CA2443437A1 - Antibodies specific for cd44v6 - Google Patents

Abstract

The present invention belongs to the field of oncology. The invention relate s to antibodies with specified sequence which are specific for an epitope whic h is coded by the variant exon v6 of the CD44 gene and to derivatives of said antibody. The invention also provides nucleic acid molecules encoding said antibody proteins. The invention furthermore pertains to methods for produci ng said antibody proteins. The invention also provides pharmaceutical compositions comprising said antibody proteins. The invention furthermore is concerned with the use in the manufacture of a medicament for the treatment of cancer.

Description

s Antibodies specific for CD44v6 Technical Field of the Invention The present invention belongs to the field of oncology. The invention relates to antibodies with ~o specified sequence which are specific for an epitope which is coded by the variant axon v6 of the CD44 gene and to derivatives of said antibody. The invention also provides nucleic acid molecules encoding said antibody proteins. The invention furthermore pertains to methods for producing said antibody proteins. The invention also provides pharmaceutical compositions comprising said antibody proteins. The invention furthermore is concerned with the use in the ~s manufacture of a medicament for the treatment of cancer.
Background Art Recently it has been shown that the expression of variants of the surface glycoprotein CD44 is 2o necessary and sufficient for causing so-called spontaneous metastatic behaviour of a non-metastasizing rat pancreatic adenocarcinoma cell line as well as a non-metastasizing rat fibrosarcoma cell line (Gunthert et al., 1991). While the smallest CD44 isoform, the standard form CD44s (or CD44std), is ubiquitary expressed in different tissues including epithelial cells, certain CD44 splice variants (CD44v, CD44var) axe expressed only on a subset of epithelial ~s cells. The CD44 variants are generated by alternative splicing in a way that the sequences of ten axons (vl-vI0) are completely excised in CD44s but can appear in the bigger variants in different combinations (Screaton et al., 1992; Tolg et al., 1993; Hofmann et al., 1991). The variants differ in that different amino acid sequences are inserted at a certain site of the extracellular part of the protein. Such variants can be detected in various human tumor cells as 3o well as in human tumor tissue. So, the expression of CD44 variants in the course of colorectal carcinogenesis has recently been investigated (Heider et al., I993a). The expression of CD44 variants is absent in normal human colon epithelium, and only a weak expression is detectable in the proliferating cells of the crypts. In later stages of the tumor progression, e.g. in adenocarcinomas, all malignancies express variants of CD44. Tissue expression of variant s CD44 on a high level has also been shown in aggressive Non-Hodgkin lymphomas (Koopman et al., 1993).
Exon v6 appears to play a special role especially in the course of metastatic spread (Rudy et al., 1993). In an animal model, antibodies against v6 specific epitopes could prevent the m settlement of metastatic cells and the growth of metastases (Seiter et al., 1993). In colon carcinomas, v6 expression correlates with tumor progression (Wielenga et al., 1993). In gastric carcinomas, v6 expression is an important diagnostic marker to distinguish tumors of the intestinal type from those of the dii~use type (Heider et al., 1993b). In the latter two publications, v6 expression has been determined using antibodies against v6 specific epitopes.
rs As CD44v6 has been shown to be a tumor-associated antigen with a favorable expression pattern in human tumors and normal tissues (Heider et al., 1995; Heider et al., 1996), it has been subject to antibody-based diagnostic and therapeutic approaches, (Heider et al., 1996; .
WO 95/33771; WO 97121104).
One serious problem that arises when using non-human antibodies for applications in humans is that they quickly raise a human anti-non-human response that reduces the efficacy of the antibody in patients and impairs continued administration. To overcome that probem, concepts of "humanising" non-human antibodies have been developed in the art. In the first approach, 2s humanization of non-human antibodies has been tried to achieve by constructing non-human/human chimeric antibodies, wherein the non-human variable regions are joined to human constant regions (Boulianne G. L., Hozumi N. and Shulman, M .J. (1984) Production of functional chimeric mouse/human antibody Nature 3I2: 643) The chimeric antibodies thus generated retain the binding specificity and affinity of the original non-human antibody.
3o However, chimeric antibodies, although significantly better than mouse antibodies, can still elicit an anti-chimeric response in humans (I,oBuglio A. F., Wheeler R. H., Trang J., Haynes A., Rogers K., Harvey E. B., Sun L., Ghrayeb J. and Khazaeli M. B. (1989) Mouse/human chimeric monoclonal antibody in man: Kinetics and immune response. Proc. Natl.
Acad. Sci.
86: 4220). This approach was later refined by further reducing the amount of non-human s sequences by grafting the complementarity determining regions (CDRs) from the non-human variable regions to human variable regions and then joining these "reshaped human" variable regions to human constant regions (Riechmann L., Clark M., Waldmann H. and Winter G.
(1988) Reshaping human antibodies for therapy. Nature 332: 323). CDR-grafted or reshaped human antibodies contain little or no protein sequences that can be identified as being derived from mouse antibodies. Although an antibody humanised by CDR-grafting may still be able to elicit some immune reactions, such as an anti-allotype or an anti-idiotypic response, as seen even with natural human antibodies, the CDR-grafted antibody will be significantly less immunogenic than a mouse antibody thus enabling a more prolonged treatment of patients.
rs However, it soon turned out that CDR-grafting alone did not result in antibodies with sufftcient binding affinity. CDR-grafted antibodies have relatively poor binding characteristics as compared to their parent non-human antibodies because more amino acids than those within the CDR's are involved in antigen binding. In consequence, CDR-grafted antibodies with poor binding affinity are not regarded to be useful in therapy. Therefore, attempts have been made 2o to create antibodies which combine the low immunogenicity of CDR-grafted antibodies with the good binding characteristics of the non-human parent antibodies. The concept was developed that, in addition to CDR-grafting, one to several amino acids in the humanized framework region have to be retained as residues of rodent donor origin for retaining binding affinity (Queen et al, (1989)Proc. Natl. Acad. Sci. 86: 10029-10033).
Because of the high potential utility such antibodies could have in diagnosis and therapy, there is a need of antibodies with improved properties which are suitable for treatment of human cancer.
3o The problem underlying the present invention was to provide an antibody with significantly better properties as compared to the known CD44v6 specific antibodies.
Summary of the invention s The above-captioned technical problem is solved by the embodiments characterized in the claims and the description. The before-mentioned disadvantages in the art are overcome by the claims and the description of the present invention.
In order to solve the problems mentioned above, the present inventors have designed and o generated a CD44v6 specific humanised antibody called BIWAB, which was both CDR-grafted and framework-mutated and had low immunogenicity combined with high affinity.
However, the inventors were able to create an antibody with even superior therapeutic utility, called BIWA4. Albeit this one has less binding affinity as compared to BIWA8, it surprisingly s shows a much more favorable biodistribution and tumor uptake when administered in vivo.
The present invention belongs to the field of oncology. The invention relates to antibodies with specified sequence which are specific for an epitope which is coded by the variant exon v6 of the CD44 gene and to derivatives of said antibody. The invention also provides nucleic acid Zo molecules encoding said antibody proteins. The invention furthermore pertains to methods fox producing said antibody proteins. The invention also provides pharmaceutical compositions comprising said antibody proteins. The invention furthermore is concerned with the use in the manufacture of a medicament for the treatment of cancer.
Zs Brief description of the figures (see also example 1) Figure 1: Evaluation of relative binding affinities tested in a competitive cell ELISA.
IC50: concentrations of cMAb and hMAbs at which binding of mMAb BIWA I to attached 3o A43I cells is reduced by 50%. IG50 values relative to BIWA 2 are indicated.
Figure 2. Biodistributions of co-injected'25I- and 1311-labeled CD44v6-specific MAbs (10 pCi, 50 ~.g) in HNX-OE xenograft-bearing mice at 3 or 4 days p.i. Three groups of mice received either (A) 1311- U36 (black bars) and lasl-BIWA I (hatched bars) (n=5), (B) 1311-BIWA 4 (black s bars) and lasl-BIWA 2 (hatched bars) (n=6) or (C) 1311-BIWA 4 (black bars) and l2sI-BIWA 8 (hatched bars) (n=6). At 3 (A) or 4 days (B,C) after injection mice were bled, sacrificed, dissected and the radioactivity levels (%11.7/g ~ s.e.m.) of tumor, blood and several organs were assessed. (Bld: blood, Tum: tumor, Liv: liver, Spl: spleen, Kid: kidney, Hrt: heart, Stm:
stomach, Ilm: ileum, Cln: colon, Bk: bladder, Str: sternum, Msc: muscle, Lng:
lung, Skn: skin, to Tng: tongue).
Figure 3. Therapeutic efficacy of 1g6 Re-labeled CD44v6-specific MAbs in HNX-OE
xenograft-bearing nude mice. Mice received 3OO pCl lg6Re-U36 ( - ~ -, Fig A), 300 p,Ci 18s Re-BIWA 1 (-~-, Fig A), 300 pCi 186Re-BIWA 4 (- ~-, Fig. B), 300 p.Ci is6Re-BIWA 2 (- ~
s -, Fig. B), 400 p.Ci 186 Re-BIWA 4 (- ~ -, Fig. C), 400 pCi 1g6 Re-BIWA 8 (-~ -, Fig. C), or saline (- ~-, Fig. A, B, G) as control. Control groups in Fig A and B are the same. The tumor size is expressed as the average tumor volume (~ s.e.m.) during treatment relative to the average tumor volume at the start of therapy.
2o Figure 4. Relationship between MAb dose administered and the AUC observed following BIWA 4 intravenous infusion to 10 patients in Part A of the study.
Figure 5. Relationship between MAb dose administered and the maximum plasma concentration observed following BIWA 4 intravenous infusion to 10 patients in Part A of the 25 study.

s Disclosure of the preferred embodiments of the invention Before the embodiments of the present invention it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies, reference to the "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing ~s of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by 2o virtue of prior invention.
The terms "antibody molecule" or "antibody protein" or "antibody" as used herein shall be considered equivalent.
"Complementarity determining regions of a monoclonal antibody" are understood to be those amino acid sequences involved in specific antigen binding according to Kabat (Kabat E. A., z5 Wu T. T., Perry H. 1VL, Gottesman K. S. and Foeller C. (1991) Sequences of Proteins of Immuraological Interest (5th Ed.). NI~I Publication No. 91-3242. U.S.
Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD.) in connection with Chothia and Lesk (Chothia and Lesk (1987) J. Mol. Biol.
196:901-917).
3o As used herein, the term "framework modifications" refers to the exchange, deletion or addition of single or multiple amino acids in the variable regions surrounding the individual complementarity determining regions. Framework modifications may have an impact on the immunogenicity, producibility or binding specificity of an antibody protein.

s The present invention provides an antibody molecule comprising a variable region of the heavy chain as characterized by the amino acid sequence as defined in SEQ D.7 No. 1 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof. Both antibodies BIWA4 and B1WA 8 comprise the variable region of the heavy chain as characterized in amino acid sequence SEQ ll~ No. 1.
A "fragment" according to the invention is a shorter antibody molecule, i.e.
any polypeptide subset, characterized in that it is encoded by a shorter nucleic acid molecule than disclosed below, however still retains its antibody binding activity.
A "functional variant" of the antibody molecule according to the invention is an antibody Is molecule which possesses a biological activity (either functional or structural) that is substantially similar to the antibody molecule according to the invention, i.e. a substantially similar substrate specificity or cleavage of the substrate. The term "functional variant" also includes "a fragment", "an allelic variant" "a functional variant", "variant based on the degenerative nucleic acid code" or "chemical derivatives". Such a "functional variant" e.g. may carry one or several point mutations, one or several nucleic acid exchanges, deletions or insertions or one or several amino acid exchanges, deletions or insertions.
Said functional variant is still retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.
A "functional variant" of the antibody molecule according to the invention is a antibody Zs molecule which possesses a biological activity (either functional or structural) that is substantially similar to the antibody molecule according to the invention, i.e. a substantially similar target molecule binding activity. The term "functional variant" also includes "a fragment", "an allelic variant" "a functional variant", "variant based on the degenerative nucleic acid code" or "chemical derivatives".
30 An "allelic variant" is a variant due to the allelic variation, e.g.
differences in the two alleles in humans. Said variant is still retaining its biological activity such as antibody target binding activity, at least in part or even going along with an improvement said biological activity.
A "variant based on the degenerative of the genetic code" is a variant due to the fact that a certain amino acid may be encoded by several different nucleotide triplets.
Said variant is still s retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.
A "fusion molecule" may be the antibody molecule according to the invention fused to e.g. a reporter such as a radiolabel, a chemical molecule such as a toxin or a fluorescent label or any other molecule known in the art.
o As used herein, a "chemical derivative" according to the invention is an antibody molecule according to the invention chemically modified or containing additional chemical moieties not normally being part of the molecule. Such moieties may improve the molecule's activity such as target destruction (e.g. killing of tumor cells) or may improve its solubility, absorption, biological half life etc.
s A molecule is "substantially similar" to another molecule if both molecules have substantially similar structures or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical.
20 For many uses of the antibodies according to the invention it is desirable to have the smallest possible antigen-binding, i.e. CD44v6-binding units. Therefore in another preferred embodiment an antibody protein according to the invention is a Fab fragment (Fragment antigen-binding = Fab). These CD44v6-specific antibody proteins according to the invention consist of the variable regions of both chains which are held together by the adjacent constant zs region. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced in the mean time by genetic engineering. In another preferred embodiment an antibody protein according to the invention is an F(ab')2 fragment, which may be prepared by proteolytic cleaving with pepsin.
Using genetic engineering methods it is possible to produce shortened antibody fragments 3o which consist only of the variable regions of the heavy (V~ and of the light chain (VL). These are referred to as Fv fragments (Fragment variable = fragment of the variable part). Tn another preferred embodiment an CD44v6-specific antibody molecule according to the invention is such an Fv fragment. Since these Fv-fragments lack the covalent bonding of the two chains by the cysteines of the constant chains, the Fv fragments are often stabilised.
It is advantageous to s link the variable regions of the heavy and of the light chain by a short peptide fragment, e.g. of to 30 amino acids, preferably 15 amino acids. In this way a single peptide strand is obtained consisting of VH and VL, linked by a peptide linker. An antibody protein of this kind is known as a single-chain-Fv (scFv). Examples of scFv-antibody proteins of this kind known from the prior art are described in Huston et al. (1988, PNAS 16: 5879-5883).
Therefore, in another ~o preferred embodiment an CD44v6-specific antibody protein according to the invention is a single-chain-Fv protein (scFv).
In recent years, various strategies have been developed for preparing scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity. In 5 order to achieve multimerisation of the scFv, scFv were prepared as fusion proteins with multimerisation domains. The multimerisation domains may be, e.g. the CH3 region of an IgG
or coiled Boil structure (helix structures) such as Leucin-zipper domains.
However, there are also strategies in which the interaction between the VH/VL regions of the scFv are used for the multimerisation (e.g. di-, tri- and pentabodies). Therefore in another preferred embodiment an 2a antibody protein according to the invention is an CD44v6-specific diabody antibody fragment.
By diabody the skilled person means a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS I6: 5879-5883). The shortening of the Linker in an scFv molecule to 5- 10 amino acids leads to the formation of homodimers in which an inter-chain VIIIVL-superimposition takes place. Diabodies may additionally be stabilised by the incorporation of disulphide bridges.
25 Examples of diabody-antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226).
By minibody the skilled person means a bivalent, homodimeric scFv derivative.
It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerisation region which is connected to the scFv via a Hihge region 30 (e.g. also from IgGl) and a Li~iker region. The disulphide bridges in the Hinge region are mostly formed in higher cells and not in prokaryotes. In another preferred embodiment an antibody protein according to the invention is an CD44v6-specific minibody antibody fragment.
Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).

s By triabody the skilled person means a: trivalent homotrimeric scFv derivative (Kortt et al.
1997 Protein Engineering 10: 423-433). ScFv derivatives wherein VH-VL are fused directly without a linleer sequence lead to the formation of trimers.
The skilled person will also be familiar with so-called miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv. The multimerisation is carned out by di-, tri-0 or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology 11:, 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28-34).
Therefore in a preferred embodiment an antibody protein according to the invention is an CD44v6-specific multimerised molecule based on the abovementioned antibody fragments and may be, for example, a triabody, a tetravalent miniantibody or a pentabody.
m In a more preferred embodiment, the invention relates to an antibody molecule wherein the variable region of the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 1.
In another preferred embodiment, the invention relates to an antibody molecule comprising a 2o variable region of the light chain as characterized by the amino acid sequence as defined in SEQ 117 No. 2 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof. Antibody BIWA4 as used herein comprises the variable region of the light chain as defined in amino acid sequence SEQ ID
No. 2.
In another more preferred embodiment, the invention relates to an antibody molecule wherein Z5 the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 2.
In another preferred embodiment, the invention relates to an antibody molecule comprising a variable region of the light chain as characterized by the amino acid sequence as defined in SEQ D7 No. 3 or a fragment, allelic variant, functional variant, glycosylation variant, fixsion 3o molecule or a chemical derivative thereof. Antibody BIWA 8 comprises the variable region of the light chain as characterized in amino acid sequence SEQ ll~ No. 3.
In another more preferred embodiment, the invention relates to an antibody molecule wherein the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ll~ No. 3.

s In another more preferred embodiment, the invention relates to an antibody molecule according to the invention comprising a variable region of the heavy chain as characterized by the amino acid sequence as defined in SEQ m No. 1 and comprising a variable region of light chain as characterized by the amino acid sequence as defined in SEQ ID No. 2 or a fragment, allelic variant, functional variant, glycosylation variant, fixsion molecule or a chemical ~o derivative thereof. Antibody BIWA4 comprises the variable region of the heavy chain as characterized in amino acid sequence SEQ B7 No. 1 and variable region of the light chain as defined in amino acid sequence SEQ ~ No. 2.
In a most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the variable region of the heavy chain consists of the amino acids as m characterized by the amino acid sequence of SEQ ID No. 1 and wherein the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ
ID No. 2.
In another more preferred embodiment, the invention relates to an antibody molecule according to the invention comprising a variable region of the heavy chain as characterized by Zo the amino acid sequence as defined in SEQ ~ No. 1 and comprising a variable region of the light chain as characterized by the amino acid sequence as defined in SEQ ID
No. 3 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof. Antibody BIWA8 comprises the variable region of the heavy chain as characterized in amino acid sequence SEQ TI~ No. I and variable region of the light chain as zs defined in amino acid sequence SEQ ~ No. 3.
In another most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the variable region of the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ~ No. I and wherein the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ
3a D7 No. 3.
In another preferred embodiment, the invention relates to an antibody molecule comprising a variable region of the heavy chain encoded by the nucleic acid sequence as defined in SEQ ll7 No. 4 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Both antibodies BIWA4 and B1WA 8 comprise the variable region of the heavy chain as characterized in nucleic acid sequence SEQ ID No. 4.
Tn another more preferred embodiment, the invention relates to an antibody molecule wherein the variable region of the heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4.
ro Tn another preferred embodiment, the invention relates to an antibody molecule comprising a variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ ID
No. 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Antibody BIWA4 as used herein comprises the variable region of the light chain as defined in nucleic acid sequence SEQ
t5 D.7 No. 5.
In another more preferred embodiment, the invention relates to an antibody molecule wherein the variable region of the light chain is encoded by the nucleic acid sequence as defined in SEQ
117 No. 5.
In another preferred embodiment, the invention relates to an antibody molecule comprising a 2o variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ 117 No. 6 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Antibody comprises the variable region of the light chain as characterized in nucleic acid sequence SEQ
D7 No. 6.
In another more preferred embodiment, the invention relates to an antibody molecule wherein the variable region of the light chain is encoded by the nucleic acid sequence as defined in SEQ
m No. 6.
In another more preferred embodiment, the invention relates to an antibody molecule 3o according to the invention comprising a variable region of the heavy chain encoded by the nucleic acid sequence as defined in SEQ DJ No. 4 and comprising a variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ ll~ No. 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Antibody BIWA4 comprises the variable region of the heavy chain as characterized in nucleic acid sequence SEQ B7 No. 4 and variable region of the light chain as defined in nucleic acid sequence SEQ m No. 5.
In another most preferred embodiment, the invention relates to an antibody molecule according to to the invention wherein the variable region of the heavy chain is encoded by the nucleic acid sequence as defined in SEQ B7 No. 4 and wherein the variable region of the light chain is to encoded by the nucleic acid sequence as defined in SEQ ID No. 5.
In another more preferred embodiment, the invention relates to an antibody molecule according to the invention comprising a variable region of the heavy chain encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and comprising a variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ ID No. 6 or a fragment, allelic m variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Antibody BIWA8 comprises the variable region of the heavy chain as characterized in nucleic acid sequence SEQ TD No. 4 and variable region of the light chain as defined in nucleic acid sequence SEQ ID No. 6.
In another most preferred embodiment, the invention relates to an antibody molecule according 20 to the invention wherein the variable region of the heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and wherein the variable region of the light chain is encoded by the nucleic acid sequence as defined in SEQ 117 No. 6.
To generate humanised CD44v6-specific antibody proteins the disclosed nucleic acid sequences were expressed (see infra and examples) by molecular biology methods known in ZS the art.
The variable regions of the antibody proteins of the present invention are typically linked to at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated i'n accordance with well-known procedures from a variety of human cells, but preferably immortalized B cells (see 3o Kabat et al., supra, and WO 87/02671). Hence the antibody proteins of the invention may contain all ox only a portion of the constant region as long as they exhibit specific binding to the CD44v6 antigen. The choice of the type and extent of the constant region depends on whether effector functions like complement fixation or antibody dependent cellular toxicity are desired, and on the desired pharmacological properties of the antibody protein. The antibody s protein of the invention will typically be a tetramer consisting of two light chain/heavy chain pairs, but may also be dimeric, i.e. consisting of a light chain/heavy chain pair, e.g. a Fab or Fv fragment.
Therefore, in a further embodiment the invention relates to antibody proteins according to the invention, characterised in that they have a variable light chain region and a variable heavy chain region, each joined to a human constant region. In particular, the variable region of the light chain was joined to a human kappa constant region and the variable region of the heavy chain was joined to a human gamma-1 constant region. Other human constant regions for chimerizing light and heavy chains are also available to the expert.
~s Humanization of the variable region of a murine antibody may be achieved employing methods known in the art. EP 0239400 discloses grafting of the CDRs of a murine variable region into the framework of a human variable region. WO 90/07861 discloses methods of reshaping a CDR-grafted variable region by introducing additional framework modifications.
WO
92/11018 discloses methods of producing humanized Ig combining donor CDRs with an 2o acceptor framework that has a high homology to the donor framework. WO

discloses the preparation of framework mutated antibodies starting from a murine antibody.
Further prior art references related to humanization of murine monoclonal antibodies are EP
0368684; EP 0438310; WO 92/07075, or WO 92/22653.
In another preferred embodiment, the invention relates to an antibody molecule according to 2s the invention characterised in that each of said variable region of the light chain and said variable region of the heavy chain region is separately joined to a human constant region.
In another more preferred embodiment, the invention relates to an antibody molecule according to the invention, wherein said human constant region of the light chain is a human kappa constant region.
3o In another more preferred embodiment, the invention relates to an antibody protein according to the invention, wherein said human constant region of the heavy chain is a human IgGl constant region.

s Preferred are also antibodies comprising the heavy chain as characterized by the amino acid sequence of SEQ ID No. 7 and/or the light chain as characterized by the amino , acid sequence of SEQ 117 No. 8 or as characterized by the amino acid sequence of SEQ ID No.
9.
Thus, another important embodiment is an antibody molecule according to the invention comprising a heavy chain as characterized by the amino acid sequence as defined in SEQ ff~
to No. 7 and comprising a light chain as characterized by the amino acid sequence as defined in SEQ ID No. 8 or a fragment, allelic variant, functional variant, glycosylation variant, fizsion molecule or a chemical derivative thereof. Antibody BIWA4 comprises the heavy chain as characterized in amino acid sequence SEQ ID No. 7 and variable region of the light chain as defined in amino acid sequence SEQ ID No. 8.
In a most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 7 and wherein the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 8. Antibody BIWA4 consists of the sequences as disclosed in amino acid sequence SEQ ZD No. 7 (heavy chain) and amino acid so sequence SEQ ID No. 8 (light chain). BIWA4 is a CDR-grafted antibody without framework modifications. Surprisingly, this antibody has, despite lower binding affinity, superior therapeutic eiFicacy, better biodistribution and tumor uptake over the framework-mutated antibody BIWA8 (see example). It is a humanised version of antibody VFF-18 (=BIWAl) mentioned above, having the complementary determining regions of the murine monoclonal Zs antibody VFF-I8 in a completely human framework, and human constant regions. It is therefore an antibody of very low immunogenicity in man, which is a favorable trait. However, as it has no murine framework residues to optimise antigen binding, it has a significanty lower antigen binding affinity as its parent antibody VFF-18, and therefore would not have been regarded as a good candidate for a therapeutic drug. Unexpectedly, it has been found that 3o BIWA4, despite its poor binding affinity, has a very favorable biodistribution and tumor uptake in vivo, making it superior to other humanised versions of VFF-18 with higher binding af~nitity.
Another important embodiment is an antibody molecule according to the invention comprising a heavy chain as characterized by the amino acid sequence as defined in SEQ ID
No. 7 and s comprising a light chain as characterized by the amino acid sequence as defined in SEQ ID No.
9 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof. Antibody BIWA8 comprises the heavy chain as characterized in amino acid sequence SEQ ID No. 7 and variable region of the light chain as defined in amino acid sequence SEQ ID No. 9.
to In a most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 7 and wherein the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 9. Antibody BIWA8 consists of the sequences as disclosed in amino acid sequence SEQ ID No. 7 (heavy chain) and amino acid ~s sequence SEQ ID No. 9 (light chain). BIWAB is a CDR-grafted antibody with framework modifications. This antibody has significant higher binding affinity than BIWA4 (see example).
Preferred are also antibodies comprising the heavy chain as encoded by the nucleic acid sequence of SEQ ID No. 10 and/or the light chain as characterized by the nucleic acid sequence of SEQ ID No. 11 or as characterized by the nucleic acid sequence of SEQ 117 No.
20 12. Said sequences include non-translated sequences and the leader sequence as cloned in vector pAD-CMV 1/pAD-CMV 19.
Therefore, another important embodiment is an antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No.
and comprising a light chain as characterized by the nucleic acid sequence as defined in ZS SEQ ll~ No. 11 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Antibody BIWA4 comprises the heavy chain as encoded by nucleic acid sequence SEQ ID No.
10 and variable region of the light chain as encoded by nucleic acid sequence SEQ I17 No. 11.
In a most preferred embodiment, the invention relates to an antibody molecule according to the 3o invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ 117 No. 10 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID
No. 11.
Another important embodiment is an antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No.
10 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ lD

s No. 12 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof Antibody comprises the heavy chain as encoded by nucleic acid sequence SEQ ID No. 10 and variable region of the light chain as encoded by nucleic acid sequence SEQ ID No. 12.
In a most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID No. 10 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID
No. 12.
Preferred are also antibodies comprising the heavy chain as encoded by the nucleic acid sequence of SEQ ID No. 13 and/or the light chain as characterized by the nucleic acid sequence of SEQ ll~ No. 14 or as characterized by the nucleic acid sequence of SEQ ID No.
Is 15. Said sequences include the leader sequence as cloned in vector NS.KGIval.
Therefore, another important embodiment is an antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ lD No.
13 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ~ No. 14 or a fragment, allelic variant, fiznctional variant, variant based on the 2o degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Antibody BIWA4 comprises the heavy chain as encoded by nucleic acid sequence SEQ 11.7 No. 13 and variable region of the light chain as encoded by nucleic acid sequence SEQ ID
No. 14.
In a most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ll7 No. 13 as and wherein the light chain is encoded by the nucleic acid sequence of SEQ
ID No. 14.
Another important embodiment is an antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ LD No.
13 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ 117 No. 15 or a fragment, allelic variant, functional variant, variant based on the degenerative 3o nucleic acid code, fusion molecule or a chemical derivative thereof.
Antibody BIWA8 comprises the heavy chain as encoded by nucleic acid sequence SEQ ID No. 13 and variable region of the light chain as encoded by nucleic acid sequence SEQ ID No. 15.

s In a most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID No. 13 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID
No. 15.
Most preferred is the antibody protein comprising the heavy and light chain as encoded by the nucleic acid sequence of SEQ ID No. 16. Said sequence includes the leader sequence as cloned ~o in vector NSKGlval.
Therefore, another highly important embodiment is an antibody molecule according to the invention comprising a heavy and light chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 16 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof. Antibody ~s B1WA4 comprises the heavy and light chain as encoded by nucleic acid sequence SEQ 117 No.
16.
In a most preferred embodiment, the invention relates to an antibody molecule according to the invention wherein the heavy and light chain is encoded by the nucleic acid sequence of SEQ ID
No. 16. This sequence is encoding the entire antibody BIWA4.
ao The antibody proteins of the invention provide a highly specific tool for targeting therapeutic agents to the CD44v6 antigen. Therefore, in a further aspect, the invention relates to antibody proteins according to the invention, wherein said antibody protein is conjugated to a therapeutic agent. Of the many therapeutic agents known in the art, therapeutic agents selected ss frorn the group consisting of radioisotopes, toxins, toxoids, inflammatogenic agents, enzymes, antisense molecules, peptides, cytokines, and chemotherapeutic agents are preferred. Among the radioisotopes, gamma, beta and alpha-emitting radioisotopes may be used as a therapeutic agent. l3-emitting radioisotopes are preferred as therapeutic radioisotopes.
186Rhenium, 188Rhenium, 131Iodine .and 9°Yttrium have been proven to be particularly useful 13-emitting 3o isotopes to achieve localized irradiation and destruction of malignant tumor cells. Therefore, radioisotopes selected from the group consisting of lg6Rhenium, 188.Rhenium, 131Iodine and Soy-ttrium are particularly preferred as therapeutic agents conjugated to the antibody proteins of the invention. For example, for the radioiodination of an antibody of the invention, a method as disclosed in WO 93/05804 may be employed.

s Thus, a more preferred aspect of the present invention is an antibody protein according to the invention, wherein said therapeutic agent is a therapeutic agent selected from the group consisting of radioisotopes, toxins, toxoids, pro-drugs and chemotherapeutic agents.
A more preferred aspect of the present invention is an antibody protein according to the invention, wherein said therapeutic agent is linked to the antibody protein via a linker selected from the group of MA.G-3 (US 5082930 A, EP 0247866 Bl (page 2 lines 55-56 -page 3 lines 1-23)); MAG-2 GABA (LJS 5681927 A, EP 0284071 Bl (page 6 lines 9-29)); and ((=phenthioate) US 4897255 A, US 5242679 A, EP 0185256 Bl (page 2, lines 38-page 3, lines 18)), all herein incorporated by reference.
The formulae of said linkers are as follows:
~s o N"
R ~
S N O
O ~N-BIW A 4 O

O~N\~iN~O ~ O~N~i\ ~O
a S R N
S S

o Phenthioate A more preferred aspect of the present invention is an antibody protein according to the invention, wherein said therapeutic agent is linked to the antibody protein via MAG-2 GABA.
A more preferred aspect of the present invention is an antibody protein according to the Zo invention, wherein said radioisotope is a I3-emitting radioisotope.
A more preferred aspect of the present invention is an antibody protein according to the invention, wherein said radioisotope is selected from the group consisting of ls6Rhenium, ~ss~e~um, 131Iodine and 9°Yttrium.
A more preferred aspect of the present invention is an antibody protein according to the ?s invention, wherein said radioisotope is ~86Rhenium.

