Lieu. Glu Ser Gly Gly Gly Lieu Val Glin Pro Lys Gly 1. 5 1O 15 Ser Lieu Lys Lieu. Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 2O 25 3O Ala Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ala Arg Ile Arg Ser Llys Tyr Asn. Asn Tyr Ala Thr Tyr Tyr Ala Asp SO 55 6 O

Ser Val Lys Asp Arg Phe Thir Ile Ser Arg Asp Asp Ser Glin Ser Ile 65 70 7s 8O Lieu. Tyr Lieu Gln Met Asn. Asn Lieu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 1OO 105 11 O Ala Tyr Trp Gly Glin Gly Thr Lieu Val Thr Val Ser Ala Gly Gly Gly 115 12 O 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glin Ala Wall Wall 13 O 135 14 O

Thr Glin Glu Ser Ala Lieu. Thir Thr Ser Pro Gly Glu Thr Val Thir Lieu. 145 150 155 160

Thr Cys Arg Ser Ser Thr Gly Ala Val Thir Thr Ser Asn Tyr Ala Asn 1.65 17O 17s