KR20040000478A - Antibodies specific for CD44v6 - Google Patents

Abstract

The present invention belongs to the field of oncology. The present invention relates to antibodies having specific sequences specific for epitopes encoded by variant exon v6 of the CD44 gene and derivatives of such antibodies. The present invention also relates to nucleic acid molecules encoding such antibody proteins. Moreover, the present invention relates to a method for producing the antibody protein. The invention also relates to a pharmaceutical composition comprising said antibody protein. Moreover, the present invention relates to the use in the manufacture of a medicament for the treatment of cancer.

Description

Antibodies specific for CD44v6

Recently, expression of variants of surface glycoprotein CD44 has been found to be sufficient and necessary to induce the so-called spontaneous metastatic behavior of non-metastatic pancreatic adenocarcinoma cell lines as well as non-metastatic rat fibrosarcoma cell lines. Gunthert et al., 1991]. While the smallest CD44 isotype, standard CD44s (or CD44std) is ubiquitous expressed in different tissues including epithelial cells, any CD44 splice variants (CD44v, CD44var) are expressed only on a subset of epithelial cells. The CD44 variant is produced by alternative splicing in such a way that 10 exon sequences (v1-v10) are completely cleaved in CD44s, but can emerge as larger 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 specific sites in the extracellular portion of the protein of interest. Such variants can be detected in various human tumor cells as well as human tumor tissues. Thus, recently, expression of CD44 variants in colorectal carcinogenesis has been studied . Heider et al. , 1993a]. The CD44 variant is not expressed in normal human colon epithelium, only a weak level of expression is detectable in proliferative cells of the aneurysms. In later stages of tumor progression, eg, adenocarcinoma, all tumors express variants of CD44. CD44 variants have been found to have high tissue expression in aggressive non-Hodgkin lymphomas (Koopman et al., 1993).

Exon v6 is believed to play a special role, particularly in the metastatic spread process (Rudy et al., 1993). In animal models, antibodies against v6 specific epitopes were able to prevent the settlement of metastatic cells and the growth of metastases . Seiter et al. , 1993]. In colon carcinoma, v6 expression is associated with tumor progression . Wielenga et al. , 1993]. In gastric carcinoma, v6 expression is an important diagnostic marker for distinguishing intestinal type tumors from diffuse type tumors . Heider et al. , 1993b]. In the latter two publications, v6 expression was determined using antibodies against v6 specific epitopes.

Since CD44v6 has been found to be a tumor-associated antigen with favorable expression patterns in human tumors and normal tissues (see Heider et al., 1995; Heider et al., 1996], which has been studied as a major challenge in antibody-utilized diagnostic and therapeutic approaches (Heider et al., 1996; WO 95/33771; WO 97/21104.

One serious problem that arises when using non-human antibodies in humans is that the antibodies rapidly elicit a human anti-non-human response, reducing the efficacy of the antibody in patients and impairing subsequent administration. Is to wear. To overcome this problem, the concept of "humanizing" non-human antibodies has been developed in the art. In the first approach, we attempted to humanize non-human antibodies by constructing non-human / human chimeric antibodies linking the non-human variable regions to human constant regions. Boulianne GL, Hozumi N. and Shulman, MJ (1984) Production of functional chimeric mouse / human antibody Nature 312 : 643. The resulting chimeric antibody retains the binding specificity and affinity of the original non-human antibody. However, although chimeric antibodies are significantly better than mouse antibodies, they can still induce anti-chimeric responses in humans. LoBuglio AF, Wheeler RH, Trang J., Haynes A., Rogers K., Harvey EB, Sun L., Ghrayeb J. and Khazaeli MB (1989) Mouse / human chimeric monoclonal antibody in man: Kinetics and immune response. Proc. Natl. Acad. Sci. 86 : 4220]. The approach later adds the amount of non-human sequence by implanting complementarity determining regions (CDRs) from non-human variable regions into human variable regions and then linking these “reshaped human” variable regions to human constant regions. Improved by 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 derived from mouse antibodies. As can be observed in native human antibodies, although antibodies adapted to humanization by CDR-transplantation can still elicit some immune responses, such as anti- allogeneic or anti-genic responses, Transplanted antibodies are significantly less immunogenic than mouse antibodies and may therefore be used for longer-term treatment of patients.

However, it was soon discovered that antibodies with sufficient binding affinity were not produced solely by CDR-transplantation alone. CDR-grafted antibodies exhibit relatively poor binding characteristics compared to their non-human parent antibodies because more amino acids are involved in antigen binding than in the CDRs. As a result, CDR-grafted antibodies with poor binding affinity are not considered useful for therapy. Thus, attempts have been made to produce antibodies that combine the low immunogenicity of CDR-grafted antibodies with the superior binding characteristics of non-human parent antibodies. In addition to CDR-transplantation, it has evolved to the theory that one to several amino acids in the humanized framework region should be retained as a residue of rodent donor origin to retain binding affinity. Queen et al, (1989) ) Proc. Natl. Acad. Sci. 86 : 10029-10033.

Because of the potential utility of such antibodies in diagnosis and treatment, antibodies with improved properties suitable for the treatment of human cancer are desired.

It is an object of the present invention to provide antibodies with significantly superior properties compared to known CD44v6 specific antibodies.

Summary of the Invention

The technical problem mentioned above is solved by the aspects characterized by the claims and the specification. The above disadvantages of the art are overcome by the specification and claims of the present invention.

In order to solve the above-mentioned problems, the inventors have devised and produced a CD44v6 specific humanized antibody named BIWA8 which is CDR-grafted, skeletal-mutated and also combines low immunogenicity and high affinity.

However, the inventors were able to produce an antibody, BIWA4, with even greater therapeutic utility. Although this is inferior in binding affinity compared to BIWA8, it surprisingly shows a much more favorable biodistribution and tumor absorption rate when administered in vivo.

The present invention belongs to the field of oncology. The present invention relates to antibodies having specific sequences specific for epitopes encoded by variant exon v6 of the CD44 gene and derivatives of such antibodies. The present invention also relates to nucleic acid molecules encoding such antibody proteins. Moreover, the present invention relates to a method for producing said antibody protein. The invention also relates to a pharmaceutical composition comprising said antibody protein. Moreover, the present invention relates to the use in the manufacture of a medicament for the treatment of cancer.

The present invention belongs to the field of oncology. The present invention relates to antibodies having specific sequences specific for epitopes encoded by variant exon v6 of the CD44 gene and derivatives of such antibodies. The present invention also relates to nucleic acid molecules encoding such antibody proteins. Moreover, the present invention relates to a method for producing said antibody protein. The invention also relates to a pharmaceutical composition comprising said antibody protein. Moreover, the present invention relates to the use in the manufacture of a medicament for the treatment of cancer.

(See Example 1)

1: Assessment of relative binding affinity tested in competitive cellular ELISA. IC50: The concentration of 50% cMAb and hMAbs is inhibited in binding of mMAb BIWA1 to attached A431 cells. IC50 values for BIWA2 are indicated.

FIG. 2: Biodistribution of ¹²⁵ I- and ¹³¹ I-labeled CD44v6-specific MAbs (10 μCi, 50 μg) in HNX-OE xenograft transplant-bearing mice 3 or 4 days after infusion. Three groups of mice were assigned (A) ¹³¹ I-U36 (black bars) and ¹²⁵ I-BIWA1 (hatched bars) (n = 5), (B) ¹³¹ I-BIWA4 (black bars) and ¹²⁵ I-BIWA2 (hatched) Parent bars) (n = 6) or (C) ¹³¹ I-BIWA4 (black bars) and ¹²⁵ I-BIWA8 (hatched bars) (n = 6). After 3 days (A) or 4 days (B, C) of injection, mice were bleeded, sacrificed and dissected, and the radioactivity levels (% ID / g ± sem) of tumors, blood and several organs were evaluated. (Bld: blood, Tum: tumor, Liv: liver, Spl: spleen, Kid: kidney, Hrt: heart, Stm: stomach, Ilm: ileum, Cln: colon, Blr: bladder, Str: sternum, Msc: muscle, Lng : Lung, Skn: Skin, Tng: Tongue).

Figure 3: Therapeutic efficacy of ¹⁸⁶ Re-labeled CD44v6-specific MAbs in HNX-OE xenograft transplant-bearing hairless mice. 300 μCi ¹⁸⁶ Re-U36 (- -, Figure A), 300 μCi ¹⁸⁶ Re-BIWA1 (- -, Figure A), 300 μCi ¹⁸⁶ Re-BIWA4 (- -, Figure B), 300 μCi ¹⁸⁶ Re-BIWA2 (- -, Figure B), 400 μCi ¹⁸⁶ Re-BIWA4 (- -, Figure C), 400 μCi ¹⁸⁶ Re-BIWA8 (- -, Figure C), or saline as a control (- A, B, and C) were administered. The control groups in Figures A and B are identical. Tumor size is expressed as the mean tumor volume (± sem) for one treatment compared to the mean tumor volume at the start of treatment.

4: Relationship between AUC observed and doses of MAb administered after intravenous infusion of BIWA 4 in 10 patients in Part A of the study.

Figure 5: Relationship between the maximum plasma BIWA 4 concentration observed and the dose of MAb administered after intravenous infusion of BIWA 4 to 10 patients in Part A of the study.

Prior to aspects of the present invention, the singular forms “a”, “an” and “the” as used in this specification and the appended claims should be understood to encompass the plural forms unless otherwise indicated. Thus, for example, "antibody" refers to a number of antibodies, and "cell" refers to one or more cells and their equivalents known to those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly recognized by one of ordinary skill in the art to which this invention belongs. Although all methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described below. All publications mentioned herein are incorporated herein by reference for the purpose of describing and describing the cell lines, vectors and methodologies reported in the publications that may be used in connection with the present invention. What is not described herein should be regarded as accepting that the present invention is not entitled to antedate the foregoing specification by virtue of prior invention.

As used herein, the term “antibody molecule” or “antibody protein” or “antibody” should be considered equivalent.

"Complementarity determining regions of monoclonal antibodies" are described in Chothia and Lesk (1987) J. Mol. Biol. 196 : 901-917, Kabat EA, Wu TT, Perry HM, Gottesman KS and Foeller C. (1991) Sequences of Proteins of Immunological Interest (5th Ed.). NIH Publication No. 91-3242. US Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD.] Should be recognized that the amino acid sequence involved in specific antigen binding.

The term “skeletal modification” as used herein refers to the exchange, deletion or addition of a single or multiple amino acids in the variable region surrounding an individual complementarity determining region. Skeletal modifications can affect the immunogenicity, productivity or binding specificity of an antibody protein.

The present invention relates to an antibody molecule comprising a variable region of a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 1, or fragments thereof, allelic variants, functional variants, glycosylation variants, fusion molecules or chemicals To derivatives. Both antibodies BIWA 4 and BIWA 8 comprise the variable region of the heavy chain as characterized by the amino acid sequence of SEQ ID NO: 1.

A “fragment” according to the invention is a shorter antibody molecule, ie all polypeptide subsets, characterized by a nucleic acid molecule shorter than that described below, but still retaining its antigen binding activity.

A “functional variant” of an antibody molecule according to the invention is an antibody molecule which has a biological activity (functional or structural) substantially similar to the antibody molecule according to the invention, ie, substantially similar substrate specificity or substrate cleavage. The term “functional variant” also includes “fragment”, “allelic variant”, “functional variant”, “variant based on degenerate nucleic acid code” or “chemical derivative”. Such "functional variants" may, for example, involve one or several point mutations, one or several nucleic acid exchanges, deletions or inserts, or one or several amino acid exchanges, deletions or inserts. can do. The functional variant still retains its biological activity, such as antibody binding activity, which may at least partly or even be accompanied by improved biological activity.

A “functional variant” of an antibody molecule according to the invention is an antibody molecule which has a biological activity (functional or structural) that is substantially similar to the antibody molecule according to the invention, ie, substantially similar target molecule binding activity. The term “functional variant” also includes “fragment”, “allelic variant”, “functional variant”, “variant based on degenerate nucleic acid code” or “chemical derivative”.

An “allelic variant” is an allelic variant, eg, a variant due to a difference in two alleles in a human. The variant still retains its biological activity, such as antibody target binding activity, which may at least partially or even be accompanied by improved biological activity.

"Variants based on the degeneracy of the genetic code" are variants due to the fact that certain amino acids may be encoded by several different nucleotide triplets. The variant still retains its biological activity, such as antibody binding activity, which may at least partially or even be accompanied by improved biological activity.

A “fusion molecule” can be, for example, an antibody molecule according to the invention fused with a reporter, such as a radiolabel, a chemical molecule, such as a toxin or fluorescent label, or any other molecule known in the art.

As used herein, a "chemical derivative" according to the invention is an antibody molecule according to the invention which contains additional chemical moieties which are chemically modified or which are normally not part of the molecule. Such residues may improve the activity of molecules such as target destruction (eg, tumor cell death) or may improve their solubility, absorption, biological half-life, and the like.

Any molecule is "substantially similar" to another molecule when both molecules have substantially similar structure or biological activity. Thus, if two molecules have similar activity, a structure is considered to be a variant as used herein, even if the structure of one of these molecules is not found in the other or the amino acid residue sequence is not identical.

For many uses of the antibodies according to the invention, it is desired to have the smallest possible antigen-binding unit, ie CD44v6-binding unit. Thus in another preferred embodiment, the 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 variable regions of both chains which are held together by adjacent constant regions. They can be formed from conventional antibodies by protease digestion, for example using papain, but similar Fab fragments may be generated during genetic engineering processing. In another preferred embodiment, the antibody protein according to the invention is an F (ab ') 2 fragment, which may be prepared by proteolytic cleavage with pepsin.

Genetic engineering methods can be used to prepare shorter antibody fragments consisting solely of the variable region (VH) of the heavy chain and the variable region (VL) of the light chain. These are referred to as Fv fragments (fragment variable = fragment of variable part). In another preferred embodiment, the CD44v6-specific antibody molecule according to the invention is said Fv fragment. Because these Fv-fragments lack covalent bonds of the two chains by the cysteine of the constant chains, the Fv fragments are often stabilized. It is advantageous to link the variable region of the heavy chain and the variable region of the light chain by short peptide fragments, for example 10-30 amino acids, preferably 15 amino acids. In this way, a single peptide chain consisting of VH and VL, which is linked by a peptide linker, is obtained. Antibody proteins of this kind are known as single-chain-Fv (scFv). Examples of scFv-antibody proteins of this kind known from the prior art are described in Houston et al., 1988, PNAS 16: 5879-5883. Thus, in another preferred embodiment, the CD44v6-specific antibody protein according to the invention is a single-chain-Fv protein (scFv).

In recent years, various strategies have been developed for producing scFv as a multimeric derivative. This is particularly intended to induce recombinant antibodies with improved binding aspiration as well as improved pharmacodynamic and biodistribution properties. To achieve multimerization of scFv, scFv was prepared as a fusion protein with the multimerization domain. The multimerization domain can be, for example, a helical coil structure (spiral structure), such as the CH3 region or the Leucin-zipper domain of IgG. However, there is also a strategy that the interaction between the VH / VL regions of the scFv is used for multimerization (eg di-, tri- and pentabodies) processes. Thus in another preferred embodiment, the antibody protein according to the invention is a CD44v6-specific diabody antibody fragment. One skilled in the art refers to a dibody divalent homo-dimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883). Shortening the linker length to 5 to 10 amino acids in the scFv molecule leads to the formation of homo-dimer in which interchain VH / VL-redundancy occurs. Diabodies can be further stabilized by incorporating disulfide bridges. Examples of diabodi-antibody proteins from the prior art are described in Perisic et al., 1994, Structure 2: 1217-1226.

One skilled in the art means a dibody, divalent homo-dimeric scFv derivative. It consists of a fusion protein containing a CH ₃ region, preferably IgG, most preferably IgG1 of an immunoglobulin as a dimerization region that is linked to the scFv via a hinge region (eg IgG1 origin) and a linker region. Disulfide bridges in the hinge region are mostly formed in higher cells and not in prokaryotic cells. In another preferred embodiment, the antibody protein according to the invention is a CD44v6-specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art are described in Hu et al., 1996, Cancer Res. 56: 3055-61.

One skilled in the art refers to triabody as a trivalent homo-trimeric scFv derivative (Kortt et al., 1997 Protein Engineering 10: 423-433). ScFv derivatives in which VH-VL is directly fused without a linker sequence lead to the formation of trimers.

Those skilled in the art will also be well aware of the so-called mini-antibodies having divalent, trivalent or tetravalent structures and derived from scFv. Multimerization can be achieved by dimer, trimer or tetrameric helical 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.

Thus, in a preferred embodiment, the antibody protein according to the invention is a CD44v6-specific multimerization molecule based on the above-mentioned antibody fragments, and can be, for example, triabodies, tetravalent miniantibodies or pentabodies.

In a more preferred aspect, the invention relates to antibody molecules, wherein the variable region of the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 1.

In another preferred embodiment, the invention provides an antibody molecule comprising a variable region of a light chain as characterized by an amino acid sequence as defined in SEQ ID NO: 2, or a fragment thereof, an allelic variant, a functional variant, a glycosylation thereof. It relates to variants, fusion molecules or chemical derivatives. Antibody BIWA 4 as used herein comprises the variable region of the light chain as defined in the amino acid sequence of SEQ ID NO: 2.

In another more preferred aspect, the invention relates to antibody molecules, wherein the variable region of the light chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 2.

In another preferred embodiment, the invention provides an antibody molecule comprising a variable region of a light chain as characterized by an amino acid sequence as defined in SEQ ID NO: 3, or fragments thereof, allelic variants, functional variants, glycosylation thereof. It relates to variants, fusion molecules or chemical derivatives. Antibody BIWA 8 comprises the variable region of the light chain as characterized by the amino acid sequence of SEQ ID NO: 3.

In another more preferred aspect, the invention relates to antibody molecules, wherein the variable region of the light chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 3.