s A further aspect of the present invention pertains to antibody proteins according to the invention, characterised in that they are labelled. Such an CD44v6-specific labelled antibody allows for the localisation and/or detection of the CD44v6 antigen i~ vitro and/or ih vivo. A
label is defined as a marker that may be directly or indirectly detectable. An indirect marker is defined as a marker that cannot be detected by itself but needs a further directly detectable marker specific for the indirect marker. Preferred labels for practicing the invention are detectable markers. From the large variety of detectable markers, a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin is most preferred.
Thus, a more preferred aspect of the present invention is an antibody protein according to the m invention, characterised in that it is labelled. More preferred is the antibody protein according to the invention, wherein said label is a detectable marker. Also more preferred is the antibody protein according to the invention, wherein the detectable marker is a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin.
A further aspect of the present invention relates to antibody proteins according to the 2o invention, characterised in that they are conjugated to an imageable agent.
A large variety of imageable agents, especially radioisotopes, are available from the state of the art. For practising the invention gamma-emitting isotopes are more preferred. Most preferred is iasIodine.
Therefore, a more preferred aspect of the present invention is an antibody protein to the 2s invention conjugated to an imageable agent. A more preferred aspect of the present invention is an antibody protein according to the invention, wherein the imageable agent is a radioisotope. A more preferred aspect of the present invention is an antibody protein according to the invention, wherein said radioisotope is a y-emitting radioisotope. A
more preferred aspect of the present invention is an antibody protein according to the invention, wherein said 3P radioisotope is lash.
Therefore, a more preferred aspect of the present invention is an antibody protein conjugated to a radioisotope as described above, wherein the antibody protein has specific activity of from about 0.5 to about 1S mCi/mg, or from about 0.5 to about 14 mCi/mg, preferably about 1 to s about 10 mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCilmg.
Another preferred embodiment of the present invention is a pharmaceutical composition containing an antibody according to the invention and a pharmaceutically acceptable carrier or excipient.
A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of an AMPA
glutamate receptor agonist, antagonist or modulator. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or is excipients (see also e.g. Remington's Pharmaceutical Sciences (1990), 18th ed. Mack Publ., Easton). One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.
In an animal or human body, it can prove advantageous to apply the pharmaceutical 20 compositions as described above via an intravenous or other route, e.g.
systemically, locally or topically to the tissue or organ of interest, depending on the type and origin of the disease or problem treated, e.g. a tumor. For example, a systemic mode of action is desired when different organs or organ systems are in need of treatment as in e.g. systemic autoimmune diseases, or allergies, or transplantations of foreign organs or tissues, or tumors that are diffuse zs or difficult to localise. A local mode of action would be considered when only local manifestations of neoplastic or immunologic action are expected, such as; for example local tumors.
The pharmaceutical compositions comprising antibody proteins of the present invention may be applied by different routes of application known to the expert, notably intravenous injection or 3o direct injection into target tissues. For systemic application, the intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred. A more local application can be effected subcutaneously, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily or directly in or near the tissue to be.treated (connective-, bone-, muscle-, nerve-, epithelial tissue). Depending on the desired duration and effectiveness s of the treatment, pharmaceutical antibody compositions may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.
For preparing suitable pharmaceutical compositions comprising antibody preparations for the la applications described above, the expert may use known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as e.g. saline or corresponding plasma protein solutions are readily available. The pharmaceutical compositions may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use m under sterile conditions, e.g. as a kit of parts. The final preparation of the antibody compositions of the present invention are prepared for injection, infusion or perfusion by mixing purified antibodies according to the invention with a sterile physiologically acceptable solution, that may be supplemented with known carrier substances or/and additives (e.g. serum albumin, dextrose, sodium bisulfite, EDTA).
2o The amount of the antibody applied depends on the nature of the disease. In cancer patients, the applied dose of a 'naked' antibody which is comprised in the pharmaceutical composition according to the invention may be between 0.1 and 100 mg/m2, preferably between 5 and 50 mg/m2 per application, preferably 10 mg/m~ to about 40 mg/mZ, preferably 10 mg/m2 to about 30 mg/m2, also preferably 20 mg/m2 to about 30 mg/m2, and most preferably about 25 mg/m2 25 body surface area. Also most preferred is an antibody protein dose of about 50 mg/m2 body surface area.
The dose of radioactivity applied to the patient per administration has to be high enough to be effective, but must be below the dose limiting toxicity (DLT). For pharmaceutical compositions comprising radiolabeled antibodies, e.g. with 186Rhenium, the maximally tolerated 3o dose (1VITD) has to be determined which must not be exceeded iri therapeutic settings.
Application of radiolabeled antibody to cancer patients may then be carried out by repeated (monthly or weekly) intravenous infusion of a dose which is below the MTD (See e.g. Welt et al. (1994) J. Clin. Oncol. 12: 1193-1203). Multiple administrations are preferred, generally at weekly intervals; however, radiolabelled materials should be administered at longer intervals, i.e., 4-24 weeks apart, preferable 12-20 weeks apart. The artisan may choose, however, to divide the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging, e.g. from 1 day to 1 week.
Furthermore, the applied radioactivity dose will be in accordance with the guidelines outlined below. In general, the radioactivity dose per administration will be between 30 and 75 mCi/m2 to body surface area (BSA). Thus, the amount of radiolabelled antibody in the pharmaceutical composition according to the invention, preferably labelled with lg6Rhenium, lgBRhenium, ssmTechnetium, 131lodine, or 9°Yttrium, most preferably labelled with 186Rhenium, to be applied to a patient is 10, 20, 30, 40, 50 or 60 mCi/m2, preferably 50 mCi/m2. In a preferred embodiment, the invention relates to a pharmaceutical composition, wherein the dose of said l5 radiolabelled antibody according to the invention is MTD, preferably 50 mCi/ma. This is extensively exemplified in clinical studies as set out in examples 3 to 6.
Preferred also is a pharmaceutical composition according to the invention comprising an antibody protein conjugated to a radioisotope according to the invention as defined supYa, wherein the antibody protein has specific activity of from about 0.5 to about 15 mCi/mg, or 20 from about 0.5 to about 14 mCi/mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.
Preferred also is a pharmaceutical composition according to the invention comprising an antibody protein conjugated to a radioisotope according to the invention as defined supra;
wherein said antibody or antibody derivative is in an aqueous solution at pH
of from about 7 to ZS about 8, and at a concentration of from about 0.5 to about 2.0 mg/ml.
A preferred embodiment is a pharmaceutical composition according to the invention, further comprising one or more radioprotectants selected from the group of ascorbic acid, gentisic acid, reductic acid, erythrorbic acid, p-aminobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2-5-dihydroxy-1,4-benzenedisulfonic acid, povidone, inositol, and/or 3o citrate.
Preferred is a pharmaceutical composition according to the invention, wherein the radioprotectantis ascorbic acid.
Another preferred embodiment is a pharmaceutical composition according to the invention, wherein said antibody protein comprises an antibody molecule selected from the group of s antibody molecules BIWA4 or BIWA8 as described supra linked to 186Rhenium via MAG-2 GABA further comprising the radioprotectant ascorbic acid.
Another preferred embodiment of the present invention is the use of an antibody protein according to the invention in the manufacture of a medicament for treatment of cancer. In a preferred embodiment the present invention relates to the use of antibody proteins according to o the invention conjugated to a therapeutic agent as described above for the treatment of cancer.
Cancer includes any disease associated with malignant growth such as solid tumors, sarcomas and leukemias. A necessary precondition for such diseases is the expression of CD44v6.
Cancer according to the invention includes, but is not limited to:
I) The treatment of epithelial carcinomas including breast, lung, colorectal, head and l5 neck, pancreatic, ovarian, bladder, gastric, skin, endometrial, ovarian, testicular, esophageal, prostatic and renal origin;
2) Bone and soft-tissue sarcomas: Osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyosarcoma;
3) Hematopoietic malignancies: Hodgkin's and non-Hodgkin's lymphomas, leukemias;
so 4) Neuroectodermal tumors: Peripheral nerve tumors, astrocytomas, melanomas;
S) Mesotheliomas.
Examples for cancerous disease states associated with solid tumors include, but are not limited to: colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian 2s cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic cancers of the brain.
Thus, a preferred embodiment is the use of an antibody protein according to the invention wherein said cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic cancers of the brain.
3o Preferably also is the use of an antibody protein according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein the amount of antibody protein per application is between 0.1 and I00 mg/m2, preferably between S and SO mg/m2, preferably 10 mg/m2 to about 40 mg/m2, preferably IO mg/m2 to about 30 mg/m2, also s preferably 20 mg/m2 to about 30 mg/m2, and most preferably about 25 mg/mz body surface area. Also most preferred is an antibody protein dose of about 50 mg/m2body surface area.
Preferred also is the use of an antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein the radioactivity dose per administration is between 30 and 75 mCi/ma body surface to area (BSA). Preferred is the use of an antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein the antibody protein according to the invention is radiolabelled with 186Rhenium, lggRhenium, 99"'Technetium, lsyodine, or 9°Yttrium, and most preferably is labelled with is6Rhenium. In yet another preferred embodiment the invention relates to the use of an is antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein to antibody dose is 10, 20, 30, 40, 50 or 60 mCi/m2, most preferably 50 mCi/ma. This is extensively exemplified in clinical studies as set out in examples 3 to 6.
Preferred also is the use of an antibody protein conjugated to a radioisotope according to the 2o invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein the antibody protein has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi/mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.
Preferred also is the use of an antibody protein conjugated to a radioisotope according to the ZS invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein said antibody or antibody derivative is in an aqueous solution at pH
of from about 7 to about 8, and at a concentration of from about 0.5 to about 2.0 mg/ml.
The invention further relates to a method of cancer treatment, wherein an antibody protein according to the invention is administered once to several times to an individual in need 3o thereof, said antibody protein selectively binds to CD44v6, destroys tumor cells via the therapeutic agent linked to the antibody protein and the therapeutic success is monitored. Said antibody protein may be present as naked/unmodified antibody protein, modified antibody protein, such as e.g. fusion protein, or antibody protein conjugated to a therapeutic agent, which comprises contacting the tumor with an effective amount of said antibodies. The method of treating tumors as described above may be effective ita vitro oY in vivo.
Cancer is any cancer as described above.
The amount of the antibody applied depends on the nature of the disease. In cancer patients, the applied dose of a 'naked' antibody may be between 0.1 and 100 mg/ma, preferably between 5 and 50 mg/m2 per application, preferably 10 mg/m2 to about 40 mg/ma, preferably 10 mg/m2 o to about 30 mg/m2, also preferably 20 mg/m2 to about 30 mg/m2, and most preferably about 25 mg/m2 body surface area. Also most preferred is an antibody protein dose of about 50 mg/m2 body surface area.
The dose of radioactivity applied to the patient per administration has be high enough to be effective, but must be below the dose limiting toxicity (DLT). For radiolabeled antibodies, e.g.
~s with's6Rhenium, the maximally tolerated dose (MTD) has to be determined which must not be exceeded in therapeutic settings. Application of radiolabeled antibody to cancer patients may then be carried out by repeated (monthly or weekly) intravenous infusion of a dose which is below the MTD (See e.g. Welt et al. (1994) J. Cli~c. Oncol. 12: 1193-1203).
Multiple administrations are preferred, generally at weekly intervals; however, radiolabelled materials 2o should be administered at longer intervals, i.e., 4-24 weeks apart, preferable 12-20 weeks apart. The artisan may choose, however, to divide the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging, e.g. from 1 day to 1 week.
Furthermore, the applied radioactivity dose will be in accordance with the guidelines outlined 25 below. In general, the radioactivity dose per administration will be between 30 and 75 mCi/ma body surface area (BSA). Thus, the amount of radiolabelled antibody, preferably labelled with ~ss~e~um' ~ss~e~um, 9g~"Technetium, 131lodine, or 9°Yttrium, mast preferably labelled with ~ss~e~um, to be applied to a patient is 10, 20, 30, 40, 50 or 60 mCi/m2, preferably 50 mCi/ma. In a preferred embodiment, the invention relates to a method of treatment, wherein 3o the radiolabelled antibody as described above is administered to a patient suffering from cancer, wherein the dose of said radiolabelled antibody is MTD, preferably SO
mCi/m2, whereby said cancer is prevented or treated. This is extensively exemplified in clinical studies as set out in examples 3 to b.

s Preferred also is a method of cancer treatment according to the invention (see above), wherein the antibody protein conjugated to a radioisotope according to the invention as defined supYa has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi/mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about S
mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.
~o Preferred also is a method of cancer treatment according to the invention (see above), wherein the antibody protein conjugated to a radioisotope according to the invention as defined supra is in an aqueous solution at pH of from about 7 to about 8, and at a concentration of from about 0.5 to about 2.0 mg/ml.
Preferably, the invention relates to a method according to the invention, wherein the tumor is a ~s tumor selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.
A further aspect of the present invention is a nucleic acid, characterised in that it codes for an antibody protein according to the invention. Said nucleic acid may be RNA or preferably DNA.
2o Said DNA molecule may be chemically synthesized. First, suitable oligonucleotides can be synthesized with methods known in the art (e.g. Gait,M.J., 1984, Oligonucleotide Synthesis. A
Practical Approach.IItL Press, Oxford, UK), which can be used to produce a synthetic gene.
Methods to generate synthetic genes are known in the art (e.g. Stemmer et al.
1995, Single-step assembly of a gene and entire plasmid ft~om large numbers of oligodeoxyr~ibonucleotides, 2s Gene 164(1): 49-53; Ye et al. 1992, Gene synthesis and expression in E.
coli for pump, a human matt~ix naetallopYOteirzase, Biochem Biophys Res Commun 186(1):143-9;
Hayden et Mandecki 1988, Gene synthesis by serial cloning of oligohucleoiides, DNA 7(8):
571-7).
These methods can be used to synthesize any DNA molecule disclosed in the present application, e.g. the DNA encoding BIWA4.
3o Preferably, too, a nucleic acid according to the invention is characterised in that it contains 5 ' or 3 ' or 5 ' and 3 ' untranslated regions. The nucleic acid according to the invention may contain other untranslated regions upstream andlor downstream. The untranslated region may contain a regulatory element, such as e.g. a transcription initiation unit (promoter) or enhancer.
Said promoter may, for example, be a constitutive, inducible or development-controlled s promoter. Preferably, without ruling out other known promoters, the constitutive promoters of the human Cytomegalovirus (CMV) and Rous sarcoma virus (RSV), as well as the Simian virus 40 (SV40) and Herpes simplex promoter. Inducible promoters according to the invention comprise antibiotic-resistance promoters, heat-shock promoters, hormone-inducible "Mammary tumour virus promoter" and the metallothioneine promoter. Preferably, too, a to nucleic acid according to the invention is characterised in that it codes for a fragment of the antibody protein according to the invention. This refers to part of the polypeptide according to the invention.
Preferably, a nucleic acid according to the invention is a nucleic acid as disclosed in SEQ 117 SEQ ID No. 4, 5, 6, 10, 11, 12, 13, 14, 15, and/or 16. Most preferred, said nucleic acid is a nucleic acid of SEQ D7 No. 16.
Another important aspect of the present invention is a recombinant DNA vector, characterised in that it contains a nucleic acid according to the invention. Preferably, said vector contains a nucleic acid as characterized in SEQ 117 No. 4, 5, 6, 10, 11, 12, 13, I4, I5, and/or I6. Most preferred, said vector contains the nucleic acid as characterized in SEQ ll~
No. 16.
2o Examples are viral vectors such as e.g. Vaccinia, Semliki-Forest-Virus and Adenovirus.
Vectors for use in COS-cells have the SV40 origin of replication and make it possible to achieve high copy numbers of the plasmids. Vectors for use in insect cells are, for example, E.
coli transfer vectors and contain e.g. the DNA coding for polyhedrin as promoter.
Another preferred aspect of the present invention is a recombinant DNA vector according to 2s the invention, characterized in that it is an expression vector.
Another preferred aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is vector pAD-CMV or a functional derivative thereof.
Such derivatives are e.g. pAD-CMVI, pAD-CMV19 or pAD-CMV25.
Another preferred aspect of the present invention is a recombinant DNA vector according to 3o the invention, characterized in that it is the of SEQ ID No. 17 or a functional derivative thereof.
Another preferred aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is the of SEQ ID No. 18 or a functional derivative thereof.

s Preferably also, said vectors comprise one or several of the nucleic acid molecules as characterized in SEQ 117 No. 4, 5, 6, 10, 11, 12, 13, 14, 15, and/or 16.
Preferred is also a vector as disclosed in US 5648267 A or US US 5733779 A
comprising a nucleotide sequence according to the invention. Preferably also, said vector comprises one or several of the nucleic acid molecules as characterized in SEQ ID No. 4, 5, 6, 10, 11, 12, 13, to 14, 15, and/or 16. Another preferred aspect of the present invention is a recombinant DNA
vector according to the invention, characterized in that it is vector NSKGIVaI
or a derivative thereof.
Another important aspect is a host, characterised in that it contains a vector according to the invention.
r5 Another important aspect is a host according to the invention, characterised in that it is a eukaryotic host cell. The eukaryotic host cells according to the invention include fungi, such as e.g. Pichia pasto~is, Saccha~omyces ce~evisiae, Schizosaccha~omyces, Trichoderma, insect cells (e.g. from Spodoptera f~ugiperda Sf 9, with a Baculovirus expression system), plant cells, e.g. from Nicotiana tabacum, mammalian cells, e.g. COS cells, BHI~, CHO or myeloma cells.
2o In descendants of the cells of the immune system in which antibody proteins are also formed in our body, the antibody proteins according to the invention are particularly well folded and glycosylated. Mammalian host cells, preferably CHO or COS cells are preferred, e.g. a CHO
DG44 (Urlaub and Chasin, Proc. Natl. Acad. Sci. U.S.A. 77(7): 4216-20 (1980)), or CHO-I~l (ATCC CCL-61) cells. Thus, another preferred aspect is a host according to the invention z5 according to the invention, characterised in that it is a BHK, CHO or COS
cell, most preferred CHO DG44 or CHO-K1 (ATCC CCL-61) cells.
Another preferred aspect is a host according to the invention, characterised in that it is a bacteriophage.
Another preferred aspect is a host according to the invention, characterised in that it is a 3o prokaryotic host cell. Examples of prokaryotic host cells are Escherichia coli, Bacillus subtilis, S"t~~eptomyces or Proteus mirabilis.
The invention further relates to a process for preparing an antibody protein according to the invention, characterized in that it comprises the following steps: a host according to the invention is cultivated under conditions in which said antibody protein is expressed by said host s cell and said antibody protein is isolated. The antibody according to the invention may be produced as follows. Nucleic acid molecules coding for the light chain and the heavy chain may be synthesised chemically and enzymatically by standard methods. First, suitable oligonucleotides can be synthesized with methods known in the art (details supra). Methods to generate synthetic genes from oligonucleotides are known in the art (details supra). These nucleic acid molecules encoding the antibody heavy and light chains may be cloned into an expression vector (either both chains in one vector molecule, or each chain into a separate vector molecule), which then is introduced into a host cell. The host cell preferably is a mamalian host cell (details supra), e.g. a COS, CHO, or BHK cell, more preferably a Chinese hamster ovary (CHO) cell, The host cell then is cultured in a suitable culture medium under is conditions where the antibody is produced, and the antibody is then isolated from the culture according to standard procedures. Procedures for production of antibodies from recombinant DNA in host cells and respective expression vectors are well-known in the art (see e.g. WO
94/11523, WO 97/9351, EP 0481790.) The invention preferably relates to a process according to the invention, characterised in that said host is a mammalian cell, preferably a CHO or COS cell.
The invention preferably relates to a process according to the invention, characterised in that said host cell is co-transfected with two plasmids which carry the expression units for the light or the heavy chain.
The following examples serve to further illustrate the present invention; but the same should zs not be construed as limiting the scope of the invention disclosed herein.
Examples Example 1 Radioimmunotherapy MATERIAY,S AND METHODS
Monoclonal antibodies. mMAb BIWA 1=VFF 18 (which is secreted by a hybridoma cell line which has been deposited on 7 June 1994 with the accession number DSM ACC2174 with the DSM-Deutsche Sammlung far Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Deutschland; see also WO 95/33771) was generated by immunizing BALBIc mice with glutathione S-transferase fusion protein containing the human CD44 domains v3-v10 (Heider et al., 1996). The epitope recognized by BIWA 1 has been mapped to amino acids 360-370 in domain v6 of CD44 (numbering according to Kugelman et al. (1992)). The batch used for the present studies was obtained after purification on protein-o G-Sepharose and dialysis against PBS.
MAb U36 (IgGI) was derived after immunization of mice with the HNSCC cell line UMt-SCC-22B and recognised a different epitope within CD44v6 as BIWA 1. U36 was purified from a concentrated tissue culture supernatant by affznity chromatography on protein-A-Sepharose and further purified on Q-Sepharose.
Generation of chimeric and humanized MAbs. mRNA was isolated from the BIWA 1 hybridoma cell line by use of the QuickPrep mRNA Purification Kit (Pharmacia, Uppsala, Sweden). cDNA from the variable heavy (VH) and variable light (VL) chain was generated by RT-PCR.
2o The fragments were cloned into the TA cloning vector pCR II (Tnvitrogen, Groningen, The Netherlands) and sequenced. Two expression vectors derived from the plasmid pAD CMV 1 (Himmler et al., 1990) were constructed carrying the constant region of human gamma-1 and the constant region of the human kappa light chain, respectively.
Subsequently, the VH and VL
fragments of BIWA 1 were cloned into the corresponding expression vectors in front of the zs constant regions. The chimeric antibody was named cMAb BTWA 2. Humanized versions of the BIWA 1 heavy and light chain variable regions (generated by CDR grafting) were cloned in front of the immunoglobulin constant regions of the above mentioned expression vectors. For the construction of humanized antibodies, the human variable regions used were derived for the heavy chain from the human immunoglobulin fragment accession number 531669 of 3o databank GenPept and for the light chain from the human immunoglobulin HUMIGKAX
(rearranged anti-myelin kappa chain), Genbank accession number M29469. The resulting MAbs were named hMAb B1WA 4 and BIWA 8, respectively. BIWA 8 contained two amino acids of the murine parent antibody within the light chain framework 2 while BIWA 4 did not contain murine residues in the framework.

s Recombinant MAbs were stably expressed in dihydrofolate reductase deficient Chinese hamster ovary cells by electroporation with heavy and light chain expression plasmids.
Cells were seeded into 96 well microtiter plates at densities of 500 and 100 cells/well in selection medium (a,-MEM with I O% dialyzed fetal calf serum). When colonies became visible (after ~ 14 days), culture supernatants were tested for their IgG content by ELISA, and the best producers were !o expanded. Gene amplification was performed by culturing in the presence of increasing concentrations of methotrexate (20-500 nM).
Laboratory scale production of chimeric and humanized MAbs was performed in a standard culture medium containing 1 % fetal calf serum. IgG fractions were purified from tissue culture supernatants by affinity chromatography on protein A sepharose.
Purity was ~s tested by SDS-PAGE and high performance size exclusion chromatography.
Evaluation of antibody affinity. Measurement of kinetic and affinity constants using recombinant antigen was performed on a BIAcore 2000 system (BIAcore AB, Uppsala, Sweden). A glutathione-S-transferase fusion protein containing domains v3-v10 of human 2a CD44 (GST/CD44v3-v10; 20 pg/mI) was immobilized on a CMS sensor chip by the amine coupling method according to the manufacturer's instructions, using 10 mM
sodium acetate pH 5.0 as coupling buffer. 35 p1 ofMAb at various concentrations (8-67 nM) in HBS (10 mM
HEPES, pH 7.4, 150 mM NaCI, 3.4 mM EDTA, 0.05% BIAcore surfactant P20) were injected over the antigen-coated surface at a flow rate of 5 ~L/min.
Dissociation of the MAb ZS was assessed for S minutes in buffer flow (HBS). Between two analyses, the surface of the chip was regenerated with a single pulse of 15 p.1 30 mM HCI. Analysis of the data and calculation of the kinetic constants were performed with BIAcore's BIAevaluation software, version 2.1. Association rates (kQ), dissociation rates (kd), and dissociation constants (Kd) were assessed for all antibodies.
3° Relative binding affinities were also evaluated by competitive cell ELISA. Human A431 cells, originating from an epidermoid carcinoma of the vulva and known to express high levels of CD44v6, were seeded in 96 well tissue culture plates in 200 ~.l per well RPMI
1640 with 10%
fetal calf serum at a density of 2.5 - Sx105 cells/ml. The plates were incubated overnight at 37°C in a humidified incubator with 5% C02 in air. After removal of the medium the cells were s washed once with PBS, fixed with 96% ethanol for 1 min, and washed again with PBS. cMAb BIWA 2, hMAb BIWA 4 and hMAb BIWA 8 (prediluted to 10 pg/ml) were applied in 1:2 serial dilutions (8 steps) in 100 ul/well in PBS/0.5%BSA/0.05% Tween 20 (assay buffer) and incubated for 30 min at room temperature. 100 ~.l prediluted mMAb BIWA 1 (20 nglml) was added and the plates were incubated for 2 h at room temperature on an orbital shaker. Control samples contained prediluted samples only, without B1WA 1 (0% control) or B1WA
1 only without any competing antibodies (100% control). After washing three times with PBS/0.05%
Tween 20 (washing buffer), 100 ~1 of the secondary antibody (peroxidase-conjugated goat anti-mouse Fc, diluted 1:15,000 in assay buffer, DAKO Copenhagen, Denmark) was added for detection of mMAb BIWA 1, and plates were incubated for 1 h at room temperature on an ~s orbital shaker. After washing three times with washing buffer, the plates were developed with 100 ~.l/well tetramethylbenzidine substrate solution (Kierkegaard and Perry Laboratories, Gaithersburg, USA). The reaction was stopped after 15 min with 50 p.l/well 1 M
phosphoric acid. Absorbance was measured in an ELISA plate reader at 450 nm (reference 610-690 nm).
zo Radioiodination of Antibodies. Iodination of MAbs was performed essentially as described by Haisma et al. (1986), using either lasl (100 mCilmL) or 1311 (200 mCi/mL), both purchased from Amersham, Aylesbury, England. One mg MAb IgG dissolved in 500 ~.1 PBS, pH=7.4, and 1 mCi lasl or 1311 were mixed in a vial coated with 75 ~g Iodogen (Pierce, Oud Bijerland, The Netherlands). After 5 minutes of incubation at room temperature, free iodine was removed ZS by gelfiltration on a PD10-column (Pharmacia-LKB, Woerden, The Netherlands). After removal of unbound 1251 or 1311 the radiochemical purity always exceeded 97%
as determined by TLC and HPLC procedures which have been described before (Van Gog et al., 1997a). No aggregates or fragments were formed as assessed by HPLC analysis.
3o Preparation of Rhenium-186-tabeied lVIAbs. 1$6Re-labeled lVIAbs were prepared according to a multistep procedure using the chelate S-benzoylmercaptoacyltriglycine (S-benzoyl-MAG3) as previously described (Van Gog et al., 1997a). In this procedure a solid-state synthesis fox the preparation of 186Re-IVIAG3 is followed by esterification with 2,3,5,6-tetrafluorophenol (TFP) and conjugation of the reactive 186Re-MAG3-TFP ester to the MAb.

After conjugation the lg6Re-labeled MAb was purified on a PD10-column. After removal of unbound 186Re the radiochemical purity always exceeded 98%.
Binding-assay for radiolabeled antibodies. In vitro binding characteristics of the labeled MAbs used in the biodistribution and therapy studies were determined in an immunoreactivity to assay essentially as described previously (Van Gog et al., 1997a). To test the binding of iodinated or 186Re-labeled MAbs, UM-SCC-11B cells fixed in 0.1% glutaraldehyde were used.
UM-SCC-11B cells were kindly provided by Dr. T.E. Carey, University of Michigan, Ann Arbor, MI. Five serial dilutions (ranging from 5 x 106 cells per tube to 3.1 x lOs cells per tube) were prepared with 1% BSA in PBS. Excess of unlabeled lVIAb IgG was added to a second tube with the lowest concentration of cells to determine non-specific binding.
IgG labeled with 10,000 cpm of lzsh 1311 or lg6Re was added to each tube and the samples were incubated overnight at 4°C. Cells were spun down, radioactivity in the pellet and supernatant was measured in a gamma counter (LKB-Wallace 1282 CompuGamma, I~abi Pharmacia, Woerden, The Netherlands), and the percentage of bound and free radioactivity was calculated. Data 2o were graphically analyzed in a modified Lineweaver Burk plot and the immunoreactivity was determined by linear extrapolation to conditions representing infinite antigen excess.
Biodistribution studies in HNSCC-bearing nude mice. For the biodistribution experiments nude mice bearing subcutaneously implanted human HNSCC xenografts (HNX-OE) were used Zs as described previously (Van Gog et al., 1997a). Female mice (Hsd: Athymic nu/nu, 25-32 g, Harlan CPB, Zeist, The Netherlands) were 8-10 weeks old at the time of the experiments.
Three biodistribution experiments were conducted with mice bearing 1 or 2 tumors ranging from 30 to 470 mm3. In the first experiment, 10 p,Ci (50 ~,g) 1311-labeled mMAb U36 were injected simultaneously with 10 p,Ci (50 p,g) lzsl-labeled mMAb BIWA 1 in mice bearing .
3o tumors of 133 ~ 28 mm3 (n=20 mice, 37 tumors). In the second experiment, 10 pCi (50 fig) fall-labeled hMAb B1WA 4 and 10 uCi (50 fig) lzsl-labeled cMAb BIWA 2 were co-injected in mice bearing tumors of 167 ~ 31 mm3 (n=21 mice, 32 tumors). In the third experiment, 10 ~.Ci (50 ~.g) lsll-labeled hMAb BIWA 4 and 10 pCi (50 ~.g) lzsl-labeled hMAb BIWA 8 were co-injected in mice with tumors of 130 ~ 21 mm3 (n=23 mice, 40 tumors).
Conjugates were s intravenously (i.v.) injected in a volume of 100 p1 after dilution in 0.9%
NaCI. To obtain a comparable blood/body clearance of the co-injected MAbs, only MAbs with an identical murine or human isotype were combined. The antibody dose (total dose 100 ~.g per mouse) was chosen high enough to prevent rapid isotype-related elimination of the MAb from the blood (Sharkey et al., 1991, Van Gog et al., 1997b), and low enough to prevent antigen to saturation in the tumor.
At indicated time points after injection, mice were anaesthetized, bled, killed and dissected. Besides the tumors, the following organs were removed: liver, spleen, kidney, heart, stomach, ileum, colon, bladder, sternum, muscle, lung, skin and tongue. After weighing, radioactivity in tumors, blood and organs was counted in a dual-isotope gamma counter (LKB-t5 Wallace 1282 CompuGamma), with automatic correction for the 1311-comptons in the lzsI
window setting. Radioactivity uptake in these tissues was calculated as the percentage of the injected dose per gram oftissue (%117/g).
Until the day of MAb administration mice were routinely housed under specific-pathogen-free conditions, in sterile cages in a humidity- and temperature controlled clean room, classification 2o 2000 according to the Federal Standard 209d. On the day of injection, mice were transported to a Radio Nuclide Center, and sterile radioimmunoconjugates were administered under aseptic conditions in a laminar flow hood.
Radioimmunotherapy studies in nude mice. Animal RIT studies were performed to 2s compare the therapeutic effcacy of the diiTerent MAbs labeled with lg6Re.
The immunoreactive fractions of the conjugates always exceeded 75 %. Three therapy experiments were conducted with mice bearing 1 or 2 I3NX-OE tumors ranging from 45 to 195 mm3. The 186Re doses were chosen at the maximum tolerated dose (MTD) level (i.e. 400 ~Ci) or lower (300 ~.Ci). The MTD level is defined as the dose resulting in 5-15% body weight loss. In the first experiment 3o mice were given a single i.v. injection with either 300 ~.Ci (100 ~.g) lg6Re-labeled mMAb U36 or 300 pCi (100 ~.g) lg6Re-labeled mIYIAb BIWA 1. In the second experiment either 300~~Ci (100 fig) IB~Re-labeled hMAb BIWA 4 or 300 ~.Ci (100 fig) 186Re-labeled cMAb were administered, and in the third experiment either 400 ~.Ci (100 pg) lasRe-labeled hMAb BIWA 4 or 400 ~Ci (100 fig) 186Re-labeled hMAb BIWA 8. Average tumor volumes were s similar for all experimental groups. Experiment 1: 95 mm3 ~ 34 mm3 (n = 7 mice, 12 tumors) for the 186Re-mMAb U3 6 treated group, 91 mm3 ~ 1 S mm3 (n = 7 mice, 12 tumors) for the ia6Re-mMAb BIWA 1 treated group, arid 99 mm3 ~ 54 mm3 (n = 6 mice, 11 tumors) for the control group. Experiment 2: 101 mm3 ~ 35 mm3 (n = 7 mice, 12 tumors) for the 186Re-hMAb BIWA 4 treated group, 92 mm3 ~ 43 ~3 (n = 7 mice, 12 tumors) for the 186Re-cMAb BIWA
l0 2 treated group, while the control group was the same as in experiment 1.
Experiment 3: 105 mm3 ~ 43 mm3 (n = 8 mice, 13 tumors) for the 186Re-hMAb BIWA 4 treated group, 100 mm3 +_ 42 mm3 (n = 8 mice, 13 tumors) for the lg6Re-hMAb BIWA 8 treated group, and 110 mm3 ~
46 mm3 (n = 7 mice, 11 tumors) for the control group. During treatment tumors were measured twice weekly and tumor volumes relative to the volume at the start of treatment IS were calculated. Toxicity was monitored by measurement of the body weight twice weekly.
Mice were sacrificed when one of the tumors exceeded 1000 mm3.
Statistics. Differences in tissue uptake between co-injected MAbs were statistically analyzed for each time point with the Student's t-test for paired data. Differences in average tumor volume between the various RTT treatment groups were statistically analyzed for each time 2o point with the Student's t-test for independent samples.
RESULTS
I~ vitr~ binding characteristics of the CD44e6-specific MAbs. The binding affinities of the five MAbs were analyzed using recombinant antigen as well as human tumor cell lines. Kinetic z5 and affinity constants were evaluated by surface plasmon resonance using GST/CD44v3-v10 as immobilized antigen. Table 1 shows the association rates (ka), dissociation rates (ka) and dissociation constants (Kd). mMAb B1WA 1 and cMAb BIWA 2, containing identical variable regions, have similar ka, kd, and Kd and show the highest affinity. In contrast, mMAb U36 and hMAb BIWA 4 have lower ka and higher k~, resulting in markedly lower dissociation constants 30 (factors 35.0 and 10.5, respectively). hIVIAb BIWA 8, containing murine residues in the light chain framework region 2, shows a marked decrease of kd resulting in increased affinity.
Table 1 Kinetics and affinity constants of MAbs directed against CD44v6.