In another more preferred aspect, the invention comprises a variable region of a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 1 and is characterized by an amino acid sequence as defined in SEQ ID NO: 2 An antibody molecule comprising a variable region of a light chain as such, or fragments, allelic variants, functional variants, glycosylation variants, fusion molecules or chemical derivatives thereof. Antibody BIWA 4 comprises a variable region of the heavy chain as characterized by the amino acid sequence of SEQ ID NO: 1 and a variable region of the light chain as defined in the amino acid sequence of SEQ ID NO: 2.

In the most preferred embodiment, the invention consists of amino acids as the variable region of the heavy chain is characterized by the amino acid sequence of SEQ ID NO: 1, and the amino acid as the variable region of the light chain is characterized by the amino acid sequence of SEQ ID NO: It relates to an antibody molecule according to the invention consisting of.

In another more preferred aspect, the invention comprises a variable region of a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 1 and is characterized by an amino acid sequence as defined in SEQ ID NO: 3 An antibody molecule, or fragment thereof, allelic variant, functional variant, glycosylated variant, fusion molecule or chemical derivative, according to the present invention comprising a variable region of a light chain as such. Antibody BIWA 8 comprises a variable region of the heavy chain as characterized by the amino acid sequence of SEQ ID NO: 1 and a variable region of the light chain as defined in the amino acid sequence of SEQ ID NO: 3.

In another most preferred embodiment, the invention consists in that the variable region of the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 1, wherein the variable region of the light chain is characterized by the amino acid sequence of SEQ ID NO: 3 It relates to an antibody molecule according to the invention consisting of the same amino acids.

In another preferred embodiment, the invention provides an antibody molecule comprising a variable region of a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, or a fragment thereof, an allelic variant, a functional variant, a degenerate nucleic acid code To variants, fusion molecules or chemical derivatives based thereon. Antibodies BIWA 4 and BIWA 8 both comprise a variable region of the heavy chain as characterized by the nucleic acid sequence of SEQ ID NO: 4.

In another more preferred aspect, the invention relates to antibody molecules in which the variable region of the heavy chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 4.

In another preferred embodiment, the invention provides an antibody molecule comprising a variable region of a light chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 5, or a fragment thereof, an allelic variant, a functional variant, a degenerate nucleic acid code. To variants, fusion molecules or chemical derivatives based thereon. Antibody BIWA 4 as used herein comprises the variable region of the light chain as defined in the nucleic acid sequence of SEQ ID NO: 5.

In another more preferred aspect, the invention relates to antibody molecules in which the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 5.

In another preferred embodiment, the invention provides an antibody molecule comprising a variable region of a light chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 6, or fragments thereof, allelic variants, functional variants, degenerate nucleic acid codes. To variants, fusion molecules or chemical derivatives based thereon. Antibody BIWA 8 comprises the variable region of the light chain as characterized by the nucleic acid sequence of SEQ ID NO: 6.

In another more preferred aspect, the invention relates to antibody molecules in which the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 6.

In another more preferred aspect, the invention comprises a variable region of a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, and a variable region of a light chain encoded by a nucleic acid sequence as defined in SEQ ID NO: An antibody molecule, or fragment thereof, allelic variant, functional variant, variant, fusion molecule or chemical derivative based on a degenerate nucleic acid code according to the invention comprising a. Antibody BIWA 4 comprises a variable region of the heavy chain as characterized by the nucleic acid sequence of SEQ ID NO: 4 and a variable region of the light chain as defined in the nucleic acid sequence of SEQ ID NO: 5.

In another most preferred aspect, the invention provides that the variable region of the heavy chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, and the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 5, It relates to an antibody molecule according to the invention.

In another more preferred aspect, the invention comprises a variable region of a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, and a variable region of a light chain encoded by a nucleic acid sequence as defined in SEQ ID NO: An antibody molecule, or fragment thereof, allelic variant, functional variant, variant, fusion molecule or chemical derivative based on a degenerate nucleic acid code according to the invention comprising a. Antibody BIWA 8 comprises the variable region of the heavy chain as characterized by the nucleic acid sequence of SEQ ID NO: 4 and the variable region of the light chain as defined in the nucleic acid sequence of SEQ ID NO: 6.

In another most preferred aspect, the invention provides that the variable region of the heavy chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, and the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 6, It relates to an antibody molecule according to the invention.

To generate human adapted CD44v6-specific antibody proteins, the described nucleic acid sequences were expressed by molecular biology methods known in the art (see below and Examples).

The variable regions of the antibody proteins of the invention typically link to one or more portions of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, preferably immortalized B cells, according to well known procedures (Kabat et al., Supra, and WO 87/02671). Thus, the antibody protein of the present invention may contain all or only part of the constant region as long as it exhibits specific binding to the CD44v6 antigen. The choice of type and extent of the constant region depends on whether effector function such as complement fixation or antibody dependent cellular toxicity is desired, and on the desired pharmacological properties of the antibody protein. Antibody proteins of the invention will typically be tetramers of two light / heavy chain pairs, but can be dimers, ie, light / heavy chain pairs such as Fab or Fv fragments.

Thus, in a further aspect, the invention relates to an antibody protein according to the invention, characterized in that it has a variable light region and a variable heavy chain region, each bound to a human constant region. In particular, the variable region of the light chain was linked to the human kappa constant region and the variable region of the heavy chain was linked to the human gamma-1 constant region. Other human constant regions for dimerizing light and heavy chains are also available to the expert.

Human adaptation of the variable regions of murine antibodies can be accomplished using methods known in the art. EP 0239400 describes the transplantation of murine variable region CDRs into the framework of human variable regions. WO 90/07861 describes a method for reshaping CDR-grafted variable regions by introducing additional framework modifications. WO 92/11018 describes a method for producing humanized Ig by combining donor CDRs with a receptor backbone having a high homology with the donor backbone. WO 92/05274 describes methods for preparing skeletal mutated antibodies starting from murine antibodies. Further prior art references on human adaptation of murine monoclonal antibodies are described in EP 0368684; EP 0438310; WO 92/07075 or WO 92/22653.

In another preferred embodiment, the invention relates to an antibody molecule according to the invention, characterized in that each of the variable region of the light chain and the variable region of the heavy chain is linked separately to a human constant region.

In another more preferred aspect, the invention relates to an antibody molecule according to the invention, wherein the human constant region of the light chain is a human kappa constant region.

In another more preferred aspect, the invention relates to an antibody protein according to the invention, wherein the human constant region of the heavy chain is a human IgG1 constant region.

And an antibody comprising a heavy chain as characterized by the amino acid sequence of SEQ ID NO: 7 and / or a light chain as characterized by the amino acid sequence of SEQ ID NO: 8 or characterized by the amino acid sequence of SEQ ID NO: 9 Is preferred.

Thus, another important aspect comprises a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 7 and a light chain as characterized by an amino acid sequence as defined in SEQ ID NO: 8. An antibody molecule, or fragment, allelic variant, functional variant, glycosylated variant, fusion molecule or chemical derivative thereof according to the present invention. Antibody BIWA 4 comprises a heavy chain as characterized by the amino acid sequence of SEQ ID NO: 7 and a variable region of the light chain as defined in the amino acid sequence of SEQ ID NO: 8.

In a most preferred embodiment, the invention is directed to the invention, wherein the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 7 and the light chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 8 It relates to the following antibody molecule. Antibody BIWA 4 consists of the sequence as described in amino acid sequence of SEQ ID NO: 7 (heavy chain) and the amino acid sequence of SEQ ID NO: 8 (light chain). BIWA 4 is a CDR-grafted antibody with no skeletal modification. Surprisingly, the antibody shows superior therapeutic efficacy, better biodistribution and tumor uptake, despite lower binding affinity than the skeletal-mutated antibody BIWA 8 (see Examples). This is the human adapted version of the above-mentioned antibody VFF-18 (BIWA1), having the complementarity determining region and the human constant region of the murine monoclonal antibody VFF-18 in the complete human skeleton. Thus, it is an antibody that exhibits extremely low immunogenicity which is a beneficial feature for humans. However, since there is no murine skeletal residue for optimizing antigen binding, the antigen binding affinity is quite low, such as the parental antibody VFF-18 above, and thus cannot be considered as a good candidate for therapeutic drugs. Unexpectedly, BIWA 4, despite poor binding affinity, is superior to other humanized versions of VFF-18 with higher binding affinity, resulting in extremely favorable biodistribution and tumor uptake in vivo.

Another important embodiment includes a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 7 and a light chain as characterized by an amino acid sequence as defined in SEQ ID NO: 9. Antibody molecule, or fragment thereof, allelic variant, functional variant, glycosylated variant, fusion molecule or chemical derivative thereof. Antibody BIWA 8 comprises a heavy chain as characterized by the amino acid sequence of SEQ ID NO: 7 and a variable region of the light chain as defined in the amino acid sequence of SEQ ID NO: 9.

In the most preferred embodiment, the invention relates to the invention, wherein the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 7 and the light chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 9 It relates to the following antibody molecule. Antibody BIWA 8 consists of the sequence as described in amino acid sequence of SEQ ID NO: 7 (heavy chain) and the amino acid sequence of SEQ ID NO: 9 (light chain). BIWA 8 is a CDR-grafted antibody with no skeletal modification. The antibody shows significantly higher binding affinity than BIWA 4 (see Examples).

Furthermore, an antibody comprising a heavy chain as encoded by a nucleic acid sequence of SEQ ID NO: 10 and / or a light chain as characterized by a nucleic acid sequence of SEQ ID NO: 11 or as characterized by a nucleic acid sequence of SEQ ID NO: 12 desirable. Such sequences include leader sequences and non-translated sequences as cloned into the vector pAD-CMV1 / pAD-CMV19.

Thus, another preferred embodiment comprises an antibody molecule according to the invention comprising a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 10 and a light chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 11. , Or fragments, allelic variants, functional variants, variants based on degenerate nucleic acid codes, fusion molecules or chemical derivatives thereof. Antibody BIWA 4 comprises the heavy region as encoded by the nucleic acid sequence of SEQ ID NO: 10 and the variable region of the light chain as encoded by the nucleic acid sequence of SEQ ID NO: 11.

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 the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 11.

Another preferred embodiment comprises an antibody according to the invention comprising a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 10 and comprising a light chain as characterized by a nucleic acid sequence as defined in SEQ ID NO. Molecule, or fragment thereof, allelic variant, functional variant, variant based on degenerate nucleic acid code, fusion molecule or chemical derivative. Antibody BIWA 8 comprises a heavy chain as encoded by the nucleic acid sequence of SEQ ID NO: 10 and a variable region of a light chain as encoded by the nucleic acid sequence of 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 the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 12.

Furthermore, an antibody comprising a heavy chain as encoded by a nucleic acid sequence of SEQ ID NO: 13 and / or a light chain as characterized by a nucleic acid sequence of SEQ ID NO: 14 or as characterized by a nucleic acid sequence of SEQ ID NO: 15 desirable. The sequence includes a leader sequence as cloned in vector N5KGlval.

Thus, another preferred embodiment includes a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 13 and comprises a light chain as characterized by a nucleic acid sequence as defined in SEQ ID NO: 14. Followed are antibody molecules, or fragments thereof, allelic variants, functional variants, variants based on degenerate nucleic acid codes, fusion molecules or chemical derivatives. Antibody BIWA 4 comprises the heavy region as encoded by the nucleic acid sequence of SEQ ID NO: 13 and the variable region of the light chain as encoded by the nucleic acid sequence of 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 ID NO: 13 and the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 14.

Another preferred embodiment comprises an antibody according to the invention comprising a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 13 and comprising a light chain as characterized by a nucleic acid sequence as defined in SEQ ID NO: 15. Molecule, or fragment thereof, allelic variant, functional variant, variant, fusion molecule or chemical derivative based on degenerate nucleic acid code. Antibody BIWA 8 comprises the heavy region as encoded by the nucleic acid sequence of SEQ ID NO: 13 and the variable region of the light chain as encoded by the nucleic acid sequence of SEQ ID NO: 15.

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 the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 15.

Most preferred are antibody proteins comprising heavy and light chains as encoded by the nucleic acid sequence of SEQ ID NO: 16. The sequence includes a leader sequence as cloned in vector N5KGlval.

Thus, another highly important aspect is an antibody molecule, or fragment, allelic variant, functional variant thereof according to the invention comprising a heavy chain and a light chain as encoded by a nucleic acid sequence as defined in SEQ ID NO: 16. , Variants, fusion molecules or chemical derivatives based on degenerate nucleic acid codes. Antibody BIWA 4 includes heavy and light chains as encoded by the nucleic acid sequence of SEQ ID NO: 16.

In a most preferred embodiment, the invention relates to an antibody molecule according to the invention, wherein the heavy and light chains are encoded by the nucleic acid sequence of SEQ ID NO: 16. The sequence encodes the entire antibody BIWA 4.

Antibody proteins of the invention relate to highly specific tools for targeting therapeutics against the CD44v6 antigen. Thus, in a further aspect, the invention relates to an antibody protein according to the invention, wherein the antibody protein is conjugated with a therapeutic agent. Among the many therapeutic agents known in the art, preferred is a therapeutic agent selected from the group consisting of radioisotopes, toxins, toxoids, inflammatory agents, enzymes, antisense molecules, peptides, cytokines and chemotherapeutic agents. Among radioisotopes, gamma, beta and alpha-emitting radioisotopes can be used as therapeutic agents. β-emitting radioisotopes are preferred as therapeutic radioisotopes. ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ¹³¹ iodine and ⁹⁰ yttrium have proven to be β-emitting radioisotopes that are particularly useful for achieving localization investigation and destruction of malignant tumor cells. Thus, radioisotopes selected from the group consisting of ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ¹³¹ iodine and ⁹⁰ yttrium are particularly preferred as therapeutic agents conjugated with the antibody proteins of the invention. For example, to radioiodize an antibody of the invention, a method as described in WO 93/05804 can be used.

Thus, a more preferred aspect of the invention is the 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.

More preferred aspects of the invention include MAG-3 (US 5082930 A, EP 0247866 B1 (page 2 lines 55-56-page 3lines 1-23), all of which are incorporated herein by reference); MAG-2 GABA (US 5681927 A, EP 0284071 B1 (page 6 lines 9-29)); And N2S2 ((= pentoate) US 4897255 A, US 5242679 A, EP 0188256 B1 (page 2, lines 38-page 3, lines 18)) to the antibody protein via a linker selected from the group. It is the following antibody protein.

The formula of the linker is as follows:

A more preferred aspect of the invention is the 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 invention is the antibody protein according to the invention, wherein the radioisotope is a β-emitting radioisotope.

A more preferred aspect of the invention is the antibody protein according to the invention, wherein the radioisotope is selected from the group consisting of ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ¹³¹ iodine and ⁹⁰ yttrium.

A more preferred aspect of the invention is the antibody protein according to the invention, wherein the radioisotope is ¹⁸⁶ rhenium.

A further aspect of the invention relates to the antibody protein according to the invention, characterized in that the antibody protein is labeled. The CD44v6-specific labeled antibody localizes and / or detects the CD44v6 antigen in vitro and / or in vivo. Labels are defined as markers that can be detected directly or indirectly. Indirect markers cannot be detected by themselves, but are defined as markers that require additional directly detectable markers specific to indirect markers. Preferred labels for practicing the present invention are detectable markers. Of the various detectable markers, the detectable markers selected from the group consisting of enzymes, dyes, radioisotopes, digoxigenin and biotin are most preferred.

Thus, a more preferred aspect of the invention is the antibody protein according to the invention, characterized in that the antibody protein is labeled. More preferred is the antibody protein according to the invention, wherein said label is a detectable marker. Further preferred is an antibody protein according to the invention, wherein the detectable marker is a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxigenin and biotin.

A further aspect of the invention relates to an antibody protein according to the invention, characterized in that the antibody protein is conjugated with an imageable agent. Various imageable agents, in particular radioisotopes, are available at the skill level of the art. Gamma-emitting radioisotopes are more preferred in practicing the present invention. ¹²⁵ iodine is most preferred.

Thus, a more preferred aspect of the invention is the antibody protein for the invention conjugated with an imageable agent. A more preferred aspect of the invention is the antibody protein according to the invention, wherein the imageable agent is a radioisotope. A more preferred aspect of the invention is the antibody protein according to the invention, wherein the radioisotope is a γ-emitting radioisotope. A more preferred aspect of the invention is the antibody protein according to the invention, wherein the radioisotope is ¹²⁵ I.

Thus, a more preferred aspect of the invention is that the antibody protein has about 0.5 to about 15 mCi / mg, or about 0.5 to about 14 mCi / mg, preferably about 1 to about 10 mCi / mg, preferably about 1 to about Antibody protein conjugated with the radioisotope as mentioned above, having a specific activity of 5 mCi / mg, and most preferably 2 to 6 mCi / mg or 1 to 3 mCi / mg.

Another preferred embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable carrier or excipient.

Pharmaceutically acceptable carriers may contain, for example, physiologically acceptable compounds that act to increase or stabilize the absorption of AMPA glutamate receptor agonists, antagonists or modulators. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. Remington's Pharmaceutical Sciences (1990), 18th ed. Mack Publ., Easton. Those skilled in the art recognize that the choice of a pharmaceutically acceptable carrier, including physiologically acceptable compounds, depends, for example, on the manner of administration of the composition.

In an animal or human body, depending on the disease or problem for which the pharmaceutical composition as mentioned above is to be treated, e.g., the origin and type of the tumor, intravenous or other pathways such as systemic, or tissue of interest Or administration locally or locally to an organ may prove advantageous. Systemic mode of action is desired when different organs or organ systems need to be treated, for example in systemic autoimmune diseases, allergies, transplantation of foreign organs or tissues, or diffuse tumors or tumors that are difficult to localize. do. Local mode of action may be contemplated where the tumor or immunological action only appears locally, for example local tumors.