Antibody , kQ (M''s') k~(s') Kd(M) Kd relative to murine BIWA

Murine BIWA 1 1.3 x I05 4.2 x 10'5 3.2 x 10'1 1.0 Murine U36 I.5 x 104 1.7 x 10~ 1.1 x 10'8 35.0 Chimeric BIWA 1.7 x 105 4.1 x 10'5 2.4 x 10-' 0.7 Humanized BIWA 6.5 x I04 2.2 x 10'4 3.4 x 10-g 10.5 Humanized BIWA 7.5 x 104 6.3 x 10'5 8.4 x 10-' 2.6 s The relative binding ai~nities of the cMAb and the hMAbs were also evaluated in a competitive cell ELISA using human A43I tumor cells (Fig. I). In accordance with the affinity measurements on recombinant antigen, cMAb BIWA 2 was the most ei~ective competitor, followed by hMAb BIWA 8 and hMAb BIWA 4. Similar results (not shown) were obtained with two other human HNSCC cell lines (FaDu and LICR-LON-HN5).
Biodistribution in HNSCC-bearing nude mice. Biodistribution studies were performed in HNX-OE xenograft bearing nude mice. Two MAbs with identical murine or human isotype were labeled with either lzsl or i3ll and injected simultaneously (50 p,g, 10 ~.Ci each). Each pair ~s ofMAbs was selected to provide a stepwise decrease in the difference in affinities: mMAb U36 has a 35.0 fold lower amity than mMAb BTWA 1 (experiment 1), hMAb BIWA 4 has a 14.0-fold lower affinity than cMAb BIWA 2 (experiment 2) and hMAb BIWA 4 has a 4.0-fold lower affuiity than hMAb B1WA 8 (experiment 3). The immunoreactive fractions of all iodinated MAbs were at least 74°J° after extrapolation (Table 2).

s Table 2 Immunoreactive fraction of iodinated MAbs determined by binding to cells.
Experiment no. Antibody Label Binding to Binding 5x106 cells (%) Extrapolateda (%) 1 Murine U36 '3'I 59.7 87.4 Murine B1WA 1 'z5I 91.1 91.1 2 Humanized BIWA 4 '3'I 77,4 82.3 Chimeric BIWA 2 'z5I 80.5 . 79.9 3 Humanized BIWA 4 '3'I 77.3 74.5 Humanized BIWA 8 'z5I 91.8 92.1 Immunoreactivity was determined by linear extrapolation to conditions representing infinite antigen excess: see Materials and Methods The biodistributions in experiment 1 were determined at day 1, 2, 3 and 7 after injection;
~o biodistributions in experiments 2 and 3 were determined at day 1, 2, 4 and 7 days after injection. The calculated average %ID/g of tumor and blood of all three experiments are given in Table 3.
Table 3 Tumor and blood levels of iodinated GD44v6-specific MAbs with different affinity after co-ts injection to HNX-OE bearing mice Conjugate A (°!°Il7/g) Conjugate B (%ID/g) Conj. A:B ratio Exp. No Time after Tumor Blood Tumor Blood Tumor ao Blood injection 1 Conj. A:'3'I-mMAb 1 d 15.7 17.9 13.0 17.8 1.2 1.0 Conj. B: 'z5I-mMAb 2 d 18.4 15.0 13.4 14.9 1.4 1.0 3 d 20.6 11.8 13.7 11.0 1.5 1.1 ~s 7 d 16.5 7.1 7.8 4.8 2.1 1.5 s 2. Conj. A;1311-hMAb BIWA 4 1 d 10.8 10.2 9.1 10.6 1.2 1.0 Conj. B:'z5I-cMAb BIWA 2 12.4 10.2 9.8 10.1 1.3 1.0 2 d 4 d 12.9 7.2 8.9 7.2 1.5 1.0 7 d 7.6 3.3 4.8 2.7 1.6 1.2 to 3. .Conj. A:'31I-hMAb 10.5 11.1 9.5 11.4 1.1 BIWA 4 1 d 1.0 Conj. B:'ZSI-hMAb BIWA10.9 I0.0 9.6 9.8 1.1 1.0 8 2 d 4 d 1 1.7 6.2 9.6 5.9 1.2 1.0 7 d 10.1 4.2 7.8 3.9 1.3 1.1 For each pair of co-injected MA.bs the uptake ratios for tumor and blood are provided. The average %Il7/g and s.e.m. of tumor, blood and various organs at 3 (experiment 1) or 4 days p.i. (experiments 2 and 3) are shown in Figure 2.
In a direct comparison of the two murine MAbs, tumor uptake of low affinity U36 was significantly higher than uptake of high affinity BIWA 1 at all time points (p < 0.001) (Table 2o 3). In contrast, no significant differences were found between the uptake values of these lVIAbs in blood and normal tissues at 1, 2 and 3 days p.i.. At day 7 p.i., BIWA 1 levels in blood and most of the organs were significantly lower (p < 0.05) than U36 levels, indicating more rapid clearance of BIWA 1 from the blood/body. A 50% higher tumor uptake of U36 in comparison with BIWA 1 at day 3 p.i, is illustrated by Figure 2A.
as Similar relationships were found in the evaluation of the two other MAb pairs. hMAb BIWA 4, while having the lower affinity, showed a significantly higher tumor uptake (p < 0.001) than cMAb BIWA 2 and hMAb BIWA 8 at all time points (Table 3). In contrast, MAb levels in blood and normal tissues were similar for these pairs of MAbs at 1, 2, and 4 days p.i.. At 7 days p.i., B1WA 2 and BIWA 8 levels in blood and most of the organs were significantly lower 30 (p < 0.05) than B1WA 4 levels, indicating more rapid clearance of these MAbs from the blood/body. A 45% higher tumor uptake of BIWA 4 in comparison with BIWA 2 is illustrated by Figure ZB, while a 20% higher tumor uptake of BIWA 4 in comparison with BIWA 8 is illustrated by Figure 2C, for the 4 days post injection time points.
Consistent results were obtained from an additional experiment (data not shown) in which the 3s radiolabels were exchanged: 'zsI-B1WA 4 versus 131I-BIWA 8 instead of 131I-BIWA 4 vef°sus 'ZSI-BIWA 8. Data from this latter experiment rule out the possibility that the type of radiolabel had influenced the pharmacokinetic behavior of the labeled MAb.
Radioimmunotherapy in HNSCC-bearing nude mice. From the three biodistribution experiments it appeared that the low affinity MAbs showed a higher and more selective tumor uptake than the high affinity MAbs, and thus might be better suited for RIT.
To test this to possibility the following treatment groups were compared in RIT studies with HNX-OE
xenograft bearing mice:
Experiment 1: 300 ~.Ci 1$6Re-U36 or 300 p,Ci 186Re-BIWA 1 or saline as control. Experiment 2: 300 ~.Ci 186Re-BIWA 4 or 300 pCi 1$6Re-BIWA 2 or saline as control.
Experiment 3: 400 pCi 186Re- BIWA 4 or 400 pCi 186Re-BIWA 8 or saline as control.
~s In Figure 3, the mean relative tumor volume (as a percentage of the tumor volume at day 0) for the control and treatment groups is plotted against time. Tumors of mice in the control group in all three experiments showed exponential growth with a tumor volume doubling time of about 7 days. In the groups treated with the lg6Re-labeled MAbs, tumors stopped growing, in some cases accompanied by tumor regression, shortly after injection of the conjugates.
2o However, all tumors ultimately regrew.
In experiment 1, administration of 300 p,Ci 1$6Re-BIWA 1 resulted in a decrease of the tumor growth rate, but not in a reduction of the mean tumor size. Administration of 300 pCi 186Re-U36, however, caused a reduction of the mean tumor volume from 185 mm3 to 120 mm3 between day 7 and day 17 post injection, after which tumors started growing again. The mean 2s relative tumor volume in the IB~Re-U36-treated group was significantly smaller (p < 0.001) than that of the 186Re-BIWA 1-treated group from day 14 on.
In experiment 2, administration of either 300 pCi lg6Re-BIWA 4 or 300 ~.Ci lg6Re-BIWA 2 resulted in tumor growth arrest at day 7 with start of regrowth at day 17 p.i.. BIWA 4 was more effective in RIT than B1WA 2 from day 14 on, but a significant difference between the 3o mean relative tumor volumes was only found at day 14 p.i. (p < 0.05).
In experiment 3, mice were treated with either 400 p,Ci lg6Re-BIWA 4 or BIWA
g, which resulted in a decrease of the relative tumor volume to a minimum of 80 ~ 62 %
and 98 ~ 81%, respectively, at day 19. Thereafter, tumors started regrowth s These data indicate that the low amity MAb BIWA 4 is more effective in RIT
than the high affinity Mabs cBIWA 2 and BIWA 8.
References Giinthert, U., Hofmann, M., Rudy, W., Reber,, S., Zoller, M., Haul3mann, L, Matzku, S., Wenzel, A., Ponta, H., and Herrlich, P. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65: 13-24 (1991).
Haisma, H.J., Hilgers, J., and Zurawski, V.R. Jr. Iodination of monoclonal antibodies for m diagnosis and therapy using a convenient one vial method. J. Nucl. Med., 27:
1890-1895, 1986.
Heider, K.-H., Hofmann, M., Horst, E., van den Berg, F., Ponta, H., Herrlich, P., and Pals, S.T. A human homologue of the rat metastasis-associated variant of CD44 is expressed in zo colorectal carcinomas and adenomatous polyps. .J. Cell Biol. 120: 227-233 (1993a).
Heider, K-H., Dammrich, J., Sl~-och-Angel, P., Muller-Hermelink, H-K., Vollmers, H-P., Herrlich, P., and Ponta, H. Differential expression of CD44 splice variants in intestinal- and diffuse-type human gastric carcinomas and normal gastric mucosa. CanceY Res.
53: 4197-4203 zs (1993b).
Heider KH, Mulder JWR, Ostermann E,Susani S, Patzelt E, Pals ST, Adolf GRA.
Splice variants of the cell surface glycoprotein CD44 associated with metastatic tumor cells are expressed in normal tissues of humans and cynomolgus monkeys. Eur. J. Cancer 31A: 2385 30 2391, 1995.
Heider KH, Sproll M, Susani S, Patzelt E, Beaumier P, Ostermann O, Ahorn H, Adolf GRA..
Characterization of a high affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas. Cancer Immunology T.~nmunotherapy 43: 245 3s 253, 1996.
Himmler, A., Maurer-Fogy, L, Kronke, M., Scheurich, P., Pfizenmaier, K., I,antz, M., Olsson, L, Hauptmann, R., Stratowa, C., and Adolf, G.R. Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis 4a binding protein. DNA & Cell Biol., 9: 705-715, 1990.
Hofmann, M., Rudy, W., Zoller, M., Tolg, C., Ponta, H., Herrlich P., and Giinthert, U. CD44 splice variants confer metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines. Cancer Res. 51: 5292-5297 (1991).

s Kugelman, L.C., Gangluly, S., Haggerty, J.G., Weissman, S.M., and Milstone, L.M. The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44. J. Invest. Dermatol., 99: 886-891, 1992.
Koopman, G., Heider, K.-H., Horts, E., Adolf, G. R., van den Berg, F., Ponta, H., Herrlich, to P., Pals, S. T. Activated human lymphocytes and aggressive Non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44. J. Exp.
Med. 177: 897-904 (1993).
Rudy, W., Hofmann, M., Schwartz-Albiez, R., Zoller, M., Heider, K.-H., Ponta, H., Herrlich, m P. The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants: Each one individually suffices to confer metastatic behaviour. Cancer Res. 53: 1262-1268 (1993).
Screaton, G.R., Bell, M.V., Jackson, D.G., Cornelis, F.B., Gerth, U., and Bell, J. I. Genomic 2o structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. U.S.A. 89: 12160-12164 (1992).
Sharkey, R.M., Natale, A., Goldenberg, D.M., and Mattes, M.J. Rapid blood clearance of immunoglobulin G2a and immunoglobulin G2b in nude mice. Cancer Res., Sl: 3102-3107, z5 1991.
Tolg, C., Hofinann, M., Herrlich, P., and Ponta, H. Splicing choice from ten variant exons establishes CD44 variability. Nucleic Acids. Res. 21: 1225-1229 (1993).
3o Van Gog, F.B., Visser, G.W.M., Stroomer, J.W.G., Roos, J.C., Snow, G.B., and Van Dongen, G.A.M.S. High dose lg6Re-labeling of monoclonal antibodies for clinical application: pitfalls and solutions. Cancer, 80: 2360-2370, 1997a.
Van Gog, F.B., Brakenhofl; R.H., Snow, G.B., and Van Dongen, G.A.M.S. Rapid elimination 35 of mouse/human chimeric monoclonal antibodies in nude mice. Cancer Immunol.
Immunother., 44: 103-111, 1997b.
Wielenga, V. J. M., Heider; K.-H., O~erhaus, G. J. A., Adolf, G. R., van den Berg, F. M., Ponta, H., Herrlich, P., Pals, S.T. Expression of CD44 variant proteins in human colorectal 4o cancer is related to tumor progression. Cancer Res. 53: 4754-4756 (1993).

s Example 2 Details of sequences This example shows the details of sequences, e.g. the position of cloning sites, leaders and untranslated regions.
to Abbreviations:
as = amino acids nt = nucleotide sequence SEQ ID No. 1 VH BIWA 4/8 as m EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSY
TYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS
SEQ ID No. 2 VL BIWA 4 as EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPAR
2o FSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK
SEQ ID No. 3 VL BIWA 8 as S GS GS GTDFTLTIS SLEPEDFAVYYCLQW S SNPLTFGGGTKVEIK
~zs SEQ ll~ No. 4 VH BIWA 4/8 nt GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTAAGACTCTCC
TGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGGCTCCGGGG
AAGGGGCTGGAGTGGGTCTCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCTAGACAGT
3o ATAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCCTGTACCTGCAAATGAA.C
AGTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGACAGGGGTTGGACTACTGGGGT
CGAGGAACCTTAGTCACCGTCTCCTCA
SEQ 117 No. 5 VL BIWA 4 nt GAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTG
TCCTGCAGTGCCAGCTCAAGTATAA.ATTACATATACTGGTACCAGCAGAAGCCAGGACAGGCT
CCTAGACTCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGC
AGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCCGTT
ao TATTACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGATT
AAA
SEQ ID No. 6 VL BIWA 8 nt as GAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTG
TCCTGCAGTGCCAGCTCAAGTATAAA.TTACATATACTGGCTCCAGCAGAAGCCAGGACAGGCT
CCTAGAATCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGC
AGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCCGTT
TATTACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGATT
so AAA.

s SEQ TD No. 7 heavy chain (variable + constant) BIWA 4l8 as EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSY
TYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
io SSGLYSLSSVVTVPSSSLGTQTYICNVNHI~PSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVI~FT1WYV17GVEVHNAKTKp REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPTEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
is SEQ ZD No. 8 light chain (variable + constant) BIWA 4 as EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPAR
FSGSGSGTDFTLTTS SLEPEDFAVYYCLQWS SNPLTFGGGTKVEIKRTVAAPSVFIFPPS
Zo DEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC
SEQ B7 No. 9 light chain (variable + constant) BIWA 8 as as EIVLTQSPATLSLSPGERATLSCSASSSINYZYWLQQKPGQAPRILIYLTSNLASGVPARF
S GS GS GTDFTLTIS SLEPEDFAVYYCLQW S SNPLTFGGGTKVEIKRT VA AP S VFIFPP SD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
3o SEQ m No. 10 heavy chain (variable + constant) BIWA 4/8 nt ; insert in pAD-CMVl/pAD-CMV 19, contains introns (lower case) between CHl-hinge, hinge-CH2, CHZ-CH3, leader sequence underlined, non-translated sequences in italic, cloning sites bold aagatttgacagacgcacaaccctggactcccaagtctttctcttcagtgacaaacacagacataggatatcaca 3s tttgcttctgacacaactgtgttcactagcagcctcaaacagacaccATGAACTTTGGGCTCAGCTTGATTTTCC
TTGTCCTAATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAG
GGTCCCTAAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGGCTC
CGGGGAAGGGGCTGGAGTGGGTCTCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCTAGACAGTATAAAGG
GCCGATTCACCATCTCCAGAGRCAATGCCAAGAACTCCCTGTACCTGCAAA.TGAACAGTCTGAGGGCTGAGGACA
ao CGGCCGTGTRTTACTGTGCAAGACRGGGGTTGGACTACTGGGGTCGAGGAACCTTAGTCACCGTCTCCTCAGCTA
GCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACA
CCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG
GCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCARCACCAAGGTGGACAAGAAAGTTggtgagaggc as cagcacagggagggagggtgtctgctggaagcaggctcagcgctcctgcctggacgcatcccggctatgcagccc cagtccagggcagcaaggcaggccccgtctgcctcttcacccggagcctctgcccgccccactcatgctcaggga gagggtcttctggctttttcccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaa ggggcaggtgctgggctcagacctgcaaagagccatatccgggaggaccctgcccctgacctaagcccaccccaa aggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctct so gcaGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAggtaagccagcccaggcctcgccctcc agctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccac ctccatctcttcctcaGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA.CCCAAGGACAC
CCTGATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACRAAGCCGCGGGAGGAGCAGTACAACAGCACGTA

s CCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGRGTACAAGTGCAA.GGTCTCCAA
CAAAGCCCTCCCAGCCCCCATCGRGAAAACCATCTCCAAAGCCAAAggtgggacccgtggggtgcgagggccaca tggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtcctacaGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGRCCTGCCT
GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAGAACTACAAGAC
~o CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCRTGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT
GTCTCCGGGTAARTGAgtgcgacggccgcgaattc SEQ m No. 11 light chain (variable + constant) BIWA 4 nt ; sequence in pAD-CMV1/pAD-~s CMVI9, leader sequence underlined, non-translated sequences in italic, cloning sites bold aagcttgatcttcaggatatcacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccATGG
ATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATGAGTGCCTCAGTCATAATGTCCAGGGGAGAAATTGTTCTCA
CCCAGTCTCCAGCARCCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTGTCCTGCAGTGCCAGCTCAAGTATAA
2o RTTACATATACTGGTACCAGCAGAAGCCAGGACAGGCTCCTAGACTCTTGATTTATCTCACATCCAACCTGGCTT
CTGGAGTCCCTGCGCGCTTCAGTGGCAGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTG
AAGATTTTGCCGTTTATTACTGCCTGCAGTGGAGTAGTAACCCGCTCRCATTCGGTGGTGGGACCAAGGTGGAGA
TTAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCTA
GCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCRAAGTACAGTGGARGGTGGATAACGCCCTCCAAT
as CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC
TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCR
CAAAGAGCTTCAACAGGGGAGAGTGTTAGgaattc SEQ 1!17 No. 12 light chain (variable + constant) BIVVA 8 nt ; sequence in pAD-CMVl/pAD-3o CMV19, leader sequence underlined, non-translated sequences in italic, cloning sites bold aagcttgatcttcaggatatcacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccATGG
ATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATGAGTGCCTCAGTCATAATGTCCAGGGGAGAAATTGTTCTCA
CCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTGTCCTGCRGTGCCAGCTCAAGTATAA
3s ATTACATATACTGGCTCCAGCAGAAGCCAGGACAGGCTCCTAGAATCTTGATTTATCTCACATCCAACCTGGCTT
CTGGAGTCCCTGCGCGCTTCAGTGGCAGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTG
AAGATTTTGCCGTTTATTACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGA
TTAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA,TCTGGAACTGCTA
GCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT
ao CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTRCAGCCTCAGCAGCACCCTGACGC
TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA
CAAAGAGCTTCAACAGGGGAGAGTGTTAGgaattc SEQ >Z7 No. 13 heavy chain (variable + constant) BIWA 4/8 nt ; sequence in NSI~Glval, no os introns contained, leader sequence underlined ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAA.GGTGTCCAGTGTGAAGTG
CAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTAAGACTCTCCTGTGCA
GCCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGGCTCCGGGGAAGGGG
so CTGGAGTGGGTCTCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCTAGACAGTATAAA.G
GGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCCTGTACCTGCAAA.TGAACAGTCTG
AGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGACAGGGGTTGGACTACTGGGGTCGAGGA
ACCTTAGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC
TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
ss CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC
CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC
ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT

s GAGCCCAAA.TCTTGTGACAA.AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAA.CCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG
to TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA.GCCAA.AGGG
CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC
AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC
rs TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT
AAATGA
SEQ m No. 14 light chain (variable + constant) BIWA 4 nt ; sequence in NSKGlval, leader sequence underlined ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTGCTCTGGCTCCCAGATACCACCGGAGAA
ATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTGTCC
TGCAGTGCCAGCTCAAGTATAAATTACATATACTGGTACCAGCAGAAGCCAGGACAGGCTCCT
AGACTCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGCAGT
zs GGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCCGTTTAT
TACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGATTAAA
CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG
GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC
3o AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAA.CACAAAGTC
TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA
GAGTGTTGA
SEQ m No. 15 light chain (variable + constant) BIWA 8 nt ; sequence in NSKGIvaI, leader 3s sequence underlined ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTGCTCTGGCTCCCAGATACCACCGGAGAA
ATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTGTCC
TGCAGTGCCAGCTCAAGTATAAATTACATATACTGGCTCCAGCAGAAGCCAGGACAGGCTCCT
4a AGAATCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGCAGT
GGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCCGTTTAT
TACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGATTAAA
CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG
as GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC
AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAA.GTC
TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA
GAGTGTTGA
so SEQ J!D No. 16 BIWA 4 in NSKGlval CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG
CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGT

s TCGCCGGCTTTCCCCGTCAAGCTCTAAA.TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTT
TACGGCACCTCGACCCCAAAA.AACTTGATTAGGGTGATGGTTCACGTAGGGTCGCGACGTACC
GGGCCCCCCCTCGATTAATTAATCGAGCTACTAGCTTTGCTTCTCAATTTCTTATTTGCATAA
TGAGAAAAAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCC
AAA.AAGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAA.CATTATTCAGAGGGAG
io TACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGT
CATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGAT
AGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTT
GCTTCTGACATAGTTGTGCCAGCATGGAGGAATCGATCCTCCATGCTTGAACAAGATGGATTG
CACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACA
~s ATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTC
AAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGTAAGTGCGGCCGCTCTAGGCCTCCAA
AAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAA
TAAAt~.AAIAATTAGTCAGCCATGCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGC
GGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATA
zo CTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATG
CTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTC
CACAGAATTAATTCCCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA
TATATGGAGTTCCGCGTTACATAACTTACGGTAA.ATGGCCCGCCTGGCTGACCGCCCAACGAC
CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT
zs TGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT
ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAA.TGGCCCGCCTGGCATTATGCCCAG
TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC
ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTT
CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAA.A.ATCAACGGGACTTT
so CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG
GTCTATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCACAGATCT
CTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTGCTCTGGCTCCCAGATACCACC
GGAGAAA.TTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACC
CTGTCCTGCAGTGCCAGCTCAAGTATAAA.TTACATATACTGGTACCAGCAGAAGCCAGGACAG
3s GCTCCTAGACTCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGT
GGCAGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCC
GTTTATTACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAG
ATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA.
TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG
ao TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC
AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC
AAAGTCTACGCCTGCGAAGTCAC
CCATCAGGGCCTGAGCTCGCCCGTCACAAA.GAGCTTCAACAGGGGAGAGTGTTGAATTCAGAT
~CCGTTAACGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCGGCCGTGATATCTACGT
as ATGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCC
TTCCTTGACCCTGG.A.A.GGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAA.TTGCATC
GCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA
GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGAC
TAGTAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAA.AAAAGGAAAATTAATTTTAAC
so ACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAA.AA.GGATGCTTTAGAGACAGTGTTCT
CTGCACAGATAAGGACAAA.CATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCTAAGCCAGT
GAGTGGCACAGCATTCTAGGGAGAAA.TATGCTTGTCATCACCGAAGCCTGATTCCGTAGAGCC
ACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAGCCAGGGCAGAGCAT
ATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTT

s GGATAGCTTGGACAGCTCAGGGCTGCGATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAA
GGCTGGTAGGATTTTATCCCCGCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTG
TCCCAAA.ATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTC
AAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAA.ACAGAATCTGGTGATTATG
GGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAA.AGGACAGAATTAATATA
~a GTTCTCAGTAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAA.GTTTGGAT
GATGCCTTAAGACTTATTGAACAACCGGAATTGGCAAGTAAA.GTAGACATGGTTTGGATAGTC
GGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACA
AGGATCATGCAGGAATTTGAAA.GTGACACGTTTTTCCCAGAAATTGATTTGGGGAA.ATATAAA
CTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAG
~s TTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCTA
AAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCAGCCTCGACTGTGCC
TTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC
CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCA
TTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAG
ao GCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGAAGCGGCCGCTCCGGATA
TGCCAAGTACGCCCCCTATTGACGTCA.ATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT
ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA
TGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC
CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTC
as CAA.AA.TGTCGTAACAACTCCGCCCCATTGACGCAAA.TGGGCGGTAGGCGTGTACGGTGGGAGG
TCTATATAAGCAGAGCTGGGTACGTCCTCACATTCAGTGATCAGCACTGAACACAGACCCGTC
GACATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAA.AGGTGTCCAGTGTGAA
GTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTAAGACTCTCCTGT
GCAGCCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGGCTCCGGGGAAG
so GGGCTGGAGTGGGTCTCAACC.ATTAGTAGTGGTGGTAGTTACA.CCTACTATCTAGACAGTATA
AAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCCTGTACCTGCAAATGAACAGT
CTGAGGGCTGAGGACACGGCCGT
GTATTACTGTGCAAGACAGGGGTTGGACTACTGGGGTCGAGGAACCTTAGTCACCGTCTCCTC
AGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG
as CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA
CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA
CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA
CGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTT
ao CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
TAATGCCAAGACAAA.GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT
CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAA.ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
as GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT
CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCAC
CGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCCGTTAACGG
so TTACCAA.CTACCTAGACTGGATTCGTGACAACATGCGGCCGTGATATCTACGTATGATCAGCC
TCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACC
CTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA.A.TGAGGAAATTGCATCGCATTGTCTG
AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAA
GACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGCGC

s TGCGATCGCCTCGAGGCCGCTACTAACTCTCTCCTCCCTCCTTTTTCCTGCAGGACGAGGCAG
CGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTG
AAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACC
TTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATC
CGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGG
~o AAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAAC
TGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATG
CCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGC
TGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTG
GCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCA
~s TCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGA
CCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTT
GGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCT
GGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAG
CATCACAA.ATTTCACAA.ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAA.CT
ao CATCAATCTATCTTATCATGTCTGGATCGCGGCCGGCCGCACCGCGGTGGAGCTTTAATTAAG
GCGCGCCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATG
GTCATAGCTGTTTCCTGTGTGAA
ATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAA.GTGTAAAGCCTGGG
GTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGG
zs GAAACCTGTCGTGCCAGCTGCA'T'TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA
TTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAG
CGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA
AGAACATGTGAGCAAAAGGCCAGCAAA.AGGCCAGGAACCGTAAAA.AGGCCGCGTTGCTGGCGT
TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC
so GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC
CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC
TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT
GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGT
CCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG
ss CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA
GGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAA.GAGTTGGTAGCT
CTTGATCCGGCAAA.CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA.GCAGCAGATTA
CGCGCAGAAAA~1AAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT
GGAACGAAA.A.CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA.AAGGATCTTCACCTAGA
ao TCCTTTTAAATTAAA.AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG
ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCA
TAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCA
GTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC
CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTA
as ATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCA
TTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC
AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC
CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGC
ATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA
so AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATA
ATACCGCGCCACATAGCAGAACTTTAA.A.AGTGCTCATCATTGGAAA.ACGTTCTTCGGGGCGAA
AACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT
GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG
CCGCAA.A.A.A.AGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAT

s ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGA
AAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA.AGTGCCACA
SEQ )D No. 17 pAD-CMV1, cloning sites in bold ~o TCGACATTGATTATTGACTAGTTRTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT
CCGCGTTACATAACTTACGGTAAA.TGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCRATAAT
GACGTATGTTCCCATAGTRACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC
CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAA.TGGCCCGC
CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT
rs TRCCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGRTAGCGGTTTGACTCRCGGGGATTTCCAAGTCT
CCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTC
CGCCCCATTGACGCAARTGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTARCTAG
AGAACCGACTGCTTAACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTTCTGCAGGTCGAC
ATCGATGGATCCGGTACCTCGAGCGCGAATTCTCTAGAGGRTCTTTGTGAAGGAACCTTRCTTCTGTGGTGTGAC
zo ATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAA
ACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGC
CTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCRACA
TTCTACTCCTCCAAAAAAGAAGAGAAA.GGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAG
TCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCRCAAAGGAAAAAGCTGCACTGCTATA
zs CAAGAAAATTATGGAAAAATATTTGATGTATAGTGCCTTGRCTAGRGATCATAATCAGCCATACCACATTTGTAG
AGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGRACCTGAAACATAAAATGAATGCAATTGTTGTTG
TTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTT
TTTCACTGCRTTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAATTCTGAGAAAC
TAGCCTTRAAGACAGACAGCTTTGTTCTAGTCAGCCAGGCAAGCATATGTAAATAAAGTTCCTCRGGGAACTGAG
3o GTTAAAAGATGTATCCTGGACCTGCCAGACCTGGCCATTCACGTAAACAGAAGATTCCGCCTCAAGTTCCGGTTA
ACAACAGGAGGCAACGRGATCTCAAATCTATTACTTCTAATCGGGTAATTAAAACCTTTCAACTAAAACACGGAC
CCACGGATGTCACCCRCTTTTCCTTCCCCGGCTCCGCCCTTCTCAGTACTCCCCACCATTAGGCTCGCTACTCCA
CCTCCACTTCCGGGCGCGACACCCACGTGCCCTCTCCCACCCGACGCTAACCCCGCCCCTGCCCGTCTGACCCCG
CCCACCACCTGGCCCCGCCCCGTTGAGGACAGAAGAARCCCCGGGCAGCCGCAGCCAAGGCGGACGGGTAGACGC
ss TGGGGGCGCTGAGGAGTCGTCCTCTACCTTCTCTGCTGGCTCGGTGGGGGACGCGGTGGATCTCAGGCTTCCGGA
AGACTGGAAGAACCGGCTCAGAACCGCTTGTCTCCGCGGGGCTTGGGCGGCGGAAGAATGGCCGCTAGACGCGGA
CTTGGTGCGAGGCATCGCAGGATGCAGAAGAGCAAGCCCGCCGGGAGCGCGCGGCTGTACTACCCCGCGCCTGGA
GCGGCCACGCCGGACTGGGCGGGGCCGGCCTGGTGGAGGCGGAGTCTGACCTCGTGGAGGCGGGGCCTCTGATGT
TCAAA.TAGGATGCTAGGCTTGTTGAGGCGTGGCCTCCGATTCACAAGTGGGRAGCAGCGCCGGGCGRCTGCAATT
ao TCGCGCCAAACTTGGGGGAAGCACAGCGTACAGGCTGCCTAGGTGATCGCTGCTGCTGTCRTGGTTCGACCGCTG
ARCTGCATCGTCGCCGTGTCCCAGAATATGGGCATCGGCAAGAACGGAGACCTTCCCTGGCCAATGCTCAGGTAC
TGGCTGGATTGGGTTAGGGAAACCGAGGCGGTTCGCTGAATCGGGTCGAGCACTTGGCGGAGACGCGCGGGCCAA
CTACTTAGGGACAGTC:ATGAGGGGTAGGCCCGCCGGCTGCTGCCCTTGCCCATGCCCGCGGTGRTCCCCATGCTG
TGCCAGCCTTTGCCCAGAGGCGCTCTAGCTGGGAGCAAAGTCCGGTCACTGGGCAGCACCACCCCCCGGACTTGC
as ATGGGTAGCCGCTGAGATGGA.GCCTGAGCACACGTGACAGGGTCCCTGTTAACGCAGTGTTTCTCTAA.CTTTCAG
GAACGAGTTCAAGTACTTCCAAAGAATGACCACCACCTCCTCAGTGGAAGGTAAACAGAACCTGGTGATTATGGG
CCGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGA
GCTCAAGGAACCACCACAAGGAGCTCATTTTCTTGCCAAAAGTCTGGACCATGCCTTAAAACTTATTGAACAACC
AGAGTTAGCAGATAAAGTGGACATGGTTTGGATAGTTGGAGGCAGTTCCGTTTACAAGGAAGCCATGAATCAGCC
so AGGCCATCTCAGACTCTTTGTGACAAGGATCATGCAGGAA.TTTGAAA.GTGACACGTTCTTCCCAGAAATTGATTT
GGAGAAATATAAACTTCTCCCAGAGTACCCAGGGGTCCTTTCTGAAGTCCAGGAGGAAAAAGGCATCAAGTATAA
ATTTGAAGTCTATGAGAAGAAAGGCTAACAGAAAGATACTTGCTGRTTGACTTCAAGTTCTACTGCTTTCCTCCT
AAAATTATGCATTTTTACAAGACCRTGGGACTTGTGTTGGCTTTAGATCCTGTGCATCCTGGGCAACTGTTGTAC
TCT.AAGCCACTCCCCAAAGTCATGCCCCAGCCCCTGTATAATTCTAAACAATTAGAATTATTTTCATTTTCATTA
ss GTCTAACCRGGTTATRTTAAATATACTTTAAGAAACACCATTTGCCATAAAGTTCTCAATGCCCCTCCCATGCAG
CCTCAAGTGGCTCCCCRGCAGATGCATAGGGTRGTGTGTGTACAAGAGACCCCAAAGACRTAGAGCCCCTGAGAG
CATGRGCTGATATGGGGGCTCATAGAGATAGGAGCTAGATGAATAAGTACAAAGGGCAGAAATGGGTTTTAACCA
GCAGAGCTAGARCTCAGACTTTAAAGAAAATTAGRTCAAAGTAGAGRCTGAATTATTCTGCACATCAGACTCTGA
GCAGAGTTCTGTTCACTCAGACAGAAAATGGGTAAATTGAGAGCTGGCTCCATTGTGCTCCTTAGAGATGGGAGC
so AGGTGGAGGATTATATAAGGTCTGGAACATTT.AACTTCTCCGTTTCTCATCTTCAGTGAGATTCCAAGGGATACT
ACAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGC

s ATGCATCTCAATTAGTCAGCRACCRGGTGTGGAARGTCCCCAGGCTCCCCAGCRGGCAGAAGTATGCAAAGCATG
CATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCRGTTCCGCC
CATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCRGAGGCCGAGGCGCCTCTGAGCTATTCCAGAAGT
AGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAT~I~lIAAGCTARTTCAGCCTGAATGGCGAATGGGACGCGCC

CTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC
ro GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTRAA.TCGGGG
GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAACTTGATTAGGGTGRTGGTTCACGT
AGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTRATAGTGGACTCTTG
TTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGRTTTTGCCGATTTCGGCC
TATTGGTTAAAP.AATGAGCTGATTTAACAAAAATTTAACGCGRATTTTAACAAAATATTAACGTTTACAATTTCA
rs GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG
CTCATGRGACRATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGRGTATTCAACATTTCCGT
GTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAA
GATGCTGARGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGT
TTTCGCCCCGAAGAACGTTTTCCAATGATGRGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT
ao GACGCCGGGCAAGAGCARCTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA
GAAARGCATCTTACGGATGGCATGACAGTAAGRGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG
GCCARCTTACTTCTGACAACGRTCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACARCATGGGGGATCATGTA
ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGRCGAGCGTGACACCACGRTGCCTGTA
GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTRCTTACTCTAGCTTCCCGGCAACAATTAATAGAC
zs TGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA
TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCRGATGGTAAGCCCTCCCGTATCGTA
GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAARTAGACAGATCGCTGAGATAGGTGCCTCRCTG
ATTARGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT
AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA
3o GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA
AGAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACGAACTCTTTTTCCGAAGGTAACT
GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCT
GTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT
ACCGGGTTGGACTCAAGACGATAGTTACCGGA'PAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG
3s CCCRGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCC
GAR.GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGRGAGCGCACGAGGGAGCTTCCAGGG
GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG
TCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCC
4o SEQ m No. 18 pAD-CMV19, cloning sites in bold TCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCRTAGCCCATATATGGAGTT
CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAAT
GACGTATGTTCCCATAGT.AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC
as CCACTTGGCAGTACATCAAGTGTATCRTATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC
CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT
TACCATGGTGATGCGGTTTTGGCAGTRCATCARTGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA.GTCT
CCACCCCATTGACGTCARTGGGAGTTTGTTTTGGCACCAAAATCARCGGGACTTTCCAAAATGTCGTAACAACTC
CGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCRGAGCTCGTTTAGTGAACCG
so TCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGG
CCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTCAGGTAAGTACCGCCTATAGAGAAGACTCTTG
GGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGCTTAACTGGC
TTATCGAAATTAATACGACTCACTATRGGGAGACCCAAGCTTCTGCAGGTCGACATCGATGGATCCGGTACCTCG
AGCGCGAATTCTCTAGAGATATCTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAA.ATT
ss TCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCT
GGATCAATTCTGAAAAACTAGCCTTAAA.GACAGACAGCTTTGTTCTAGTCAGCCAGGCAAGCATATGTAAATAAA
GTTCCTCAGGGAACTGAGGTTAAAAGATGTATCCTGGACCTGCCAGACCTGGCCATTCACGTAAACAGAAGRTTC
CGCCTCAAGTTCCGGTTAACAACAGGAGGCRACGAGATCTCAAATCTATTACTTCTAATCGGGTAATTAAAACCT
TTCAACTAAAACACGGACCCACGGATGTCACCCACTTTTCCTTCCCCGGCTCCGCCCTTCTCAGTACTCCCCACC
so ATTAGGCTCGCTACTCCACCTCCACTTCCGGGCGCGACACCCACGTGCCCTCTCCCACCCGACGCTAACCCCGCC
CCTGCCCGTCTGACCCCGCCCACCACCTGGCCCCGCCCCGTTGAGGACAGAAGAAACCCCGGGCAGCCGCAGCCA
AGGCGGACGGGTAGACGCTGGGGGCGCTGAGGAGTCGTCCTCTACCTTCTCTGCTGGCTCGGTGGGGGACGCGGT

s GGATCTCAGGCTTCCGGAAGACTGGAAGAACCGGCTCAGAACCGCTTGTCTCCGCGGGGCTTGGGCGGCGGAAGA
ATGGCCGCTAGACGCGGACTTGGTGCGAGGCATCGCAGGATGCAGAAGAGCAAGCCCGCCGGGAGCGCGCGGCTG
TACTACCCCGCGCCTGGAGCGGCCRCGCCGGACTGGGCGGGGCCGGCCTGGTGGAGGCGGAGTCTGACCTCGTGG
AGGCGGGGCCTCTGATGTTCAAATAGGATGCTAGGCTTGTTGAGGCGTGGCCTCCGATTCACAAGTGGGAAGCAG
CGCCGGGCGACTGCAATTTCGCGCCAAACTTGGGGGAAGCACAGCGTACAGGCTGCCTAGGTGATCGCTGCTGCT
ro GTCATGGTTCGACCGCTGAACTGCATCGTCGCCGTGTCCCAGAATATGGGCRTCGGCAAGAACGGAGACCTTCCC
TGGCCAATGCTCAGGTACTGGCTGGATTGGGTTAGGGAAACCGAGGCGGTTCGCTGAATCGGGTCGAGCACTTGG
CGGAGACGCGCGGGCCAACTACTTAGGGACAGTGATGAGGGGTRGGCCCGCCGGCTGCTGCCCTTGCCCATGCCC
GCGGTGATCCCCRTGCTGTGCCRGCCTTTGCCCAGRGGCGCTCTAGCTGGGAGCAAAGTCCGGTCACTGGGCAGC
ACCACCCCCCGGACTTGCATGGGTAGCCGCTGAGATGGAGCCTGAGCACACGTGACAGGGTCCCTGTTAACGCAG
rs TGTTTCTCTAACTTTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACCACCTCCTCAGTGGAAGGTAAACA
GAACCTGGTGATTRTGGGCCGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAA
TATAGTTCTCAGTAGAGRGCTCAAGGAACCACCACAAGGAGCTCATTTTCTTGCCAAAAGTCTGGACCATGCCTT
AAAACTTATTGAACAACCAGAGTTAGCAGATAAAGTGGACATGGTTTGGATAGTTGGRGGCAGTTCCGTTTACAA
GGAAGCCATGAATCAGCCAGGCCRTCTCAGACTCTTTGTGACAAGGATCATGCAGGARTTTGAAAGTGACACGTT
za CTTCCCAGAAATTGATTTGGAGAAATATAAACTTCTCCCAGAGTACCCAGGGGTCCTTTCTGAAGTCCAGGAGGA
AAAAGGCATCAAGTATAAATTTGAAGTCTATGAGAAGAAAGGCTAACAGAAAGATRCTTGCTGRTTGACTTCAAG
TTCTACTGCTTTCCTCCTAAAATTATGCATTTTTACAAGACCATGGGACTTGTGTTGGCTTTAGATCCTGTGCAT
CCTGGGCAACTGTTGTACTCTAAGCCACTCCCCAAAGTCATGCCCCAGCCCCTGTATAATTCTAAACAATTAGAA
TTATTTTCRTTTTCATTAGTCTAACCRGGTTATATTAAATATACTTTAAGAAACACCATTTGCCATAAAGTTCTC~
2s AATGCCCCTCCCRTGCAGCCTCAAGTGGCTCCCCAGCAGATGCATAGGGTAGTGTGTGTACAAGAGACCCCAAAG
ACATAGAGCCCCTGAGAGCATGAGCTGATATGGGGGCTCATAGAGATAGGAGCTAGATGAATAAGTACAAAGGGC
AGAAATGGGTTTTAACCAGCAGAGCTAGAACTCAGACTTTAAAGAAAATTAGATCAAAGTAGAGACTGAATTATT
CTGCACATCAGACTCTGAGCAGAGTTCTGTTCACTCAGACAGAAAATGGGTAAATTGAGAGCTGGCTCCATTGTG
CTCCTTAGAGATGGGAGCAGGTGGAGGATTATATAAGGTCTGGAACATTTAACTTCTCCGTTTCTCATCTTCAGT
3o GAGATTCCAAGGGATACTACAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCA
GGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC
AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA
ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCRTGGCTGACTAATTTTTTTTATTTRTGCRGAGGCCGAGGCGCCT
CTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAAGCTAATTCAGCCTGAA
3s TGGCGAATGGGAAATTGTAAACGTTAATRTTTTGTTAAAA.TTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTT
TAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCC
AGTTTGGRACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAA.GGGCGAAAAACCGTCTATCAGGGCGA
TGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCT
AAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAA
ao GGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCG
CCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATA
CATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATG
AGTATTCAACATTTCCGTGTCGCCCTTRTTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAA
ACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCRCGAGTGGGTTACATCGAACTGGATCTCAACAGC
as GGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGC
GCGGTATTRTCCCGTATTGACGCCGGGCARGAGCAACTCGGTCGCCGCATACACTRTTCTCAGAATGACTTGGTT
GAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTARCCGCTTTTTTGCAC
AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGT
so GACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCC
CGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC
TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCRTTGCAGCACTGGGGCCAGATGGT
AAGCCCTCCCGTATCGTRGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCT
GAGATAGGTGCCTCACTGATTARGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA
ss AAA.CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGT
GAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAARGATCAAA.GGATCTTCTTGAGATCCTTTTTTTCTGCGC
GTARTCTGCTGCTTGCAAACAAAAAP.ACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACT
CTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC
CACCACTTCAAGAACTCTGTAGCACCGCCTACATRCCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGT
so GGCGATAAGTCGTGTCTTRCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG
GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGARCGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGA
GAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACRGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC

s ACGAGGGAGCTTCCRGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCRCCTCTGACTTGAGCGT
CGRTTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCAGCTGC
Example 3 Clinical study 1170.1 m 1. LIST OF
ABBREVIATIONS
AND DEFINITION
OF TERMS

ADGC Antibody Dependent Cell-mediated Cytotoxicity AE Adverse Event ALT Alanine Amino Transferase a.p. Anterior posterior AP Alkaline phosphatase AST Aspartate Amino Transferase AUC Area Under the concentration-time Curve Bq Becquerel, SI unit for radioactivity Becquerel (1Bq = one decay /s) BSA Bovine Serum Albumin CD44v6 CD44 variant isoform v6 CDS Corporate Drug Safety cGy Centi Gray measure for radioactivity CHO Chinese Hamster Ovary cells Ci Curie; unit for radioactivity; 1 Ci = 37 x 109 decays / s = 37 GBq CL Total body clearance cMAb Chimeric Monoclonal Antibody cm Centimetre CmaX Maximum drug concentration observed cpm Count per minute CRF Gase Report/Record Form CT (scan) Computed Tomography CTC Common Toxicity Criteria CV Coefficient of Variation DLT Dose Limiting Toxicity ECG Electrocardiogram ELISA Enzyme-Linked Immuno-Sorbent Assay ENT Ear Nose Throat f Female FDA Food and Drug Administration .

g Gram GBq SI unit for radioactivity Giga Becquerel (lGBq = 109 decays /s) GCP Good Clinical Practice GGT Gamma Glutaryl Transpeptidase GMP Good Manufacturing Practice Gy Gray HAHA Human-Anti-Human-Antibody Hb Haemoglobin HER2 Human Epidermal growth factor Receptor 2 hMAb Humanised Monoclonal Antibody HNSCC Head and Neclc Squamous Cell Carcinoma HPLC High Performance Liquid Chromatography hr / h Hour hrs Hours Ht Haematocrit i3~I Iodine-131 (half life 8.05 days) ICH International committee on harmonisation ID Injected Dose IEC Independent Ethics Committee IgG Immunoglobulin G

INN International Non proprietary Name IRB Institutional Review Board ITT Intent-To-Treat i.v. Intravenous(ly) KeV Kilo electron volt 1 / L Litre m Male ma Square metres MAb Monoclonal Antibody MAG2GABA-TFP Mercaptoacetylglycylglycyl-gamma-aminobutyrate-tetrafluorophenol ester (MAG2GABA-TFP), chelate used for coupling lg6Re to monoclonal antibodi~

(future method) MAG3 Mercaptoacetyltriglycine. Chelate used for coupling of 99~'Tc and lg6Re to monoclonal antibodies MBq SI unit for radioactivity; Mega Becquerel (1 MBq = 106 decays/s) mCi Milli Curie; unit for radioactivity; 1 mCi, = 37 million decays / s = 37 MBq MCV Mean Corpuscular Volume mGy lVIilli Gray p,g Microgram mg Milligram min Minute ml / mL Millilitre mMAb Murine Monoclonal Antibody mmHg Millimetre mercury ~.mol Micromol mmol Millimol MRI Magnetic Resonance Imaging MRT Mean Residence Time mSv Milli Sievert MTD Maximum Tolerated Dose NA / n.a. Not Applicable NCI US National Cancer Institute ND Not Done ng Nanogram No / N Number nos Not other specified NSCLC Non Small Cell Lung Cancer n.y.r. Not yet recovered p.a. Posterior anterior PBS Phosphate Buffered Saline p.i. Post infusion p.o. Per os PP Per Protocol Pt. / Pat Patient Pts. Patients iasRe ~e~um-186, radionuclide; half life of 3.7 days (tissue penetration of 13-particles of about 1.2 mm) Recov. Recovered RES Reticulo Endothelial System RIS Radioimmunoscintigraphy RIT Radioimmunotherapy ROI Region of Interest S~ Serious Adverse Event SCC Squamous Cell Carcinoma sCD44v6 Soluble CD44v6 SD Stable Disease sGOT Serum Glutamic Oxalacetic Transaminase sGPT Serum Glutamic Pyruvic Transaminase SOC System Organ Class SOP Standard Operating Procedure SPECT Single Photon Emission Computed Tomography tls Elimination half life 99mTC Technetium 99m (half life 6 hours) TLC Thin Layer Chromatography TmaX Time point at which the maximum drug concentration is observed TNM Tumour Node Metastasis system for staging tumours s TSH Thyroid Stimulating Hormone UICC Union Internationale Contre 1e Cancer (international union against cancer) u~ Unknown Vss Apparent volume of distribution under steady-state conditions VZ Apparent volume of distribution during the terminal phase WBC White Blood Count WHO World Health Organisation 2. Study Objectives 2.1 GENERAL AIM / CLINICAL OBJECTIVE
The general aim of the present study was to assess the safety and tolerability of intravenously administered 99mTc and lg6Re-labelled hlVIAb BIWA 4, to confirm preferential accumulation in to the tumour of 99mTc-labelled hMAb BIWA 4, to determine the maximum tolerated radiation dose of 186Re-labelled hMAb BIWA 4 and to propose a safe dose for phase II
development. In order to reach this goal the study was divided into two parts:
Part A:
Objectivese m ~ To determine the safety and tolerability of a single infusion of intravenously administered ssmTc-labelled hMAb BIWA 4 in patients with advanced squamous cell carcinoma of the head and neck.
~ To determine the biodistribution of a single infusion of 99'°Tc-labelled hMAb BIWA 4 at different BIWA 4 dose levels in patients with advanced squamous cell carcinoma of the 1o head and neck.
~ To study the pharmacokinetics of a single infusion of 99"'Tc-labelled hMAb Part B:
Objectives ~ To determine the qualitative and quantitative toxic effects of 186Re-labelled hMAb BIWA 4 and to study the predictability, onset, duration, intensity, reversibility and dose-relationship of the toxic side effects.
~ To determine the maximum tolerated radiation dose of intravenously administered i86Re-labelled hMAb BIWA 4 in head and neck cancer patients.
1o ~ To study the pharmacokinetics of intravenously administered 186Re-labelled hMAb BIWA 4 in patients with squamous cell carcinoma of the head and neck.
~ A secondary objective was to determine the preliminary therapeutic effects of 186Re-labelled hMAb BIWA 4.
The following objectives outlined in the protocol could not be addressed during the ~s performance of the trial. The development of the programme with the linker chelate mercaptoacetyltriglycine (MAG3), used for coupling of 99~"Tc and 186Re to monoclonal antibodies was discontinued and thus the trial was finished. The further development with BIWA 4 will continue by using the linker mercaptoacetylglycylglycl-gamma-aminobutyrate-tetrafluorophenol ester (MAG2GABA-TFP).
2o ~ The MTD for single dose treatment was to be identified first before more patients were entered to define the MTD for a second treatment with 186Re-labelled hMA.b BIWA 4.
~ To propose a safe dose for first and consecutive infusions with 186Re-labelled hMAb BIWA
4 for further studies.
~ To obtain initial results on a dose schedule for repeated dosing.
ZS 2.2 PRIMARY VARIABLES
Safety: clinical laboratory tests, human-anti-human-antibody (HAHA) assessments, vital signs measurements and adverse events.
~ Efficacy: biopsy biodistribution data (Part A only) and radioimrnunoscintigraphic images (Part A and B including dosimetry for Part B of the trial).
30 ~ Pharmacokinetic results.
2.3 SECONDARY VARIABLES
A secondary variable for the study was tumour response (Part B only).

3. INVESTIGATIONAL PLAN
3.1 OVERALL STUDY DESIGN AND PLAN - DESCRIPTION
The trial was performed in two parts. Part A evaluated the optimal dose of cold BIWA 4 and quantified tumour uptake while Part B investigated the maximum tolerated dose of 186Re-BIWA 4.
to 3.1.1 Part A:
This part of the clinical trial was an uncontrolled, rising dose sequential group study. It was designed to provide initial data on the safety and tolerability of a single infusion of 99mTc-labelled hMAb BIWA 4, to investigate the pattern and level of biodistribution and to establish the pharmacokinetic profile of hMAb BIWA 4 in patients with head and neck cancer. Three protein doses of hMAb BIWA 4 were used in this Part A of the study with three patients planned to be treated at each dose level.
Patients were routinely investigated by the Department of Otolaryngology to determine the extent of the tumour. This included physical examination, computed tomography (CT) or MRI
scans of the head and neck and panendoscopy (optional). During these procedures samples of ao tissues, suspect of tumour, were taken and investigated for the presence of squamous cell carcinoma. Based on the results of these investigations, the patients were destined to undergo surgery including neck dissection.
Radiolabelled antibody was injected and the patient was observed for occurrence of adverse events. Radioimmunoscintigraphic scans were performed 21 hours post infusion (p.i.) prior to 25 surgery. Patients underwent surgery 48 hours (hrs) after the infusion .of the radioactively labelled hMAb BIWA 4. The pathologist investigated the neck dissection specimen to determine the exact tumour load. Moreover, the amount Of 99="TC ~n biopsies from tumour sites) and normal tissues in the surgical specimen was measured. Tumour sites and tumour infiltrated nodes were examined for the presence of CD44v6 antigen by immunohistochemical 3o techniques. The first three patients were administered 2 mg hMAb BIWA 4 labelled with 20 mCi 99"'Tc combined with 23 mg unlabelled hMAb BIWA 4. The second group of three evaluable patients was administered 2 mg hMAb BIWA 4 labelled with 20 mCi 99"'Tc combined with 48 mg unlabelled hMAb BIWA 4 and the third group was administered 2 mg of the labelled antibody combined with 98 mg of unlabelled antibody.
3s Pharmacokinetic assessments were done at the specified timepoints.
3.1.2 Part B:
This part of the clinical trial was an open uncontrolled, dose escalation study. It was designed to assess the safety and tolerability of 186Re-labelled hMAb BIWA 4, to determine the maximum tolerated dose (MTD) of intravenously administered ~g6Re-labelled hMAb 4o and to determine the preliminary therapeutic effects of lg6Re-labelled hMAb B1WA 4 in head and neck cancer patients for whom no curative options were available. In addition the pharmacokinetic profile of 186Re-labelled hMAb BIWA 4 was assessed.
All patients entering into this part of the trial received the dose of hMAb BTWA 4, which was selected basing on the results of Part A of the study. The hMAb BIWA 4 was labelled with escalating doses of 186Re. At the lower dose levels (toxicity observed did not exceed grade 1) two patients were entered per dose group and at the higher levels (>_ grade 2 toxicity) a minimum of three patients were entered per dose group. All patients were evaluated to determine the safety of the administered hMAb BIWA 4.
Patients were routinely investigated by the Department of Otolaryngology to determine the extent of the tumour. This included physical examination and CT or MRI
scanning of tumour locations.
issRe-labelled antibody was injected with escalating radiation doses: patients in the first dose group received a radiation dose of 20 mCi/m2, after which the dose for patients in the subsequent dose groups was escalated by 10 mCi/m2 increments until the MTD was reached.
Patients were observed for occurrence of adverse events.
Radioimmunoscintigraphic scans zo were performed.
3.1.3 STUDY PROCEDURES AT EACR VISIT
3.1.3.1 Visit Schedule a) Screening visit Before entry into the study each patient was screened for eligibility.
Demographics and zs medically relevant history were recorded, concomitant therapy was recorded and a general physical examination was done and the body weight was measured. The I~arnofsky performance score was determined. Written informed consent had to be obtained.
A pregnancy test was required for women with childbearing potential. A 12-lead electrocardiogram (ECG) was made and blood as well as urine safety laboratory assessments were done. A
blood sample 3o for _H_AHA_-testing was also taken. A Chest X-ray was made and a full disease assessment (CT/MRI, ear-nose-throat [ENTJ examination) was performed.
At the end ~ of this visit the results of the required investigations were evaluated, the inclusion and exclusion criteria verified and arrangements for surgery (Part A only) were made.
b) Visit 2: Study days 1-7 3s Parts A and B:
On the first study day, which was not more than 3 weeks after the screening visit, the patient was admitted. to the hospital. All required baseline laboratory assessments not obtained at the screening visit had to be done and evaluated prior to the infusion and the eligibility criteria had to be met. The body weight was measured. All concomitant therapy had to be recorded. Every adverse event starting after the signing of the written informed consent had to be recorded in the patient file and the case report form (GRF).

On the day of treatment, before antibody administration a blood sample was collected for pharmacokinetics (including the assessment of soluble CD44v6) and the vital signs were recorded. Urine was collected from 0-4 hrs, 4-8 hrs, 8-12 hrs and 12-24 hrs during the first 24 hrs and then in 24 hrs intervals up to 48 and 96 hrs post infusion for Part A and B, respectively.
!e The antibody was administered at the department ofNuclear Medicine or in a designated room in the hospital. The antibody had to be administered through a peripheral upper extremity vein as proximally as possible. In all cases, the infusion was given-over 5 minutes, after a 10-mL
saline flush, through a freely flowing line. In order to obtain reproducible pharmacokinetic results a syringe pump had to be used. Therefore, dilution of the volume with NaCI 0.9 % was ~s required to 20 mL. This antibody infusion was followed by a 10 mL saline flush. Any suspected extravasation was documented in the CRF and the patient was imaged.
An emergency unit available with resuscitation equipment, anti-histamines, corticosteroids and epinephrine was within reach to counteract possible anaphylaxis. Adverse events and changes in the concomitant therapy were recorded daily.
20 Part A:
Vital signs were recorded at 10, 60 and 120 minutes p.i. Blood samples for safety and soluble CD44v6 were collected 21, 48 and 144 hrs post infusion.
Blood samples for pharmacokinetics were drawn at the end of the infusion and at 5, 10, 30 minutes and 1, 2, 4, 16, 21, 48, 72 hrs after the end of the infusion, and on day 7 (144-hrs p.i.
zs including a serum sample for _H_A_H_A_ and soluble CD44v6 assessment).
Urine for pharmacokinetics was collected from 0-4 hrs, 4-8 hrs, 8-12 hrs and 12-24 hrs during the first 24 hrs and a 24-hr sample until 48 hrs p.i.
Whole body scintigraphic images were made directly after the infusion and 21 hours after the infusion. Twenty-one hours after infusion, in addition to the whole body image, single photon 3o emission computed tomography (SPELT) and planar images of the head and neck region were made.
The patient was operated at 48 hrs p.i. and stayed in the hospital for post-operative care.
On day 7 (144 hrs p.i.) safety urine samples were collected.
Adverse events and changes in the concomitant therapy were recorded daily.
3s Part B:
Vital signs were recorded at 10, 60, 120 and 240 minutes post infusion. Blood samples for safety and for soluble CD44v6 were collected 21, 48 and 144 hrs post infusion.
A blood sample for HAHA, assessment was collected 144 hrs after the infusion. A urine sample for safety was also collected 144 hrs p.i.

Blood samples for pharmacokinetics were drawn at the end of the infusion and at 5 and 30 minutes and 1, 2, 4, 16, 21, 48, 72 hours after the end of the infusion and on day 7 (144-hrs p.i.) after infusion. The originally planned sample at 10 minutes was omitted as per amendment I.
Urine for pharmacokinetics was collected from 0-4 hrs, 4-8 hrs, 8-12 hrs and 12-24 hrs during to the first 24 hrs and in 24-hr samples until 96 hrs p.i.
Whole body scintigraphic images were made directly after, at 21, 48, 72 and 144 hrs p.i. and optionally at two weeks p.i. if counting statistics permitted.
Planar images of the head and neck region were made at 21, 48 (optional), 72 and 144 hrs p.i.
and optionally at two weeks p.i., if counting statistics permitted.
Is SPECT imaging was only performed seventy-two hours after the infusion.
Adverse events and changes in the concomitant therapy were recorded daily.
Patients were allowed to leave the hospital after three days.
c) Visit 3: Follow-up visits Part A:
2o Patients visited the outpatient clinic six weeks after the infusion. A
physical examination was performed, safety blood and urine samples collected, body weight and vital signs measured and adverse events recorded. Blood samples for pharmacokinetics, uaua assessment and soluble CD44v6 were also obtained. A pregnancy test was required for women with childbearing potential. Follow-up of adverse events was recorded. Any changes in the concomitant therapy ~s were recorded.
Part B:
Patients visited the outpatient clinic weekly for at least six weeks for recording of adverse events and collection of safety blood samples. Blood samples for pharmacokinetics were drawn at 240 hours and 336 hours after infusion. The originally planned sample at 6 weeks p.i. was 30 omitted as per amendment 1. Any changes in the concomitant therapy were also recorded. The disease assessment performed at baseline was repeated six weeks after the infusion and thereafter if indicated (the assessment was done after six weeks rather than after four weeks as mentioned once in the protocol). Six weeks after the infusion blood samples for safety, HAHA
assessment and soluble CD44v6 were collected, vital signs recorded and body weight 3s measured. A physical examination was done during this visit. A urine sample for safety was collected. A pregnancy test was required for women with childbearing potential.

d) Visits 4 and 5: second treatment (Part B) A second treatment as outlined in the original protocol was not performed due to the premature termination of the trial because of the linker change. Instead patients who responded to the first dose of 1$6Re-BIWA 4 were eligible for a second administration.
They underwent the same visit schedule as for the first administration.
to 3.2 DISCUSSION OF STUDY DESIGN,1NCLUDING THE
CHOICE OF CONTROL GROUPS
The aim of the present study was to assess the preferential accumulation of 99mTc-labelled BIWA 4 in the tumour (Part A) and to evaluate the maximum tolerated dose of lg6Re-BIWA 4 (Part B) as well as the pharmacokinetics of BIWA 4 in patients suffering from advanced head is and neck cancer (Part A and B).
An open design was used as is general practice in these types of Phase I
trials in oncology. A
minimum of two patients in the lower radiation dose tiers and three patients in the higher radiation dose tiers were included in Part B of the trial. In case of occurrence of drug-related Common Toxicity Criteria (CTC) grade 4 haematology and grade 3 non-haematology toxicity Zo a further three patients were treated with the respective dose tier (for further dosing details refer to section 3.4.4. and 3.4.5).
The dose of BIWA 4 administered was based on previous results with mMAb BIWA 1 indicating that a dose of 50 mg yielded a high and selective uptake in tumour tissue with a low uptake in non-tumour tissues and the results from Part A of the trial (see also section 3.4.4.1).
2s The starting dose level for radioactivity chosen was based on previous data suggesting that a dose of 20 mCi/m2 may be a safe dose (see also section 3.4.4.2).
The criteria for efficacy which applied as well as the criteria for assessing tolerability are well-established for this patient population and can also be evaluated in an open design.
3.3 SELECTION OF STUDY POPULATION
30 3.3.1 Inclusion criteria Patients with histological confirmation of squamous cell carcinoma in the head and neck.
- Patients destined for surgery by means of a neck dissection (Part A) or:
- Patients with either local and/or regional recurrent disease for which curative treatment 35 options were not available, or distant metastases. The tumour deposits had to be measurable either clinically or by one or more radiological techniques) (CT, MIZI, bone scintigraphy).

Because RIT was expected to be more effective in smaller size tumour deposits, patients with lesions measuring < 3 cm in greatest dimension were preferred (Part B).
- Patients over 18 years of age.
- Patients younger than 80 years of age.
- Patients who had given "written informed consent".
to - Patients with a life expectancy of at least 3 months.
Patients with a good performance status: Karnofsky > 60.
3.3.2 Exclusion criteria - Life-threatening infection, allergic diathesis, organ failure (bilirubin >
30 ~mol/1 and/or creatinine > 150 ~.mol/1) or evidence of a recent myocardial infarction on ECG
or unstable 15 angina pectoris.
Pre-menopausal women (last menstruation _< 1 year prior to study start):
~ Not surgically sterile (hysterectomy; tubal ligation) and ~ Not practising acceptable means of birth control, (or not planned to be continued throughout the study). Acceptable methods of birth control include oral, implantable or ao injectable contraceptives.
- Women with a positive serum pregnancy test at baseline.
- Chemotherapy or radiotherapy within 4 weeks before inclusion in the study.
- White blood cell count < 3000/mm3, granulocyte count < 1500/mm3 or platelet count < 100,000 /mm3.
zs - Haematological disorders, congestive heart failure, bronchial asthma, alimentary or contact allergy, severe atopy or allergy.
3.3.3 Removal of subjects from therapy or assessment 3.3.3.1 Criteria for Stopping Subject Treatment The infusion had to be terminated immediately if the patient developed tachycardia (pulse rate 3o greater than 120 per minute), hypotension (blood pressure less than 100 mm Hg systolic), respiratory distress, chest pain, or any symptoms intolerable to the patient.
3.3.3.2 Dropouts and Withdrawals The subjects were free to discontinue their participation in this study at any time.
Evaluation of radiolabelled hMAb BIWA 4 was considered not to be. feasible if the patient was prematurely removed from the study because of voluntary withdrawal. A case was considered not evaluable if adequate follow-up information was not available. Any unevaluable patients were planned to be replaced.
If the patient discontinued early from the study, the reason had to be documented on the CRF.
If a patient did not return for the post-infusion blood samples for ua_u_a_ determination or pharmacokinetics, the reason had to be documented. If a patient developed a serious adverse event the study schedule had to be followed as closely as possible depending on the serious adverse events (SAE).
~s 3.4 TREATMENTS
3.4.1 Treatments administered Patients in Part A were administered 99mTc-BIWA 4 at a radioactivity dose of 20 mCi. The dose of BIWA 4 administered was 2S mg, SO mg or 100 mg for three patients each. The drug was administered intravenously as a single dose.
2o Patients in Part B received SO mg BIWA 4 labelled with Rhenium 186. The lowest radioactivity dose was 20 mCi/m2 which was increased in dose tiers of 10 mCi/m2. The trial drug was administered intravenously as a single dose.
3.4.2 Identity of investigational product Part A:
2s Substance (INl~: BIWA 4 (bivatuzumab) Pharmaceutical form: solution for injection Chiffre number: BIWA 4 LOI 99 1D 1A

Batch number: B981101 Source: Boehringer Ingelheim Pharma KG

3o Unit strength: S mg / mL

Daily dose: 2S mg Duration of use: single dose Route of administration:intravenous Posology: infusion over five minutes 3s Substance (INl~: BIWA 4 (bivatuzumab) Pharmaceutical form: solution for injection Chiffre number: BIWA 4 LOI 99 1D 1A

Batch number: B981101 Source: Boehringer Ingelheim Pharma KG

Unit strength: S mg l mL

Daily dose: 50 mg Duration of use: single dose Route of administration: intravenous Posology: infusion over five minutes Substance (INN): BIWA 4 (bivatuzumab) m Pharmaceutical form: solution for injection Chiffre number: BIWA 4 LOI 991D 1A

Batch number: B981101 Source: Boehringer Ingelheim Pharma I~G

Unit strength: 5 mg / mL

Is Daily dose: 100 mg Duration of use: single dose Route of administration:intravenous Posology: infusion over five minutes BIWA 4 was administered as radioconjugate finked with 99mTc. Linker molecule was MAG3.
2o MAG3 was purchased from Mallinckrodt, Petten, The Netherlands.
99mTc was ordered locally via the laboratory where the radioimmunoconjugate was prepared (laboratory of Prof. Dr. van Dongen, Section Tumor Biology, Department of Otorhinolaryngology / Head and Neck Surgery, T~r~e Uhiversiteit University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands).
Z5 Part B:
Substance (TNN): BIWA 4 (bivatuzumab) Pharmaceutical form: solution for injection Chiffre number: BIWA 4 LOI 99 1D 1A

Batch number: 8981101 3o Source: Boehringer Ingelheim Pharma I~G

Unit strength: 5 mg / mL

Daily dose: 50 mg Duration of use: single dose Route of administration:intravenous 3s Posology: infusion over five minutes BIWA 4 was administered as radioconjugate linked with lg6Re. Linker molecule was MAG3.
MAG3 was purchased from Mallinckrodt, Petten, The Netherlands.
issRe was ordered locally via the laboratory where the radioimmunoconjugate was prepared (laboratory of Prof. Dr. van Dongen, Section Tumor Biology, Department of do Otorhinolaryngology l Head and Neck Surgery, T~Yije Unive~siteit University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands).
3.4.2.1 Characteristics and quality of the,trial drug Antibody characteristics The antigen recognised by hMAb BIWA 4 is a transmembrane glycoprotein located on the outer cell surface and is only to a small extent internalised (< 20 %).
Further analysis revealed that hMA.b BIWA 4 recognises an epitope encoded by variant exon v6 of CD44.
The antigen was shown to be expressed by all primary head and neck tumours (n=54) and by the majority of cells within these tumours. A comparable expression was observed for sixty-eight tumour infiltrated lymph nodes from neck dissection specimens (R97-2054). The reactivity pattern of hMAb B1WA 4 in human normal tissues is provided in Appendix TII of the protocol. Reactivity of hMAb BIWA 4 was found to be essentially restricted to squamous epithelia.
As demonstrated by the previous RIS study with murine monoclonal antibody (mMAb) BIWA 1, ~s reactivity with normal squamous epithelium was not a limiting factor for utility in tumour targeting with respect to tumour uptake.
Quality control of 9~"'Tc- or 186Re-labelled hMAb BIWA 4 The antibody was labelled with 99mTc or 186Re according to a method described by Fritzberg et al. (R96-2106: See protocol Appendix IV) which was modified according to Visser et al.
2o (R96-2094) and Van Gog et al. (R96-2111).
The procedures for radiolabelling hMAb BIWA 4 with 99mTc and lg6Re had been validated with respect to the final quality of the prepared conjugate. In five independent labelling experiments, performed according to the procedure described in appendix IV of the protocol, the percentage of label bound to the antibody was found to be 96-99%.
zs In this clinical investigation the radiochemical purity of each 99~'Tc- or 186Re-labelled antibody batch prepared was assessed by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) and passage through a PD-10 gel filtration column, and had to be more than 90% to allow administration to a patient.
The immunoreactive fraction of each 99"'Tc/ig6Re-labelled antibody batch was checked after 3o administration by use of validated methods and had to be above 60%. In brief, analyses were essentially performed according to a procedure as described by Lindmo et al.
(R96-2104).
UM-SCC 11B cells (human larynx carcinoma), containing the CD44v6 antigen, were fixed in 0.1% gtutaraldehyde. Six dilutions, ranging from 5 x 106 cells per tube to 3.1 x 105 cells per tube, were made with 1% bovine serum albumine (BSA) in phosphate buffered saline (PBS).
35 To the tubes, 80,000 counts per minute (cpm) of the 99'"Tc- or 10,000 of the lg6Re-labelled hMAb BIWA 4 were added and incubated overnight at room temperature.
To the last sample, excess unlabelled hlVIAb BIWA 4 was added to determine non-specific binding. Cells were spun down and the radioactivity in pellet and supernatant were determined in a gamma counter and the percentage bound and free radiolabelled MAb calculated (LKB-4a Wallac 1218 CompuGamma).

Data were graphically analysed in a modified Lineweaver Burk plot and the immunoreactive fraction was determined by linear extrapolation to conditions representing infinite antigen excess.
In case the discharge level of 60% was not reached in this binding assay the preparation for infusion was re-evaluated in a second binding assay. For this purpose the antibody preparation was labelled with 1311. The immunoreactive fraction in at least one of both assays, had to be larger than 60% for each patient to be evaluable and for the study to continue with the next patient.
Antibod, BIWA 4 used for radiolabelling with 99mTc and 186Re in this study is a well-characterised ~s monoclonal antibody product which was produced by Chinese hamster ovary (CHO)-cell culture fermentation. A master cell bank was established under Good Manufacturing Practice (GMP) conditions and thoroughly examined for microbiological status (bacteria, fungi, mycoplasma) as well as viral status (adventitious viruses, retroviruses). With the exception of endogenous retroviruses, which were known to be present in most CHO cells, no contaminants zo were detected.
In accordance with the Food and Drug Administration (FDA) 1994 "Points to consider"
document on MAbs used in clinical phase I studies in cancer patients, a standardised downstream purification process, which has been validated for e~cient virus removal, was applied to the purification of BIWA 4 material. Four model viruses were included in the 25 validation (MuLV, PsRV, REO-3, SV 40). The concentrated bulk harvest was examined for the retrovirus titer to ensure that the subsequent downstream purification process was capable of adequately removing the retrovirus. Data on removal of endotoxins were within the acceptance limits (5 0.01 EU/mg) and were available for the bulk product.
Furthermore pyrogenicity testing according to European Pharmacopoea guidelines had been successfully 3o performed.
The final hMAb BIWA 4 to be used for further radiolabelling was analysed in great detail and was proven to be a highly pure (SDS page, isoelectric focusing), sterile solution containing minimal endotoxin levels (< 0.01 EU/mg). Results of testing for pyrogenicity were conform to European Pharmacopoea standard. The equality of the B1WA 4 product from the manufacture 35 for pre-.clinical and clinical supplies was demonstrated by analytical results.
Radiolabelling of hMAb BIWA 4 with 99mTc or lg6Re was performed by the department of Otolaryngology and Head & Neck Surgery of the VY~e Universiteit University Medical Center according to Standard Operating Procedures (SOPs). Sterility of the final product was guaranteed. Absence of endotoxins was tested during validation runs. For drug administration Qe to a patient in the University Hospital Nijmegen the proper amount of radiolabelled hMAb BIWA 4 was transported in a special container directly after preparation to the study centre in Nijmegen. Twenty-four hours were allowed between labelling of the compound and administration to the patient.

See also appendix 16.1.6 for an allocation of the individual Rhenium batches to the patients.
3.4.2.2 Packaging, Labelling and Supply BIWA 4 was supplied by Boehringer Ingelheim The Netherlands. It was produced by Boehringer Ingelheim, Germany using a GMP manufacturing and purification process and filled in vials as a sterile, non-pyrogenic solution containing 25 mg hMAb BIWA 4 in 5 mL
~o isotonic PB S, pH 7.2. Examples of the vial labels of the native and also of the labelled antibody were included in the Clinical Trial Manual.
One batch with a total amount of 27 g of hMAb BIWA 4 had been prepared and filled into vials. Labelling of hlVIAb BIWA 4 with 9~"'Tc or lg6Re was performed in a class B certified nuclear laboratory of the hrije Universiteit University Medical Center, Amsterdam.
~s 3.4.2.3 Storage Conditions The unlabelled hMAb BIWA 4 had to be stored in the hospital pharmacy in a limited access area for study materials at a monitored temperature between +2 and +8°C.
3.4.3 Method of assigning subjects to treatment groups No randomisation was used. Patients were assigned to the different dose groups according to 2o the sequence of inclusion.
3.4.4 Selection of doses in the study 3.4.4.1 Selection of the BIWA 4 doses Part A:
Since hMAb BIWA 4 had never been administered to patients before, it was essential to be 2s informed about its safety and biodistribution before starting RIT trials.
Its biodistribution might strongly depend on the MAb dose used for tumour targeting and would need careful consideration. On the basis of MAb protein dose escalation studies with the low aff'xnity anti-CD44v6 mMAb U36 and the high affinity anti-CL~44v6 mMAb BIWA l, it was anticipated that the optimal dose was in the range of 25-100 mg, with 50 mg being optimal for mMAb 3o U36.
To confirm that this dose also was adequate for the intermediate affinity of hMAb BIWA 4 and to obtain initial information on the variability of the tumour uptake the biodistribution was assessed at 25, 50 and 100 mg total hMAb BIWA 4 and three evaluable patients were planned to be treated at each of these dose levels.
3s All evaluable patients received a single intravenous infusion of 2 mg hMAb BIWA 4 labelled with 20 mCi 99'"Tc, measured by a radiation calibration system just prior to administration.
Patients scheduled to receive 25 mg, 50 mg or 100 mg received 23 mg, 48 mg or 98 mg, respectively of unlabelled hMAb BIWA 4 administered together with this 2 mg hMAb BIWA 4 labelled with 20 mCi 99mTc.
Selection of the B1WA 4 dose for Part B' On theoretical grounds a hMAb BIWA 4 dose of 50 mg was calculated as most suited for development. Part A of this study was performed to confirm the tumour preferential uptake of to hMAb BIWA 4 at the three dose levels tested (25 mg, 50 mg and 100 mg). It was expected that the tumour uptake (expressed as percent injected dose per kilogram, %
ID/kg) and tumour to non-tumour uptake ratio for these three dose levels would not differ much.
If this was the case the hMAb B1WA 4 dose to be used for Part B of the study was 50 mg.
However, if there was a clinically relevant difference between these dose levels, which favoured one over the ~s other levels, the dose level with the best pattern of biodistribution would have been selected.
In this case a clinically relevant difference was defined as a difference of the tumour (mean of the tumour uptake) to bone marrow uptake ratio (mean of the bone marrow uptake = the cellular fraction and the supernatant) of more than 50 %.
Finally the dose selected was 50 mg hMAb BIWA 4 basing on the results of Part A.
20 3.4.4.2 Selection of the radiation starting .dose Part B:
The hMAb BIWA 4 dose selected in Part A of the study was labelled with escalating doses of Re.
It has been reported that the maximum tolerated dose of 186Re-labelled marine MAb NR-LU-ZS 10 was 90 mCi/m2 for heavily pre-treated patients, while dose-limiting myelosuppression was observed at 120 mCi/m2. For chimeric NIAb NR-LU-13, recognising the same antigen as NR
LU-10, reversible rriyelosuppression occurred at 60 mCi/m2. In a study with cMAb U36, performed at the Tl~~e U~rive~siteit University Medical Center dose limiting myelosuppression was observed at 41 mCi/m2.
3o In the light of the above-mentioned results a radiation dose of 20 mCi/m2 administered as ls6Re-labelled hMAb BIWA 4 was considered a safe starting dose.
The dose was escalated with 10 mCi/m2 increments. Two evaluable patients were entered at the lower dose levels. When >_ grade 2 drug-related toxicity according to the CTC was observed a minimum of three patients were treated per dose level.

s Before entering patients at a next higher dose level it had to be sure that the patients at the ongoing dose level did not experience dose limiting toxicity (DLT) defined as:
drug-related CTC grade 3 non-haematologic toxicity or drug-related CTC grade 4 haematologic toxicity excluding nausea and vomiting without adequate antiemetic treatment. For this purpose all patients at such an ongoing dose level had to be observed long enough to ensure that possibly to induced toxicity was reversible.
When at the ongoing dose level one patient experienced DLT the number of patients treated at that dose level was increased to a total of six patients maximum. When 1 out of 6 patients experienced DLT the dose was escalated to the next level. When two or more of the patients experienced DLT, the next lower dose level was expanded (if not previously done) to a total of ~s six patients in order to establish MTD and a safe recommended dose for phase II.
It was originally planned that in case of acceptable toxicity (less than two patients with DLT) at that dose level additional patients could be entered who received this dose and an escalating, lower second dose. This plan was not pursued due to the change of the linker.
MAG3 will be replaced by MAG2GABA-TFP, a comparable linker, but with a more convenient linkage 2o procedure. The present development programme with MAG3 has been finished.
Alternatively patients who responded to and tolerated the first administration of 186Re-BIWA 4 were eligible for a second administration with a dose of 50 mCi/m2 las.Re-BIWA
4.
3.4.5 Selection and timing of dose for each subject 3.4.5.1 Dosage and Treatment Schedule zs The investigators were allowed to administer the trial drug at any time.
The time elapsed between radiolabelling and administration, however, should not exceed 24 hours.
3.4.6 Blinding This was an open uncontrolled trial. No blinding was done.
3.4.7 Prior and concomitant therapy or procedures 30 3.4.7.1 Rescue Medication and Additional Treatments) Anaphylaxis was considered to be the most serious potential side effect and would have mandated immediate cessation of antibody infusion and the institution of appropriate resuscitative measures. Precautions to be taken were: Resuscitation equipment, within reach:
anti-histaminics, corticosteroids and epinephrine. Any patient experiencing this type of adverse 3s reaction was not allowed to receive additional monoclonal antibody.
Patients were allowed to receive other treatments) either for the study indication or for unrelated illnesses providing the inclusion and exclusion criteria were met.
All concomitant therapy had to be recorded in the CRF.

In case of serious haematologic toxicity (CTC grade 4) cytokine intervention or other methods for alleviating bone marrow toxicity were allowed.
3,4.7.2 Restrictions Additional chemotherapy or radiotherapy was not allowed and the last chemotherapy or radiotherapy should have stopped more than 4 weeks before inclusion in this study. All prior chemotherapy and radiotherapy was to be recorded in the CRF.
3.4.8 Treatment compliance The study medication was given as a single intravenous infusion. The compliance was verified with pharmacokinetic assessments and with radioimmunoscintigraphic images.
3.5 EFFICACY / CLINICAL PHARMACOLOGY AND SAFETY
IS VARIABLES
3.5.1 Efficacy / pharmacodynamics and safety measurements assessed and flow chart Flow Charts:
Part A
2o The investigations performed in Part A and the respective times are detailed in the flow chart given below. A description of the investigations is given in the following sections:

TABLE 3.5.1: 1 FLOW CHART: Part A: Biodistribution study with 99"'Tc-labelled hMAb BIWA 4 Visit Visit Visit screeningDay Day Day Day Day Follow-Up 1* 2 3 4 7 visit* Week pre-inj.Post-inj. 6 Signed informed x consent Demographics x Conc. Therapy x x x x x x x X

Med-ical Historyx Physical Examinationx x ECG x Chest X-ray x Blood safety x' x' xl x1 xi x' analysis Urine safety x analysis x x Pregnancy test x x HAHA assessment x2 2 2 x x ENT examination x CT or MRI x In-/exclusion x criteria Adverse events x x x x x x X

Vital SLgriS x3 x3 x3 g x body weight x x x Immunoscintigraphy:

- Whole Body x4 x4 Scan - Planar Scan xs - SPECT Scan xs Pharmacokinetics:

BlOOd Samples x6 x6 x6 x6 x6 x6 6 Urine COlIHCttOn x6 x6 x6 x6 Serum soluble x' x' x' x~ 6 CD44v6 Surgery *~ I fIP TTP_W .v..1..
FW nW v nn ..L-..1.1 ..

- --.- i--- .-u...,.v.1 ~.~woomww ouvulu vccut ttv 111c11c uiall 3 weeKS pnor to trie actual miizsion.
xl: Blood samples were tested for: Glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, serum glutamic oxalacetic transaminase (sGOT), serum glutamic pyruvic transaminase (sGPT), Alkaline ~o Phosphatase, Gamma Glutaryl Transpeptidase (GGT), bilirubin, urea, uric-acid, thyroid stimulating hormone (TSH), haemoglobin (H6), haematocrit (Ht), mean corpuscular volume (MCV), reticulocytes, leucocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets at the screening visit or on day 1 pre-infusion at 21, 48 and 144 hrs p.i. and at six weeks post infusion (p.i.).
x2: HAHA was assessed in a serum sample obtained at the screening visit, after one week (144 hrs) and after six weeks is p.i.
x3: Vital signs were assessed at the screening visit, pre-infusion, at 10, 60 and 120 minutes post infusion and after 6 weeks.
x4: Whole body scan was done immediately after infusion and 21 hrs p.i.
xs: The SPECT scan and planar scan was done once 21 hrs p.i.
ao x6; Blood samples for pharmacokinetics were obtained pre-infusion, at the end of the infusion, at 5, 10, 30 minutes; 1, 2, 4, 16, 21, 48, 72 and 144 hrs post end of infusion and six weeks p.i. Blood samples were drawn for enzyme-linked s immuno-sorbent assay (ELISA) and for measurement of radioactivity (for details see section 9.5.4.1). Urine was collected from 0-4 hrs, 4-8 hrs, 8-12 lus, and 12-24 hrs during the first 24 hrs and in a 24-hr sample for the remaining time until 48 hrs p.i.
x': Soluble CD44v6 was measured from a serum sample obtained pre-infusion, at 21, 48 and 144 hrs p.i and six weeks p.i.
jo 6 week six Part B
The investigations performed and the respective times are given in the flow chart below. The individual investigations are described in more detail in the following sections.
TABLE 3.5.1: 2 FLOW CHART Part B: Dose escalation study with lg6Re-labelled hMAb Visit Visit Visit ScreeningDay Day Day Day Day Follow-Up 1* 2 3 4 7 visit* Week No.
pre-inj.Post-inj.

Signed informed x consent Demographics x Conc. Therapy x x x x x x x 2, 3, 4, 5, Medical History x Physical Examinationx 6 ECG x Chest X-ray x Blood safety x' x1 x' x' xt 2, 3, analysis 4, 5, Urine safety x x 6 analysis Pregnancy test x 6 ~

HAHA assessment x2 x2 6 ENT examination x 6 x8 CT or MRI x 6 xs CT thorax x8 In-/exclusion x criteria Adverse events x x x x x x 2, 3, 4, 5, Vital signs x3 x3 x3 6 Body weight x x 6 Immunoscintigraphy:

- Whole Body x4 x~ x4 x4 x4 Scan - Planar Scan x5 (x5) xs xs - SPECT Scan xs Pharmacokinetics:

B100d SaIllpleS X6 X6 x6 x6 x6 x6 6 UTlIIe COlleCtlOn x6 X6 x6 x6 x6 Senun soluble x' x' x' x' 6 CD44v6 *' The pre-infusion he infusion.
assessment had actual to occur no more than 3 weeks prior to t x1: Blood samples were tested for: Glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, sGOT, sGPT, Alkaline Phosphatase, GGT, bilirubin, urea, uric-acid, TSH, Hb, Ht, MCV, reticulocytes, leucocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets at the screening visit or on day 1 pre-infusion, at 21 hrs p.i., at 48 hrs p.i. and at 144 hrs p.i.. During weeks 2-6 p.i. safety blood samples were io obtained at least weekly.
x2: HAHA was assessed in a serum sample obtained at the screening visit, at 144 hrs and six weeks post infusion.
x3: Vital signs were assessed once at the screening visit, pre-infusion, at 10, 60,120 and 240 minutes post infusion and after 6 weeks.
x°: Whole body scan was done immediately p.i., at 21, 48, 72, 144 hrs p.i, and optionally at two weeks p.i.
~s xs: The planar scan was done at 21, 48 (optional), 72, 144 hrs p.i. and optionally at two weeks p.i. The SPECT scan was done at 72 hrs p.i.
x6: Blood samples for pharmacokinetics were obtained pre-infusion, at the end of the infusion, at 5, 30 minutes; 1, 2, 4, 16, 21, 48, 72, 144 , 240 and 336 hrs post end of infusion. Blood samples were drawn for ELISA and for measurement of radioactivity. Urine was collected from 0-4 hrs, 4-8 hrs, 8-12 hrs, and 12-24 hrs during the first 24 zo hrs and in 24-hr samples for the remaining time until 96 hrs p.i.
x': Soluble CD44v6 was measured from a serum sample obtained pre-infusion, at 21, 48 and 144 hrs p.i and six weeks p.i.
x8: Disease assessment was to be repeated every 6 weeks until disease progression or loss to follow-up. CT thorax was done at baseline and repeated at follow-up if there were abnormalities.
2s 3.5.1.1 Radioimmunoscintigraphy and dosimetry The data obtained from radioimmunoscintigraphy (RIS) were presented qualitatively for both Part A and B (i.e. uptake in tumour, bone marrow, liver, lung, intestine, kidney and additional organs expressed as low, medium or high) and quantitatively for Part B.
For qualitative assessment the rating scale was transformed into numbers (0 being no uptake, 1 3o being low uptake, 2 being medium uptake and 3 being high uptake). Mean values were calculated and presented.
For Part B the quantitative presentation of RIS results used dosimetric calculations as outlined in section 5.1.4 of the protocol by including the following parameters:
~ Actual organ activity at a given time point expressed in MBq.
3s ~ The residence time of radioactivity in organs expressed in hours.
~ The absorbed (radiation) dose expressed in mGy/1VVlBq.
~ The effective (radiation) dose expressed in mSv.
More details concerning the analysis of dosimetry can be found in the dosimetry report (dated November 21, 2001).
4o e) Radioimmunoscintigraphic Procedures A description of the methods used for Part A and B and the time points are given in the following section:
Part A
With a large field of view dual headed gamma camera equipped with a low energy collimator, digital whole body images (anterior posterior (a.p.) and posterior anterior (p.a.)) were obtained l0 directly after and 21 hrs after infusion. At 21 hrs p.i, planar and SPELT
images of the head and neck were acquired. A calibration source also was acquired. Data was stored to enable quantitative analysis.

Part B
With a large field of view dual headed gamma camera equipped with a low energy collimator, digital whole body images (a.p. and p.a.) were obtained directly after, at 21, 48, 72 and 144 hrs after administration of the radioimmunoconjugate. Additional planar images of the head and neck region were made at 21, 48 (optional), 72, and 144 hrs p.i. Optionally additional imaging m (whole body and planar) were performed two weeks p.i. A calibration source (i.e. an aliquot with a known fraction of the injected dose in a 10 millilitre (ml) vial), inserted in an Adams phantom, had to be placed between the lower legs of the patient during the whole body scan.
The initial activity of the calibration source had to be 100-200 MBq 186Re and this source had to be used during all whole body imaging studies. The energy-window and peak settings, the IS scanspeed, the scanlength, the scanning date, the time of starting the scan and the scan duration had to be reported. The anterior-posterior thickness of the neck and of the abdomen had to be measured, while the patient was in supine position on the scanning table.
At each imaging session, anterior and planar static images had to be taken and just before or just after this image a static image had to be acquired from the calibration source in the Za Adams-phantom.
At 72 hr. p.i. a SPECT study of the neck had to be performed. The methods used for reconstruction and the filter functions with cut offfrequencies had to be reported.
Single Photon Emission Computed Tomograph~(SPECT) SPECT images were obtained using a double headed rotating gamma camera equipped with a 2s low energy collimator. Acquisition required at least thirty minutes. Twenty percent symmetric windows were centred at the 137 keV photon peaks.
Planar and SPECT data acquisition parameters Planar imaging included the following minimal requirements: matrix 128x128 (detail) or 256 x 256 (whole body) and a minimum of 400000 counts with a maximum acquisition time of 10 3o minutes for detail and 60 minutes for whole body.
SPECT imaging included the following minimal requirements: 64 images, matrix size 64 x 64, 360 degree circular orbit, 60 second acquisitions per angle.
Analysis of the data At the 21 hr p.i. anterior whole body images, rectangular regions of interest (ROI's) had to be 35 drawn around the whole body and the calibration source. Also irregular ROI's around the organs which accumulated 186Re (e.g. liver, spleen and left kidney) and around the tumour had to be drawn. One or more representative background regions had to be drawn.
These regions had to be mirrored to the posterior images. Originally it was planned to draw a ROI around the sacrum on the posterior image. This plan was not pursued during the trial. All regions had to do be saved on the computer, in order to project these ROI's on images at other time points. The number of pixels and the counts per pixel in each region had to be reported.
The number of counts in the regions for all imaging time points had to be recorded digitally in a spreadsheet.
Calculation of the absorbed dose in the organs The amount of activity in the organs, tumour and the total body was estimated from the geometric mean counts in the ROI's of the anterior and posterior views.
Background and m attenuation correction were applied when indicated. The activity in the urine was not used to estimate the absorbed dose in the bladder as originally planned. Instead the dynamic bladder model was used. The residence times in the organs and the rest of the body were calculated and imported in the MIR1DOSE3 program.
Required quality control tests rs Planar Imaging Routine quality controls were performed at the department of nuclear medicine weekly:
1) 100M flood table.
2) Extrinsic 57Co flood with the low energy collimator was obtained each week.
SPECT imaging 2o Quality control of the SPECT imaging system had to include center of rotation determination.
Tomographic processing A filtered back projection algorithm was used for tomographic image reconstruction using a ramp filter.
Data storage 2s All planar images and tomographic data had to be stored permanently on magnetic tape or optical disc. For tomographic studies, original projecting images and reconstructed studies had to be written to a back up tape.
Copies of images and reports Copies of all relevant images, pathology and surgery reports were required for all patients.
3o Also copies of MRI and CT reports were required. ' 3.5.1.2 Biopsy biodistribution of 99"~Tc-labelled hMAb BItUA 4 (Part A) Patients entered in Part A of the study underwent surgery 48 hrs after infusion of the radiolabelled hMAb BIWA 4. Biopsies from tumour sites) and from normal tissues (as many as possible) in the surgical specimen were taken. Then also under general anaesthesia a bone 3s biopsy and a bone marrow aspirate was taken. The bone marrow aspirates were centrifixged to assess the radioactivity in the supernatant (plasma) and in the sediment (cellular fraction).

All biopsies were weighed and the amount of 9smTc was measured. Specific uptake of radioactivity into tumour was evaluated by comparing %m/kg tumour with %)D/kg normal tissue.

CD44v6 antigen expression was assessed by immunohistochemistry using cryostats sections, which were first incubated with mMAb BIWA 1, followed by anti-mouse Immunoglobulin G
(IgG) secondary reagent. The surgical specimen and the biopsies were investigated histo/cytopathologically.
Evaluation of sur ical specimen and biodistribution to After receiving the surgical specimen it was processed in the following chronological order.
Pictures were taken of the specimen. A Polaroid from the front and slides from the front and back.
2. The size of the surgical specimen was assessed.
3. Biopsies of primary tumour, suspect lymph node, and if possible from normal tissues in the surgical specimen like normal mucosa, normal lymph node, fat and muscle were taken. All biopsies were weighed and the amount of 99"'Tc was measured. All data were converted to percentages injected dose/kilogram tissue. Specific uptake of radioactivity into tumour was evaluated by comparing %ID/kg tumour with %ID/kg normal tissue.
4. Next the specimen was nailed to a board and fixed in formaldehyde 4% for at least 36 2o hours.
After dissecting the sternocleidomastoid muscle (a structure with a high radiodensity), a specimen radiograph was made to show the exact size and location of the lymph nodes involved. This radiograph was made while the specimen was being immersed in ethanol 96%, which has the same X-ray absorption as fat.
2s 6. All the nodes visualised with the X-ray were indicated on the Polaroid and specimen radiograph.

7. All the nodes found by examining the surgical specimen and by X-ray were dissected from the specimen.

8. All macroscopically negative nodes were entirely processed for microscopy and one 3o single section was evaluated. Of all macroscopically positive nodes two or more slices were made. Macroscopical evidence for the presence or absence of tumour necrosis were recorded.

9. Furthermore, the number of nodes enclosed and the number, localisation and lymph node level (according to the Memorial Sloan Kettering Cancer Center Classification) of 3s tumour containing nodes were recorded.

3.5.1.3 Tumour Response (Part B) The e~cacy parameter for the radioimmunotherapy treatment was tumour response.
Tumour response was assessed with tumour measurements as assessed clinically and/or with CT, MRI
or bone scintigraphy investigations. Evaluation was done according to response criteria of the World Health Organisation (WHO). See Appendix VI of the protocol, (R96-0941).
3.5.1.4 Physical Examination Before inclusion in the study the disease status of each patient was evaluated by ENT-examination (palpation included) and by CT or MRI of the tumour site(s).
All head and neck lesions were described per neck side.
Palpation m All patients were examined at baseline by an otolaryngologist/head and neck surgeon. This clinical investigator assessed the number, size, location and mobility of all palpable lymph nodes in the head and neck area. The character of the lymph nodes was described as: not suspected, suspected or tumour infiltrated. The status of the neck lymph nodes was classified according to the Tumour Node Metastasis system for staging tumours (TNM) of the Union 2o Internationals Contre 1e Cancer (UICC) at diagnosis.
Radiological examination Depending on the localisation of the tumour site(s), one or more of the following examinations were required: CT, MRI, bone scintigraphy. For tumour involvement of the head and neck region MRI was generally preferred. In case there were bone lesions, CT was preferred. For Zs Part B all radiological disease assessment parameters obtained at baseline were repeated six weeks after the infusion and every six weeks until progression or lost to follow-up. For patients entered in Part B of the study a CT thorax was obtained at baseline, and was repeated at follow-up if there were tumour lesions in the thorax. Ultrasound might have been employed as additional technique of tumour imaging. Guidelines for the investigations are given below:
3o Primary tumour / loco-regional recurrence For patients participating in Part A of the study and patients with a loco-regional recurrence (Part B) CT scan and/or MRI of the head and neck region had to be performed.
Computed Tomog-raphy Computed tomography of the Head and Neck region: Dynamic CT was preferred over spiral 3s CT. However, spiral CT might have been helpful in patients who were unable to cooperate.
The patient had to be examined in supine position, the neck slightly hyperextended, the head immobilised and the shoulders relaxed and pushed downwards. The patient had to breath quietly with use of abdominal rather than chest muscles. The area to be scanned was determined from the initial overview made in the lateral projection. The plane of the scan had to be parallel to that of the vocal cords. Contiguous three millimetre-slices had to be used routinely. Imaging had to be performed from skull base to upper mediastinum.
Magnetic resonance ima ig-ng The patients had to be examined in supine position, the neck slightly hyperextended and the re head immobilised. Quiet breathing was mandatory during the examination with the use of abdominal rather than the chest muscles. The appropriate images to demonstrate the neck anatomy and to assess the extent of the primary lesion and the presence of lymph node spread was obtained. They had to be obtained from the skull base to the upper mediastinum.
Distant metastases is For all patients participating in Part B of the study CT scanning of the thorax was done at baseline. Additional investigations to visualise metastases, like bone scintigraphy or CT
abdomen, were optional.
CT thorax Spiral CT-scans were obtained. Patients were examined in supine position and had to raise 2o their arms above their heads. Patients had to breath quietly with use of abdominal rather than chest muscles. The patient was scanned from just above the lungs to the level of the adrenal glands. Images had to be photographed in mediastinal and lung setting.
CT abdomen Spiral scans were obtained. Patients were examined in supine position and had to raise their ZS arms above their heads. Patients had to breath quietly. Oral contrast was used in all patients.
The area to be scanned ran from just above the diaphragm to the symphysis.
Images had to be photographed in abdominal and liver setting.
Bone scintigraphy After intravenous infusion of maximally 600 MBq of 9smTc-labelled HDP/MDP, the patient 3o was asked to consume enough fluids and to void frequently. Static views were obtained 3 hours after infusion: one whole body view and if necessary 3 more detailed views. Suspect lesions might have needed closer analysis by CT and/or MRI.
Reporting of response of bone metastases were planned to be conducted according to a separate set of response criteria. No data was provided.
35 3.5.1.5 Soluble CD44v6 Soluble CD44v6 (sCD44v6) had to be measured in serum. Concentrations were determined by means of a validated enzyme-linked immuno-sorbent assay (ELISA) that was based on a commercially available test kit and conducted in accordance with current international s guidelines at the Boehringer Ingelheim Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany. Blood samples (to be processed to serum) of S mL were obtained pre-infusion, at 21, 48 and 144 hrs p.i. and six weeks after the infusion. Samples were allowed to clot and centrifuged to prepare serum. Storage and shipment conditions: The sample for ELISA measurement was put in cryotubes, labelled carefully to enable unique identification, stored at -20°C until radioactivity has decreased (186Re: 4 weeks, 99'"TC: 3 days) and sent to BI
Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany in batches every four weeks. The serum samples were sent on dry ice.
3.5.1.6 Safety Protection of subjects Is All patients were monitored carefully during and after administration of the radiolabelled monoclonal antibody. For this purpose at least one of the investigators was present.
Laboratory Assessments Blood samples were collected for glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, serum glutamic oxalacetic transaminase (sGOT), serum glutamic 2o pyruvic transaminase (sGPT), alkaline phosphatase (AP), gamma glutaryl transpeptidase (GGT), bilirubin, urea, uric-acid, thyroid stimulating hormone (TSI~, haemoglobin (Hb), haematocrit (Ht), median corpuscular volume (MCV), reticulocytes, leucocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets at the following time points:
2s ~ At the screening visit or on day one pre-infusion and ~ At 21, 48 and 144 hrs post infusion.
~ For Part A at 6 weeks post infusion. For Part B: During weeks 2, 3, 4, S and 6 of the study at least once weekly (more often in case of toxicity).
Baseline laboratory assessments pre-infusion could have been obtained either at the screening 3o visit or on day one of the study, provided the samples were obtained less then 21 days before the infusion of radiolabelled hMAb BIVUA 4 and all required assessments were done. All required laboratory test results had to be reviewed and checked for eligibility by the responsible physician prior to the antibody infusion. The same also applied to a second hMAb BIWA 4 administration in Part B of the study.
3s Urine samples were collected for standard hospital screening (protein, blood, and glucose) at the time points:
~ Screening visit ~ One week post infusion (144 hrs p.i.) ~ Six weeks post infusion A pregnancy test was required for women with childbearing potential at the screening visit and at the study end.
Human-Anti-Human-Antibody Assessment The presence and/or the development of _H_A_H_A_ was evaluated in serum samples. Therefore ~o blood samples (to be processed to serum) of 5 mL were taken at the screening visit, at one week (144 hrs p.i.) and at six weeks after infusion of the antibody. For patients receiving two BIWA 4 administrations in Part B of the study an additional IHA_H_A_ sample was collected before the second administration. Serum level was evaluated by means of validated ELISA
methods.
~s Samples were allowed to clot and centrifuged to prepare serum.
Storage and shipment conditions: The samples for ELISA measurement were to be put in cryotubes, labelled carefully to enable unique identification, stored at -20°C until radioactivity had decreased (lg6Re: 4 weeks, 99"'Tc: 3 days) and sent toBI Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany in batches every four weeks. The serum samples 2o had to be sent on dry ice.
Any elevations in human anti-human antibody levels were compared casewise carefully with the patient's possible adverse events. This data was collected and analysed retrospectively during the course of the study.
Adverse Events z5 All adverse events were recorded in the CRF. The events were graded according to the US
National Cancer Institute CTC (NCI-CTC) version 2.0 (a version of this document had to be downloaded from the Internet at the site: http://ctep.info.nih.~ov/CTC3/ctc htm. All SAEs had to be reported.
Immuno enicity and toxicity 3o Before performance of this trial no human data existed with respect to safety and immunogenicity of hMAb BIWA 4. In the previous study performed with the parental murine antibody B1WA 1 no clinically significant toxicity was encountered. No toxicity was expected from the naked hMAb BIWA 4 antibody alone (no antibody dependent cell-mediated cytotoxicity (ADCC) effector functions in vitro; no interference with proliferation of antigen-3s expressing cells expected). It was demonstrated that hMAb BIWA 4 could be safely administered to mice (xenograft studies) and monkeys (toxicity studies). Good local tolerability was demonstrated in three local toxicity studies.
Murine and chimeric MAb U36 (anti-CD44v6-epitope with defined overlapping epitope specificity) was so far safe in HIvTSCC patients; no toxicities except for myelotoxicity (caused by 1$6Re-labels) were seen in a Phase I dose-escalation RIT study with cMAb U3 6. It was theoretically possible that hypersensitivity reactions to radiolabelled hMAb BIWA 4 might occur. Monoclonal antibodies have been administered to several thousands of patients for diagnostic applications.
Although unlikely, potential reactions to intravenously administered BIWA 4 might include hypotension, transient fever and chills, skin rashes, dyspnoea, itching, nausea, and anaphylaxis.
Vital signs (blood pressure, temperature, pulse rate and respiratory rate) were therefore recorded at the screening visit and at .the following time points: pre-infusion and I 0, 60, 120 minutes post-infusion and after 6 weeks. Additionally vital signs were recorded at 240 minutes post-infusion for patients participating in Part B only.
~s In this study radionuclides were used. The radiation burden associated with the gamma emitting radionuclide 99mTc (in the current study 20 mCi) was similar to that encountered in many routine nuclear medicine procedures, and was known to be small. To minimise radiation exposure to the bladder and kidneys the patients were well hydrated and asked to void at frequent intervals during the first 48 hours (96 hours after infusion with j86Re-labelled 2o antibody).
For the beta- and gamma-emitting radionuclide 186Re, Breitz et al. (R98-2459) found a maximum tolerated dose of lg6Re-labelled MAb TgG in heavily pretreated patients of 90 mCi/m2. One of the aims of this study was to investigate the toxicity of 186Re-labelled hMAb BIWA 4 in patients who either had local and/or regional recurrent disease for which curative 2s treatment options were not available, or distant metastases. Because bone marrow, thyroid, kidney and liver toxicity might occur, blood samples for organ function testing were taken as described in section 3.5.1.6.
To monitor the occurrence of weight loss or weight gain the body weight was recorded at the screening visit, before the infusion on day 1 and after 6 weeks.
3o Patients were monitored for the occurrence of local toxicity. Any sign of local toxicity was recorded as adverse event.
3.5.2 Appropriateness of measurements The measurements used are well-accepted for this type of trial.
3.5.3 Primary efficacy / pharmacodynamic variables(s) 3s 3.5.3 .1 Part A of the trial The primary efficacy variables determined in Part A of the trial were the biopsy distribution and the radioimmunoscintigraphy. Description of the methods how to obtain the data is given in section 3.5.1.1 and 3.5.1.2.

The uptake in the normal tissue and tumour was measured in biopsies of the surgical specimen and the uptake expressed as percent of the injected dose per kg (% 117/kg),The time course of uptake in the tumour and other tissues was evaluated and compared concerning the different doses of BIWA 4.
3.5.3.2 Part B of the trial The primary parameters for ei~cacy were the analysis of radioimmunoscintigraphy and dosimetry.
3.5.4 Drug concentration measurements / pharmaco'netics Blood concentrations of radiolabelled BIWA 4 were determined in both Part A
and Part B of the trial.
~s 3.5.4.1 Methods and timing of sample collection Patient blood samples were collected and handled as follows: 7 mL of blood (3.5 mL in potassium EDTA containing tubes and 3.5 mL in coagulation tubes: amount of millilitres requested are given as per amendment 1) were sampled from a peripheral vein of the arm opposite to the infusion site at the designated time points (i.e. just prior to antibody 2o administration, at the end of the infusion and at 5, 10, 30 minutes; l, 2, 4, 16, 21, 48, 72 hrs post-end of infusion and on day 7 p.i. (144 hrs) and 6 weeks after the infusion). End of infusion was regarded as t = 0.
The planned samples at 10 minutes and 6 week p.i. were omitted for Part B of the trial, Instead samples were collected at 240 and 366 hour p.i. as per amendment 1.
ZS Urine was collected during 48 hrs after the 99mTc-labelled hMAb BIWA 4 infusion and during 96 hrs after the lg6Re-labelled hMAb BI~A 4 infusion. Urine was collected from 0-4 hrs, 4-8 hrs, 8-12 hrs, and 12-24 hrs during the first 24 hrs and in 24-hr samples for the remaining time.
Radioactivity of urine samples was counted to determine the excretion of radioactivity.
Blood samples were centrifuged to prepare serum. Before processing, the radioactivity in 30 whole blood was measured. Radioactivity was also measured in serum.
Refrigerated storage of blood samples was obligatory, though samples had not to be frozen down. Using the aliquot retained from the conjugate preparation, a weighted dilution of the injected patient dose was prepared as standard. The counting of the patient samples and standards were performed according to local SOPs. Results were recorded in the appropriate sections of the CRF. The 35 radionuclide content was reported as a percentage of the injected doses (expressed as % ID/kg blood or serum). Apart from radioactivity counting all samples were analysed for presence of immune complexes by means of I~'LC analysis.
All sample tubes were labelled with the following information: Trial number, patient number, sample ident~cation (i.e., serum, plasma, or blood), time relative to infusion, actual time, 4o actual date and isotope (i.e., 9smTc or 186Re). The volume of each urine sample was measured and recorded and all urine samples were labelled with the following information: Trial number, patient number, total sample volume, collection interval, actual time, date and isotope (i.e., 99mTc or ls6Re).
Dates, times and radioactivity measurement results of all pharmacokinetic samples were recorded in the CRF.
m The plasma samples for ELISA measurement were transferred in cryotubes, labelled carefully to enable unique identification, stored at -20°C until radioactivity had decreased (186Re: 4 weeks, 99mTc: 3 days) and sent to Boehringer Ingelheim Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany in batches every four weeks. The plasma samples were sent on dry ice.
15 3.5.4.2 Analytical determinations Plasma samples were measured by validated ELISA methods at the Boehringer Ingelheim Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany.
Radioactivity counting of the samples were performed in full blood, serum and urine by the investigator.
3.5.4.3 Parameters and evaluation 2o The following pharmacokinetic parameters using WinNonlin 3.1 Professional (Pharsight Corporation, Mountain View, CA) were determined from full blood, plasma, serum levels and imaging data as described below:
The primary pharmacokinetic parameters are the time point at which the maximum drug concentration is observed (Tm~), maximum drug concentration observed (Cm~), area under the zs concentration-time curve (AUC)o~~, terminal elimination half life (t1,2), volume of distribution (terminal phase and predicted steady state), total body clearance (CL), and mean residence time (MRT)o~~.
The primary objectives of pharmacokinetic analysis were:
To determine and compare the disposition of total radioactivity and immunoreactive hMAb 3o BIWA 4 following administration of single doses of 99mTc-labelled (Part A) and Ig6Re-labelled hMAb (Part B) BIWA 4.
To calculate, from imaging data and blood disposition data, the fraction of the injected radioactivity reaching the liver and spleen during the studies. To achieve this objective, the WinNonlin~ software package was used to perform non-compartmental pharmacokinetic 3s analysis on the plasma concentration vs. time profiles of immunoreactive hMA.b BTWA 4 and of total blood radioactivity levels, as a function of time, following intravenous administration.
The results are reported as summary statistics.