Pharmaceutical compositions comprising the antibody proteins of the invention can be administered by different routes of administration known to those skilled in the art, in particular by direct injection or intravenous injection into the target tissue. For systemic administration, the intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral or intravesicular route is preferred. More preferred topical administration can be performed subcutaneously, intracutaneously, intracardiac, lobe, intramedullary, intrapulmonary, or directly in or near the tissue to be treated (connection, bone, muscle, nerve, epithelial tissue). Depending on the duration of treatment and efficacy desired, the pharmaceutical antibody composition may be administered once or several times, or intermittently, for example, based on different dosages, for example on days, weeks, or months. do.

To prepare pharmaceutical compositions comprising antibody formulations suitable for the administration described above, one skilled in the art can use known sterile solvents, which are injectable and physiologically acceptable. Aqueous isotonic solvents, such as saline or corresponding plasma protein solvents, for preparing ready-to-use solutions for parenteral injection or injection are readily available. The pharmaceutical composition may be present under sterile conditions, for example, as part of a kit, as a lyophilized or dry preparation, which may be reconstituted with a known injectable solvent immediately before use. The final formulation of the antibody composition of the present invention is present in combination with a sterile physiologically acceptable solution, which may be supplemented with known carrier materials and / or additives (e.g., serum albumin, dextrose, sodium sulfite, EDTA). Prepared for injection, infusion or perfusion by mixing the purified antibodies according to the invention.

The amount of antibody administered depends on the nature of the disease. For cancer patients, the dose of 'naked' antibody included in the pharmaceutical composition of the present invention is 0.1 to 100 mg / body surface area 1 m 2, preferably 5 to 50 mg / m 2, Preferably 10 to about 40 mg / m 2, preferably 10 to about 30 mg / m 2, also preferably 20 to about 30 mg / m 2, and most preferably about 25 mg / m 2. The most preferred antibody protein dose is also about 50 mg / m 2.

The dose of radioactivity administered to a patient per dose should be high enough to be effective, but below the dose limit toxicity (DLT). For example, for pharmaceutical compositions comprising an antibody labeled radioactively with ¹⁸⁶ rhenium, the maximum tolerated dose (MTD) should be determined not to exceed the therapeutic setting. Subsequently, administration of the radiolabeled antibody to a cancer patient may be performed by intravenous infusion of a dose of MTD or less (monthly or weekly). Welt et al. (1994) J. Clin. Oncol. 12 : 1193-1203. It is generally preferred to administer several times at weekly intervals, but the radiolabelled material should be administered at longer intervals, i.e. at intervals of 4 to 24 weeks, preferably 12 to 20 weeks. However, those skilled in the art can perform the administration in two or more times, which may shorten the administration interval or may be administered at predetermined intervals, for example, from 1 day to 1 week.

Moreover, the dose administered will be in accordance with the guidelines summarized below. In general, the radiation dose per dose will be between 30 and 75 mCi / body surface area (BSA) m 2. Thus, the amount of radiolabeled antibody in the pharmaceutical composition according to the invention, preferably labeled ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ^99m technetium, ¹³¹ iodine or ⁹⁰ yttrium, most preferably ¹⁸⁶ rhenium, is 10, 20, 30, 40, 50 or 60 mCi / m 2, preferably 50 mCi / m 2 patients. In a preferred embodiment, the invention relates to a pharmaceutical composition wherein the dose of said radiolabeled antibody according to the invention is MTD, preferably 50 mCi / m 2. This is broadly exemplified in clinical studies as presented in Examples 3-6.

In addition, the antibody protein is about 0.5 to about 15 mCi / mg, or 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 preferred Preferred is a pharmaceutical composition according to the invention comprising an antibody protein conjugated with a radioisotope according to the invention as defined above, with a specific activity of 2 to 6 mCi / mg or 1 to 3 mCi / mg. Do.

And an antibody protein conjugated with a radioisotope according to the invention as defined above, wherein said antibody or antibody derivative is present in an aqueous solution at about 7 to about 8 pH and at a concentration of about 0.5 to about 2.0 mg / ml. Preference is given to pharmaceutical compositions according to the invention comprising:

Preferred embodiments include ascorbic acid, gentisic acid, redductic acid, erythorbic acid, p-aminobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2,5-dihydroxy-1,4-benzenedisulfonic acid, A pharmaceutical composition according to the present invention further comprising at least one radioprotective agent selected from the group of povidone, inositol and / or citrate.

Preference is given to pharmaceutical compositions according to the invention wherein the radioprotectant is ascorbic acid.

Another preferred embodiment comprises an antibody molecule further selected from the group of antibody molecules BIWA 4 or BIWA 8 as defined above, wherein the antibody protein further comprises a radioprotectant ascorbic acid, linked to ¹⁸⁶ rhenium via MAG-2 GABA, Pharmaceutical compositions according to the invention.

Another preferred aspect of the invention is the use of an antibody protein according to the invention in the manufacture of a medicament for the treatment of cancer. In a preferred embodiment, the invention relates to the use of an antibody protein according to the invention conjugated with a therapeutic agent as mentioned above for the treatment of cancer. Cancer includes all diseases associated with malignant growth, such as solid tumors, sarcomas and leukemias. A prerequisite for the disease is the expression of CD44v6. Cancers according to the present invention include, but are not limited to:

1) treatment of epithelial carcinoma including breast, lung, colorectal, head and neck, pancreas, ovary, bladder, stomach, skin, endometrium, ovary, testes, esophagus, prostate and kidney origin;

2) bone and soft-tissue sarcoma: osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyosarcoma;

3) hematopoietic tumors: Hodgkin's and non-Hodgkin's lymphoma, leukemia;

4) neuroectodermal tumors: peripheral nerve tumors, astrocytomas, melanoma;

5) mesothelioma.

Examples of 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 cancer, lung cancer, bladder cancer, pancreatic cancer, and metastatic cancer of the brain.

Thus, a preferred embodiment is the use of an antibody protein according to the invention, wherein the 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 cancer of the brain. to be.

In addition, the amount of antibody protein per administration is 0.1 to 100 mg / body surface area m 2, preferably 5 to 50 mg / m 2, preferably 10 to about 40 mg / m 2, preferably 10 to about 30 mg / m 2. Preference is also given to the use of the antibody protein according to the invention as defined above in the manufacture of a medicament for the treatment of cancer, which is also preferably from 20 to about 30 mg / m 2, and most preferably about 25 mg / m 2. Most preferably, the antibody protein dose is about 50 mg / body surface area m 2.

Also preferred is the use of an antibody protein conjugated with a radioisotope according to the invention as defined above in the manufacture of a medicament for the treatment of cancer, wherein the radioactive dose per dose is 30 to 75 mCi / body surface area (BSA) m 2. . The antibody protein according to the invention is preferably radiolabeled with ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ^99m technetium, ¹³¹ iodine or ⁹⁰ yttrium, and most preferably radiolabeled with ¹⁸⁶ rhenium, as defined above in the preparation of a medicament for the treatment of cancer. The use of antibody proteins conjugated with radioisotopes according to the present invention as is preferred. In another preferred embodiment, the invention provides a composition as defined above in the manufacture of a medicament for the treatment of cancer, wherein the antibody dose is 10, 20, 30, 40, 50 or 60 mCi / m 2, most preferably 50 mCi / m 2. The use of an antibody protein conjugated with a radioisotope according to the invention. This is broadly exemplified in clinical studies as presented in Examples 3-6.

In addition, the antibody protein is about 0.5 to about 15 mCi / mg, or 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 preferred Preferably the use of an antibody protein conjugated with a radioisotope according to the invention as defined above in the manufacture of a medicament for the treatment of cancer, which has a specific activity of 2 to 6 mCi / mg or 1 to 3 mCi / mg, is preferred. .

Also in accordance with the invention as defined above in the manufacture of a medicament for the treatment of cancer, the antibody or antibody derivative is present in an aqueous solution at about 7 to about 8 pH and at a concentration of about 0.5 to about 2.0 mg / ml. The use of antibody proteins conjugated with radioisotopes is preferred.

The invention further comprises administering the antibody protein according to the invention once or several times to an individual in need thereof, wherein said antibody protein selectively binds CD44v6, destroying tumor cells via a therapeutic agent linked to said antibody protein, and treating It relates to a method of treating cancer, which monitors the success of medicine. The antibody protein may be present as an exposed / unmodified antibody protein, eg, a modified antibody protein such as a fusion protein, or an antibody protein conjugated with a therapeutic agent, which includes contacting a tumor with the antibody in the effective amount. do. Tumor treatment methods as mentioned above may be effective in vitro or in vivo. The cancer is any cancer as mentioned above.

The amount of antibody administered depends on the nature of the disease. For cancer patients, the dose of 'exposed' antibody is 0.1 to 100 mg / m 2 body surface area, preferably 5 to 50 mg / m 2, preferably 10 to about 40 mg / m 2, preferably one dose. Preferably from 10 to about 30 mg / m 2, also preferably from 20 to about 30 mg / m 2, and most preferably from about 25 mg / m 2. The most preferred antibody protein dose is also about 50 mg / m 2.

The dose of radioactivity administered to a patient per dose should be high enough to be effective, but below the dose limit toxicity (DLT). For example, for antibodies radiolabeled with ¹⁸⁶ rhenium, the maximum tolerated dose (MTD) should be determined not to exceed the therapeutic setting. Subsequently, administering the radiolabeled antibody to a cancer patient may be performed by repeated (monthly or weekly) intravenous infusion of a sub-MTD dose. See, Welt et al. (1994) J. Clin. Oncol. 12 : 1193-1203. It is generally preferred to administer several times at weekly intervals, but the radiolabelled material should be administered at longer intervals, i.e. at intervals of 4 to 24 weeks, preferably at intervals of 12 to 20 weeks. However, those skilled in the art can carry out the administration in two or more times, which may shorten the administration interval or may be administered at predetermined intervals, for example, from one day to one week.

Moreover, the dose administered will be in accordance with the guidelines summarized below. In general, the radiation dose per dose will be between 30 and 75 mCi / body surface area (BSA) m 2. Thus, the amount of radiolabeled antibody to be administered to a patient, preferably labeled ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ^99m technetium, ¹³¹ iodine or ⁹⁰ yttrium, most preferably ¹⁸⁶ rhenium, is 10, 20, 30, 40, 50 Or 60 mCi / m 2, preferably 50 mCi / m 2. In a preferred embodiment, the invention relates to a therapeutic method for preventing or treating a cancer by administering to a cancer patient a radiolabeled antibody having a dose of MTD, preferably 50 mCi / m 2, as mentioned above. will be. This is broadly exemplified in clinical studies as presented in Examples 3-6.

In addition, the antibody protein conjugated with the radioisotope according to the present invention as defined above is about 0.5 to about 15 mCi / mg, or about 0.5 to about 14 mCi / mg, preferably about 1 to about 10 mCi / mg, Preference is given to the method for treating cancer according to the invention (see above), which preferably has a specific activity of about 1 to about 5 mCi / mg, and most preferably 2 to 6 mCi / mg or 1 to 3 mCi / mg. .

Furthermore, according to the invention, the antibody protein conjugated with the radioisotope according to the invention as defined above is present in an aqueous solution at about 7 to about 8 pH and is present at a concentration of about 0.5 to about 2.0 mg / ml. Cancer treatment methods (see above) are preferred.

Preferably, the invention is in accordance with the invention, wherein the tumor is a cancer 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 cancer of the brain. To a method of treatment.

A further aspect of the invention is a nucleic acid characterized by encoding an antibody protein according to the invention. The nucleic acid may be RNA or preferably DNA. The DNA molecule can be synthesized chemically. First, suitable oligonucleotides can be synthesized using methods known in the art, which can be used to generate synthetic genes. Gait, MJ, 1984, Oligonucleotide Synthesis. A Practical Approach. IRL Press, Oxford, UK]. Methods for generating synthetic genes are known in the art. See Stemmer et al. 1995, Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides , Gene 164 (1): 49-53; Ye et al. 1992, Gene synthesis and expression in E. coli for pump, a human matrix metalloproteinase , Biochem Biophys Res Commun 186 (1): 143-9; Hayden et Mandecki 1988, Gene synthesis by serial cloning of oligonucleotides , DNA 7 (8): 571-7]. These methods can be used to synthesize any DNA molecule described herein, eg, DNA encoding BIWA 4.

Also preferably, the nucleic acid according to the invention is characterized by containing 5 'or 3', or 5 'and 3' non-toxic regions. Nucleic acids according to the present invention may contain other top end regions and / or bottom ends. The non-toxic region may contain regulatory elements such as, for example, transcription initiation units (promoters) or enhancers. The promoter can be, for example, a constitutive, inducible or developmentally-controlled promoter. Preferably, the constitutive promoters of monkey virus 40 (SV 40) and herpes simplex promoter, as well as other known promoters, human cytomegalovirus (CMV) and Raus sarcoma virus (RSV), are not excluded. Inducible promoters according to the present invention include antibiotic-resistant promoters, heat-sensitive promoters, hormone-induced "breast tumor virus promoters" and metallothionein promoters. Also preferably, the nucleic acid according to the invention is characterized by encoding a fragment of the antibody protein according to the invention. It refers to part of the polypeptide according to the invention.

Preferably, the nucleic acid according to the invention is a nucleic acid as described in SEQ ID NOs: 4, 5, 6, 10, 11, 12, 13, 14, 15 and / or 16. The nucleic acid is most preferably the nucleic acid of SEQ ID NO: 16.

Another important aspect of the invention is a recombinant DNA vector, characterized by containing a nucleic acid according to the invention. Preferably, the vector contains a nucleic acid as characterized by SEQ ID NOs: 4, 5, 6, 10, 11, 12, 13, 14, 15 and / or 16. Most preferably, the vector contains a nucleic acid as characterized by SEQ ID NO: 16.

Examples thereof are viral vectors such as vaccinia, Semliki-Forest-virus and adenovirus, for example. Vectors for use in COS-cells have an SV 40 origin of replication and can achieve high copy number plasmids. Vectors for use in insect cells are described, for example, in E. coli. E. coli is a transition vector and contains, for example, a DNA encoding a polyhedrin as a promoter.

Another preferred aspect of the invention is a recombinant DNA vector according to the invention, characterized in that it is an expression vector.

Another preferred aspect of the invention is a recombinant DNA vector according to the invention, characterized in that it is a vector pAD-CMV or a functional derivative thereof. The derivative is, for example, pAD-CMV1, pAD-CMV19 or pAD-CMV25.

Another preferred aspect of the invention is the recombinant DNA vector according to the invention, characterized in that SEQ ID NO: 17 or a functional derivative thereof.

Another preferred aspect of the invention is the recombinant DNA vector according to the invention, characterized in that SEQ ID NO: 18 or a functional derivative thereof.

Also preferably, said vector comprises one or several nucleic acid molecules as characterized by SEQ ID NOs: 4, 5, 6, 10, 11, 12, 13, 14, 15 and / or 16.

Also preferred are vectors as described in US 5648267 A or US 5733779 A comprising a nucleotide sequence according to the invention. Also preferably, said vector comprises one or several nucleic acid molecules as characterized by SEQ ID NOs: 4, 5, 6, 10, 11, 12, 13, 14, 15 and / or 16. Another preferred aspect of the invention is the recombinant DNA vector according to the invention, characterized in that it is a vector N5KG1val or a derivative thereof.

Another important aspect is a host characterized by containing a vector according to the invention.

Another important aspect is the host according to the invention, characterized in that it is a eukaryotic host cell. Eukaryotic host cells according to the present invention include fungi such as Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces , Trichoderma , and insects. Cells (eg Spodoptera frugiperda Sf-9 origin, using a baculovirus expression system), eg plant cells such as Nicotiana tabacum origin, e.g. For example, mammalian cells such as COS cells, BHK, CHO or myeloma cells are included.

In the progeny of immune system cells, where the antibody protein also forms in the body, the antibody protein according to the invention is particularly well folded and glycosylated. Mammalian host cells, preferably CHO or COS cells, for example CHO DG44 [Urlaub and Chasin, Proc. Natl. Acad. Sci. U. S. A. 77 (7): 4216-20 (1980)], or CHO-K1 (ATCC CCL-61) cells. Thus, another preferred aspect is the host according to the invention, characterized in that it is BHK, CHO or COS cells, most preferably CHO DG44 or CHO-K1 (ATCC CCL-61) cells.

Another preferred aspect is the host according to the invention, characterized in that it is a bacteriophage.

Another preferred aspect is the host according to the invention, characterized in that it is a prokaryotic host cell. Examples of the prokaryotic host cell is Escherichia cha coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces) or Proteus Mira Billy's (Proteus mirabilis).

The invention further comprises the steps of culturing a host according to the invention under conditions in which said host cell expresses said antibody protein; And it relates to a method for producing an antibody protein according to the invention, characterized in that it comprises the step of separating the antibody protein. Antibodies according to the invention can be produced as follows. Nucleic acid molecules encoding light and heavy chains can be synthesized chemically and enzymatically by standard methods. First, suitable oligonucleotides can be synthesized using methods known in the art (see above), and methods for generating synthetic genes from oligonucleotides are known in the art (see above). These nucleic acid molecules encoding antibody heavy and light chains are cloned into an expression vector (cloning both chains into one vector molecule, or cloning each chain into separate vector molecules) and then introduced into the host cell. The host cell is preferably a mammalian host cell (see above details), for example a COS, CHO or BHK cell, more preferably a Chinese hamster ovary (CHO) cell. The host cell is then incubated in a suitable culture medium under conditions that produce the antibody in question, and then the antibody is isolated from the culture according to standard procedures. The production of antibodies from recombinant DNA in host cells and respective expression vectors is well known in the art (eg, WO 94/11523, WO 97/9351, EP 0481790).

The invention preferably relates to a method according to the invention, characterized in that the host is a mammalian cell, preferably a CHO or COS cell.

The invention preferably relates to a method according to the invention, characterized in that the host cell is co-transfected with two plasmids carrying expression units for the light or heavy chain.

The following examples are provided to further illustrate the invention but do not limit the scope of the invention described herein.

Example 1 Radioimmunotherapy

Material and method:

Monoclonal Antibodies: mMAb BIWA 1 = VFF18, which was assigned to the DSM ACC2174 on June 7, 1994 to the Depositary (the DSM-Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Deutschland). Secreted by deposited hybridoma cell lines; See also WO 95/33771, which was generated by immunizing BALB / c mice with glutathione S-transferase fusion proteins containing human CD44 domain v3-v10. Epitopes recognized by BIWA 1 have been mapped to amino acids 360-370 in domain v6 of CD44 (numbered according to Kugelman et al., (1992)). The batch used in this study was obtained after purification on Protein-G-Sepharose and dialysis against PBS.

MAb U36 (IgG1) was induced after immunizing mice with the HNSCC cell line UM-SCC-22B and recognized different epitopes in CD44v6 as BIWA 1. U36 was purified from the tissue culture supernatant concentrated by affinity chromatography on Protein-A-Sepharose and further purified on Q-Sepharose.

Generation of chimeric and human adapted MAbs: mRNA was isolated from the BIWA 1 hybridoma cell line by using the QuickPrep mRNA Purification Kit (Pharmacia, Uppsala, Sweden). CDNAs were generated from the variable heavy (V _H ) and variable light (V _L ) using RT-PCR.

The fragment was cloned into the TA cloning vector pCR II (Invitrogen, Groningen, The Netherlands) and sequenced. Two expression vectors derived from plasmid pAD CMV1 (Himmler et al., 1990) were constructed with the constant region of human gamma-1 and the constant region of human kappa light chain, respectively. Subsequently, the V _H and V _L fragments of BIWA 1 were cloned before the constant region into the corresponding expression vectors. The chimeric antibody was named cMAb BIWA 2. Human adapted versions of the BIWA 1 heavy and light chain variable regions (generated by the CDR transplantation process) were cloned before the immunoglobulin constant regions of the aforementioned expression vectors. For the construction of human adapted antibodies, the human variable region used was derived from human immunoglobulin fragments (Accession No. S31669 of database GenPept for the heavy chain) and human immunoglobulin HUMIGKAX (rearranged anti- Myelin kappa chain) (GenBank Accession No. M29469). The resulting MAbs were named hMAb BIWA 4 and BIWA 8, respectively. BIWA 8 contains the two amino acids of the murine parental antibody in the light chain framework 2, whereas BIWA 4 does not contain any murine residues in the framework.

Recombinant MAbs were stably expressed by electroporation with heavy and light chain expression plasmids on dihydrofolate reductase deficient Chinese hamster ovary cells. Cells were seeded into 96 well microtiter plates at densities of 500 and 100 cell counts / well in selection medium (α-MEM with 10% dialyzed fetal calf serum). Once colonies were visible (after approximately 14 days), the culture supernatants were tested for their IgG content by ELISA and the best producers were doubled. Gene amplification was performed by incubating in the presence of increasing concentrations of methotrexate (20-500 nM).

Laboratory-scale production of chimeric and human adapted MAbs was performed in 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 tested by SDS-PAGE and high performance size sorting chromatography.

Antibody Affinity Assessment: Determination of kinetics and affinity constants using recombinant antigens was performed on a BIAcore 2000 system (BIAcore AB, Uppsala, Sweden). Glutathione-S-transferase fusion protein (GST / CD44v3-v10; 20 μg / ml) containing the domain v3-v10 of human CD44, using 10 mM sodium acetate pH 5.0 as coupling buffer, according to the manufacturer's instructions Immobilized on the CM5 sensor chip by the coupling method. 35 μl of MAb at various concentrations (8 to 67 nM) in HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% BIAcore surfactant P20) was injected across the antigen-coated surface at a flow rate of 5 μl / min. It became. Dissociation of the MAb for 5 minutes in buffer flow rate (HBS) was assessed. Between the two analytes, the chip surface was regenerated using a single pulse of 15 μl 30 mM HCl. Data analysis and dynamic constant estimation were performed using BIAcore's BIAevaluation software, version 2.1. Association rate (k _a ), dissociation rate (k _d ) and dissociation constant (K _d ) for all antibodies were evaluated.

Relative binding affinity was also assessed by competitive cellular ELISA. Human A431 cells, derived from epidermal carcinoma of the vulva and known to express high levels of CD44v6, have 200 μl of RPMI 1640 20 μl / containing 10% fetal calf serum at a density of 2.5-5 × 10 ⁵ cells / ml. The wells were seeded into 96 well tissue culture plates. The plates were incubated overnight at 37 ° C. in a hygroscopic incubator with 5% CO ₂ in air. After removing the medium, the cells were washed once with PBS, fixed with 96% ethanol for 1 minute, and then washed again with PBS. cMAb BIWA 2, hMAb BIWA 4 and hMAb BIWA 8 (prediluted at 10 μl / ml) were diluted 1: 2 serially in 100 μl / well in PBS / 0.5% BSA / 0.05% Tween 20 (assay buffer) Step) and incubated at room temperature for 30 minutes. 100 μl of prediluted mMAb BIWA 1 (20 ng / ml) was added and the plate was incubated for 2 hours at room temperature on an orbital shaker. The control sample contains only BIWA 1 without BIWA 1 (0% control) or contains only BIWA 1 without any competitive antibody (100% control). After three washes with PBS / 0.05% Tween 20 (wash buffer), 100 μl of secondary antibody (peroxidase-conjugated Gout anti-mouse Fc, 1 in assay buffer for detection of mMAb BIWA 1) : Diluted to 15,000, DAKO Copenhagen, Denmark) was added and the plate was incubated for 1 hour at room temperature on an orbital shaker. After washing three times with wash buffer, the plates were developed with 100 μl / well tetramethylbenzidine substrate solution (Kierkegaard and Perry Laboratories, Gaithersburg, USA). The reaction was stopped after 15 minutes with 50 μl / well 1 M phosphoric acid. Absorbance at 450 nm was measured in an ELISA plate reader (reference 610-690 nm).

Radioiodination of Antibodies: Iodide of MAbs was essentially performed using either ¹²⁵ I (100 mCi / ml) or ¹³¹ I (200 mCi / ml) (both purchased from Amersham, Aylesbury, England), see Haisma et al. (1986). 1 mg of MAb IgG and 1 mCi ¹²⁵ I or ¹³¹ I dissolved in 500 μl PBS, pH 7.4 were mixed in vials coated with 75 μg Iodogen (Pierce, Oud Bijerland, The Netherlands). Five minutes after incubation at room temperature, free iodine was removed by gel filtration on PD10-column (Pharmacia-LKB, Woerden, The Netherlands). After removal of unbound ¹²⁵ I or ¹³¹ I, radiochemical purity always exceeded 97% as determined by the TLC and HPLC procedures described previously in Van Gog et al., 1997a. HPLC analysis showed no aggregates or fragments to form at all.

Preparation of Rhenium- ¹⁸⁶ -labeled MAbs : ¹⁸⁶ Re-labeled MAbs were chelate S-benzoylmercaptoacyltriglycine (S-benzoyl-MAG3) as previously described in Van Gog et al., 1997a. It was prepared according to a multi-step process using. In this process, a solid phase synthesis is performed to prepare ¹⁸⁶ Re-MAG3 by esterifying with 2,3,5,6-tetrafluorophenol (TFP) and conjugating a reactive ¹⁸⁶ Re-MAG3-TFP ester to the MAb. It became. After conjugation, ¹⁸⁶ Re-labeled MAbs were purified on PD10-columns. After removal of unbound ¹⁸⁶ Re, the radiochemical purity always exceeded 98%.

Binding Assays for Radiolabeled Antibodies: The in vitro binding characteristics of the labeled MAbs used in biodistribution and therapeutic studies are essentially an immunoreactive assay as described previously in Van Gog et al., 1997a. It was decided. To test the binding of iodinated or ¹⁸⁶ Re-labeled MAbs, UM-SCC-11B cells immobilized in 0.1% glutaraldehyde were used. UM-SCC-11B cells are friendly to the following sources [Dr. TE Carey, University of Michigan, Ann Arbor, MI. Five serial dilutions (range from 5 × 10 ⁶ cells / tubes to 3.1 × 10 ⁵ cells / tubes) were prepared using 1% BSA in PBS. Excess of unlabeled MAb IgG was added to the tube containing the lowest concentration of cells to determine non-specific binding. 10,000 cps of IgG labeled ¹²⁵ I, ¹³¹ I or ¹⁸⁶ Re were added to each tube and the samples were incubated overnight at 4 ° C. Cells were radiated and the radioactivity in the pellet and supernatant was measured with a gamma counter (LKB-Wallace 1282 CompuGamma, Kabi Pharmacia, Woerden, The Netherlands), and the binding and free radioactivity ratios were calculated. Data was graphically analyzed with modified Lineweaver Burk plots, and immunoradioactivity was determined by linear extrapolation to conditions indicating infinite antigen overload.

Biodistribution Studies in HNSCC-bearing Hairless Mice: For biodistribution experiments, hairless mice carrying a subcutaneously implanted human HNSCC xenograft implant (HNX-OE) were previously described in Van Gog et al., 1997a. Was used as described. Female mice (Hsd: athymic nu / nu, 25-32 g, Harlan CPB, Zeist, The Netherlands) were 8 to 10 weeks old at the time of the experiment. Three biodistribution experiments showed one or two tumors ranging from 30 to 470 mm 3. In the first experiment, 10 μCi (50 μg) ¹³¹ I-labeled mMAb U36 was 133 ± simultaneously with 10 μCi (50 μg) ¹²⁵ I-labeled mMAb BIWA 1 Mice bearing tumors of 28 mm 3 (n = 20 mice, 37 tumors) were injected in the second experiment, 10 μCi (50 μg) ¹³¹ I-labeled hMAb BIWA 4 and 10 μCi (50 μg) ¹²⁵ I-labeled cMAb BIWA 2 together was injected into mice bearing 167 ± 31 mm 3 tumors (n = 21 mice, 32 tumors) In the third experiment, 10 μCi (50 μg) ¹³¹ I-label Both hMAb BIWA 4 and 10 μCi (50 μg) ¹²⁵ I-labeled hMAb BIWA 8 were injected into mice bearing 130 ± 21 mm 3 tumors (n = 23 mice, 40 tumors). After dilution in 0.9% NaCL, the conjugates were injected intravenously (iv) in a volume of 100 μl, with the same murine or human isotypes to obtain comparable blood / in vivo clearance of MAbs injected together. Only MAbs were combined, high enough to prevent the rapid removal of the MAb from the blood with respect to the isotype (Sharkey et al., 1991, Van Gog et al., 1997b) and antigens in tumors Low doses of antibody (total 100 μg dose per mouse) were chosen to prevent saturation.

Mice were euthanized, bled, killed and dissected at the indicated time points after injection. In addition to the tumor, the following organs were removed: liver, spleen, kidney, heart, stomach, ileum, colon, bladder, sternum, muscle, lung, skin and tongue. After gravimetry, radioactivity in tumors, blood and organs was counted with a double-isotope gamma counter (LKB-Wallace 1282 CompuGamma) with automatic calibration for ¹³¹ I-Comptons at ¹²⁵ I window settings. Radioactivity uptake in the tissue was calculated as the ratio (% ID / g) of dose injected per gram of tissue.

By the day of MAb administration, mice were commonly bred under specific-pathogen free conditions in sterile cages in a humidity and temperature controlled clean room of classification 2000 according to Federal Standard 209d. On the day of injection, mice were transported to a Radio Nuclide Center and sterile radioimmunoconjugates were administered in a laminar flow hood under sterile conditions.

Radioimmunotherapy Study in Hairless Mice: Animal RIT studies were performed to compare the therapeutic efficacy of different MAbs labeled with ¹⁸⁶ Re. The immunoreactive fraction of the conjugates always exceeded 75%. Three treatment experiments were performed using mice with one or two HNX-OE tumors ranging from 45-195 mm 3. ^{The 186} Re dose was selected at the maximum tolerated dose (MTD) level (ie 400 μCi) or lower (300 μCi). MTD levels are defined as doses that result in 5-15% weight loss. In the first experiment, 300μCi (100 ㎍) ¹⁸⁶ Re--labeled mMAb U36 or 300 μCi (100 ㎍) ¹⁸⁶ Re--labeled mMAb BIWA 1 was a single intravenous injection to the mice. In the second experiment, 300 μCi (100 μg) ¹⁸⁶ Re-labeled hMAb BIWA 4 or 300 μCi (100 μg) ¹⁸⁶ Re-labeled cMAb BIWA 2 was administered, and in the third experiment, 400 μCi (100 μg) ¹⁸⁶ Re -Labeled hMAb BIWA 4 or 400 μCi (100 μg) ¹⁸⁶ Re-labeled hMAb BIWA 8 was administered. Mean tumor volume was similar for all experimental groups. Experiment 1: ¹⁸⁶ Re-mMAb U36 for the treated group 95 ㎣ ± 34 ㎣ (n = 7 mice, 12 tumors) ^{and, 186 Re-mMAb BIWA 1 91} ㎣ ± 15 ㎣ for the treated group (n = 7 mice, 12 tumors) and 99 ㎣ ± 54 ㎣ (n = 6 mice, 11 tumors) for the control group. Experiment 2: ¹⁸⁶ Re-hMAb BIWA 4 for the treated group 101 ㎣ ± 35 ㎣ (n = 7 mice, 12 tumors) and, ¹⁸⁶ Re-cMAb BIWA 2 for the treated group 92 ㎣ ± 43 ㎣ (n = 7 mice, 12 tumors), whereas the control group was the same as in Experiment 1. Experiment 3: ¹⁸⁶ Re-hMAb BIWA 4 for the treated groups 105 ㎣ ± 43 ㎣ (n = 8 mice, 13 tumors) ^{and, 186 Re-hMAb BIWA 8 100} ㎣ ± 42 ㎣ for the treated group (n = 8 mice, 13 tumors), and 110 mm ± 46 mm (n = 7 mice, 11 tumors) for the control group. Tumors were measured twice weekly during treatment, and tumor volumes were calculated as compared to tumor volumes at the start of treatment. Toxicity was monitored by weighing twice a week. Mice were sacrificed when one of the tumors exceeded 1000 mm 3.

Statistics: Tissue uptake differences between co-injected MAbs were statistically analyzed at each time point using the Student's t-test on paired data. Mean tumor volume differences between the various RIT treatment groups were statistically analyzed at each time point using Student's t-test on independent samples.

result

In vitro binding characteristics of CD44v6-specific MAbs: The binding affinity of the five MAbs was analyzed using recombinant tumors as well as human tumor cell lines. The kinetics and affinity constants were assessed by surface plasmon resonance using GST / CD44v3-v10 as immobilized antigen. Table 1 shows the association rate (k _a ), dissociation rate (k _d ) and dissociation constant (K _d ). Containing the same variable region, mMAb BIWA 1 and cMAb BIWA 2 have similar k _a , k _d and K _d and show the highest affinity. In contrast, mMAb U36 and hMAb BIWA 4 have lower k _a and higher k _d , resulting in significantly lower dissociation constants (factors 35.0 and 10.5, respectively). HMAb BIWA 8, which contains murine residues in light chain framework region 2, shows a marked decrease in k _d but results in increased affinity.

Dynamics and Affinity Constants of MAbs Directed for CD44v6 Antibodies K _a (M ^-1 s ^-1 ) K _d (s^-One) K _d (M) K _a for rat and biwa 1 Rat and BIWA 1 1.3 × 10 ⁵ 4.2 × 10 ^-5 3.2 × 10 ^-10 1.0 Rat U36 1.5 × 10 ⁴ 1.7 × 10 ^-4 1.1 × 10 ^-8 35.0 Chimera BIWA 2 1.7 × 10 ⁵ 4.1 × 10 ^-5 2.4 × 10 ^-10 0.7 Person adapted BIWA 4 6.5 × 10 ⁴ 2.2 × 10 ^-4 3.4 × 10 ^-9 10.5 Person adapted BIWA 8 7.5 × 10 ⁴ 6.3 × 10 ^-5 8.4 × 10 ^-10 2.6

Relative binding affinity of cMAb and hMAbs was also assessed in competitive cellular ELISA using human A431 tumor cells (FIG. 1). According to affinity measurements for recombinant antigens, cMAb BIWA 2 was the most effective 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 hairless mice: Biodistribution studies were performed in hairless mice carrying HNX-OE xenografts. Two MAbs with the same murine or human isotypes were labeled with either ¹²⁵ I or ¹³¹ I and injected simultaneously (50 μg, 10 μCi, respectively). Each pair of MAbs was screened to reduce the affinity difference step by step: mMAb U36 had a 35.0 times lower affinity than mMAb BIWA 1 (Experiment 1) and hMAb BIWA 4 had a 14.0 times lower affinity than cMAb BIWA 2 HMAb BIWA 4 has 4.0 times lower affinity than hMAb BIWA 8 (Experiment 3). The immunoreactive fraction of all iodinated MAbs was more than 74% after extrapolation (Table 2).

Immunoreactive Fraction of Iodized MAbs Determined by Binding to UM-SCC-11B Cells Experiment number Antibodies sign Binding to 5 × 10 ⁶ cells (%) Extrapolated ^a join (%) One Rats U36 Rats BIWA 1 ¹³¹ I ¹²⁵ I 59.191.1 87.491.1 2 Person-Adapted BIWA 4 Chimera BIWA 2 ¹³¹ I ¹²⁵ I 77.480.5 82.379.9 3 Person Adapted BIWA 4 Person Adapted BIWA 8 ¹³¹ I ¹²⁵ I 77.391.8 74.592.1

a: Immune responsiveness was determined by linear extrapolation to conditions indicating infinite antigen overload (see Materials and Methods).

Biodistribution in experiment 1 was determined 1, 2, 3 and 7 days after injection, and biodistribution in experiments 2 and 3 was determined 1, 2, 4 and 7 days after injection. The average% ID / g calculated for the blood and tumors of all three experiments is shown in Table 3.