3.5.5 Pharmacokinetic / pharmacodynamic relationship A compartmental pharmacokinetic model was initially intended to describe the distribution and metabolism of lg6Re-labelled BIWA 4 in humans. It was intended to combine data from BIWA 4 plasma levels, full blood and serum radioactivity measurements, radioactivity from whole body images and specific regions of interest, the administered dose 186Re-labelled ~o BIWA 4, the dose of unlabelled BIWA 4, soluble CD44v6 levels and the assessment of the radiolabelled antibody prior to infusion.
The results were to be reported as summary statistics. However, variability was too high to be used for a reasonable model.
3.5.6 Primary Safety variable ~5 Determination of maximum tolerated dose and assessment of safety were the primary endpoints in Part B of the trial. The measurements done are described in section 3.5.1.6.
3.5.6.1 Dose Limiting Toxicity and Maximum Tolerated Dose DLT was defined as drug-related CTC grade 3 non-haematologic toxicity or drug-related CTC
grade 4 haematologic toxicity, excluding nausea and vomiting without adequate antiemetic 2o treatment.
The MTD was defined as the dose level at which less than two out of six patients developed drug-related DLT.
3.6 DATA QUALITY ASSUIIANCE
The trial was in general conducted according to the principles of Good Clinical Practice as ZS specified in the appropriate regulations and in the company standard operating procedures reflecting these regulations.
Throughout the course of the study, a representative from Boehringer Ingelheim The Netherlands was contacting and/or visiting the study site to monitor the progress of the study.
There were frequent contacts with the investigator and onsite visits for the purpose of data 3o audits, including the comparison of source documents with Case Report Forms and drug accountability checks. The investigator or his/her designee had to be available to the Boehringer Ingelheim The Netherlands representative during these onsite visits.
Duly filled in CRFs were collected on a regular basis. Data were double-data entered in-house.
Review of the data was done and implausibilities questioned to the investigator.
3s Serious adverse events were reported according to the guidelines defined in the protocol and CRF and according to Boehringer Ingelheim SOPs.

3.7 STATISTICAL METHODS PLANNED IN THE PROTOCOL AND
DETERMINATION OF SAMPLE SIZE
3.7.1 Statistical and analytical plans 3.7.1.1 Statistical design / model The designs used in this trial are commonly used in Phase I oncology trials.
~o Part A
Part A of this phase I trial was an uncontrolled, rising dose sequential group study to determine the safety, tolerability, biodistribution and pharmacokinetics of a single infusion of 99mTc-labelled hMAb BIWA 4 in patients with advanced squamous cell carcinoma of the head and neck.
~s Three hMAb BIWA 4 dose levels were used in this part of the study with three patients planned at each dose level. All patients had a proven tumour of the head and neck and were destined for surgery.
Part B
Part B of this phase I trial was an open uncontrolled dose escalation study to determine the ao safety, tolerability, MTD, pharmacokinetics and preliminary therapeutic effects of a single infusion of I86Re-labelled hMAb BIWA 4 in patients with advanced squamous cell carcinoma of the head and neck for whom no curative treatment options were available.
Tn this part of the study the radiation dose was escalated. Two patients were treated at dose levels where < grade 2 CTC toxicity was seen. If > grade 2 toxicity was seen at least three 2s patients were treated per dose group.
Safety and tolerability evaluation (Parts A + B) Safety and tolerability of the study were assessed in terms of changes in laboratory parameters, vital signs, development of HAHA and the incidence of adverse experiences. The results were reported for each dose level separately as well as in terms of overall means.
3o Biodistribution evaluation (Part A) The biodistribution of 99mTc-labelled hMAb BIWA 4 in solid tissue was assessed by radioimmunoscintigraphy (see sections 3.5,1.1) and by measurements of radioactivity of biopsy specimens (see sections 3.5. 1. l and 3.5.1.2). Because only a maximum of 3 patients were entered per dose level only descriptive statistics were possible.
3s Immunoscinti~raphic imagine evaluation (Parts A+B~

At given time-points after the administration of radio-labelled antibody immunoscintigraphic images were obtained (see section 9.5.1.1). Again only descriptive statistics were applicable.

Pharmacokinetic eyaluation (Parts A + B) The following pharmacokinetic parameters were determined from plasma or serum, urine levels and imaging data as described below:
Primary parameters (non-compartmental) Tmax, Cmax~ AUC (0-infinite time), terminal elimination half life, volume of distribution (terminal 1o phase VZ and predicted steady state V~), CL, and MRT (0-infinite time).
Cumulative urinary excretion of radioactivity over time was determined from total urine output.
Secondary_parameters fcompartmental) A compartmental pharmacokinetic model was planned to be developed to define the distribution and metabolism of lg6Re-labelled hMAb BIWA 4 in humans. The model collated ~s data from serum and urine radioactivity measurements, radioactivity from whole body images arid specific regions of interest, the administered dose 1$6Re-labelled hMAb BIWA 4, the cold-loading dose of hMAb BIWA 4, soluble CD44v6 levels and the assessment of the radiolabelled antibody prior to infusion.
The results were planned to be reported for each dose level separately and by means of o summary statistics.
Therapeutic ei~cacy evaluation (Part B) Tumour response was the parameter for therapeutic e~cacy. Tumour response was assessed according to the WHO guidelines. The sum of the products of the largest perpendicular diameters of all measured tumour lesions were the primary parameter for tumour response.
25 There were separate criteria for the assessment of response of bone lesions.
3.7.1.2 Null and alternative hypotheses All analyses in this trial were descriptive and exploratory by nature. Any statistical tests were performed only to provide a statistical framework from which to view the results and providing aid for planning further studies. No formal statistical inferences were foreseen and, 3o accordingly, no statistical tests were performed.
3.7.1.3 Planned analyses Two populations were distinguished:
~ The intention to treat subset consists of all patients who received an infusion of radiolabelled hMAb BIWA 4 and for whom data after baseline were available.
3s ~ The per protocol subset consists of evaluable patients. A patient was evaluable if the following criteria were met:

Part A:
1. Fulfilling the criteria for the intention to treat subset.
2. The follow-up in terms of adverse events, vital signs and safety blood and urine samples was adequate.
3. The immunoreactive fraction in at least one of both performed assays was larger than 60%.
to 4. Adequate biopsies and scintigraphic images were available to assess the biodistribution.
Part B:
1. Fulfilling the criteria for the intention to treat subset.
2. The follow-up in terms of adverse events, vital signs and safety blood and urine samples was adequate.
~s 3. The immunoreactive fraction in at least one of both performed assays was larger than 60%.
4. Response: Patients (Pts.) were evaluable for response if adequate tumour measurements were obtained at baseline and if patients were followed over at least a six week period. If disease progression was observed within this six weeks period, patients were also evaluable for response.
2o Primary Analyses The primary analysis was performed for the per protocol subset.
Non evaluable patients were replaced. For Part A therefore the evaluable subset was planned to consist of 9 patients and the intent to treat subset consisted of at least 9 patients. For Part B
the number of patients depended on the toxicity encountered. Patients Who were evaluable for 2s toxicity but not for response were not replaced.
Pharmacokinetic data:
The analysis of pharmacokinetic data was performed by Dr. Thomas R. MacGregor, Boehririger Ingelheim Pharmaceuticals Inc. Ridgefield CT, USA.
The primary objectives of pharmacokinetic analysis were:
30 1. To determine and compare the disposition of total radioactivity and immunoreactive hMAb BIWA 4 following administration of single doses of 99°'Tc-labelled hMAb BIWA 4 and nssRe-labelled hMAb BIWA 4.
2. To identify an appropriate cold-loading dose of hMAb B1WA 4.

3. To assess the disposition of a second dose of 186Re-labelled hMAb BIWA 4 4. To calculate, from imaging data and blood disposition data, the fraction ofthe injected radioactivity reaching the liver and spleen during the studies. To achieve this objective, the WinNonLin~ software package was used to perform noncompartmental pharmacokinetic analysis on the plasma concentration vs. time profiles of immunoreactive hMAb to and of total blood radioactivity levels, as a function of time, following intravenous administration. Any measurements involving radioactivity were normalised to the fraction of the radiolabel that was protein-bound and immunoreactive prior to infusion.
The specific pharmacokinetic parameters that were generated include: T",~, Cm~, AUC (0-infinite time), terminal elimination half life, volume of distribution (terminal phase and predicted is steady state), total body clearance, and mean residence time (0- infinite time). Cumulative urinary excretion of radioactivity over time were determined from total urine output.
The secondary objective of the pharmacokinetic analysis to develop a pharmacokinetic model to describe the distribution and metabolism of lg6Re-labelled hMAb BIWA 4 in humans was not achieved due to the high variability of the data (especially the radioscintigraphic data).
o Secondar~Analyses The secondary analyses were performed for the intention to treat subset of patients participating in Part B of the study.
The secondary analyses were restricted to key endpoints: e.g.: Response rate, duration of response, time to progression.
as Interim Anal, sis No interim analyses were made.
3.7.1.4 Handling of missing data In this phase I study it was anticipated that most data were available for analysis. In case of missing data it was most likely that the reason for missing was not outcome related. No 3o missing data were imputed.
3.7.2 Determination of sample size In Part A three patients per dose level provide some information on safety of the hMAb B1WA
4 and on the anticipated correct dose of SO mg unlabelled hMAb BIWA 4.
In Part B six patients were regarded sufficient to determine DLT. TABLE
3.7.2:1 exhibits the 3s probabilities of 2 or more out of 6 patients to be observed with DLT for some assumed underlying rates of DLT in the population of all patients.

TABLE 3.7.2: 1 Assumed Population Rates of DLT
0.40 0.42 0.45 Probability of observing DLT 0.77 0.80 0.84 in two or more out of six atients Source: Yrobabihties calculated from the cumulative distribution function of the binomial distribution with n=6 and varying p's.
With the escalation scheme in this trial, there was a probability of at least 80% for two or more patients to exhibit DLT, if the underlying individual probability for a patient to reach DLT is 42 to % or larger.
3.8 CHANGES IN THE CONDUCT OF THE STUDY OR PLANNED ANALYSES
The following changes from protocol were implemented via an amendment.
Time points for collection of blood samples were adjusted by implementation of amendment No 1 dated 1 September 1999. The samples at 10 minutes and 6 weeks after infusion were ~s dropped while additional samples were collected after 240 and 336 hours post end of infusion in Part B of the trial. It had turned out that the original blood sampling schedule was not su~cient to adequately cover an assumed half life of 50 hours which was observed in Part A of the trial. A total of 3.5 mL blood was collected in potassium EDTA tubes and 3.5 mL in coagulation tubes.
2o The protocol stated that patients could be administered a second dose of lg6Re-BIWA 4 after having determined the MTD. This procedure was modified by amendment 2.
Patients were considered eligible for a second treatment cycle in case they responded to the first administration of lg6Re-BIWA 4, were free of or had recovered from adverse events and had no HAHA. antibodies. Response was defined as stable disease (i.e. no change), partial zs remission or complete remission. The second dose administered was always 50 mCi/m2. Due to the change in the linker the trial was concluded before having investigated a second dose with the dosing regimen described in the protocol. To allow patients who responded to be treated anyhow, amendment 2 was issued.
The trial was completed and terminated after having reached the MTD due to the change of 3o the linker. MAG3 will be replaced by MAG2GABA-TFP in the future development of the BIWA 4 project.
Samples were available for sCD44v6 determination for more time points than originally planned.
The following changes were made after having evaluated the data and after discussion within 3s the trial team.

The ratio of %)17/kg tumour versus %)17/kg non-tumour tissue was performed for bone marrow only, due to the variability of the results.
The same holds true for the evaluation of the tumour size. Measurements were incomplete thus jeopardising a reasonable evaluation. Therefore, no formal analysis was done.

S 4. STUDY SUBJECTS
4.1 DISPOSITION OF SUBJECTS
Of the 33 patients screened three patients discontinued the trial due to adverse events before administration of the trial drug (TABLE 6.1: 1). These three patients are not included in the analyses. A total of 30 patients were entered and treated in this trial.
~o Part A
Ten patients of the 30 patients treated were included in Part A with 3 each receiving 25 mg and 100 mg 99mTc-BIWA 4, respectively, while four patients received 50 mg 99mTc-BIWA 4.
All patients in Part A completed the trial.
Part B
IS Two out of the 20 patients treated in Part B discontinued due to adverse events (two patients died). Three patients were administered a second dose of 50 mCi/ma.
TABLE 6.1: 1 Disposition of Patients Part Part Total A B

99'T'T'c-BIWA 186 Ci/m2]
4 Re-BIWA

[m mg mg mg BIWA BIWA BIWA

Screened 33 ~

Entered 3 4 3 2 4 3 6 5 30 Treated 3 4 3 2 4 3 6 5 30 Completed3 4 3 2 3 2 6 5 28 Discontinue d due to 0 0 0 0 1 1 0 0 2 AE

due to lack of efficacy due to other reason Source Appendix 16.1.9.2 TABLE 1.1 4.2 PROTOCOL DEVIATIONS
2o The majority of protocol violations were deviations (> ~ 10%) from the time windows provided in the protocol concerning blood and/or urine sampling. The patients affected were not excluded from the analyses but the actual times of collecting blood or urine were used for determination of blood, plasma, serum and urine concentrations.

5. EFFICACY / CLINICAL PgIAI~MACOLOGY EVALUATION
Results from Part A confirmed a dose of 50 mg BIWA 4 as the optimal dose for treatment.
Data from Part B indicate that patients may clinically benefit from lg6Re-BIWA
4 therapy at a radiation dose below that dose at which DLT was observed.
Concentrations of BIWA 4 measured were dose-proportional and a moderate amount of the dose administered was excreted via the kidneys.
5.1 DATA SETS ANALYSED
All patients who had received at least one dose of the trial drug were included in the intent-to-treat analysis. Seven patients from Part A were included in the per protocol analysis. No per protocol analysis was performed for Part B.
~s 5.2 DEMOGRAPHICS AND OTHER BASELINE CHARACTERISTICS
The mean age of all patients included was 56 years (range 37 to 78). Nineteen patients were males and eleven were females (TABLE 5.2: 1).
Three patients treated in Part A never smoked while seven were current smokers (pack years range 19 - 47). Frequent alcohol consumption was reported in eight patients while two were 2a non drinkers (TABLE 5.2: 1).
All patients in Part B were either ex-smokers or current smokers (TABLE 5.2:
1) with a mean pack-year of 35 (range 10 to 86). Six patients were non-drinkers while the remaining patients were frequent consumers of alcohol (TABLE 5.2: 1).
Stage of the disease was local operable for all patients included in Part A
while patients from 2s Part B had recurrent disease (15 patients) and/or metastases (3 patients).
One patient had local inoperable disease. In one patient the information is missing.
Concomitant diseases or relevant medical history were reported in 28 patients (TABLE 5.2: 1).
Concomitant therapy was required by all patients. The most often used medications were analgesics, sedatives, lactulose, H2-blockers, anti-emetics and antimicrobial agents.

s TABLE 5.2: 1 Demographics and baseline characteristics. Given are means for age. All other figures denote numbers of patients Part A Part B Total 9gn'Tc-BIWA 4 ~ 'g6 Re-BIWA 4 [mCi/m2]
25 mg 50 mg 100 mg 20 30 40 50 60 n 3 4 3 2 4 3 6 5 30 age [mean]45.7 58.5 58.7 57.0 61.0 53.0 57.0 60.4 56.4 male/female2/1 2/2 1/2 1/1 3/1 2/1 5/1 3/2 19/11 conc.disease3 3 2 2 4 3 6 5 28 concom. 3 4 3 2 4 3 6 5 therapy smoker 2 4 1 0 1 0 3 1 12 ex-smoker0 0 0 2 3 3 3 4 15 Alcohol 3 3 2 0 3 3 6 2 22 yes Initial diagnosis of disease was known in patients from Part A about one month before inclusion while patients included in Part B were ill since two years on average (range 0. I to To 17.5 years; TABLE 5.2: 2). The Karnofsky score ranged from 70 to 100 in patients from Part A. Patients from Part B had Karnofsky scores of 70 to 90 (TABLE 5.2: 2).
Metastases at diagnosis were reported for one patient in Part B of the trial.
All patients in Part B had undergone previous radio- and / or chemotherapy while patients in Part A had neither previous chemo- nor radiotherapy reported (TABLE 5.2: 2).
Cisplatin, ~s methotrexate and fluorouracil were the most often systemic anticancer drugs used).
Previous surgery was radical in eleven of the 13 patients in Part B who had a previous surgery.
Only one patient from Part A, had undergone previous surgery (TABLE 5.2: 2).
Lesion sizes (including metastases) of the tumours ranged from 14 mm2 to 10304 mma. The primary tumour site was moderately differentiated in the majority of the patients. Lymph nodes zo were affected in 13 patients (TABLE 5.2: 2).

s TABLE 5.2: 2 Demographics and baseline characteristics. Given are means for years of known disease and Karnofsky score. All other figures denote numbers of patients Part Part A B

s9n'Tc-BIWA I86 mCilm2]
4 Re-BIWA

[

25 50 mg 100 mg mg BIWA BIWA BIWA

n 3 4 3 2 4 3 6 5 years of known 0.11 0.08 0.07 9.30 2.13 1.17 0.80 1.16 disease Karnofsky 90 90 100 80 75 76.7 85 80 score radical surgery lymph nodes affected previous 0 0 0 1 3 1 4 1 chemotherapy previous 0 0 0 2 4 3 6 5 radiotherapy 5.3 MEASUREMENTS OF TREATMENT COMPLIANCE
~o All medication was administered under the supervision of the investigator or his/her delegate.
Blood was collected to determine the pharmacokinetics of BIWA 4. All patients had detectable blood levels of BIWA 4.
5.4 EFFICACY / CLINICAL PHARMACOLOGY RESULTS
Three out of six patients experienced stable disease at the maximum tolerated dose of ~s 50 mCi/m2. The dose of SO mg BIWA 4 was confirmed in Part A of the trial to be the optimal dose concerning blood concentrations and selective tumour uptake . The plasma concentrations of BIWA 4 were dose-proportional in the range of 25 mg to 100 mg BIWA 4 in Part A and peaked at 0.9 hours with a terminal elimination half life of 54 -74 hours and 94 hours for Part A and Part B, respectively.
2o 5.4.1 Analysis of efficacy / pharmacodynarnics 5.4.1.1 Primary endpoints Primary endpoint for effcacy was the biodistribution of 99'~'Tc-BIWA 4 in Part A. Further primary endpoints were the analyses of the radioirnmunoscintigraphic images and the pharmacokinetics (described in section 5.4.2) for Part A and B of the trial, respectively.
Dosimetry was done for Part B only.
Biodistribution of 99"'Tc-BIWA 4 in Part A
Tissue samples were obtained during surgery and the uptake of 9~"'Tc-BIWA 4 expressed as injected dose (ID)/kg tissue.
Intent-to-treat (ITT) subset: The relative biodistribution of 99~"Tc-BIWA 4 was highest in the tumour in all three dosing groups except for patient 1 (25 mg BIWA 4), patient 5 (50 mg BIWA 4, no tumour cells) and patient 9 (100 mg BIWA 4), respectively. The uptake in tumour ranged from 6 to 17 % m/kg, 5 to 28 % m/kg and 13 to 17 % 1D/kg for the dose group 25 mg, 50 mg and 100 mg, respectively. The calculated mean ratio of tumour uptake versus !s uptake in bone marrow was 1.7, 2.6 and 2 in the 25 mg, 50 mg and 100 mg dose group, respectively, (TABLES 7.2: 1 and 7.2: 2).
Per-protocol (PP) subset: The relative biodistribution of 99~'Tc-BIWA 4 was highest in the tumour in aII three dosing groups except for patient 1 (25 mg BIWA 4). The uptake in tumour ranged from 6 to 17 % JD/kg, 23 to 28 % m/kg and 16 % m/kg for the dose group 25 mg, 20 50 mg and 100 mg, respectively. The calculated mean ratio of tumour uptake versus uptake in bone marrow was 1.7, 3.2 and 2.5 in the 25 mg, 50 mg and 100 mg dose group, respectively, (TABLES 7.2: 1 and 7.2: 3).
A trend to greater uptake with smaller tumour size appeared to be present for the 25 mg BIWA 4 dose group. A similar evaluation was not possible for the 50 mg and 100 mg BIWA 4 25 dose groups due to the limited number of patients (TABLE 7.2: 4).
High %m/kg uptake was observed in bone marrow supernatant (up to 17 %ll7/kg) and plasma or blood (up to 13 %lD/kg) which was always below the uptake in the tumour in the per protocol population except for patient 1 (25 mg BIWA 4). Uptake in skin and mucosa was always below the uptake in primary tumour in the per protocol subset.
3o Radioimmunoscintigraphic images of 99"'Tc-BIWA 4 in Part A
While no radioactivity uptake was found in the tumour directly after infusion, a medium uptake of 1.9 was recorded after 21 hours. Uptake of radioimmunoconjugate appeared to be low in bone marrow and kidney both directly after infusion and 21 hours after infusion. Uptake was low to medium in lung while liver revealed a higher uptake.
~s A similar pattern was observed for the per protocol population. The only difference appeared to be a higher uptake in kidney while the uptake was lower in lung as compared to the intent-to-treat population.

Radioimmunoscintigraphic images of lasRe-BIWA 4 in Part B
Radioiummunoscintigraphic examinations were done immediately after infusion and 21, 48, 72, 144 and 336 hours after infusion. Hardly any uptake in tumour was observed directly after the infusion. Relative uptake in tumour appeared to be dose-dependent and increased over time reaching medium and high uptake after 72 to 144 hours with a decline after 336 hours.
~o Biodistribution of radioactivity was similar in bone marrow, lung, liver, kidney and intestine and did not reveal the same dose dependent effect (except for the intestine).
Moreover, relative uptake of radioactivity appeared to be constant or modestly decreased over time and was sinular for all doses of radioactivity. Uptake in bone marrow was lowest for most treatment groups.
~s 5.4.1.2 Secondary endpoints) Secondary endpoints of e~cacy were tumour response to therapy in Part B and the level of CD44v6 expression in tumour in Part A, respectively. Soluble CD44v6 expression was determined for both Part A and Part B of the trial.
CD44v6 expression in tumour in Part A
2o Data of determination of CD44v6 antigen expression is available for seven patients. CD44v6 antigen expression was observed in more than 90% and 80% of the tumour cells in five and two patients, respectively, while CD44v6 antigen expression was detected in more than 90% of the cells of lymph node metastases in the four patients for which lymph node was available for evaluation. Homogenous CD44v6 antigen expression was observed in all patients in the ZS mucosa.
Tumour response in Part B
Patients treated with doses of up to 40 mCilm2 ~s6Re-BIWA 4 did not experience a clinical tumour response. All patients experienced progressive disease. One patient was not evaluable due to intercurrent death.
3o Three of the six patients treated with 50 mCi/m2 iasRe-BIWA 4 developed stable disease or no change after the first cycle. Two of these three patients who were treated with another cycle with 50 mCilm2 lssRe-BIWA 4 had progressive disease after the second cycle.
Progressive disease was observed after a total of 148 days (patient 109) and 127 days (patient 116) after first administration of the trial drug, respectively.
3s One patient treated with 60 mCi/m2 lssRe-BIWA 4 experienced stable disease after the first cycle. This patient went into progressive disease after a second cycle with 50 mCilm2 is6Re-BIWA 4 (173 days after the first administration of the trial drug).
The overall time to progression for patients who did not respond to therapy ranged from 0 to 55 days with a mean of about five to six weeks irrespective of the treatment group.

Soluble CD44v6 in Part A and B
Soluble CD44v6 was determined in all patients treated with BIWA 4 in the present trial.
The amount of soluble CD44v6 detected tended to increase for the 186Re-BIWA 4 treated patients (TABLE 7.2: 6) during the trial in Part B while for patients treated with 99mTc in Part A no such trend could be observed.
l0 5.4.2 Drug dose, drug concentration, and relationship to response The pharmacokinetics of BIWA 4 will be presented separately for Parts A and B
because the radiolabel was different (Part A: 99"'Tc; Part B: 186Re).
5.4.2.1 Analytical performance with regard to BIWA 4, HAHAs and CD44v6 Validated assays were used for analysis of plasma samples. Analysis of quality control samples ~s produced results of high accuracy and precision.
Analytical performance with regard to gamma counting of samples The signal linearity of the scintillation crystal in the gamma counter was not tested. It can be assumed based on the type of instrument at least in the energy range that was used in the current study. Calibration samples showed typically 40000-200000 counts per minute vs less 2a than 50-100 counts per minute in background samples. The precision of the seven measurements per calibration standard was regularly within 2%. The same applies for the precision of the individual triplicate measurements in blood, serum and urine.
Blood and serum samples showed typically activities between 2000 and 100000 counts per minute.
There were no quality control samples for radioactivity counting. The precision of repeated 2s measurements of calibration standards and samples derived from the current study indicate that the precision of triplicate measurements was at least better than 2 %.

s 5.4.2.1 Pharmacokinetics Part A.

Part A consisted 25 to I00 of 10 patients mg receiving a single BIWA
4 dose ranging from with a constant iolabelled 99mTc. armacohinetic rad dose The ph parameters of in 20 plasma mCi for BIWA 4 measured short by ELISA following intravenous a infusion are given in TABLE

~0 5.4.2.2: I.

TABLE 5.4.2.2: Pharmacokin etic plasmam Part A
I parameters fro of the for BIWA
4 in study Patient BIWA tmax Cmax Half AUC;"f Vz CL MRT

Dose (min) (ng/mL)Life (~.g'h/mL)(L) (mL/min)(h) (mg) (h) 1 25 0 6634 44.1 387.4 4.1 1.076 58.0 2 25 30 6640 67.0 534.7 4.5 0.779 94.8 3 25 10 9590 53.3 542.9 3.5 0.768 71.0 4 50 29 1490064.9 1112.3 4.2 0.749 84.8 S 50 121 15500102.8 1580.2 4.7 0.527 135.2 8 50 5 1390053.0 831.0 4.6 1.003 71.7 50 41 1670083.8 1461.6 4.1 0.570 110.0 6 100 4 3050085.0 2548.9 4.8 0.654 112.0 7 100 17 3060076.0 2709.6 4.0 0.615 110.4 9 100 30 2450044.6 1708.1 3.8 0.976 61.1 Geometric mean 25 mg . 7503 54.0 482.7 4.0 0.863 73.1 50 mg 29 1521673.8 1208.8 4.4 0.689 97.5 I00 mg 13 2838366.0 2276.4 4.2 0.732 91.1 The increase in C",~ BIWA 4 plasma concentrations observed and the area under the curve were proportional to the dose administered (FI(yURES 4 and 5).
Serum concentrations of the radiolabel (9s"'Tc) were also determined and the pharmacokinetic parameters are presented in TABLE 5.4.2.2: 2.

s TABLE 5.4.2.2: 2 Pharmacokinetic parameters for 99mTc-BIWA 4 in serum from Part A of the study Patient 99"'Tc-BIWATmaxCmax Half AUC;I,fVz CL MRT
4 Life Dose (mCi) (min)(%117/kg)(h) (%IDh/kg)(kg)(kg/min)(h) 1 19.0 mCi 117 52.66 44.6 3376.9 1.90.030 63.8 2 20.0 mCi 10 28.08 55.6 2151.7 3.70.048 79.1 3 21.4 mCi 10 39.11 27.5 1445.3 2.70.072 41.5 4 19.5 mCi 29 31.84 31.3 1432.8 3.20.072 43.8 19.1 mCi 121 32.29 39.6 -1934.83.00.054 56.3 8 18.8 mCi 114 28.41 42.8 1620.1 3.80.060 60.8 19.3 mCi 41 40.31 43.1 2247.1 2.80.042 62.3 6 19.3 mCi 120 30.45 45.3 1946.5 3.40.054 65.2 7 19.8 mCi 77 35.79 36.0 1628.6 3.20.060 51.1 9 18.3 mCi 238 31.94 35.5 1805.6 2.80.054 50.6 Harmonic mean 38.7 Geometric mean 57.934.5 39.4 1898.0 3.00.053 56.5 The geometric mean half life for BIWA 4 measured by ELISA in plasma following intravenous infusion ranged from 54 to 73.8 hours for the three treatment groups studied in Part A. The geometric mean half life fox the 99mTc-radiolabeled BIWA 4 for the same samples was shorter at 39.4 hours in serum and 46.4 hours in blood. The discrepancy between the two mean estimates was attributed to longer sampling times possible with BIWA 4 due to radioactivity assay restrictions.
Radioactivity was excreted into the urine in the amounts collected in TABLE
5.4.2.2: 3. Due rs to incomplete collections over the 48 hours (i.e. cumulative data may not always be comparable because of varying duration of collection), no further assessment was made of this urinary excretion data. For six patients with complete 48-hour collections, the mean amount of radioactivity collected was 10% of the initial dose.

s TABLE 5.4.2.2: 3 Amount of radioactivity (99mTc) excreted (as percent of initial dose) into urine in Part A
Collection Interval (h) Patient0-4 4-8 8-12 12-24 24-48 Cumulative Amount (%ID) 1 2.80 10.13 5.08 n.a. n.a. 18.01 2 1.94 0.97 0.52 3.09 3.23 9.75 3 0.29 1.44 1.19 n.a. n.a. 2.92 4 1.68 1.42 1.95 1.14 2.21 8.40 2.20 1.60 1.02 3.96 3.98 12.76 8 1.29 2.45 0.88 3.67 3.00 11.29 n.a. n.a. n.a. n.a. n.a.

6 2.55 1.59 1.80 1.89 2.00 9.83 7 n.a. 3.14 n.a. 1.79 3.13 8.06 9 1.54 3.34 0.00 1.05 2.31 8.24 n.a. = not applicable, no data available Part B:
Part B consisted of 20 patients receiving BIWA 4 doses of 50 mg with varying radiolabelled doses of lB~Re-BIWA 4 ranging from 20-60 mCi/mz. Three patients received a second intravenous infusion while the other 17 patients received only a single course of therapy.
Graphically, there was consistency between plasma BIWA 4 concentrations and serum radioactivity concentrations for each patient and within the three patients that received ~86Re-IS BIWA 4 on two occasions. This consistency observed graphically contrasts with the perception of a longer half life for radioactivity when the data was modelled (noncompartmental evaluation). The geometric mean half life of 122 hours for the radioactive portion of the 186Re-BIWA 4 was longer than the ELISA-determined plasma half life of 94 hours for BIWA 4.
Grouping (TABLES 5.4.2.2: 6 and 5.4.2.2: 7) the pharmacokinetic parameters by amount of 2o radioactivity administered showed some trends of longer exposure with increased radioactivity dosed, but the numbers of individuals in each dose group are too small to make any consistent conclusions.

s TABLE 5.4.2.2: 6 Geometric means for plasma BIWA 4 pharmacokinetic parameters grouped by amount of radioactivity given.
'86Re Dosen Cmax Half AUC;"f Vz CL MRT
Life (mCi/m2) (n~~') Ch) (wgh/mL) (L) (mL/h) (h) 20 2 12160 106.5 1217 6.3 41.1 130.3 30 4 15239 86.2 1399 4.4 35.7 116.2 40 3 11121 90.6 1201 5.4 41.7 125.1 50 9'~ 12982 92.4 1055 6.3 47.4 117.4 60 5 11927 99.8 939 7.7 53.2 115.9 * patients who received two doses of 50 mCi/m2 are included TABLE 5.4.2.2: 7 Geometric means for serum 186Re-BIWA 4 pharmacokinetic parameters grouped by amount of radioactivity given.
'$6Re Dosen Cmax Half AUC;~,f Vz CL MRT
Life (mCi/m2) (%m/kg) (h) (%n7h/kg)(kg) (lcg/h)(h) 20 2 30.88 I14.4 3296 5.01 0.030 154.6 30 4 40.09 98.8 4365 3.27 0.023 134.5 40 3 28.82 126.4 3856 4.73 0.026 172.4 50 9* 40.22 123.3 4182 4.25 0.024 162.5 60 4 32.10 145.2 3493 6.00 0.029 185.5 * patients who received two doses of 50 mCi/m2 are included 5.4.2.2 Pharmacokinetic / pharmacodynamic analysis A pharmacokinetic/pharmacodynamic analysis was not performed.

s 5.4.3 Statistical / analytical issues The analysis was performed as originally planned. Details are described in section 3.7.1.3.
5.4.3.1 Primary endpoints) Not applicable.
5.4.3.2 Secondary endpoints) to Not applicable.
5.4.4 Drug-drug and drug-disease interactions No tests concerning drug-drug or drug-disease interactions were done.
5.4.5 By-subject displays For details see section 6.4.2.2 and 6.4.2.3.
~s 5.5 EFFICACY / CLINICAL PAARIVrACOLOGY CONCLUSIONS
Part A:
Results from Part A confirmed a dose of 50 mg BIWA 4 as the optimal dose for treatment based on blood concentrations and tissue uptake level. The distribution as assessed by radioscintigraphy and biopsy measurements was in almost all cases highest in the tumour as zo compared to other tissues. Uptake of radioactivity increased in the tumour over time. CD44v6 expression was present in more than 80 % and 90% of the cells of primary tumour and lymph node metastases, respectively, and in all mucosa specimens obtained.
The amount of soluble CD44v6 appeared to be constant before and after 99mTc-administration.
ZS Concentrations of BIWA 4 measured were dose-proportional in the range of 25 mg to 100 mg BIWA 4. A moderate amount of the dose administered was excreted via the kidneys. The B7 excreted in urine was similar for all dosing groups.
Part B:
Data from Part B indicate that patients may clinically benefit from 186Re-BIWA
4 therapy at 3o MTD. One out of five patients treated with 60 mCi/m2 had stable disease.
Three out of six patients experienced stable disease at the maximum tolerated dose of 50 mCi/m2. Time until progression ranged between 127 and 173 days in those patients who received a second dose of 50 mCi/ma. Radioimmunoscintigraphy indicates uptake of radioactivity in tumour tissue.
Biodistribution was comparable in other tissues irrespective of dose.
Dosimetric analysis did 35 not reveal unexpected high absorbed doses in tissues other than the tumour except for the testes. However, the relevance of the observed testes dose for the impairment of fertility is currently unclear.
The amount of soluble GD44v6 detected tended to increase for the 186Re-BIWA 4 treated patients.
The plasma concentrations of BIWA 4 peaked at 0.92 hours and the antibody was eliminated o with a geometric mean half life of 94 hours for BIWA 4 determined by ELISA.
C",~ and AUC
values (ELISA measurement) were similar to those obtained in Part A of the trial for the 50 mg BIWA 4 dose group.