Tumor and blood levels of iodinated CD44v6-specific MAbs showing different affinity after injection together in HNX-OE bearing mice Conjugate A (% ID / g) Conjugate B (% ID / g) Conjugate A: B Ratio Experiment number Time after injection tumor blood tumor blood tumor 1.Conjugate A: ¹³¹ I-mMAb U36 Conjugate B: ¹²⁵ I-mMAb BIWA 1 1d 15.7 17.9 13.0 17.8 1.2 1.0 2d 18.4 15.0 13.4 14.9 1.4 1.0 4d 20.6 11.8 13.7 11.0 1.5 1.1 7d 16.5 7.1 7.8 4.8 2.1 1.5 2. Conjugate A: ¹³¹ I-mMAb BIWA 4 Conjugate B: ¹²⁵ I-mMAb BIWA 2 1d 10.8 10.8 10.2 9.1 1.2 1.0 2d 12.4 12.4 10.2 9.8 1.3 1.0 4d 12.9 12.9 7.2 8.9 1.5 1.0 7d 7.6 7.6 3.3 4.8 1.6 1.2 3. Conjugate A: ¹³¹ I-mMAb BIWA 4 Conjugate B: ¹²⁵ I-mMAb BIWA 8 1d 10.5 105 11.1 9.5 1.1 1.0 2d 10.9 10.9 10.0 9.6 1.1 1.0 4d 11.7 11.7 6.2 9.6 1.2 1.0 7d 10.1 10.1 4.2 7.8 1.3 1.1

For each pair of co-injected MAbs, the absorption ratio to tumor and blood is provided. The average% ID / g and s.e.m. of tumors, blood and various organs after 3 days (Experiment 1) or 4 days (Experiments 2 and 3) of injection are shown in FIG.

Directly comparing the two rats with MAbs, the tumor affinity of U36 with low affinity was significantly higher than that of BIWA 1 with high affinity at all time points (p <0.001) (Table 3). In contrast, no substantial difference was found between the uptake of the MAbs in blood and normal tissues 1, 2 and 3 days after injection. After 7 days of infusion, BIWA 1 levels in blood and most organs are significantly lower than U36 levels (p <0.05), indicating that BIWA 1 is cleaned more quickly from blood / body. Three days after injection, 50% higher tumor uptake of U36 compared to BIWA 1 is shown in Figure 2A.

Similar relationships were found in evaluating two other MAb pairs. hMAb BIWA 4 showed a lower affinity, but 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 tissue were similar for the pair of MAbs 1, 2 and 4 days after injection. Seven days after infusion, BIWA 2 and BIWA 8 levels in blood and most organs were significantly lower than BIWA 4 levels (p <0.05), indicating that the MAbs were cleaned more quickly from blood / body. Four days after injection, 45% higher tumor uptake of BIWA 4 compared to BIWA 2 is shown in FIG. 2B, while 20% higher tumor uptake of BIWA 4 compared to BIWA 8 is shown in FIG. 2C. It is.

Consistent results were obtained from additional experiments where radiolabels were exchanged (data not shown): ¹³¹ I-BIWA 8 vs. ¹²⁵ I-BIWA instead of ¹²⁵ I-BIWA 4 vs. ¹³¹ I-BIWA 4 8. The latter experiment The data from excludes the possibility that the type of radiolabel affects the pharmacokinetic behavior of labeled MAbs.

Radioimmunotherapy in HNSCC-bearing hairless mice: From these three biodistribution experiments, low affinity MAbs are higher than high affinity MAbs and are considered to be more suitable for RIT as they show more selective tumor uptake. To test this possibility, the following treatment groups were compared in RIT studies in HNX-OE xenograft-bearing mice:

Experiment 1: 300 μCi ¹⁸⁶ Re-U36 or 300 μCi ¹⁸⁶ Re-BIWA 1, or saline as a control. Experiment 2: 300 μCi ¹⁸⁶ Re-BIWA 4 or 300 μCi ¹⁸⁶ Re-BIWA 2, or saline as a control. Experiment 3: 400 μCi ¹⁸⁶ Re-BIWA 4 or 400 μCi ¹⁸⁶ Re-BIWA 8, or saline as a control.

In Figure 3, the mean relative tumor volume (% of tumor volume on day 0) for the control and treatment groups was plotted against time. In all three experiments, tumors of control group mice grew exponentially and took about 7 days to double tumor volume. ^In the group treated with ¹⁸⁶ Re-labeled MAbs, tumors stopped growing, in some cases followed by tumor regression immediately after conjugate injection. But all tumors eventually grew again.

In Experiment 1, administration of 300 μCi ¹⁸⁶ Re-BIWA 1 resulted in tumor growth rate but no decrease in mean tumor size. However, 7-17 days after injection with the administration of 300 μCi ¹⁸⁶ Re-U36, the average tumor volume decreased from 185 mm 3 to 120 mm 3, after which the tumor began to grow again. The mean relative tumor volume in the ¹⁸⁶ Re-U36-treated group was significantly smaller than the ¹⁸⁶ Re-BIWA 1-treated group from day 14 (p <0.001).

In Experiment 2, the growth of either 300 μCi ¹⁸⁶ Re-BIWA 4 or 300 μCi ¹⁸⁶ Re-BIWA 2 resulted in inhibition of tumor growth 7 days after injection, but began to grow again from day 17. BIWA 4 was more effective for RIT than BIWA 2 from day 14, but only significant differences between mean relative tumor volumes were found 14 days after injection (p <0.05).

In Experiment 3, mice were treated with either 400 μCi ¹⁸⁶ Re-BIWA 4 or BIWA 8, which on day 19 reduced the relative tumor volume to at least 80 ± 62% and 98 ± 81%, respectively. After that, the tumor began to grow again. These data indicate that low affinity MAb BIWA 4 is more effective for RIT than high affinity MAbs cBIWA 2 and BIWA 8.

Reference

Example 2: Details of Sequences

This example shows details about the sequence, such as the location of the cloning site, leader and non-toxic region.

Abbreviation:

aa = amino acid

nt = nucleotide sequence

SEQ ID NO 1: VH BIWA 4/8 aa

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSS

SEQ ID NO 2: VL BIWA 4 aa

EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK

SEQ ID NO 3: VL BIWA 8 aa

EIVLTQSPATLSLSPGERATLSCSASSSINYIYWLQQKPGQAPRILIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIK

SEQ ID NO 4: VH BIWA 4/8 nt

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTCTCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCTAGACAGTATAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCCTGTACCTGCAAATGAACAGTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGACAGGGGTTGGACTACTGGGGTCGAGGAACCTTAGTCACCGTCTCCTCA

SEQ ID NO: 5: VL BIWA 4 nt

GAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTGTCCTGCAGTGCCAGCTCAAGTATAAATTACATATACTGGTACCAGCAGAAGCCAGGACAGGCTCCTAGACTCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGCAGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCCGTTTATTACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGATTAAA

SEQ ID NO: 6: VL BIWA 8 nt

GAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTGTCCTGCAGTGCCAGCTCAAGTATAAATTACATATACTGGCTCCAGCAGAAGCCAGGACAGGCTCCTAGAATCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGCAGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCCGTTTATTACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGATTAAA

SEQ ID NO: 7: Heavy chain (variable + constant) BIWA 4/8 aa

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISSGGSYTYYLDSIKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGLDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 8 light chain (variable + constant) BIWA 4 aa

EIVLTQSPATLSLSPGERATLSCSASSSINYIYWYQQKPGQAPRLLIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSLSNSVQQKSL

SEQ ID NO: 9: Light chain (variable + constant) BIWA 8 aa

EIVLTQSPATLSLSPGERATLSCSASSSINYIYWLQQKPGQAPRILIYLTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQWSSNPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSLSESVTEQKKKKVIDKSL

SEQ ID NO: 10 heavy chain (variable + constant) BIWA 4/8 nt; Inserts in pAD-CMV1 / pAD-CMV19, introns between CH1-hinge, hinge-CH2, CH2-CH3 (lower case), leader sequence (underlined), non-translated sequence (italic), cloning site (in bold) Contained

aagctt tgacagacgcacaaccctggactcccaagtctttctcttcagtgacaaacacagacataggatatcacatttgcttctgacacaactgtgttcactagcagcctcaaacagacacc ATGAACTTTGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGTCCAGTGT gtgcgacggccgc gaattc

SEQ ID NO: 11: light chain (variable + constant) BIWA 4 nt; Sequences within pAD-CMV1 / pAD-CMV19 contained leader sequences (underlined), non-translated sequences (italics), cloning sites (in bold)

aagctt gatcttcaggatatcacatttgcttctgacacaactgtgttcactagcaacctcaaacagacacc ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATGAGTGCCTCAGTCATAATGTCCAGGGGA gaattc

SEQ ID NO: 12 Light chain (variable + constant) BIWA 8 nt; Sequences within pAD-CMV1 / pAD-CMV19 contained leader sequences (underlined), non-translated sequences (italics), cloning sites (in bold)

aagctt gatcttcaggatatcacatttgcttctgacacaactgtgttcactagcaacctcaaacagacacc ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATGAGTGCCTCAGTCATAATGTCCAGGGGA gaattc

SEQ ID NO: 13: Heavy chain (variable + constant) BIWA 4/8 nt; The sequence in N5KG1val contained no introns and contained a leader sequence (underlined)

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGT

SEQ ID NO: 14 Light chain (variable + constant) BIWA 4 nt; The sequence in N5KG1val contained the leader sequence (underlined)

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTGCTCTGGCTCCCAGATACCACCGGA

SEQ ID NO: 15 Light chain (variable + constant) BIWA 8 nt; The sequence in N5KG1val contained the leader sequence (underlined)

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTGCTCTGGCTCCCAGATACCACCGGA

BIWA 4 in SEQ ID NO: 16: N5KG1val

CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGGGTCGCGACGTACCGGGCCCCCCCTCGATTAATTAATCGAGCTACTAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACATAGTTGTGCCAGCATGGAGGAATCGATCCTCCATGCTTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGTAAGTGCGGCCGCTCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGGCGGAGAATGGGCGGAACTGGGCGGAG TTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGCCATCACAGATCTCTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTGCTCTGGCTCCCAGATACCACCGGAGAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTGTCCTGCAGTGCCAGCTCAAGTATAAATTACATATACTGGTACCA GCAGAAGCCAGGACAGGCTCCTAGACTCTTGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGCAGTGGGTCTGGAACCGACTTCACTCTCACAATCAGCAGCCTGGAGCCTGAAGATTTTGCCGTTTATTACTGCCTGCAGTGGAGTAGTAACCCGCTCACATTCGGTGGTGGGACCAAGGTGGAGATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC

CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAATTCAGATCCGTTAACGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCGGCCGTGATATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGACTAGTAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCGATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCTGCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGG TGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGAAGCGGCCGCTCCGGATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAA AATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGGTACGTCCTCACATTCAGTGATCAGCACTGAACACAGACCCGTCGACATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTCTCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCTAGACAGTATAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCCTGTACCTGCAAATGAACAGTCTGAGGGCTGAGGACACGGCCGT

GTATTACTGTGCAAGACAGGGGTTGGACTACTGGGGTCGAGGAACCTTAGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCCGTTAACGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCGGCCGTGATATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGCGCTGCGATCGCCTCGAGGCCGCTACTAACTCTCTCCTCCCTCCTTTTTCCTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCT CCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATCTATCTTATCATGTCTGGATCGCGGCCGGCCGCACCGCGGTGGAGCTTTAATTAAGGCGCGCCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAA

ATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATA CTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACA

SEQ ID NO: 17 pAD-CMV1, cloning site in bold

AAGCTT CTGCAGGTCGACATCGATGGATCCGGTACCTCGAGCGC GAATTC

SEQ ID NO: 18 pAD-CMV19, cloning site (in bold)

AAGCTT CTGCAGGTCGACATCGATGGATCCGGTACCTCGAGCGC GAATTC

Example 3: Clinical Study 1170.1

1. List of abbreviations and term definitions

ADCC antibody dependent cell-mediated cytotoxicity

AE side effects

ALT Alanine Amine Group Transferase

a.p. Post-war (contrast)

AP alkaline phosphatase

AST Aspartate Amine Group Transferase

Area under the AUC concentration-time curve

Bq Becquerel, SI unit for radioactive Becquerel (1 Bq = 1 decay / s)

BSA bovine serum albumin

CD44v6 CD44 Variants Isoform v6

CDS Corporation Drug Safety

Centi Gray measurement of cGy radioactivity

CHO Chinese Hamster Ovary Cells

Ci Curie, unit for radioactivity; 1 Ci = 37 x 10 ⁹ Disintegration / s = 37 GBq

CL total body cleaning rate

cMAb chimeric monoclonal antibodies

cm centimeter

C _max Maximum drug concentration observed

cpm counts per minute

CRF Case Report / Record Form

Scan (CT) computed tomography

CTC Common Toxicity Criteria

CV variation coefficient

DLT Dose Limiting Toxicity

ECG ECG

ELISA enzyme-linked immunosorbent assay

ENT Otolaryngology

f female

FDA Food and Drug Administration

g gram

SI units for GBq radioactive gigabytes (1 GBq = 10 ⁹ decay / s)

GCP Superior Clinical Trial Criteria

GGT gamma glutaryl amine group transferase

GMP excellent pharmaceutical manufacturing standard

Gy gray

HAHA person-anti-person-antibody

Hb hemoglobin

HER2 human epidermal growth factor receptor 2

hMAb human adapted monoclonal antibodies

HNSCC Head and Neck Squamous Cell Carcinoma

HPLC high performance liquid chromatography

hr / h time

hrs times

Ht hematocrit

¹³¹ I Iodine-131 (8.05 days)

ICH International Leveling Committee

ID injection dose

IEC Independent Ethics Committee

IgG immunoglobulin G

INN international generic name

Review Board in IRB Research Facility

ITT Intensive Care

i.v. Intravenous

KeV kilo electron volt

l / L liter

m male

㎡ square meter

MAb monoclonal antibody

MAG2GABA-TFP mercaptoacetylglycosylglycosyl-gamma-aminobutyrate-tetrafluorophenol ester (MAG2GABA-TFP). Chelate used to couple ¹⁸⁶ Re with monoclonal antibodies (future method)

MAG3 mercaptoacetyltriglycine. Chelate used to couple ^99m Tc and ¹⁸⁶ Re with monoclonal antibodies

SI units for MBq radioactivity; Mega Becurel (1 MBq = 10 ⁶ Decay / s)

mCi Milli Curie; Units for radioactivity; 1 mCi = 37 x 10 ⁶ Collapse / s = 37 MBq

MCV mean red blood cell volume

mGy Milly Gray

Μg microgram

mg milligram

min min

ml / mL milliliters

mMAb murine monoclonal antibodies

mmHg mm mercury

μmol micromol

mmol mmol

MRI magnetic resonance imaging

MRT average residence time

mSv Millie Sievert

MTD Maximum Immunity Capacity

NA / n.a. Inapplicable

NCI US National Cancer Institute

Do not perform ND

ng nanogram

NO / N number

nos unless otherwise specified

NSCLC non-small cellular lung cancer

n.y.r. Recovery undecided

p.a. Postwar

PBS Phosphate Buffered Saline

p.i. After injection

p.o. oral-

According to PP protocol

Pt./Pat Patient

Pts patients

¹⁸⁶ Re rhenium- ¹⁸⁶ , radionuclide; Half-life 3.7 days (tissue penetration of β-particles of about 1.2 mm)

Recov. Recovered

RES reticulocyte

RIS radioimmunoflash angiography

RIT Radioimmunotherapy

ROI area of interest

SAE serious side effects

SCC squamous cell carcinoma

sCD44v6 Availability CD44v6

SD stable disease

sGOT Serum Glutamic Oxal Acetic Acid Amine Group Transferase

sGPT serum glutamic pyruvate amine transferase

SOC systemic organ classification

SOP Standard Surgery Course

SPECT single proton radiography computed tomography

t _1/2 elimination half-life

^99m Tc Technetium 99m (6 hours half life)

TLC thin layer chromatography

When T _max maximum drug concentration is observed

Timing of tumors according to TNM primary tumor (T), regional lymph nodes (N), and metastasis (M)

TSH Thyroid Stimulating Hormone

UICC International Cancer Society

unk Not known

Apparent volume of distribution under V _ss stationary conditions

Apparent volume of distribution above the V _z terminus

WBC Leukocyte Count

WHO World Health Organization

2. Research Purpose

2.1 General Goal / Clinical Purpose

The general goal of this study was to assess the safety and tolerability of intravenously administered ^99m Tc and ¹⁸⁶ Re-labeled hMAb BIWA 4, to identify preferential accumulation in tumors of ^99m Tc-labeled hMAb BIWA 4, ¹⁸⁶ To determine the maximum tolerated radioactivity dose of Re-labeled hMAb BIWA 4 and to suggest a safe dose for phase II development. To achieve this goal, the study was divided into two parts.

Part A

purpose:

To determine the safety and tolerability of a single infusion of ^99m Tc-labeled hMAb BIWA 4 administered intravenously in patients with head and neck squamous cell carcinoma.

To determine the biodistribution of a single infusion of ^99m Tc-labeled hMAb BIWA 4 under different BIWA 4 dose levels in patients with head and neck squamous cell carcinoma.

To study the pharmacokinetics of a single infusion of ^99m Tc-labeled hMAb BIWA 4.

Part B:

purpose:

To determine the qualitative and quantitative toxic effects of ¹⁸⁶ Re-labeled hMAb BIWA 4 and to study the predictability, initiation, duration, intensity, reversibility and dose-relationship of the toxic side effects.

To determine the maximum tolerated radioactivity dose of ¹⁸⁶ Re-labeled hMAb BIWA 4 administered intravenously in head and neck cancer patients.

To study the pharmacokinetics of ¹⁸⁶ Re-labeled hMAbBIWA 4 administered intravenously in patients with head and neck squamous cell carcinoma.

Secondary goal was to determine the preliminary treatment effect of ¹⁸⁶ Re-labeled hMAb BIWA 4.

The following objectives, summarized in the protocol, were not addressed during the conduct of this trial. The development of a program using linker chelate mercaptoacetyltriglycine (MAG3), used to couple ^99m Tc and ¹⁸⁶ Re with monoclonal antibodies, was discontinued, thus completing the trial. Further development with BIWA 4 will continue by using the linker mercaptoacetylglycigylsilyl-gamma-aminobutyrate-tetrafluorophenol ester (MAG2GABA-TFP).

The MTD for single dose treatment must first be identified before involving more patients to define the MTD for the second treatment with ¹⁸⁶ Re-labeled hMAb BIWA 4.

To suggest a safe dose for the first and subsequent infusions with ¹⁸⁶ Re-labeled hMAb BIWA 4 for further study.

To obtain initial results on a dose schedule for repeated administrations.

2.2 Primary Variables

Safety: Clinical Trials, Human-Anti-Human Antibody (HAHA) Assessment, Vital Signs Measurement and Side Effects.

Efficacy: biopsy biodistribution data (Part A only) and radioimmunoascopy angiography images (Parts A and B, including dose determination for Part B of this trial).

Pharmacokinetic results.

2.3 Secondary Variables

The secondary variable for this study is tumor response (Part B only).

3. Study plan

3.1 Overall Study Design and Plan-Description

This test was conducted in two parts. Part A evaluated the optimal dose of cold BIWA 4 and quantified tumor absorption, while Part B studied the maximum tolerated dose of ¹⁸⁶ Re-BIWA 4.

3.1.1 Part A:

This clinical trial part was a study of a group of sequentially increasing doses in an uncontrolled manner. It is designed to provide initial data on the safety and tolerability of a single infusion of ^99m Tc-labeled hMAb BIWA 4, to study biodistribution patterns and levels, and to establish the pharmacokinetic profile of hMAb BIWA 4 in head and neck cancer patients. It became. Three protein doses of hMAb BIWA 4 were used in Part A of this study with three patients planned to be treated at each dose level.

Patients were commonly studied in otolaryngology to determine tumor extent. This included physical examination of the head and neck, computed tomography (CT) or MRI scan, and universal endoscopic (optional). During this process, tissue samples that were presumed to be tumors were taken and examined for the presence of squamous cell carcinoma. Based on the results of this study, the patient underwent surgical intervention, including cervical incision.

Radiolabeled antibodies were injected and it was observed whether the patient had developed side effects. Prior to surgery, radioimmunoassensitivity scans were performed 21 hours after injection. Patients were operated 48 hours after infusion of radiolabeled hMAb BIWA 4. The pathologist studied a cervical incision sample to determine the exact tumor load. Moreover, ^99m Tc amounts were measured in biopsies from normal tissues and tumor sites in surgical specimens. Tumor sites and tumor infiltrating sections were examined for the presence of CD44v6 antigen by immunochemical techniques. The first three patients received 2 mg hMAb BIWA 4 labeled with 20 mCi ^99m Tc in combination with 23 mg of unlabeled hMAb BIWA 4. The second group of three measurable patients received 2 mg hMAb BIWA 4 labeled with 20 mCi ^99m Tc in combination with 48 mg of unlabeled hMAb BIWA 4 and the third group received 2 mg of the labeled antibody. It was administered in combination with 98 mg of the above unlabeled antibody.

At certain time points, pharmacokinetic evaluations were performed.

3.1.2 Part B:

This clinical trial part is a study of a group of sequentially increasing uncontrolled doses in an open manner. Re- ¹⁸⁶ which evaluate the safety and tolerance of the labeled hMAb BIWA 4, and the intravenous administration of ¹⁸⁶ Re- determine the maximum tolerated dose (MTD) of the labeled hMAb BIWA 4 and, at this time there is no cure and head cancer patients from ¹⁸⁶ Re-labeled hMAb BIWA 4 was designed to determine the preliminary therapeutic effect. In addition, the pharmacokinetic profile of ¹⁸⁶ Re-labeled hMAb BIWA 4 was evaluated.

A dose of hMAb BIWA 4 was administered to all patients participating in this test part, which was selected based on the results of Part A of this study. The hMAb BIWA 4 was labeled with an increased dose of ¹⁸⁶ Re. At lower dose levels (observed toxicity did not exceed grade 1), two patients per dose group participated, and at higher dose levels (≧ grade 2 toxicity), at least three patients per dose group participated. All patients were evaluated to determine the safety of the administered hMAb BIWA 4.

Patients were commonly studied in otolaryngology to determine tumor extent. This included physical examination and CT or MRI scanning of tumor localization.

¹⁸⁶ Re-labeled antibodies were injected with increasing radiation dose: after administration of a radioactive dose of 20 mCi / m 2 to a patient in the first dose group, the dose for the patient in the subsequent dose group was 10 mCi until reaching the MTD. Increased by / ㎡. It was observed that adverse events occurred in the patient. Radioimmune scintillation scans were performed.

3.1.3 Research process at each visit

3.1.3.1 Visit Schedule

a) screening visit

Before participating in this study, each patient was screened for eligibility. Demographic and medically relevant histories were recorded, current treatments recorded, total physical examinations, and weighed. Karnofsky performance scores were determined. A written informed consent had to be obtained. For women of childbearing potential, a pregnancy test was required. Twelve lead electrocardiograms (ECGs) were made and in vitro safety assessments were performed on urine as well as blood. HAHA-test blood samples were also taken. Chest X-rays were taken and a full disease assessment (CT / MRI, ENT investigation) was performed.

At the end of the visit, the required study results were evaluated, inclusion and exclusion criteria were identified, and surgical preparation (part A only) was made.

b) Visit 2: Study 1-7

Parts A and B:

On the first study day within three weeks after the screening visit, patients were asked to visit a hospital. All necessary baseline laboratory evaluation procedures, which were not obtained at the screening visit, must be performed, evaluated prior to infusion, and the eligibility criteria must be met. Body weight was measured. All current treatments had to be documented. All side effects that began after signing a written informed consent form had to be recorded in the form of a patient file and case report (CRF).

On the treatment day, blood samples were taken for pharmacokinetic testing (including evaluation of soluble CD44v6) and the vital signs were recorded prior to administration of the antibody. For each of parts A and B, urine after infusion is collected at 0-4 hours, 4-8 hours, 8-12 hours and 12-24 hours for the first 24 hours and at 24-hour intervals up to 48 and 96 hours after injection. It became.

The antibody was administered in a designated room in the Department of Nuclear Medicine or Hospital. The antibody should be administered as close to the peripheral upper vein as possible. In all cases, after 5 minutes of infusion, 10 ml of saline is ejected through a freshly flowing line. In order to obtain reproducible pharmacokinetic results, a syringe pump had to be used. Therefore, dilution with 0.9% of the volume of NaCl required 20 ml. Following injection of the antibody, 10 ml saline is ejected. All putative extravasation was recorded in CRT and the patient was imaged.

Emergency units that can be used with respiratory equipment, anti-histamines, corticosteroids and epinephrine are within the scope to counteract anaphylaxis that may occur. Side effects and changes in current treatments were recorded daily.

Part A:

Vital signs were recorded 10, 60 and 120 minutes after injection. Blood samples for safety and solubility CD44v6 testing were collected 21, 48 and 144 hours after infusion.

By the end of the infusion and 5, 10, 30 minutes and 1, 2, 4, 16, 21, 48, 72 hours, and 7 days after the end of the infusion (144 hours post-injection with serum samples for HAHA and soluble CD44v6 evaluation) Blood samples for pharmacokinetic tests were taken.

Pharmacokinetic urine was collected in 24-hour interval samples from 0 to 4 hours, 4 to 8 hours, 8 to 12 hours and 12 to 24 and 48 hours for the first 24 hours post-infusion.

Systemic scintillation images were made immediately after and at 21 hours post-infusion. In addition to whole body imaging at 21 hours post-injection, single proton radiography computed tomography (SPECT) and planar images of the head and neck regions are made.

The patient was operated 48 hours after infusion and hospitalized during surgical recovery.

On day 7 (144 hours after injection), urine samples for safety testing were collected.

Side effects and changes in current treatments were recorded daily.

Part B:

Vital signs were recorded 10, 60, 120 and 240 minutes after injection. Blood samples for safety and solubility CD44v6 testing were collected 21, 48 and 144 hours after infusion. Serum samples for HAHA evaluation were collected 144 hours after infusion. Urine samples for safety testing were also collected 144 hours after infusion.

Blood samples for pharmacokinetic testing were taken at the end of the infusion and at 5 and 30 minutes and at 1, 2, 4, 16, 21, 48, 72 hours, and 7 days (144 hours after injection) at the end of the infusion. The original planned sample at 10 minutes was omitted according to calibration I.

Pharmacokinetic urine was collected in 24-hour interval samples up to 76 hours, with 0-4 hours, 4-8 hours, 8-12 hours and 12-24 hours for the first 24 hours post-infusion.

Systemic scintillation images were made immediately after infusion and 21, 48, 72 and 144 hours post-injection, and optionally 2 weeks post-injection if statistical calculations were allowed.

21, 48 (optional), 72 and 144 hours post-infusion, and optionally, planar images of the head and neck regions are made 2 weeks post-injection if statistical calculations are allowed.

At 2 hours, only SPECT imaging was performed 7 times after injection.

Side effects and changes were recorded daily in current treatments.

After 3 days the patient was discharged.

c) Visit 3: Follow-up Visit

Part A:

The patient visited an outpatient clinic six weeks after infusion. Physical examinations were performed, safety blood and urine samples were collected, body weights and vital signs were measured, and side effects were recorded. Blood samples for pharmacokinetics, HAHA test and soluble CD44v6 test were also collected. For women of childbearing potential, a pregnancy test was required. The side effects that occurred were then recorded. All changes in current treatments have been recorded.

Part B:

Patients visited the outpatient clinic weekly for at least 6 weeks to record adverse events and collect safety blood samples. Blood samples for pharmacokinetic examination were taken 240 and 336 hours after infusion. Samples six weeks after the originally planned injection were omitted according to Calibration I. All changes in current treatments have been recorded. Disease assessments performed at baseline were repeated for 6 weeks after infusion and, if indicated, afterwards (the evaluation was performed after 6 weeks, not 4 weeks after, as mentioned once in this protocol). Six weeks after infusion, blood samples for safety, HAHA assessment, and soluble CD44v6 testing were collected, vital signs were recorded, and body weights were measured. Physical examinations were performed during this visit. Urine samples for safety testing were collected. For women of childbearing potential, a pregnancy test was required.

d) Visits 4 and 5: Second Treatment (Part B)

A second treatment, as summarized in the original protocol, was not performed due to premature termination of the test due to linker changes. Instead, patients responding to the first dose of ¹⁸⁶ Re-BIWA 4 were eligible for the second dose. They had the same visit schedule as for the first dose.

3.2 Discussion of Study Design Including Selection of Control Group

The goal of this study was to assess preferential accumulation of ^99m Tc-labeled BIWA tetravalents in tumors (Part A) and to evaluate the maximum tolerated dose of ¹⁸⁶ Re-BIWA 4 in patients with advanced head and neck cancer (Part B) as well as BIWA. To evaluate the pharmacokinetics of 4 (parts A and B).

In the oncology field an open design was used, as generally carried out on these types of Phase I trials. In Part B of this study, at least 2 patients were included at the lower radiation dose titer and 3 patients at the higher radiation dose. In the event of drug-related Common Toxicity Criterion (CTC) Grade 4 hematology and Grade 3 non-hematologic toxicity, an additional three patients were treated with each dose threshold (see section for further administration details). See 3.4.4 and 3.4.5).

The dose of BIWA 4 administered was based on previous results with mMAb BIWA 1, indicating that administration of 50 mg doses are selectively absorbed at high levels in tumor tissues and at low levels in non-tumor tissues. This is the result from Part A of this test (see also Section 3.4.4.1).

The starting dose level for the selected radioactivity is based on the above data, suggesting that a dose of 20 mCi / m 2 may be a safe dose (see also section 3.4.4.2).

Criteria for evaluating resistance as well as criteria for applied efficacy are well established for this patient population, which may be evaluated as an open design.

3.3 Screening of Study Groups

3.3.1 Inclusion Criteria

-Patients whose head and neck squamous cell carcinoma are histologically confirmed;

Patients who are expected to undergo surgery through a neck incision (Part A); or

-Patients with local and / or local relapse disease or distant metastasis that currently have no treatment. Tumor deposits should have been able to be measured clinically or by one or more radiomedical techniques (CT, MRI, bone sclerography).

Since RIT is expected to be more effective for smaller sized tumor deposits, it was preferred for patients with lesions determined to have a largest size of <3 cm (Part B).

Patients over 18 years old.

Patients under 80 years old.

-The patient has submitted a "written informed consent".

Patients with a life span extension of 3 months or longer.

Patients exhibiting good performance: Karnovsky> 60.

3.3.2 Exclusion Criteria

Symptoms of life-threatening infections, allergic constitutions, organ failure (bilirubin> 30 μmol / l and / or creatinine> 150 μmol / l), or recent myocardial infarction or unstable angina to ECG.

Pre-menopausal women (more than a year after the last menstruation prior to the start of the study):

· Without undergoing infertility surgery (hysterectomy, fallopian ligation)

Women who have not implemented acceptable contraceptive measures (or have not been planned consistently throughout the study). Acceptable contraceptive methods include oral, implantable or injectable contraceptives.

Women who were positive in serum pregnancy test at baseline.

Patients undergoing chemotherapy or radiation therapy 4 weeks prior to joining this study.

-Patients with a white blood cell count <3000 / dl, granulocyte count <1500 / dl or platelet count <100,000 / dl.

-Hematological disorders, congestive heart failure, bronchial asthma, dietary or contact allergies, severe atopic or allergic patients.

3.3.3 Withdrawal of Subjects from Therapy or Evaluation

3.3.3.1 Criteria for Discontinuing Subject Treatment

The infusion is terminated immediately if the patient exhibits anemia (pulse rate exceeds 120 per minute), hypotension (blood pressure less than 100 mmHg systolic pressure), shortness of breath, chest pain, or any symptom unacceptable to the patient. Had to be

3.3.3.2 Dropout and Withdrawal (Stop Dosing)

The subject was free to discontinue participation in this study at any time.

If the patient voluntarily withdrew before the study was completed, the evaluation of radiolabeled hMAb BIWA 4 was considered inoperable. If no appropriate subsequent information is available, the case was considered not evaluable. It was planned to replace any patient that could not be evaluated.

If the patient discontinued early in the study, the reason must be recorded on the CRF. If the patient did not return after injecting a HAHA crystal or pharmacokinetic blood sample, the reason must be recorded. If a serious adverse event occurred in the patient, the study schedule should be carried out as close as possible in accordance with the SAE.

3.4 Therapies

3.4.1 Treatment administered

Patients of Part A received ^99m Tc-BIWA 4 at a radioactivity dose of 20 mCi. The dose of BIWA 4 administered was 25 mg, 50 mg or 100 mg for each of three patients. This drug was administered intravenously as a single dose.

Patients in Part B received 50 mg of BIWA 4 labeled Rhenium 186. The lowest radioactivity was 20 mCi / m 2, increased to a capacity titer of 10 mCi / m 2. The test drug was administered intravenously as a single dose.

3.4.2 The identity of the research product

Part A:

Substance (INN): BIWA 4 (bivatuzumab)

Pharmaceutical Forms: Injectable Solvents

Chipper number: BIWA 4 LOI 99 1D 1A

Batch Number: B981101

Source: Boehringer Ingelheim Pharma KG

Unit Strength: 5 mg / ml

Daily dose: 25 mg

Term of use: single dose

Route of Administration: Intravenous

Dosage: infusion over 5 minutes

Substance (INN): BIWA 4 (bivatuzumab)

Pharmaceutical Forms: Injectable Solvents

Chipper number: BIWA 4 LOI 99 1D 1A

Batch Number: B981101

Source: Boehringer Ingelheim Pharma KG

Unit Strength: 5 mg / ml

Daily dose: 50 mg

Term of use: single dose

Route of Administration: Intravenous

Dosage: infusion over 5 minutes

Substance (INN): BIWA 4 (bivatuzumab)

Pharmaceutical Forms: Injectable Solvents

Chipper number: BIWA 4 LOI 99 1D 1A

Batch Number: B981101

Source: Boehringer Ingelheim Pharma KG

Unit Strength: 5 mg / ml

Daily dose: 100 mg

Term of use: single dose

Route of Administration: Intravenous

Dosage: infusion over 5 minutes

BIWA 4 was administered as a radioconjugate linked with ^99m Tc. The linker molecule was MAG3. MAG3 was purchased from a source (Mallinckrodt, Pettern, The Netherlands).

^99m Tc was ordered locally through the laboratory where the radioimmunoconjugate was prepared (laboratory of Prof. Dr. van Dongen, Section Tumor Biology, Department of Otorhinolaryngology / Head and Neck Surgery, Vrije Universiteit University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands).

Part B:

Substance (INN): BIWA 4 (bivatuzumab)

Pharmaceutical Forms: Injectable Solvents

Chipper number: BIWA 4 LOI 99 1D 1A

Batch Number: B981101

Source: Boehringer Ingelheim Pharma KG

Unit Strength: 5 mg / ml

Daily dose: 50 mg

Term of use: single dose

Route of Administration: Intravenous

Dosage: infusion over 5 minutes

BIWA 4 was administered as a radioconjugate linked with ¹⁸⁶ Re. The linker molecule was MAG3. MAG3 was purchased from a source (Mallinckrodt, Pettern, The Netherlands).

¹⁸⁶ Re was ordered locally through the laboratory where the radioimmunoconjugate was prepared (laboratory of Prof. Dr. van Dongen, Section Tumor Biology, Department of Otorhinolaryngology / Head and Neck Surgery, Vrije Universiteit University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands).