Dose limiting toxicity occurred at a dose of 60 mCi/m2while a dose of 50 mCi/ma is6Re-BIWA 4 turned out to be the maximum tolerated dose in the present trial. The dose limiting toxicity were adverse events from the bone marrow i.e. thrombocytopenia and leucopenia. -s 6.1 EXTENT OF EXPOSURE
All ten patients treated in Part A of the trial were administered one single dose of 99"~Tc-B1WA 4.
Doses of BIWA 4 were either 25 mg, 50 mg or 100 mg while 99mTc was 20 mCi.
In Part B, 20 patients were administered one single dose of ls6Re-BIWA 4.
Three patients received a second dose of 50 mCilma due to stable disease (no change). The dose of BIWA 4 was kept stable at 50 mg while the radioactivity of ls6Re was increased in 10 mCi/m2 body surface increments.
The dose was calculated according to body surface area (TABLE 6.1: 1).
TABLE 6.1: 1 Mean dose of radioactivity administered Part A Part B

99ITC-BIWA 4 '86 Re-BIWA 4 [mCilm2]

25 mg 50 mg 100 20 30 40 50 60 mg 20.11. 19.20. 19.10.
mC199mTC

2 3 8 n.a n.a n.a n.a n.a mCi'$6Re n.a. n.a n.a 33.1~1. 82.4~5.6 70.615. 87.2~8. 97.2~8.

n.a. = not applicable, mean values and standard deviation are given rs The radiochemical purity of the drug was more than 95 % and the immunoreactive fraction was higher than 80 %.
An observation period of at least 6 weeks followed the administration of the trial drug.
6.2 ADVERSE EVENTS (AEs) 2o Adverse events were reported in all patients treated. Two patients discontinued the observation period in Part B due to an AE. No action was taken with the trial drug since treatment had been completed.

Part A:
The majority of patients in Part A experienced adverse events from body as a whole (50 %) which may be also due to the underlying surgery and the associated pain. No special pattern of adverse events was reported and none of the patients developed CTC criteria 3 or 4 which were judged to be drug-related (TABLES 6.2.2: 1 and 2).
Three patients experienced serious adverse events which are described in section 6.3.1.
Part B:
Adverse events of the system organ class (SOC)'body as a whole' were reported in 65 % of the to patients, followed by adverse events from the system organ class 'platelet, bleeding and clotting disorders' (60 % ofthe patients) and 'white cell and reticulo endothelial system disorders' (50 % of the patients). Drug-related thrombocytopenias and leucopenias occurred in 11 (55 %) and 10 (50 %) patients on average 21 days after start of radioimmunotherapy and were generally reversible after 6 weeks. A dose-response was observed for these events (TABLE
6.2.2: 3).
m Haematological dose-limiting toxicity defined as drug-related CTC grade 4 was reported in three patients.
Non-haematological dose-limiting toxicity defined as drug-related CTC grade 3 or 4 non-haematological toxicity occurred in 4 patients (at 30 mCi/m2one patient experienced rash and Quincke's oedema, two patients experienced fever at 60 mCi/m2and one patient treated with 60 mCi/m2 experienced fatigue.
Six of the nine patients who experienced serious adverse events died. Deaths and serious adverse events are described in section 12.3.1.
6.2.2 Display of adverse events Part A:
2s The following adverse events sorted by SOC, preferred term and the three dose groups of B1WA 4 were reported in more than one patient in Part A of the trial (TABLE
6.2.2: 1). More details as well as the preferred terms can be found in section 7.3.1.
TABLE 6.2.2: 1: Patients with adverse events in Part A (pre-surgical treatment with a single dose of 20 mCi 99mTc-BIWA 4) 99mTc-BIWA 4 25 mg B1WA 4 50 mg BIWA 4 100 mg BIWA 4 total number of patients (% ofpatients 3 (100) 4 (100) 3 (100) IO (100) treated) number of patients with any adverse 3 (100) 4 (100) 3 (100) IO (I00) event application site disorders 2 (66.7) 0 (0) 0 (0) 2 (20.0) body as a whole 2 (66.7) 2 (50.0) 1 (33.3) 5 (50.0) Pin 1 (33.3) 1 (25.0) 1 (33.3) 3 (30.0) metabolic and nutritional1 (33.3) 2 (50.0) 1 (33.3) 4 (40.0) disorders weight decrease 1 (33.3) 1 (25.0) 0 (0) 2 (20.0) resistance mechanism 0 (0) 4 (100) 0 (0) 4 (40.0) disorder Infection 0 (0) 3 (75.0) 0 (0) 3 (30.0) vascular disorders I (33.3) 1 (25.0) 1 (33.3) 3 (30.0) Haemorrhage (not specified)1 (33.3) 1 (25.0) 0 (0) 2 (20.0) given are SOCs and preferred terms in case they were reported in more than one patient; figure in brackets denote percentage of patients treated The events which were considered drug-related by the investigator graded according to CTC
s criteria are listed in TABLE 6.2.2: 2. The events were grade 1 according to CTC.
TABLE 6.2.2: 2 Severity of drug-related (as judged by the investigator) adverse events according to CTC criteria reported in Part A (pre-surgical treatment with a single dose of 20 mCi 99mTc-BIWA 4).
99"'Tc-BIWA 4 25 mg BIWA50 mg 100 mg total number of patients 3 4 3 10 Number of adverse events considered drug-related Liver and biliary system0 2 0 2 disorder sGOT increased 0 1 0 1 sGPT increased 0 1 0 1 sGOT = serum glutamic oxalacetic transaminase ; sGPT = senlin glutamic pyruvic transaminase Part B:
TABLE 6.2.2: 3 presents the number of patients with adverse events sorted by SOC, preferred term and the radiation dose groups of 186Re- BIWA 4.

iasRe-BIWA 4 first and second doses considered) ~86Re-BIWA 4 [mCi/m2]
20 30 40 50 60 total number of patients treated 2 (100) 4 (100) 3 (100) 6 (100) 5 (100) 20 (100) number of patients with adverse 2 (100) 4 (100) 3 (100) 6 (100) 5 (100) 20 (100) events body as a whole 2 (100)3 2 (66.7)2 (33.3)4 (80.0)13 (65.0) (75.0) allergic reaction 0 (0) 1 0 (0) 0 (0) 1 (20.0)2 (10.0) (25.0) Fatigue 0 (0) 0 0 (0) 1 (16.7)I (20.0)2 (10.0) (0) Fever 0 (0) 0 2 (66.7)0 (0) 2 (40.0)4 (20.0) (0) oedema mouth 1 (50.0)0 0 (0) 1 (16.7)0 (0) 2 (10.0) (0) Pain 0 (0) 2 0 (0) 1 (16.7)1 (20.0)4 (20.0) (50.0) central and peripheral0 (0) 1 2 (66.7)1 (16.7)0 (0) 4 (20 nervous (25.0) 0) system disorder .

gastro-intestinal 0 (0) 1 1 (33.3)2 (33.3)3 (60.0)7 ( 35.0) system disorder (25,0) Mucositis 0 (0) 0 0 (0) 1 (16,7)3 (60.0)4 (20.0) (0) Stomatitis 0 (0) 0 0 (0) 1 (16,7)1 (20.0)2 (10.0) (0) liver and biliary 1 (50.0)0 0 (0) 0 (0) 1 (20.0)2 (10.0) system disorders (0) sGPT increased 1 (50.0)0 0 (0) 0 (0) 1 (20.0)2 (10.0) (0) metabolic and nutritional1 (50.0)0 1 (33.3)1 (16.7)1 (20.0)4 (20.0) disorders (0) Neoplasm 0 (0) 0 2 (66.7)0 (0) 2 (40.0)4 (20.0) (0) neoplasm malignant 0 (0) 0 1 (33.3)0 (0) 2 (40.0)3 (15.0) aggravated (0) platelet, bleeding 0 (0.0)1 1 (33.3)5 (83.3)5 (100)12 (60 and clotting (25.0) 0) disorders .

Thrombocytopenia 0 (0.0)1 1 (33.3)4 (66.7)5 (100)11 (55.0) (25.0) red blood cell disorder0 (0) 0 0 (0) 2 (33.3)3 (60.0)5 (25.0) (0) Anaemia 0 (0) 0 0 (0) 2 (33.3)3 (60.0)5 (25.0) (0) respiratory system 0 (0) 1 1 (33.3)0 (0) 2 (40.0)4 (20.0) disorder (25.0) Bronchitis 0 (0) 1 0 (0) 0 (0) 1 (20.0)2 (10.0) (25.0) I~yspnoea 0 (0) 0 0 (0) 0 (0) 2 (40.0)2 (I0.0) (0) Rhinitis 0 (0) 0 1 (33.3)0 (0) 1 (20.0)2 (10.0) (0) skin and appendages 1 (50.0)1 1 (33.3)0 (0) 1 (20.0)4 (20.0) disorder (25.0) Rash 0 (0) 1 0 (0) 0 (0) 1 (20.0)2 (10.0) (25.0) white cell and RES 0 (0) 0 1 (33.3)4 (66.7)5 (100)10 (50.0) disorder (0) Granulocytopenia 0 (0) 0 0 (0) 1 (16.7)2 (40.0)3 (15.0) (0) Leucopenia 0 (0) 0 1 (33.3)4 (66.7)5 (100)10 (50.0) (0) given are SOCs and preferred terms in case they were reported in more than one patient; sGPT = serum glutatnic pyruvic transaminase ; RES = reticulo endothelial system, figure in brackets denote per centage of patients treated s Drug-related adverse events were reported in 75 % of the patients. Drug-related leueopenia was reported in 50 % and thrombocytopenia in 55 % of the patients, respectively.
Adverse events considered drug-related are provided in TABLE 6.2.2: 4 graded according to the CTC.

CTC criteria reported in Part B (first and second dose considered) adverse event 186Re-BIWA

~mCi/m2~

20 30 40 50 60 total CTC grade 1 0 2 6 5 14 face oedema 1 n.a.0 0 0 1 leucopenia 0 n.a.1 2 0 3 moniliasis 0 n.a.0 0 1 . 1 mucositis 0 n.a.0 0 1 1 platelets abnormal0 n.a.0 1 0 1 purpura 0 n.a.0 0 1 1 stomatitis 0 n.a.0 1 0 1 thrombocytopenia0 n.a.1 2 2 5 CTC grade 0 1 0 7 7 15 anaemia n.a. 0 n.a. 1 1 2 gout n.a. 0 n.a. 1 0 1 leucopenia n.a. 0 n.a. 1 2 3 mucositis n.a. 0 n.a. 2 3 5 stomatitis n.a. 0 n.a. 0 1 1 taste Loss n.a. 0 n.a. I 0 1 thrombocytopenian.a. 1 n.a. I 0 2 CTC grade 0 2 0 2 8 12 3 .

allergic reactionn.a. 1 n.a. 0 0 1 fatigue n.a. 0 n.a. 0 1 1 fever n.a. 0 n.a. 0 2 2 leucopenia n.a. 0 n.a. 1 1 2 rash n.a. 1 n.a. 0 0 1 thrombocytopenian.a. 0 n.a. 1 4 5 CTC grade 0 0 0 3 5 8 anaemia n.a. n.a.n.a. 0 1 1 granulocytopenian.a. n.a.n.a. 1 2 3 leucopenia n.a. n.a.n.a: 1 2 3 thrombocytopenian.a. n.a.n.a. 1 0 1 given is the number of adverse events; n.a. = not applicable s 6.2.3 Analysis of adverse events Part A:
None of the adverse events occurring in Part A of the trial were considered drug-related except for one patient experiencing mild and reversible CTC grade 1 elevation of AST
and ALT.
No difference in AE profile was observed when comparing the different B1WA 4 doses.
ro Part B:
Thrombocytopenia and leucopenia were dose-dependent (TABLES 6.2.2: 3 and 6.2.2: 4) and dose-limiting. Time course is provided in section 6.4.2.1.

causing dose-limiting toxicity (TABLES 6.2.2: 3 and 6.2.2: 4) An allergic reaction was reported in two patients, one experiencing drug-related Quineke's oedema and rash (30 mCi/m2) and one patient (60 mCi/m2) had an allergic reaction after a thrombocyte concentrate transfusion, which was not considered drug-related.
One patient 20 mCi/m2) had urticaria which also was not considered drug-related and one patient (20 mCi/m2) experienced drug-related face oedema.
_H_AHAs were detected in two patients (see section 12.4.3).
Discontinuations due to adverse events occurred in two patients. Both patients died.
Adverse events required initiation of concomitant therapy in 17 patients.
These patients are shortly described in section 6.3.1.
6.3 DEATHS, OTHER SERIOUS ADVERSE EVENTS, AND
OTHER SIGNIFTCANT ADVERSE EVENTS
Serious adverse events were reported in 12 patients. Of those six patients died in the course of the is trial or follow-up. AlI patients who died were treated in Part B of the trial. None of the patients treated in Part A died. All fatalities were due to disease progression.
Two patients in Fart B discontinued the trial prematurely due to adverse events (death in both patient 112 and 105).
Adverse events requiring therapy were most often thrombocytopenia, leucopenia and fever.

INCLUDED IN THE TEXT
7.1 DEMOGRAPHIC DATA
Details of demographics are not included.
s 7.2 EFFICACY / PHA.RMACODYNAMIC DATA
TABLE 7.2: 1 Tumour to bone marrow uptake ratio - Part A of the trial TABLE 7.2: 2 Tumour to bone marrow uptake ratio (ITT subset) -Part A
of the trial TABLE 7.2: 3 Tumour to bone marrow uptake ratio (PP subset) - Part A
to of the trial TABLE 7.2: 4 Tumour size and % uptake of antibody - Part A of the trial (per protocol population) TABLE 7.2: 5 CD44v6 antigen expression in tumour and other tissue - Part A of the trial fs TABLE 7.2: 6 Soluble CD44v6 in serum. Mean values are given in ng/mL

Part A
99n"TC-BIWA 4 patient No o uptake tumour optake bone marrow ratio tumour / bone ( /o1D/kg~ calculated) ( /o1D/kg~ calculated) marrow (calculated) 25 mg BIWA 4 1* 6.14 5.58 1,10 2* 15.78 ~ 7.49 2.11 3 * 16.78 8.63 1.95 50 mg BIWA 4 4* 28.10 6.98 4.03 8* 27.76 7.57 3.67 10* 22.63 11.54 1.96 100 mg BIWA 4 6* 15.89 6.47 2.46 7 16.99 7.70 2.21 9 13.31 10.11 1.32 No = number, * = included in per protocol analysis; °fo 11.7 = %
injected dose; calculations were done by taking different weights of samples into account s TABLE 7.2: 2 Tumour to bone marrow uptake ratio (ITT subset) - Part A of the trial Part A

99mTc BIWA 4 ratio tumour / bone marrow (calculated) 25 mg BIWA 1.72 50 mg BIWA 2.57 100 mg BIWA 1.99 mean values are given; TTT = intent-to-treat; calculations were done by taking different weights of samples into account TABLE 7.2: 3 Tumour to bone marrow uptake ratio (PP subset) - Part A of the trial Part A

99"'Tc-BIWA 4 ratio tumour / bone marrow (calculated) 25 mg BIWA 1.72 50 mg BIWA 3.22 100 mg BIWA 2.46 mean values are given; PP = per protocol; calculations were done by taking different weights of samples into account !0 population) Part A
99"'Tc-BIWA 4 patient No tumour sizeuptake tumour (o>Z7/kg;
[mm2]

calculated) 25 mg BIWA 4 1 1350 6.14 2 900 15.78 3 225 16.78 50 mg BIWA 4 4 n.a. 28.10 8 1504 27.76 1400 22.63 100 mg BIWA 4 6 800 15.89 No = patient number, n.a.
= not applicable, only length or width given but not length and width; calculations were done by taking different.weights of samples into account s TABLE 7.2: 5 CD44v6 antigen expression in tumour and other tissue - Part A of the trial Part A

99"'Tc-BIWA

CD44v6 antigenCD44v6 expressionCD44v6 expression patient No expression m mucosa in lymph in tumour node metastases 25 mg BIWA 4 I* n.a. n.a. n.a.

2* ++/+++; > +++ ++/+++;
80 % > 90 3 * -h+-+-; > +++ +-I-+-;
90 % > 90 50 mg BIWA 4 4* -H-+; > 90 +-H- +++; > 90 %

8* n.a. n.a. n.a.

10* ++.; > 90 +++ +++; > 90 %

100 mg BIWA 4 6* -H-+; > 80 +++ n.a.
%

7 +++; > 90 +-I+ n.a.
%

9 +++; > 90 +++ n.a.
%

No = number; * = included in per protocol analysis, n.a. = not applicable Part Part A B

99"'Tc-BIWA 186 4 Re-BIWA

[mCi/mz]

25 50 mg 100 20 30 40 50 60 mg mg BIWA BIWA

BIWA

pre-dose 203 337 174 408 262 346 171 160 21 hours 223 301 179 396 177 403 199 170 48 hours 200 275 157 384 198 423 199 174 72 hours 140 386 n.a. n.a. n.a. n.a. n.a. n.a.

144 hours188 282 182 444 284 516 233 195 240/336 n.a. n.a. n.a. 445 337 n.a. n.a. n.a.
hours week 6 203 310 210 289 220 487 226 162 n.a. = not applicable, data include the second dosing s Efficacy results: Part A:
Results from Part A confirmed a dose of 50 mg BIWA 4 as the optimal dose for treatment based on blood concentrations and tissue uptake levels. The distribution as assessed by radioscintigraphy and biopsy measurements was in almost all cases highest in the tumour as compared to other tissues. Uptake of radioactivity increased in the tumour over time. CD44v6 expression was present in more than 80 % and 90% of the cells of primary tumour and lymph node metastases, respectively, and in all mucosa specimens obtained.
No correlation was observed between tumour size and uptake of radioactivity in the 50 mg BIWA 4 dose group. The amount of soluble CD44v6 appeared to be constant before and after 99""fc-BIWA 4 administration.
Concentrations of BIWA 4 measured were dose proportional in the range of 25 mg to 100 mg BIWA 4. A moderate amount of the dose administered was excreted via the kidneys. The percent injected dose (% ID) excreted in urine was similar for all dosing groups.
Part B:
Data from Part.B indicate that patients may clinically benefit from 186Re-BIWA
4 therapy at maximum tolerated dose (MTD). One out of five patients treated with 60 mCi/m2 had stable disease. Three out of six patients experienced stable disease at the maximum tolerated dose of 50 mCi/m2. Time until progression ranged between 127 and 173 days in those patients who received a second dose of 50 mCi/m2. Radioimmunoscintigraphy indicates uptake of radioactivity in tumour tissue. Biodistribution was comparable in other tissues irrespective of dose. Dosimetric analysis did not reveal unexpected high absorbed doses in tissues other than the tumour except for the testes. The amount of soluble CD44v6 detected tended to increase for the'86Re-BIWA 4 treated patients.
The plasma concentrations of BIWA 4 peaked at 0.92 hours and the antibody was eliminated with a geometric mean half life of 94 hours for BIWA 4 determined by enzyme-linked immuno-sorbent assay (ELISA) measurement.
Cm~ and AUC values (ELISA) were similar to those obtained in Part A of the trial for the 50 mg BIWA 4 dose group.

Tolerability of single dose BIWA 4 coupled to low radiation dose of Technetium-99 was acceptable. Two of the three serious adverse events were due to complications as a result of surgery.
Part B:
The maximum tolerated dose (MTD) was 50 mCilm2'ssRe-BIWA 4.
Dose-limiting adverse events were dose dependent reversible reductions in thrombocyte and leucocyte count with subsequent fever in individual patients.
Clinical symptoms of thrombocytopenia were mild petechiae.
Mucositis was observed in patients treated with higher radiation doses but was not dose-limiting.
No relevant changes in thyroid stimulating hormone (TSI~ values were observed during the course of the trial.
Twelve patients experienced a serious adverse event. Of those, six patients died during the course of Part B of the trial mainly due to progression of the underlying disease.
Allergic reactions were observed rarely with one serious drug-xelated Quincke's oedema. No allergic reactions occurred during or shortly after infusion of the drug.
Two patients developed HAHAs. Repeated dosing did not induce HAHA
development.
Conclusions: Results indicate uptake of 1$6Re-BIWA 4 in tumour tissue and clinical benefit in patients with advanced squamous cell carcinoma of the head and neck. The safety profile appears to be acceptable. BIWA 4 showed dose-proportional pharmacokinetics and tumour uptake did not change relevantly between doses of 50 mg and 100 mg BIWA4.

Example 4 Introduction. Patients with an advanced stage of head and neck squamous cell carcinoma s (HNSCC) have an increased risk for development of locoregional recurrent tumors and/or distant metastases. For these patients, development of an effective adjuvant systemic treatment is needed.
Knowing that HNSCC are intrinsically radiosensitive, targeting of radionuclides selectively to HNSCC by use of monoclonal antibodies as a form of radioimmunotherapy, might contribute to a more effective therapy.
Objective. To determine the safety, maximum tolerated dose (MTD), immunogenicity and efficacy of radioimmunotherapy with Rhenium-186 (lg6Re)-labelled humanised monoclonal antibody BIWA4 in patients with HNSCC.
Patients and methods. A phase I dose escalation study was conducted in HNSCC
patients for whom no curative therapeutic options were available. In a total of 20 patients 186Re-labelled m BIWA4 was administered intravenously in doses of 20, 30, 40, 50 or 60 mCi/m2. Three patients received, at least 3 months after a dose of 50 or 60 mCi/m2, a second dose of 50 mCi/m~.
Results. All single as well as repeated administrations were well tolerated and no signs of acute adverse events were observed. The only significant manifestations of toxicity at the higher doses were oral mucositis and dose-limiting myelotoxicity consisting of thrombo- and leucocytopenia.
2o The MTD was established at 50 mCi/m2. One patient developed a human-anti-human response after one single administration. Stable disease, lasting 4 to 19 weeks, was observed in 5 patients treated at the highest dose levels.
Conclusion. Radioimmunotherapy with 186Re-labelled BIWA4 in HNSCC patients seems to be safe and tumoricidal doses can be reached. Moreover, due to the low rate of immunogenicity, 2s repeated administrations appear possible. The results of this phase I study encourage the further development of radioimmunotherapy with Rhenium-186 (186Re)-labelled humanised monoclonal antibody BIWA4 towards an adjuvant therapy for head and neck cancer patients.

SEQUENCE LISTING

<110> Boehringer Ingelheim International GmbH

s <120> Antibodies specific for CD44v6 <130> 1-1212 PCT

<140> not yet assigned ~o <141> 2002-05-16 <150> EP 01112237.1 <151> 2001-05-18 is <150> US 60/325147 <151> 2001-09-26 <160> 18 zo <170> PatentIn Ver. 2.1 <210> 1 <211> 114 <212> PRT

zs <213> Artificial Sequence <220>

<223> Description of ArtificialSequence:
humanized antibody sequence <400> 1 Glu Val Gln Zeu Val Glu Ser Gly GlyLeu Val Zys Pro Gly Gly Gly 3s Ser Leu Arg Zeu Ser Cys Ser GlyPhe Thr Phe Ser Ser Ala Ala Tyr Asp Met Ser Trp Val Arg Gln Pro GlyLys Gly Zeu Glu Trp Ala Val ao Ser Thr Ile Ser Ser Gly Gly Tyr ThrTyr Tyr Leu Asp Ser Ser Ile Zys Gly Arg Phe Thr Ile Ser Asp AsnAla Lys Asn Ser Zeu Arg Tyr as 65 70 75 80 Zeu Gln Met Asn Ser Leu Arg Glu AspThr Ala Val Tyr Tyr Ala Cys so Ala Arg Gln Gly Zeu Asp Gly ArgGly Thr Zeu Val Thr Tyr Trp Val Ser Ser ss <210> 2 <211> 106 <212> PRT
so <213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized antibody sequence <400> 2 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly s Glu Arg A1a Thr Leu Ser Cys Ser Ala Ser Ser Ser Ile Asn Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr to 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser is Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe A1a Val Tyr Tyr Cys Leu Gln Trp Ser Ser Asn Pro Leu Thr zo Phe Gly Gly Gly Thr Lys Val Glu Ile Lys as<210> 3 <211> 106 <212> PRT