3.4.2.1 Characteristics and Quality of the Test Drug

Antibody Features

The antigen recognized by hMAb BIWA 4 is a transmembrane glycoprotein localized on the outer cell surface, which was only internalized to a low extent (<20%). Further analysis revealed that hMAb BIWA 4 recognizes an epitope encoded by variant exon v6 of CD44. The antigen was found to be expressed by all primary head and neck tumors (n = 54) and the majority of cells in these tumors. Comparable expression was observed for 68 tumor infiltrated lymph nodes from cervical incision specimens (R97-2054). Reactivity patterns of hMAb BIWA 4 in human normal tissues are provided in Appendix III of this protocol. The reactivity of hMAb BIWA 4 was found to be essentially limited to squamous epithelium. As demonstrated by previous RIS studies with murine monoclonal antibody (mMAb) BIWA 1, reactivity with normal squamous epithelium was not a limiting factor in its use in tumor targeting compared to tumor uptake.

^99m Tc or ¹⁸⁶ Quality control of re-labeled hMAb BIWA 4

Antibodies are described in Visser et al. (R96-2094) and Van Gog et al. (R96-2111), Fritzberg et al. (R96-2106: See protocol Appendix IV) according to the method described as ^99m Tc or ¹⁸⁶ Re.

Radiolabeling of hMAb BIWA 4 with ^99m Tc and ¹⁸⁶ Re was effective compared to the final quality of the prepared conjugate. In five independent labeling experiments performed according to the procedure described in Appendix IV of the protocol, it was found that the percentage of label bound to the antibody was 96-99%.

In this clinical study, the radiochemical purity of each ^99m Tc- or ¹⁸⁶ Re-labeled antibody batch prepared was evaluated by passing into thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) and PD-10 gel filtration column. This must be at least 90% to be available to the patient.

The immunoreactive fraction of each ^99m Tc / ¹⁸⁶ Re-labeled antibody batch was examined using an effective method after administration. In brief, the analysis is essentially described in Lindmo et al. (R96-2104)]. UM-SCC 11B cells (human laryngeal carcinoma) containing CD44v6 antigen were fixed in 0.1% glutaraldehyde. Six dilutions ranging from 5 x 10 ⁶ cells or 3.1 x 10 ⁵ cells per tube were made using 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS).

To the tube, 80,000 cpm ^99m Tc- or 10,000 cpm ¹⁸⁶ Re-labeled hMAb BIWA 4 was added and incubated overnight at room temperature.

To the last sample, excess unlabeled hMAb BIWA 4 was added to determine non-specific binding. Cells were spun off, the radioactivity in the pellet and supernatant was determined with a gamma counter, and the bound and free radiolabeled MAb ratios were calculated (LKB-Wallace 1282 CompuGamma).

Data was analyzed graphically with a modified Lineweaver Burk plot, and the immunoreactive fraction was determined by linear extrapolation to conditions indicating infinite antigen overload.

If the release level did not reach about 60% of the release level, the preparation for infusion was reevaluated in the second binding assay. For this purpose, antibody preparations were labeled with ¹³¹ I. The immunoreactive fraction in at least one of the two assays had to exceed 60% for each patient to be evaluable and to continue to study the next patient.

Antibody safety

BIWA 4, used for radiolabeling with ^99m Tc and ¹⁸⁶ Re in this study, is a well characterized monoclonal antibody product produced by Chinese hamster ovary (CHO) -cell culture fermentation. Master cell banks were established under Good Manufacturing Practice (GMP) conditions and thoroughly examined for microbial conditions (bacteria, fungi, mycoplasmas) as well as viral conditions (egenological viruses, retroviruses). No contaminants were detected at all, with the exception of endogenous retroviruses known to be present in most CHO cells.

According to the Food and Drug Administration (FDA) 1994 "Points to Consider" document for MAbs used in clinical Phase I studies in cancer patients, a standardized downstream purification method suitable for efficient virus removal was applied to the purification of BIWA 4 material. . Four model viruses (MuLV, PsRV, REO-3, SV 40) were included in this ratification. The concentrated bulk harvest was tested for retroviral titers to ensure that subsequent downstream purification methods can properly remove retroviruses. Data on endotoxin removal was within acceptable limits (≦ 0.01 EU / mg) and was available for bulk products. Moreover, pyrogenic tests in accordance with the European Pharmacopoeia Directive have been successfully performed.

The final hMAb BIWA 4 to be used for further radiolabeling was analyzed in more detail and proved to be a highly pure (SDS page, isoelectric focusing) sterile solution containing minimal endotoxin levels (≦ 0.01 EU / mg). The exothermic test results met European Pharmacopoeia standards. An equivalent amount of BIWA 4 product from preparations for pre-clinical and clinical feed was demonstrated by the analytical results.

Radiolabeling of hMAb BIWA 4 with ^99m Tc or ¹⁸⁶ Re was performed by the Department of Otorhinolaryngology and Head & Neck Surgery of the Vrije Universiteit University Medical Center according to standard surgical procedures (SOPs). The sterility of the final product was guaranteed. It was tested for the absence of endotoxin during the ratification run. For drug administration to the patient at the University Hospital Nijmegen, an appropriate amount of radiolabeled hMAb BIWA 4 was transferred to a research center in Nijmegen in a special container immediately after preparation. Allow 24 hours for the compound to be labeled and administered to the patient.

The assignment of individual rhenium batches to patients can be found in Annex 16.1.6.

3.4.2.2 Packaging, Labeling and Supply

BIWA 4 was provided by Boehringer Ingelheim The Netherlands. It is a sterile, non-pyrogenic solution prepared by the following company (Boehringer Ingelheim, Germany) using GMP preparation and purification methods and containing 25 mg hMAb BIWA 4 in 5 ml isotonic PBS (pH 7.2) and filled into vials. It became. Examples of original vial labels and vial labels of labeled antibodies have been included in the clinical trial manual.

One batch with a total amount of 27 g of hMAb BIWA 4 was prepared and filled into vials. Labeling hMAb BIWA 4 with ^99m Tc or ¹⁸⁶ Re was performed in a Class B notarized nuclear laboratory at the following institution ( Vrije Universiteit University Medical Center, Amsterdam).

3.4.2.3 Storage Conditions

Unlabeled hMAb BIWA 4 must be stored in a hospital pharmacy within an area with limited access to the study material at a temperature monitored between +2 and +8 ° C.

3.4.3 How to assign a subject to a treatment group

Do not do it randomly. Patients were assigned to different dose groups according to the participation (inclusive) order.

3.4.4 Dose Screening in this Study

3.4.4.1 BIWA 4 Capacity Screening

Part A:

Since hMAb BIWA 4 has never been administered to patients, it was essential to obtain information about its safety and biodistribution before starting the RIT test. Its biodistribution will depend largely on the MAb dose used for tumor targeting and will need to be carefully considered. Based on the increased dose of MAb protein studies with low affinity anti-CD44v6 mMAb U36 and high affinity anti-CD44v6 mMAb BIWA 1, the optimal dose range is 25 to 100 mg, the optimal range for mMAb U36 Was expected to be 50 mg.

In order to confirm that the dose is adequate for the moderate affinity of hMAb BIWA 4, and to obtain initial information on the change in tumor absorption rate, biodistribution was evaluated at a total of 25, 50 and 100 mg of hMAb BIWA 4, 3 Two evaluable patients were planned to be treated at each of these dose levels.

All evaluable patients received a single intravenous infusion of 2 mg hMAb BIWA 4 labeled with 20 mCi ^99m Tc, measured by a radiocalibration system immediately prior to dosing. Patients scheduled to receive 25 mg, 50 mg or 100 mg received 23 mg, 48 mg or 98 mg, respectively, of unlabeled hMAb BIWA 4 administered with the 2 mg hMAb BIWA 4 labeled 20 mCi ^99m Tc, respectively.

BIWA 4 dose screening for Part B:

In theory, a dose of 50 mg hMAb BIWA 4 was estimated to be the most suitable for development. Part A of this study was performed to confirm preferential uptake of tumors at three dose levels (25 mg, 50 mg and 100 mg) where hMAb BIWA 4 was tested. Tumor uptake rate (expressed as% injected per kg, expressed as% ID / kg) and tumor to non-tumor uptake ratio for these three dose levels was not expected to be very different. If so, the hMAb BIWA 4 dose to be used for Part B of this study was 50 mg. However, if there is a clinically relevant difference between these dose levels, where one level is preferred over the other, the dose level that exhibits the best biodistribution pattern will be selected.

In this case, the clinically relevant difference was defined as the difference of the tumor (average tumor uptake) to bone marrow uptake ratio (mean bone marrow uptake = cellular fraction and supernatant) of at least 50%.

Finally, the selected dose was 50 mg hMAb BIWA 4 based on the results of Part A.

3.4.4.2 Screening for Radiation Initiation Concentrations

Part B:

The hMAb BIWA 4 dose selected in Part A of this study was labeled with increasing concentration of ¹⁸⁶ Re.

The maximum tolerated dose of ¹⁸⁶ Re-labeled murine MAb NR-LU-10 was 90 mCi / m 2 for the pretreatment group, whereas dose-limiting myelosuppression was observed at 120 mCi / m 2. In the case of chimeric MAb NR-LU-12, which recognizes the same antigen as NR-LU-10, reversible myelosuppression occurred at 60 mCi / m 2. In a study with cMAb U36, conducted at the following institution ( Vrije Universiteit University Medical Center), dose-limiting myelosuppression was observed at 41 mCi / m 2.

In view of the above mentioned results, a radiation dose of 20 mCi / m 2 administered as ¹⁸⁶ Re-labeled hMAb BIWA 4 was considered to be a safe starting dose.

The capacity was increased by 10 mCi / m 2. Lower dose levels were injected in two evaluable patients. If drug-related toxicity greater than grade 2 according to CTC was observed, at least three patients per dose level were treated.

Prior to injecting the patient with the next higher dose level, at the starting dose level, the drug-related CTC grade 4 hematologic toxicity or drug-related CTC grade 3 non-blood, in which the patient excluded vomiting and nausea without proper antiemetic treatment Do not experience dose limiting toxicities (DLT), such as toxicological effects. To do this, all patients under this starting dose level had to be observed for a time long enough that the toxicity that could be induced was reversible.

When one patient experienced DLT at the starting dose level, the number of patients treated at that dose level increased to a total of up to six. When one of six patients experienced DLT, the dose increased to the next level. When two or more of the patients experienced DLT, the next lower dose level was extended to a total of six patients (if not performed earlier) to establish the safe dose and MTD recommended for phase II.

Originally, in case of acceptable toxicity at the dose level (less than two patients experienced DLT), additional patients were administered with the dose administered and incrementally lower second doses planned to participate. . The plan was not carried out due to changes in the linker. MAG3 will be replaced by a comparable linker, MAG2GABA-TFP, but with a more convenient linking process. The current development program using MAG3 is completed.

Alternatively, ¹⁸⁶ Re-BIWA 4 in response to the first dosage and the patient is tolerant were eligible for a second administration to 50 mCi / ㎡ capacity of ¹⁸⁶ Re-BIWA 4.

3.4.5 Timing of Dosing and Dosing for Each Subject

3.4.5.1 Dosage and Treatment Schedule

The researchers allowed the study drug to be administered at any time. However, the elapsed time between radiolabeling and administration should not exceed 24 hours.

3.4.6 Blinding

This is an uncontrolled open test. No blinding was performed.

3.4.7 Prior and Concurrent Therapy or Procedure

3.4.7.1 Rescue Dosage and Additional Treatment (s)

Anaphylaxis was considered to be the most serious potential side effect and could direct the immediate discontinuation of antibody injection and the establishment of appropriate ventilation measures. The warning to be taken was that ventilation equipment and anti-histamines, corticosteroids and epinephrine should be in the vicinity. No additional monoclonal antibody can be administered to any patient who has experienced this type of side effect.

As long as the participation and exclusion criteria are met, the treatment or other treatment (s) for diseases not related to this study may be administered to the patient. At the same time, all accompanying treatments had to be recorded in the CRF.

In cases of severe hematological toxicity (CTC Grade 4), other methods for reducing cytokine involvement or bone marrow toxicity are permitted.

3.4.7.2 Restrictions

No additional chemotherapy or radiotherapy is allowed, and the last chemotherapy or radiotherapy must be discontinued at least four weeks prior to participation in this study. All existing chemotherapy and radiotherapy had to be recorded in the CRF.

3.4.8 Treatment Compliance

This study dose is given as a single intravenous infusion. Compliance was confirmed using pharmacokinetic evaluation and radioimmuno scintillation imaging.

3.5 Efficacy / Clinical Pharmacological and Safety Variables

3.5.1 Efficacy / evaluated pharmacodynamic and safety measures and flow charts

Flow Chart:

Part A

The study performed in Part A and each timing are described in detail in the flow chart provided below. A description of this study is presented in the following sections:

Flowchart: Part A: Biodistribution Study Using ^99m Tc-labeled hMAb BIWA 4 Visit1 Visit2 Visit3 Screening visit 1 day* 2 days 3 days 4 days 7 days 6th week next visit Before injection After injection Written Information Agreement x Demographics x Current treatments x x x x x x x x case history x Physical examination x x ECG x Chest x-ray x Blood analysis for stability test x ¹ x ¹ x ¹ x ¹ x ¹ x ¹ Urine analysis for stability testing x x x Pregnancy test x x HAHA rating x ² x ² x ² ENT inspection x CT or MRI x Inclusion / Exclusion Criteria x Side Effect x x x x x x x Life signs x ³ x ³ x ³ x ³ weight x x x Immunofluorescence: Full body scan x ⁴ x ⁴ Planarity Scan x ⁵ -SPECT scan x ⁵ Pharmacokinetics: Blood sample x ⁶ x ⁶ x ⁶ x ⁶ x ⁶ x ⁶ 6 Urine collection x ⁶ x ⁶ x ⁶ x ⁶ Serum Soluble CD44v6 x ⁷ x ⁷ x ⁷ x ⁷ 6 Operation x

*: Pre-injection assessments should be performed three weeks prior to the actual infusion.

x ¹ : screening visit or 1 day pre-infusion day, 21, 48 and 144 hours after infusion and 6 weeks after infusion, blood samples were glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, Serum glutamic oxal acetic acid amine transferase (sGOT), serum glutamic pyruvate amine transferase (sGPT), alkaline phosphatase, gamma glutamyl transpeptidase (GGT), bilirubin, urea, uric acid, thyroid stimulating hormone (TSH), hemoglobin (Hb), hematocrit (Ht), mean erythrocyte volume (MCV), reticulocytes, leukocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets.

x ² : HAHA was evaluated in serum samples obtained at the screening visit, 1 week after infusion (144 hours) and 6 weeks after.

x ³ : Vital signs were assessed at screening visits, pre-injection, 10, 60 and 120 minutes and 6 weeks after infusion.

x ⁴ : Whole body scan was performed immediately after infusion and 21 hours after infusion.

x ⁵ : SPECT scan and planar scan were performed once 21 hours after injection.

x ⁶ : pharmacokinetic blood samples were pre-injected, at the end of the infusion, 5, 10, 30 minutes after infusion; 1, 2, 4, 16, 21, 48, 72 and 144 hours after completion of injection and 6 weeks after injection. Blood samples were taken for enzyme-linked immunosorbent assay (ELISA) and radioactivity measurements (see section 9.5.4 for details). Urine was collected at 0 to 4 hours, 4 to 8 hours, 8 to 12 hours and 12 to 24 hours for the first 24 hours after infusion and at 24 hour intervals up to 48 hours.

x ⁷ : Soluble CD44v6 was measured from serum samples obtained at pre-injection, 21, 48 and 144 hours and 6 weeks after infusion.

6: 6 weeks.

Part B

The studies performed and the respective time periods are presented in the flow chart below. Individual studies are described in more detail in the following sections:

*: Pre-injection assessments should be performed three weeks prior to the actual infusion.

x ¹ : 21, 48 and 144 hours after the screening visit or 1 day pre-injection, infusion, blood samples were glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, sGOT, sGPT, alkaline phosphatase, GGT, bilirubin, urea, uric acid, TSH, Hb, Ht, MCV, reticulocytes, leukocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets. For 2-6 weeks after infusion, blood samples for safety testing were obtained at least weekly.

x ² : HAHA was evaluated in serum samples obtained at the screening visit, 144 hours and 6 weeks after injection.

x ³ : Vital signs were assessed at 10, 60, 120 and 240 minutes and 6 weeks after the screening visit, pre-injection, infusion.

x ⁴ : Systemic scans were performed immediately after infusion, 21, 48, 72, 144 hours post infusion, and optionally 2 weeks post infusion.

x ⁵ : Planar scans were performed at 21, 48 (optional), 72, 144 hours after injection and optionally, 2 weeks after injection. SPECT scans were performed 72 hours after injection.

x ⁶ : blood samples for pharmacokinetic examination were pre-injected, at the end of infusion, 5, 30 minutes after infusion; Obtained at 1, 2, 4, 16, 21, 48, 72, 144, 240 and 336 hours after completion of infusion. Blood samples were taken for ELISA and for radioactivity measurement. Urine was collected at 0-4 hours, 4-8 hours, 8-12 hours and 12-24 hours for the first 24 hours post-infusion, at 24-hour intervals up to 96 hours.

x ⁷ : Soluble CD44v6 was measured from serum samples obtained at pre-injection, 21, 48 and 144 hours and 6 weeks after infusion.

x ⁸ : Disease assessments should be repeated every six weeks until disease progresses or is not affected by subsequent stages. The CT chest was performed at baseline and then repeated if abnormal.

3.5.1.1 Radioimmunoascopy and Dose Determination

Data obtained from radioimmunoastreopography (RIS) has been qualitatively expressed for both Part A and Part B (ie low, medium or high uptake of tumors, bone marrow, liver, lungs, intestines, kidneys and additional organs). Part B is shown quantitatively.

For qualitative evaluation, the grades were converted to numerical values (0 is not absorbed at all, 1 is low absorption, 2 is medium absorption, 3 is high absorption). Average values were calculated and displayed.