<213> Artificia l Sequence so<220>

<223> Descripti on of Sequence:
Artificial humanized antibody sequence <400> 3 ssGlu Ile LeuThr SerPro AlaThrLeu SerLeuSer ProGly Val Gln 1 5 ' 10 15 Glu Arg ThrLeu CysSer AlaSerSer SerIleAsn TyrIle Ala Ser ao Tyr Trp GlnGln ProGly GlnAlaPro ArgIleLeu IleTyr Leu Lys Leu Thr AsnLeu SerGly ValProAla ArgPheSer GlySer Ser Ala as50 55 60 Gly Ser ThrAsp ThrLeu ThrIleSer SerLeuGlu ProGlu Gly Phe 65 70 75 gp soAsp Phe ValTyr CysLeu GlnTrpSer SerAsnPro LeuThr Rla Tyr Phe Gly GlyThr ValGlu IleLys Gly Lys 7.00 105 ss <210> 4 <211> 342 <212> DNA
so <213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized antibody sequence <400> 4 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctaagactc 60 tcctgtgcag cctctggatt cactttcagt agctatgaca tgtcttgggt tcgccaggct 120 s ccggggaagg ggctggagtg ggtctcaacc attagtagtg gtggtagtta cacctactat 180 ctagacagta taaagggccg attcaccatc tccagagaca atgccaagaa ctccctgtac 240 ctgcaaatga acagtctgag ggctgaggac acggccgtgt attactgtgc aagacagggg 300 ttggactact ggggtcgagg aaccttagtc accgtctcct ca 342 ~o ~s <210> 5 <211> 318 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized antibody sequence zo <400> 5 gaaattgttc tcacccagtc tccagcaacc ctgtctctgt ctccagggga gagggccacc 60 ctgtcctgca gtgccagctc aagtataaat tacatatact ggtaccagca gaagccagga 120 caggctccta gactcttgat ttatctcaca tccaacctgg cttctggagt ccctgcgcgc 180 ttcagtggca gtgggtctgg aaccgacttc actctcacaa tcagcagcct ggagcctgaa 240 as gattttgccg tttattactg cctgcagtgg agtagtaacc cgctcacatt cggtggtggg 300 accaaggtgg agattaaa 31g <210> 6 as <211> 318 <212> DNA
<213> Artificial Sequence <220>
3s <223> Description of Artificial Sequence: humanized antibody sequence <400> 6 gaaattgttc tcacccagtc tccagcaacc ctgtctctgt ctccagggga gagggccacc 60 ao ctgtcctgca gtgccagctc aagtataaat tacatatact ggctccagca gaagccagga 120 caggctccta gaatcttgat ttatctcaca tccaacctgg cttctggagt ccctgcgcgc 180 ttcagtggca gtgggtctgg aaccgacttc actctcacaa tcagcagcct ggagcctgaa 240 gattttgccg tttattactg cctgcagtgg agtagtaacc cgctcacatt cggtggtggg 300 accaaggtgg agattaaa 318 as <210> 7 <211> 444 <212> PRT
so <213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized antibody sequence ss <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 60 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Ser Ile s Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Rsn Ser Leu Tyr 65 70 75 gp Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys IO
Rla Arg Gln Gly Leu Asp Tyr Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser A1a Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser is 115 12 0 12 5 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys zo Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser G1y Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu zs Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val so 195 200 205 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro as Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp~Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Rsp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 4s 275 280 285 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr so Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Rla Pro Ile Glu Lys Thr Ile Ser Lys Ala ss Lys Gly Gln Pro Rrg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly so 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln s 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His to Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 8 !s <211> 213 <212> PRT
<213> Artificial Sequence <220>
zo <223> Description of Artificial Sequence: humanized antibody sequence <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly as 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Ile Asn Tyr Ile 3o Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 3s Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Rla Val Tyr Tyr Cys Leu Gln Trp Sex Ser Asn Pro Leu Thr ao 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro as Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys so Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser ss 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala so Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys <210> 9 <211> 213 s <212> PAT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized ro antibody sequence <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly IS
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Ile Asn Tyr Ile Tyr Trp Leu Gln Gln Lys Pro Gly Gln Ala Pro Arg Ile Leu Ile Tyr zo 35 40 45 Leu Thr Ser Asn Leu A1a Ser Gly Val Pro Ala Arg Phe Ser Gly Ser as Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 3s 115 120 125 Rla Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys ao Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 4s Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe so 195 200 205 Asn Arg Gly Glu Cys ss so <210> 10 <211> 2135 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized antibody sequence <400> 10 aagctttgac agacgcacaa ccctggactc ccaagtcttt ctcttcagtg acaaacacag 60 acataggata tcacatttgc ttctgacaca actgtgttca ctagcagcct caaacagaca 120 ccatgaactt tgggctcagc ttgattttcc ttgtcctaat tttaaaaggt gtccagtgtg 180 s aagtgcagct ggtggagtct gggggaggct tagtgaagcc tggagggtcc ctaagactct 240 cctgtgcagc ctctggattc actttcagta gctatgacat gtcttgggtt cgccaggctc 300 cggggaaggg gctggagtgg gtctcaacca ttagtagtgg tggtagttac acctactatc 360 tagacagtat aaagggccga ttcaccatct ccagagacaa tgccaagaac tccctgtacc 420 tgcaaatgaa cagtctgagg gctgaggaca cggccgtgta ttactgtgca agacaggggt 480 ro tggactactg gggtcgagga accttagtca ccgtctcctc agctagcacc aagggcccat 540 cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg gccctgggct 600 gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca ggcgccctga 660 ccagcggcgt gcacaccttc CCggCtgtCC taCagtCCt C aggaCtCtaC tCCCtCagCa 720 gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc aacgtgaatc 780 rs acaagcccag caacaccaag gtggacaaga aagttggtga gaggccagca cagggaggga 840 gggtgtctgc tggaagcagg ctcagcgctc ctgcctggac gcatcccggc tatgcagccc 900 cagtccaggg cagcaaggca ggccccgtct gcctcttcac ccggagcctc tgcccgcccc 960 actcatgctc agggagaggg tcttctggct ttttcccagg ctctgggcag gcacaggcta 1020 ggtgccccta acccaggccc tgcacacaaa ggggcaggtg ctgggctcag acctgccaag 1080 zo agccatatcc gggaggaccc tgcccctgac ctaagcccac cccaaaggcc aaactctcca 1140 ctccctcagc tcggacacct tctctcctcc cagattccag taactcccaa tcttctctct 1200 gcagagccca aatcttgtga caaaactcac acatgcccac cgtgcccagg taagccagcc 1260 caggcctcgc cctccagctc aaggcgggac aggtgcccta gagtagcctg catccaggga 1320 CaggCCCCag ccgggtgctg acacgtccac CtCCatCtCt tCC'tCagCaC ctgaactcct 1380 as ggggggaccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg 1440 gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt 1500 caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca 1560 gtacaacagc acgtaccggg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 1620 tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac 1680 so catctccaaa gccaaaggtg ggacccgtgg ggtgcgaggg ccacatggac agaggccggc 1740 tcggcccacc ctctgccctg agagtgaccg ctgtaccaac ctctgtccta cagggcagcc 1800 ccgagaacca caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt 1860 cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag 1920 caatgggcag ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc 1980 3s cttcttcctc tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt 2040 ctcatgctcc gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct 2100 gtctccgggt aaatgagtgc gacggccgcg aattc 2135 ao <210> 11 <211> 785 <212> DNA
<213> Artificial Sequence as <220>
<223> Description of Artificial Sequence: humanized antibody sequence <400> 11 so aagcttgatc ttcaggatat cacatttgct tctgacacaa ctgtgttcac tagcaacctc 60 aaacagacac catggatttt caggtgcaga ttttcagctt cctgctaatg agtgcctcag 120 tcataatgtc caggggagaa attgttctca cccagtctcc agcaaccctg tctctgtctc 180 caggggagag ggccaccctg tcctgcagtg ccagctcaag tataaattac atatactggt 240 accagcagaa gccaggacag gctcctagac tcttgattta tctcacatcc aacctggctt 300 ss ctggagtccc tgcgcgcttc agtggcagtg ggtctggaac cgacttcact ctcacaatca 360 gcagcctgga gcctgaagat tttgccgttt attactgcct gcagtggagt agtaacccgc 420 tcacattcgg tggtgggacc aaggtggaga ttaaacggac tgtggctgca ccatctgtct 480 tcatcttccc gccatctgat gagcagttga aatctggaac tgctagcgtt gtgtgcctgc 540 tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac gccctccaat 600 so cgggtaactc ccaggagagt gtcacagagc,aggacagcaa ggacagcacc tacagcctca 660 gcagcaccct gacgctgagc aaagcagact acgagaaaca caaagtctac gcctgcgaag 720 tcacccatca gggcctgagc tcgcccgtca caaagagctt caacagggga gagtgttagg 780 aattc <210> 12 <211> 785 <212> DNA
s <213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized antibody sequence ro <400> 12 aagcttgatc ttcaggatat cacatttgct tctgacacaa ctgtgttcac tagcaacctc 60 aaacagacac catggatttt caggtgcaga ttttcagctt cctgctaatg agtgcctcag 120 tcataatgtc caggggagaa attgttctca cccagtctcc agcaaccctg tctctgtctc 180 is caggggagag ggccaccctg tcctgcagtg ccagctcaag tataaattac atatactggc 240 tccagcagaa gccaggacag gctcctagaa tcttgattta tctcacatcc aacctggctt 300 ctggagtccc tgcgcgcttc agtggcagtg ggtctggaac cgacttcact ctcacaatca 360 gcagcctgga gcctgaagat tttgccgttt attactgcct gcagtggagt agtaacccgc 420 tcacattcgg tggtgggacc aaggtggaga ttaaacggac tgtggctgca ccatctgtct 480 zo tcatcttccc.gccatctgat gagcagttga aatctggaac tgctagcgtt gtgtgcctgc 540 tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac gccctccaat 600 cgggtaactc ccaggagagt gtcacagagc aggacagcaa ggacagcacc tacagcctca 660 gcagcaccct gacgctgagc aaagcagact acgagaaaca caaagtctac gcctgcgaag 720 tcacccatca gggcctgagc tcgcccgtca caaagagctt caacagggga gagtgttagg 780 zs aattc 785 <210> 13 <211> 1392 30 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized as antibody sequence <400> 13 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgtgaa 60 gtgcagctgg tggagtctgg gggaggctta gtgaagcctg gagggtccct aagactctcc 120 4o tgtgcagcct ctggattcac tttcagtagc tatgacatgt cttgggttcg ccaggctccg 180 gggaaggggc tggagtgggt ctcaaccatt agtagtggtg gtagttacac ctactatcta 240 gacagtataa agggccgatt caccatctcc agagacaatg ccaagaactc cctgtacctg 300 caaatgaaca gtctgagggc tgaggacacg gccgtgtatt actgtgcaag acaggggttg 360 gactactggg gtcgaggaac cttagtcacc gtctcctcag ctagcaccaa gggcccatcg 420 4s gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc 480 ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 540 agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 600 gtggtgaccg tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac 660 aagcccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac 720 so acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 780 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 840 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 900 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 960 gtCCt CdCCg tCCtgcaCCa ggactggctg aatggcaagg agtacaagtg caaggtctcc 1020 ss aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1080 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1140 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1200 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1260 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1320 co tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1380 ccgggtaaat ga 1392 <210> 14 <211> 702 <212> DNA
<213> Artificial Sequence s ~ <220>
<223> Description of Artificial Sequence: humanized antibody sequence <400> 14 ~o atggaagccc cagctcagct tctcttcctc ctgctgctct ggctcccaga taccaccgga 60 gaaattgttc tcacccagtc tccagcaacc ctgtctctgt ctccagggga gagggccacc 120 ctgtcctgca gtgccagctc aagtataaat tacatatact ggtaccagca gaagccagga 180 caggctccta gactcttgat ttatctcaca tccaacctgg cttctggagt ccctgcgcgc 240 ttcagtggca gtgggtctgg aaccgacttc actctcacaa tcagcagcct ggagcctgaa 300 is gattttgccg tttattactg cctgcagtgg agtagtaacc cgctcacatt cggtggtggg 360 accaaggtgg agattaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 42O
gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 zo agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ga 702 <210> 15 zs <211> 702 <212> DNA
<213> Artificial Sequence <220>
so <223> Description of Artificial Sequence: humanized antibody sequence <400> 15 atggaagccc cagctcagct tctcttcctc ctgctgctct ggctcccaga taccaccgga 60 ss gaaattgttc tcacccagtc tccagcaacc ctgtctctgt ctccagggga gagggccacc 120 ctgtcctgca gtgccagctc aagtataaat tacatatact ggctccagca gaagccagga 180 caggctccta gaatcttgat ttatctcaca tccaacctgg cttctggagt ccctgcgcgc 240 ttcagtggca gtgggtctgg aaccgacttc actctcacaa tcagcagcct ggagcctgaa 300 gattttgccg tttattactg cctgcagtgg agtagtaacc cgctcacatt cggtggtggg 360 ao accaaggtgg agattaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 4s agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ga 702 <210> 16 <211> 9568 so <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: humanized ss antibody sequence <400> 16 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120 so ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 180 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtaggg 240 tcgcgacgta ccgggccccc cctcgattaa ttaatcgagc tactagcttt gcttctcaat 300 ttcttatttg cataatgaga aaaaaaggaa aattaatttt aacaccaatt cagtagttga 360 ttgagcaaat gcgttgccaa aaaggatgct ttagagacag tgttctctgc acagataagg 420 acaaacattattcagagggagtacccagagctgagactcctaagccagtgagtggcacag480 cattctagggagaaatatgcttgtcatcaccgaagcctgattccgtagagccacaccttg540 gtaagggccaatctgctcacacaggatagagagggcaggagccagggcagagcatataag600 gtgaggtaggatcagttgctcctcacatttgcttctgacatagttgtgccagcatggagg660 s aatcgatcctccatgcttgaacaagatggattgcacgcaggttctccggccgcttgggtg720 gagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtg780 ttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgcc840 ctgaatgaactgcaggtaagtgcggccgctctaggcctccaaaaaagcctcctcactact900 tctggaatagctcagaggccgaggcggcctcggcctctgcataaataaaaaaaattagtc960 !oagccatgcatggggcggagaatgggcggaactgggcggagttaggggcgggatgggcgga1020 gttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttctgcct2080 gctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgc1140 atacttctgcctgctggggagcctggggactttccacaccctaactgacacacattccac1200 agaattaattcccctagttattaatagtaatcaattacggggtcattagttcatagccca1260 ~statatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaac1320 gacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggact1380 ttccattgacgtcaatgggtggactatttacggtaaactgcccacttggcagtacatcaa1440 gtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctgg1500 cattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtatta1560 sogtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcgg1620 tttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgg1680 caccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatg1740 ggcggtaggcgtgtacggtgggaggtctatataagcagagctgggtacgtgaaccgtcag1800 atcgcctggagacgccatcacagatctctcaccatggaagccccagctcagCttCtCttC1860 zsctcctgctgctctggctcccagataccaccggagaaattgttctcacccagtctccagca1920 accctgtctctgtctccaggggagagggccaccctgtcctgcagtgccagctcaagtata1980 aattacatatactggtaccagcagaagccaggacaggctcctagactcttgatttatctc2040 acatccaacctggcttctggagtccctgcgcgcttcagtggcagtgggtctggaaccgac2100 ttcactctcacaatcagcagcctggagcctgaagattttgccgtttattactgcctgcag2160 30tggagtagtaacccgctcacattcggtggtgggaccaaggtggagattaaacgtacggtg2220 gctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcc2280 tctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg2340 gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggac2400 agcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaa2460 ssgtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac2520 aggggagagtgttgaattcagatccgttaacggttaccaactacctagactggattcgtg2580 acaacatgcggccgtgatatctacgtatgatcagcctcgactgtgccttctagttgccag2640 ccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccact2700 gtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctatt2760 aoctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcat2820 gctggggatgcggtgggctctatggaaccagctgggactagtagctttgcttctcaattt2880 cttatttgcataatgagaaaaaaaggaaaattaattttaaaccaattcagtagttgatt2940 c gagcaaatgcgttgccaaaaaggatgctttagagacagtgttctctgcacagataaggac3000 aaacattattcagagggagtacccagagctgagactcctaagccagtgagtggcacagca3060 asttctagggagaaatatgcttgtcatcaccgaagcctgattccgtagagccacaccttggt3120 aagggccaatctgctcacacaggatagagagggcaggagccagggcagagcatataaggt3180 gaggtaggatcagttgctcctcacatttgcttctgacatagttgtgttgggagcttggat3240 agcttggacagctcagggctgcgatttcgcgccaaacttgacggcaatcctagcgtgaag3300 gctggtaggattttatccccgctgccatcatggttcgaccattgaactgcatcgtcgccg3360 sotgtcccaaaatatggggattggcaagaacggagacctaccctggcctccgctcaggaacg3420 agttcaagtacttccaaagaatgaccacaacctcttcagtggaaggtaaacagaatctgg3480 tgattatgggtaggaaaacctggttctccattcctgagaagaatcgacctttaaaggaca3540 gaattaatatagttctcagtagagaactcaaagaaccaccacgaggagctcattttcttg3600 ccaaaagtttggatgatgccttaagacttattgaacaaccggaattggcaagtaaagtag3660 ssacatggtttggatagtcggaggcagttctgtttaccaggaagccatgaatcaaccaggcc3720 accttagactctttgtgacaaggatcatgcaggaatttgaaagtgacacgtttttcccag3780 aaattgatttggggaaatataaaCttCtCCCagaataCCCdggCgtCCtCtctgaggtcc3840 aggaggaaaaaggcatcaagtataagtttgaagtctacgagaagaaagactaacaggaag3900 atgctttcaagttctctgctcccctcctaaagctatgcatttttataagaccatgggact3960 sotttgctggctttagatcagcctcgactgtgccttctagttgccagccatctgttgtttgc4020 CCC'tCCCCCgtgCCttCCttgaccctggaaggtgccactcCCaCtgtCCtttCCtaataa4080 aatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtg4140 gggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtg4200 ggctctatggaaccagctggggctcgaagcggccgctccggatatgccaagtacgccccc4260 tattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatg4320 ggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcg4380 gttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtct4440 ccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaa4500 s atgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggt4560 ctatataagcagagctgggtacgtcctcacattcagtgatcagcactgaacacagacccg4620 tcgacatggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagt4680 gtgaagtgcagctggtggagtctgggggaggcttagtgaagcctggagggtccctaagac4740 tctcctgtgcagcctctggattcactttcagtagctatgacatgtcttgggttcgccagg4800 ioctccggggaaggggctggagtgggtctcaaccattagtagtggtggtagttacacctact4860 atctagacagtataaagggccgattcaccatctccagagacaatgccaagaactccctgt4920 acctgcaaatgaacagtctgagggctgaggacacggccgtgtattactgtgcaagacagg4980 ggttggactactggggtcgaggaaccttagtcaccgtctcctcagctagcaccaagggcc5040 catcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgg5100 ~sgctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccc5160 tgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctca5220 gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtga5280 atcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaa5340 ctcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctct5400 zotccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtgg5460 tggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgg5520 aggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtgg5580 tcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaagg5640 tctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagc5700 ascccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccagg5760 tcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggaga5820 gcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggct5880 ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtct5940 tctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccc6000 3otgtctccgggtaaatgaggatccgttaacggttaccaactacctagactggattcgtgac6060 aacatgcggccgtgatatctacgtatgatcagcctcgactgtgccttctagttgccagcc6120 atctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgt6180 cctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattct6240 ggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgc6300 3stggggatgcggtgggctctatggaaccagctggggctcgacagcgctgcgatcgcctcga6360 ggccgctactaactctctcctccctcctttttcctgcaggacgaggcagcgcggctatcg6420 tggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcggga6480 agggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgct6540 cctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccg6600 aogctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatg6660 gaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagcc6720 gaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccat6780 ggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgac6840 tgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatatt6900 4sgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgct6960 cccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactc7020 tggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattcca7080 ccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatga7140 tcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcag7200 socttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttttt7260 cactgcattctagttgtggtttgtccaaactcatcaatctatcttatcatgtctggatcg7320 cggccggccgcaccgcggtggagctttaattaaggcgcgccagctccagcttttgttccc7380 tttagtgagggttaatttcgagcttggcgtaatcatggtcatagctgtttcctgtgtgaa7440 attgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcct7500 ssggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttcc7560 agtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcg7620 gtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttc7680 ggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcag7740 gggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaa7800 soaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc7860 gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcccc7920 ctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccg7980 cctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagtt8040 cggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgacc8100 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 8160 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 8220 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 8280 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 8340 s ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 8400 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 8460 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 8520 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 8580 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 8640 ~o ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 8700 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 8760 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 8820 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 8880 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 8940 ~s gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 9000 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 9060 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 9120 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 9180 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 9240 20 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 9300 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 9360 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 9420 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 9480 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa.ataggggttc 9540 as cgcgcacatt tccccgaaaa gtgccaca 9568 <210> 17 <211> 6414 so <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:plasmid <400> 17 tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 60 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 120 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 180 4o gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 240 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 300 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 360 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 420 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 480 as ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 540 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag 600 agaacccact gcttaactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660 cttctgcagg tcgacatcga tggatccggt acctcgagcg cgaattctct agaggatctt 720 tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta cagagattta 780 so aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat 840 tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 900 ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac 960 tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga 1020 ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc 1080 ss ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga 1140 aaaatatttg atgtatagtg ccttgactag agatcataat cagccatacc acatttgtag 1200 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga 1260 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata 1320 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 1380 so aactcatcaa tgtatcttat catgtctgga tcaattctga gaaactagcc ttaaagacag 1440 acagctttgt tctagtcagc caggcaagca tatgtaaata aagttcctca gggaactgag 1500 gttaaaagat gtatcctgga cctgccagac ctggccattc acgtaaacag aagattccgc 1560 ctcaagttcc ggttaacaac aggaggcaac gagatctcaa atctattact tctaatcggg 1620 taattaaaac ctttcaacta aaacacggac ccacggatgt cacccacttt tccttccccg 1680 gctccgcccttctcagtactccccaccattaggctcgctactccacctccacttccgggc1740 gCgaC3CCCaCgtgCCCtCtCCCaCCCgaCgCtaaCCCCgCCCCtgCCCgtCtgaCCCCg1800 cccaccacctggccccgccccgttgaggacagaagaaaccccgggcagccgcagccaagg1860 cggacgggtagacgctgggggcgctgaggagtcgtcctctaccttctctgctggctcggt1920 s gggggacgcggtggatctcaggcttccggaagactggaagaaccggctcagaaccgcttg1980 tctccgcggggcttgggcggcggaagaatggccgctagacgcggacttggtgcgaggcat2040 cgcaggatgcagaagagcaagcccgccgggagcgcgcggctgtactaccccgcgcctgga2100 gcggccacgccggactgggcggggccggcctggtggaggcggagtctgacctcgtggagg2160 cggggcctctgatgttcaaataggatgctaggcttgttgaggcgtggcctccgattcaca2220 roagtgggaagcagcgccgggcgactgcaatttcgcgccaaacttgggggaagcacagcgta2280 caggctgcctaggtgatcgctgctgctgtcatggttcgaccgctgaactgcatcgtcgcc2340 gtgtcccagaatatgggcatcggcaagaacggagaccttccctggccaatgctcaggtac2400 tggctggattgggttagggaaaccgaggcggttcgctgaatcgggtcgagcacttggcgg2460 agacgcgcgggccaactacttagggacagtcatgaggggtaggcccgccggctgctgccc2520 rsttgcccatgcccgcggtgatccccatgctgtgccagcctttgcccagaggcgctctagct2580 gggagcaaagtccggtcactgggcagcaccaccccccggacttgcatgggtagccgctga2640 gatggagcctgagcacacgtgacagggtccctgttaacgcagtgtttctctaactttcag2700 gaacgagttcaagtacttccaaagaatgaccaccacctcctcagtggaaggtaaacagaa2760 cctggtgattatgggccggaaaacctggttctccattcctgagaagaatcgacctttaaa2820 aoggacagaattaatatagttctcagtagagagctcaaggaaccaccacaaggagctcattt2880 tcttgccaaaagtctggaccatgccttaaaacttattgaacaaccagagttagcagataa2940 agtggacatggtttggatagttggaggcagttccgtttacaaggaagccatgaatcagcc3000 aggccatctcagactctttgtgacaaggatcatgcaggaatttgaaagtgacacgttctt3060 cccagaaattgatttggagaaatataaacttctcccagagtacccaggggtcctttctga3120 zsagtccaggaggaaaaaggcatcaagtataaatttgaagtctatgagaagaaaggctaaca3180 gaaagatacttgctgattgacttcaagttctactgctttcctcctaaaattatgcatttt3240 tacaagaccatgggacttgtgttggctttagatcctgtgcatcctgggcaactgttgtac3300 tctaagccactccccaaagtcatgccccagcccctgtataattctaaacaattagaatta3360 ttttcattttcattagtctaaccaggttatattaaatatactttaagaaacaccatttgc3420 3ocataaagttctcaatgcccctcccatgcagcctcaagtggctccccagcagatgcatagg3480 gtagtgtgtgtacaagagaccccaaagacatagagcccctgagagcatgagctgatatgg3540 gggctcatagagataggagctagatgaataagtacaaagggcagaaatgggttttaacca3600 gcagagctagaactcagactttaaagaaaattagatcaaagtagagactgaattattctg3660 cacatcagactctgagcagagttctgttcactcagacagaaaatgggtaaattgagagct3720 3sggctccattgtgctccttagagatgggagcaggtggaggattatataaggtctggaacat3780 ttaacttctccgtttctcatcttcagtgagattccaagggatactacaattctgtggaat3840 gtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagc3900 atgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcaga3960 agtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgccc4020 40atCCCCJCCCCtaaCtCCg'CCCagttCCgCCCattCtCCgCCCCatggCtgactaattttt4080 tttatttatgcagaggccgaggcgcctctgagctattccagaagtagtgaggaggctttt4140 ttggaggcctaggcttttgcaaaaaagctaattcagcctgaatggcgaatgggacgcgcc4200 ctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacact4260 tgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgc4320 4scggetttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttt4380 acggcacctcgaccccaaaaacttgattagggtgatggttcacgtagtgggccatcgccc4440 tgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttg4500 ttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggatt4560 ttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaat4620 sotttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcgga4680 acccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataa4740 ccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgt4800 gtcgcccttattcecttttttgcggcattttgccttcctgtttttgctcacccagaaacg4860 ctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactg4920 ss.gatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatg4980 agcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagag5040 caactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcaca5100 gaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatg5160 agtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaacc5220 sogcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctg5280 aatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacg5340 ttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagac5400 tggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctgg5460 tttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg5520 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 5580 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 5640 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 5700 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 5760 s ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 5820 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 5880 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 5940 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 6000 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 6060 ~o gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 6120 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 6180 ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg gagaaaggcg 6240 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 6300 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 6360 is tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcc 6414 <210> 18 <211> 6062 ao <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:plasmid as <400> 18 tcgacattga ttattgactagttattaatagtaatcaattacggggtcattagttcatag60 cccatatatg gagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcc120 caacgacccc cgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagg180 3o gactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtaca240 tcaagtgtat catatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgc300 ctggcattat gcccagtacatgaccttatgggactttcctacttggcagtacatctacgt360 attagtcatc gctattaccatggtgatgcggttttggcagtacatcaatgggcgtggata420 gcggtttgac tcacggggatttccaagtctccaccccattgacgtcaatgggagtttgtt480 ss ttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgca540 aatgggcggt aggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccg600 tcagatcgcc tggagacgccatccacgctgttttgacctccatagaagacaccgggaccg660 atccagcctc cgcggccgggaacggtgcattggaacgcggattccccgtgccaagagtca720 ggtaagtacc gcctatagagaagactcttgggtttctgataggcactgactctctctgcc780 0o tattggtctattttcccacccttaggctgctggtgcttaactggcttatcgaaattaata840 cgactcacta tagggagacccaagcttctgcaggtcgacatcgatggatccggtacctcg.900 agcgcgaatt ctctagagatatcttgtttattgcagcttataatggttacaaataaagca960 atagcatcac aaatttcacaaataaagcatttttttcactgcattctagttgtggtttgt1020 ccaaactcat caatgtatcttatcatgtctggatcaattctgaaaaactagccttaaaga1080 as cagacagctttgttctagtcagccaggcaagcatatgtaaataaagttcctcagggaact1140 gaggttaaaa gatgtatcctggacctgccagacctggccattcacgtaaacagaagattc1200 cgcctcaagt tccggttaacaacaggaggcaacgagatctcaaatctattacttctaatc1260 gggtaattaa aacctttcaactaaaacacggacccacggatgtcacccacttttccttcc1320 ccggctccgc ccttctcagtactccccaccattaggctcgctactccacctccacttccg1380 so ggcgcgacacccacgtgccctctcccacccgaCC~'CtaaCCCCCJCCCCtgCCCgtCtgaCC1440 ccgcccacca cctggccccgccccgttgaggacagaagaaaccccgggcagccgcagcca1500 aggcggacgg gta.gacgctgggggcgctgaggagtcgtcctctaccttctctgctggctc1560 ggtgggggac gcggtggatctcaggcttccggaagactggaagaaccggctcagaaccgc1620 ttgtctccgc ggggcttgggcggcggaagaatggccgctagacgcggacttggtgcgagg1680 ss catcgcaggatgcagaagagcaagcccgccgggagcgcgcggctgtactaccccgcgcct1740 ggagcggcca cgccggactgggcggggccggcctggtggaggcggagtctgacctcgtgg1800 aggcggggcc tctgatgttcaaataggatgctaggcttgttgaggcgtggcctccgattc1860 acaagtggga agcagcgccgggcgactgcaatttcgcgccaaacttgggggaagcacagc1920 gtacaggctg cctaggtgatcgctgctgctgtcatggttcgaccgctgaactgcatcgtc1980 so gccgtgtcccagaatatgggcatcggcaagaacggagaccttccctggccaatgctcagg2040 tactggctgg attgggttagggaaaccgaggcggttcgctgaatcgggtcgagcacttgg2100 cggagacgcg cgggccaactacttagggacagtcatgaggggtaggcccgccggctgctg2160 cccttgccca tgcccgcggtgatccccatgctgtgccagcctttgcccagaggcgctcta2220 gctgggagca aagtccggtcactgggcagcaccaccccccggacttgcatgggtagccgc2280 tgagatggag cctgagcacacgtgacagggtccctgttaacgcagtgtttctctaacttt2340 caggaacgag ttcaagtacttccaaagaatgaccaccacctcctcagtggaaggtaaaca2400 gaacctggtg attatgggccggaaaacctggttctccattcctgagaagaatcgaccttt2460 aaaggacaga attaatatagttctcagtagagagctcaaggaaccaccacaaggagctca2520 s ttttcttgccaaaagtctggaccatgccttaaaacttattgaacaaccagagttagcaga2580 taaagtggac atggtttggatagttggaggcagttccgtttacaaggaagccatgaatca2640 gccaggccat ctcagactctttgtgacaaggatcatgcaggaatttgaaagtgacacgtt2700 cttcccagaa attgatttggagaaatataaacttctcccagagtacccaggggtcctttc2760 tgaagtccag gaggaaaaaggcatcaagtataaatttgaagtctatgagaagaaaggcta2820 to acagaaagatacttgctgattgacttcaagttctactgctttcctcctaaaattatgcat2880 ttttacaaga ccatgggacttgtgttggctttagatcctgtgcatcctgggcaactgttg2940 tactctaagc cactccccaaagtcatgccccagcccctgtataattctaaacaattagaa3000 ttattttcat tttcattagtctaaccaggttatattaaatatactttaagaaacaccatt3060 tgccataaag ttctcaatgcccctcccatgcagcctcaagtggctccccagcagatgcat3120 ~s agggtagtgtgtgtacaagagaccccaaagacatagagcccctgagagcatgagctgata3180 tgggggctca tagagataggagctagatgaataagtacaaagggcagaaatgggttttaa3240 ccagcagagc tagaactcagactttaaagaaaattagatcaaagtagagactgaattatt3300 ctgcacatca gactctgagcagagttctgttcactcagacagaaaatgggtaaattgaga3360 gctggctcca ttgtgctccttagagatgggagcaggtggaggattatataaggtctggaa3420 zo catttaacttctccgtttctcatcttcagtgagattccaagggatactacaattctgtgg3480 aatgtgtgtc agttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaa3540 agcatgcatc tcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggc3600 agaagtatgc aaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccg3660 CCCatCCCgC CCCtaaCtCCgCCCagttCCgcccattctccgccccatggctgactaatt3720 zs ttttttatttatgcagaggccgaggcgcctctgagctattccagaagtagtgaggaggct3780 tttttggagg cctaggcttttgcaaaaaagctaattcagcctgaatggcgaatgggaaat3840 tgtaaacgtt aatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttt3900 taaccaatag gccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagg3960 gttgagtgtt gttccagtttggaacaagagtccactattaaagaacgtggactccaacgt4020 3o caaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatc4080 aagtttttgg ggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccga4140 tttagagctt gacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaa4200 ggagcgggcg ctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacaccc4260 gccgcgctta atgcgccgctacagggcgcgtcaggtggcacttttcggggaaatgtgcgc4320 ss ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa4380 taaccctgat aaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttc4440 cgtgtcgccc ttattcccttttttgcggcattttgccttcctgtttttgctcacccagaa4500 acgctggtga aagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaa4560 ctggatctca acagcggtaagatccttgagagttttcgccccgaagaacgttttccaatg4620 ao atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa4680 gagcaactcg gtcgccgcatacactattctcagaatgacttggttgagtactcaccagtc4740 acagaaaagc atcttacggatggcatgacagtaagagaattatgcagtgctgccataacc4800 atgagtgata acactgcggccaacttacttctgacaacgatcggaggaccgaaggagcta4860 accgcttttt tgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag4920 as ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca4980 acgttgcgca aactattaactggcgaactacttactctagcttcccggcaacaattaata5040 gactggatgg aggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc5100 tggtttattg ctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagca5160 ctggggccag atggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca5220 so actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg5280 taactgtcag accaagtttactcatatatactttagattgatttaaaacttcatttttaa5340 tttaaaagga tctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgt5400 gagttttcgt tccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagat5460 cctttttttc tgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtg5520 ss gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga5580 gcgcagatac caaatactgtccttctagtgtagccgtagttaggccaccacttcaagaac5640 tctgtagcac cgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt5700 ggcgataagt cgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag5760 cggtcgggct gaacggggggttcgtgcacacagcccagcttggagcgaacgacctacacc5820 so gaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaag5880 gcggacaggt atccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca5940 gggggaaacg cctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt6000 cgatttttgt gatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcagct6060 gc 6062

Claims (80)

claims

1. Antibody molecule comprising a variable region of the heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 1 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

2. Antibody molecule wherein the variable region heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 1.

3. Antibody molecule comprising a variable region light chain as characterized by the amino acid sequence as defined in SEQ ID No. 2 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

4. Antibody molecule wherein the variable region light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 2.

5. Antibody molecule comprising a variable region light chain as characterized by the amino acid sequence as defined in SEQ ID No. 3 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

6. Antibody molecule wherein the variable region light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 3.

7. Antibody molecule according to claim 1 and 3 comprising a variable region heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 1 and comprising a variable region light chain as characterized by the amino acid sequence as defined in SEQ ID
No. 2 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

8. Antibody molecule according to claim 2 and 4 wherein the variable region heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ
ID No. 1 and wherein the variable region light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 2.

9. Antibody molecule according to claim 1 and 5 comprising a variable region heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 1 and comprising a variable region light chain as characterized by the amino acid sequence as defined in SEQ ID
No. 3 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

10. Antibody molecule according to claim 2 and 6 wherein the variable region heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ
ID No. 1 and wherein the variable region light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 3.

11. Antibody molecule comprising a variable region heavy chain encoded by the nucleic acid sequence as defined in SEQ ID No. 4 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

12. Antibody molecule wherein the variable region heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4.

13. Antibody molecule comprising a variable region light chain encoded by the nucleic acid sequence as defined in SEQ ID No. 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

14. Antibody molecule wherein the variable region light chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 5.

15. Antibody molecule comprising a variable region light chain encoded by the nucleic acid sequence as defined in SEQ ID No. 6 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

16. Antibody molecule wherein the variable region light chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 6.

17. Antibody molecule according to claim 11 and 13 comprising a variable region heavy chain encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and comprising a variable region light chain encoded by the nucleic acid sequence as defined in SEQ ID
No. 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

18. Antibody molecule according to claim 12 and 14 wherein the variable region heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and wherein the variable region light chain is encoded by the nucleic acid sequence as defined in SEQ
ID No. 5.

19. Antibody molecule according to claim 11 and 15 comprising a variable region heavy chain encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and comprising a variable region light chain encoded by the nucleic acid sequence as defined in SEQ ID
No. 6 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

20. Antibody molecule according to claim 12 and 16 wherein the variable region heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and wherein the variable region light chain is encoded by the nucleic acid sequence as defined in SEQ
ID No. 6.

21. An antibody molecule according to any one of claims 1 to 20 characterised in that each of said variable light chain and said variable heavy chain region is separately joined to a human constant region.

22. The antibody molecule of claim 21, wherein said human constant region of the light chain is a human kappa constant region.

23. The antibody molecule of claim 21 or 22, wherein said human constant region of the heavy chain is a human IgGl constant region.

24. Antibody molecule according to any one of claims 1 to 23 comprising a heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 7 and comprising a light chain as characterized by the amino acid sequence as defined in SEQ ID No. 8 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

25. The antibody molecule according to any one of claims 1 to 24 wherein the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ
ID No. 7 and wherein the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 8.

26. The antibody molecule according to any one of claims 1 to 25 comprising a heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 7 and comprising a light chain as characterized by the amino acid sequence as defined in SEQ ID No. 9 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.

27. The antibody molecule according to any one of claims 1 to 26 wherein the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ
ID No. 7 and wherein the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 9.

28. The antibody molecule according to any one of claims 1 to 27 comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 10 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ID No.
11 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

29. The antibody molecule according to any one of claims 1 to 28 wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID No. 10 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID No. 11.

30. The antibody molecule according to any one of claims 1 to 29 comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 10 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ID No.
12 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code,fusion molecule or a chemical derivative thereof.

31. The antibody molecule according to any one of claims 1 to 30 wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID No. 10 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID No. 12.

32. The antibody molecule according to any one of claims 1 to 31 comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 13 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ID No.
14 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

33. The antibody molecule according to any one of claims 1 to 32 wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID No. 13 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID No. 14.

34. The antibody molecule according to any one of claims 1 to 33 comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 13 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ID No.
15 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

35. The antibody molecule according to any one of claims 1 to 34 wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID No. 13 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID No. 15.

36. The antibody molecule according to any one of claims 1 to 35 comprising a heavy and light chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 16 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.

37. The antibody molecule according to any one of claims 1 to 36 wherein the heavy and light chain is encoded by the nucleic acid sequence of SEQ ID No. 16.

38. An antibody protein according to any one of claims 1 to 37, wherein said antibody protein is conjugated to a therapeutic agent.

39. The antibody protein of claim 38, wherein said therapeutic agent is a therapeutic agent selected from the group consisting of radioisotopes, toxins, toxoids, inflammatory agents and chemotherapeutic agents.

40. The antibody protein of claim 39, wherein said therapeutic agent is linked to the antibody protein via a linker selected from the group of MAG-3 GABA, MAG-2 GABA and N2S2.

41. The antibody protein of claim 40, wherein said therapeutic agent is linked to the antibody protein via MAG-2 GABA.

42. The antibody protein of any one of claims 38 to 41, wherein said radioisotope is a 13-emitting radioisotope.

43. The antibody protein of claim 42, wherein said radioisotope is selected from the group consisting of 186Rhenium, 188Rhenium,131lodine and 90Yttrium.

44. The antibody protein of claim 43, wherein said radioisotope is lg6Rhenium.

45. The antibody protein according to any one of claims 39 to 44, wherein the antibody protein has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi/mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.

46. The antibody protein according to any one of claims 1 to 37, characterised in that it is labelled.

47. The antibody protein of claim 46, wherein said label is a detectable marker.

48. The antibody protein of claim 47, wherein the detectable marker is a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin.

49. An antibody protein according to any one of claims 1 to 37 conjugated to an imageable agent.

50. The antibody protein of claim 49, wherein the imageable agent is a radioisotope.

51. The antibody protein of claim 50, wherein said radioisotope is a y-emitting radioisotope.

52. The antibody protein of claim 51, wherein said radioisotope is 125L,

53. A pharmaceutical composition containing an antibody protein according to any one of claims 1 to 45 and a pharmaceutically acceptable carrier or excipient.

54. Pharmaceutical composition according to claim 53, wherein the antibody protein is conjugated to a radioisotope according to any one of claims 39 to 44 and has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi/mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.

55. Pharmaceutical composition according to claim 53 or 54, wherein the amount of radiolabelled antibody in the pharmaceutical composition to be applied to a patient is 10, 20, 30, 40, 50 or 60 mCi/m2.

56. Pharmaceutical composition according to any one of claims 53 to 55, wherein the amount of radiolabelled antibody in the pharmaceutical composition to be applied to a patient is 5O
mCi/m2.

57. Pharmaceutical composition according to any one of claims 53 to 56, further comprising one or more radioprotectant selected from the group of ascorbic acid, gentisic acid, reductic acid, erythrorbic acid, p-aminobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2-5-dihydroxy-1,4-benzenedisulfonic acid, povidone, inositol, and/or citrate.

58. Pharmaceutical composition according to any one of claims 53 to 57, wherein the radioprotectant is ascorbic acid.

59. Pharmaceutical composition according to any one of claims 53 to 58, wherein said antibody protein comprises an antibody molecule selected from the group of antibody molecules of claims 8, 10, 18, 20, 24, 25, 28, 29, 32, 33, 36, 37 linked to 186Rhenium via further comprising the radioprotectant ascorbic acid.

60. Use of an antibody protein according to any one of claims 1 to 45 in the manufacture of a medicament for treatment of cancer.

61. Use of an antibody protein according to claim 60 wherein said cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic cancers of the brain.

62. Use of an antibody protein according to any one of claims 60 or 61, wherein the antibody protein is conjugated to a radioisotope according to any one of claims 39 to 44 and has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi/mg, preferably about I to about IO mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.

63. Use of an antibody protein according to claim 62, wherein the amount of radiolabelled antibody in the pharmaceutical composition to be applied to a patient is 10, 20, 30, 40, 50 or 60 mCi/m2.

64. Use of an antibody protein according to claim 62, wherein the amount of radiolabelled antibody in the pharmaceutical composition to be applied to a patient is 50 mCi/m2.

65. Method of cancer treatment, wherein an antibody protein according to any one of claims 1 to 45 is administered once to several times to an individual in need thereof, said antibody protein selectively binds to CD44v6, destroys tumor cells via the therapeutic agent linked to the antibody protein and the therapeutic success is monitored.

66. The method of claim 65, wherein the tumor is a tumor selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.

67. Nucleic acid, characterised in that it codes for an antibody protein according to one of claims 1 to 37.

68. Recombinant DNA vector, characterised in that it contains a nucleic acid according to claim 67.

69. Recombinant DNA vector according to claim 68, characterized in that it is an expression vector.

70. Recombinant DNA vector according to claim 68 or 69, characterized in that it is vector pAD-CMV or a functional derivative thereof.

71. Recombinant DNA vector according to any one of claims 68 to 70, characterized in that it is vector N5KG1 Val or a derivative thereof.

72. Host, characterised in that it contains a vector according to any one of claims 68 to 71.

73. Host according to claim 72, characterised in that it is a eukaryotic host cell.

74. Host according to claim 72 or 73, characterised in that it is a mammalian cell.

75. Host according to any one of claims 72 to 74, characterised in that it is a BHK, CHO or COS
cell.

76. Host according to claim 72, characterised in that it is a bacteriophage.

77. Host according to claim 72, characterised in that it is a prokaryotic host cell.

78. Process for preparing an antibody protein according to any one of claims 1 to 37, characterized in that it comprises the following steps: a host according to one of claims 72 to 77 is cultivated under conditions in which said antibody protein is expressed by said host cell and said antibody protein is isolated.

79. Process according to claim 78, characterised in that said host is a mammalian cell, preferably a CHO or COS cell.

80. Process according to claim 78 or 79, characterised in that said host cell is co-transfected with two plasmids which carry the expression units for the light or the heavy chain.