For Part B, the quantitative representation of the RIS results used the dose determination method as summarized in section 5.1.4 of the protocol by including the following parameters:

Actual organ activity (expressed in MBq) at a given point in time.

Radioactive residence time in the engine (expressed in hours)

Absorption (radiation) capacity (expressed in mGy / MBq).

Effective (radiation) capacity (in mSv).

More details on the capacity determination analysis can be found in the Capacity Determination Report (November 21, 2001).

e) radioimmuno flash imaging process

A description of the method and timing used in Parts A and B is provided in the next section.

Part A

Using a large double head gamma camera equipped with a low energy collimator, digital whole body images (a.p. and a.p.) were obtained immediately after the injection and 21 hours after the injection. At 21 hours after injection, planar and SPECT images of the head and neck were obtained. Correctors were also obtained. Data was stored for quantitative analysis.

Part B

Using a large field of view dual head gamma camera equipped with a low energy collimator, digital whole body images (ap and pa) were obtained at 21, 48, 72 and 144 hours immediately after administration of the radioimmunoconjugate. Additional planar images of the head and neck were made 21, 48 (optional), 72 and 144 hours after infusion. Two weeks after injection, optional additional imaging (full body and planar) was performed. The calibrator (ie, equivalent to a known fraction of the injection dose in a 10 ml vial), inserted into Adams plantom, must be placed between the lower extremities of the patient during a systemic scan. The initial activity of the calibration source should be between 100 and 200 MBq ¹⁸⁶ Re and the source should be used during all systemic imaging studies. The energy-window and peak settings, scan speed, scan length, scanning date, scan start time and scan duration must be recorded. The anterior and posterior thickness of the neck and abdomen should be measured, during which the patient had to lie flat on the scanning table.

At each imaging operation time, anterior and planar still images are taken and a still image must be obtained from the corrector of Adams-Plantom immediately before or immediately after the image.

72 hours after infusion, cervical SPECT studies should be performed. The filter function using the cut off frequency and the method used for the reconstruction should be recorded.

Single Proton Radiographic Tomography (SPECT)

SPECT images were obtained using a dual head rotary gamma camera equipped with a low energy collimator. I had to catch at least 30 minutes. The 20% symmetry window is centered at the 137 keV proton peak.

Planar and SPECT Data Acquisition Parameters

Planar imaging requires a minimum of 400000 coefficients, which require at least a matrix of 128 x 128 (detail) or 256 x 256 (full body) and a maximum acquisition time of 10 minutes in detail and 60 minutes in the whole body.

SPECT imaging requires at least 64 images, a matrix size of 64 x 64, a 360 degree circular orbit, and 60 second acquisition / imprint.

Data analysis

Twenty-one hours after infusion, anterior whole body image, ROI's of interest should be taken around the body and the orthodontist. In addition, irregular ROI's around the organ where ¹⁸⁶ Re accumulates (eg liver, spleen and left kidney) and around the tumor should be taken. One or more representative background areas should be taken. These areas should be mirror image symmetric with the posterior image. Originally it was planned to take the ROI around the sacrum on the posterior image. The plan was not performed during this test. In order to drive these ROI's for images at other viewpoints, all regions were stored on a computer. The number of pixels and the count per pixel in each area should be reported. The counts in the area for all imaging points should be recorded digitally in a spreadsheet.

Estimation of Absorption Capacity

The amount of radiation in organs, tumors and whole bodies was evaluated from the geometric mean counts of ROI's from the anterior and anterior perspectives. Background and attenuation corrections were applied whenever indicated. Urine activity was not used to assess the absorption capacity in the bladder as originally planned. Instead, a dynamic bladder model was used. Retention time in the trachea and in the rest of the body was estimated and involved in the MIRDOSE3 program.

Required quality control test

Planar imaging

Normal quality control was conducted weekly in the Department of Nuclear Medicine:

1) 100M float table.

2) Exogenous ⁵⁷ Co floes with low energy collimators were obtained weekly.

SPECT Imaging

Quality control of the SPECT imaging system had to include a rotation decision center.

Tomography process

The filtered back projection algorithm was used for tomographic image reconstruction using a lamp filter.

Data storage

All planar images and tomography data must be permanently stored on magnetic tape or optical disks. For tomography studies, the original projection image and the reconstructed study should be recorded on the back-up tape.

Copy video and reports

Copies of all relevant imaging, pathology and surgical reports were required for all patients. Copies of MRI and CT reports were also required.

3.5.1.2 Biopsy Biodistribution Diagram of ^99m Tc-labeled hMAb BIWA 4 (Part A)

Patients in Part A of this study underwent surgery 48 hours after infusion of radiolabeled hMAb BIWA 4. Biopsies were taken from the tumor site (s) and normal tissue (as much as possible) in the surgical specimens. Bone biopsies and bone marrow aspirates were then also taken under general anesthesia. Bone marrow aspirates were centrifuged to assess radioactivity in supernatants (plasma) and sediments (cellular fractions). All biopsies were weighed and the amount of ^99m Tc was measured. Specific uptake of radioactivity into tumors was assessed by comparing% ID / kg tumors with% ID / kg normal tissue.

CD44v6 antigen expression was assessed by immunohistochemistry using a cryo section, first incubated with mMAb BIWA 1 and then incubated with anti-mouse immunoglobulin G (IgG) secondary reagents. Surgical specimens and biopsies were studied histological / cytopathological.

Evaluation of Surgical Specimens and Biodistribution Charts

After obtaining a surgical specimen, it was processed in the following time order:

1. A photograph of the specimen was taken. Anterior polaroid and anteroposterior slide.

2. The size of the surgical specimen was evaluated.

3. Biopsies of normal tissue in surgical specimens such as primary tumors, dragon lymph nodes, and, if possible, normal mucosa, normal lymph nodes, fat and muscle. All biopsies were weighed and the amount of ^99m Tc was measured. All data are converted to% injection dose / kg of tissue. Specific uptake of radioactivity in tumors was assessed by comparing% ID / kg tumors with% ID / kg normal tissue.

4. The specimen is then fixed to the plate with a nail and held in formaldehyde 4% for at least 36 hours.

5. After incision of thoracic mastoid muscles (structures with high radiation density), a sample X-ray was taken to show the exact size and localization of the involved lymph nodes. Made while immersed, it shows the same X-ray absorbance as fat.

6. All sections visualized by X-rays were indicated on polaroid and sample X-rays.

7. All the sections were examined and found by surgical specimen and cut out from the specimen by X-ray.

8. All visually negative sections were processed entirely under the microscope and one single section was evaluated. Of all the sections that were visually benign, two or more flakes were made. Visual evidence of whether tumor necrosis is present or absent was recorded.

9. Furthermore, the number of enclosed sections, and the number of tumors containing sections, localization and lymph node levels (according to the Memorial Sloan Kettering Cancer Center Classification) were recorded.

3.5.1.3 Tumor Response (Part B)

The efficacy parameter for radioimmunotherapy treatment was tumor response. Tumor response was assessed using tumor measurements and / or CT, MRI or bone scintillation studies as clinically evaluated. The evaluation was carried out in accordance with the World Health Organization's response criteria (see Annex VI of this protocol (R96-0941)).

3.5.1.4 Physical Inspection

Before participating in the study, the disease state of each patient was assessed by ENT-test (including promotion) and CT or MRI of the tumor site.

All head and neck lesions are listed per neck side.

Palpation

All patients were examined baseline by an otolaryngologist / head and neck surgeon. The clinical researchers evaluated the number, size, localization and mobility of all palpable lymph nodes in the head and neck region. The characteristics of the lymph nodes were described as not suspected, suspected or tumor infiltrated. The status of cervical lymph nodes was classified according to the following criteria at diagnosis: Tumor Node Metastasis system for staging tumours (TNM) of the Union Internationale Cancer Cancer (UICC).

Radiological examination

Depending on the localization of the tumor site (s), one or more of the tests, such as CT, MRI, and bone sclerography, are required. MRI is generally preferred for determining tumor involvement in the head and neck regions. CT is preferred if bone lesions are present. For Part B, all radiological disease assessment parameters obtained at baseline were repeated 6 weeks after infusion and every 6 weeks until disease progressed or not affected by subsequent steps. For patients participating in Part B of this study, the CT thorax was obtained with a baseline and continued repeated until tumor lesions existed within the thorax. Ultrasound may also be used as an additional technique of tumor imaging. Guidelines for this study are given below:

Primary tumor / local recurrence

For patients participating in Part A of this study and for locally relapsed patients (Part B), CT scans and / or MRI of the head and neck areas should be performed.

Computed tomography

Computed tomography of the head and neck region: Dynamic CT is preferred over spiral CT. However, spiral CT may help patients who cannot cooperate.

The patient should be in a supine position, with the cervix slightly overstretched, the neck fixed, the shoulders relaxed, and slightly lowered. The patient should breathe quietly using the abdomen rather than the pectoral muscles. The area to be scanned was determined from the schematic initial observations made in the side projection. The scan plane should be placed alongside the vocal cords. Three successive milliliter-flakes should normally be used. Imaging should be performed from the skull to the upper mediastinum.

Magnetic resonance imaging

The patient should be placed in a supine position with the neck slightly extended and the neck fixed. During the test, it is desirable to breathe quietly using your abdomen rather than your chest muscles. Images suitable for demonstrating cervical anatomy and assessing the extent of primary lesions and the presence of lymph node diffusers were obtained. They were obtained from the skull to the upper mediastinum.

Remote metastasis

For all patients participating in Part B of this study, CT scanning of the rib cage was performed as baseline. Additional studies to visualize metastases, such as bone scintillation or CT abdomen, were optional.

CT rib cage

Helical CT-scans were obtained. The patient was examined in a supine position with his arms raised above his head. The patient had to breathe quietly using the abdomen rather than the pectoral muscles. The patient was scanned from just above the lung to the adrenal level. Images were taken with mediastinum and lung settings.

CT abdomen

A spiral scan was obtained. The patient was examined in a supine position with his arms raised above his head. The patient should breathe quietly. Oral contrast was used for all patients. The site to be scanned was from just above the diaphragm to fibrocartilage. Video was taken with abdominal and liver settings.

Bone Sclerography

After intravenous infusion of up to 600 MBq of ^99m Tc-labeled HDP / MDP, the patient was asked to consume enough fluid and then discard it frequently. At 3 hours post infusion, one systemic phase and, if necessary, three or more phases of stationary phase were obtained. Suspicious lesions will need to be analyzed more closely by CT and / or MRI.

Reporting on bone metastasis reactions is planned to be performed according to a separate set of response criteria. No data was provided.

3.5.1.5 Availability CD44v6

Soluble CD44v6 (sCD44v6) should be measured in serum. Concentrations were determined via a validated enzyme-linked immunosorbent assay (ELISA), performed in accordance with current international guidelines (Boehringer Ingelheim Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany) based on commercially available test kits. 5 ml of blood samples (to be treated for serum) were obtained pre-injection, 21, 48 and 144 hours after infusion and 6 weeks after infusion. Samples were coagulated and centrifuged to produce serum. Storage and Shipping Conditions: Place the sample for ELISA measurement in a cold bath, carefully label to identify the original entity, store at -20 ° C until radioactivity is reduced ( ¹⁸⁶ Re: 4 weeks, ^99m Tc: 3 The batches were sent to the next department (BI Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany) every four weeks. The serum sample was sent on dry ice.

3.5.1.6 Safety

Object protection

All patients were carefully monitored during and after administration of radiolabeled monoclonal antibodies. For this purpose, at least one researcher had to be present at all times.

Laboratory evaluation

Blood samples include glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, serum glutamic oxalacetic acid amine transferase (sGOT), serum glutamic pyruvate amine transferase (sGPT), alkaline phosphatase ( AP), gamma glutaryl transpeptidase (GGT), bilirubin, urea, uric acid, thyroid stimulating hormone (TSH), hemoglobin (Hb), hematocrit (Ht), mean red blood cell volume (MCV), reticulocyte, leukocyte, Neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets were collected at the following time points:

1 day prior to screening visit or infusion and

21, 48 and 144 hours after injection.

6 weeks after injection for Part A. For Part B, at least once per week for study 2, 3, 4, 5, and 6 weeks (more frequently for toxicity).

Pre-injection samples for baseline laboratory evaluation can be obtained on the screening visit or on day 1 of the present study, provided that the samples were obtained 21 days prior to injection of radiolabeled hMAb BIWA 4 and all required assessments had to be done. All required laboratory test results were reviewed and the competent physician should be checked for eligibility prior to antibody injection. This also applies to the second hMAb BIWA 4 administration in Part B of this study.

Urine samples were collected at standard time points for standard hospital screening (protein, blood and glucose):

Screening visit

1 week after injection (144 hours after injection)

6 weeks after injection.

For women of childbearing potential, a pregnancy test was required at the screening visit and at the conclusion of the study.

Person-anti-person-antibody evaluation

The presence and / or occurrence of HAHA was assessed in serum samples. Thus, 5 ml of blood samples (to be treated for serum) were taken at the screening visit, 1 week (144 hours) and 6 weeks after antibody injection. For patients administered BIWA 4 twice in Part B of this study, additional HAHA samples were collected prior to the second dose. Serum levels were assessed using the ratified ELISA method.

Serum was prepared by coagulation and centrifugation of the sample.

Storage and Shipping Conditions: Place the sample for ELISA measurement in a cold bath, carefully label to identify the original entity, store at -20 ° C until radioactivity is reduced ( ¹⁸⁶ Re: 4 weeks, ^99m Tc: 3 (B) every four weeks in batches to the next department (BI Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany). The serum sample was sent on dry ice.

Any increase in human-anti-human antibody levels was carefully compared step by step with possible side effects in patients. The data were collected and retrospectively analyzed during the study.

Side Effect

All adverse events were recorded as CRF. The side effects were graded according to the US National Cancer Institute CTC (NCI-CTC) version 2.0, which was downloaded from the Internet site http://ctep.info.nih.gov/CTC3/ctc.htm . All SAEs were to be recorded.

Immunogenicity and Toxicity

Prior to performing this test, no data on the safety and immunogenicity of hMAb BIWA 4 in humans were available. In previous studies conducted with the murine parental antibody BIWA 1, no clinically real toxicity was encountered. No toxicity was expected from the exposed hMAb BIWA 4 antibody alone (no antibody dependent cell-mediated cytotoxicity (ADCC) effector function and no interference with proliferation of antigen-expressing cells in vitro). Became). hMAb BIWA 4 has been shown to be safely administered to mice (xenograft tissue studies) and monkeys (toxicity studies). Good local resistance has been demonstrated in three local toxicity studies.

Murine and chimeric MAb U36 (anti-CD44v6-epitope with defined overlapping epitope specificities) has been safe so far for HNSCC patients and has no toxicity except myelotoxicity (induced by ¹⁸⁶ Re-labels), cMAb U36 Was not observed in the Phase I dose-increasing RIT study with. It was theoretically possible that a hypersensitivity reaction to radiolabeled hMAb BIWA 4 could occur. Monoclonal antibodies have been administered to thousands of patients for diagnostic purposes.

Although different, potential responses to intravenously administered BIWA 4 may include hypotension, transient fever and chills, skin rash, dyspnea, pruritus, nausea and anaphylaxis.

Thus, vital signs (blood pressure, body temperature, beat rate and respiratory rate) were recorded at the screening visit and at the next time points, 10, 60 and 120 minutes and 6 weeks before and after infusion. In addition, vital signs were recorded 240 minutes after infusion in patients who participated in Part B only.

Radionuclides were used in this study. The cumulative amount of radiation associated with gamma radionuclides ^99m Tc (20 mCi in the present study) is similar and less known than that encountered in many conventional nuclear medicine procedures. To minimize exposure of the bladder and kidneys to radiation, patients were asked to drink plenty of water and then requested frequent intervals at intervals of the first 48 hours (96 hours after injection with ¹⁸⁶ Re-labeled antibody).

For beta and gamma-radioactive radionuclides ¹⁸⁶ Re, see Breitz et al. (R98-2459) describes the maximum tolerated dose of ⁸⁶ Re-labeled hMAb IgG in patients pretreated with a large amount of 90 mCi / m 2. One of the aims of this study was to study the toxicity of ¹⁸⁶ Re-labeled hMAb BIWA 4 in patients with local and / or local relapse disease or distant metastasis that currently have no treatment. Because bone marrow, thyroid, kidney and liver toxicity may occur, blood samples for organ function testing were taken as described in section 3.5.1.6.

To monitor whether weight loss or weight gain occurred, body weights were recorded at the screening visit, one day before infusion, and six weeks after infusion.

Patients were monitored for local toxicity. All local toxicity signs were recorded as side effects.

3.5.2 Compliance of the methods of measurement

The measures used are widely accepted for this type of test.

3.5.3 Primary Efficacy / Pharmacokinetic Variable (s)

3.5.3.1 Part A of this test

The primary efficacy variables determined in Part A of this study were biopsy distribution and radioimmunoassay. A description of how to obtain the data is given in sections 3.5.1.1 and 3.5.1.2.

Absorption rates in normal tissues and tumors were measured in biopsies of surgical specimens, which were expressed as% of injected dose per kg (% ID / kg). Absorption rates in tumors and other tissues over time were assessed and compared with different doses of BIWA 4.

3.5.3.2 Part B of the Test

Primary parameters for efficacy were analyzed by radioimmunoascent scintigraphy and dose determination.

3.5.4 Drug Concentration Methods / Pharmacokinetics

Blood concentrations of radiolabeled BIWA 4 were determined in both Part A and Part B of this test.

3.5.4.1 How and when to collect samples

Patient blood samples were collected and manipulated as follows: 7 ml of blood (3.5 ml in a potassium EDTA containing tube and 3.5 ml in a coagulation tube: the requested milliliter amount is provided according to calibration 1) at a designated time point (ie, immediately prior to antibody administration). At the end of the infusion, and at 5, 10, 30 minutes post injection: 1, 2, 4, 16, 21, 48, 72 hours and 7 days (144 hours) post injection and 6 weeks post injection) Sampled from the peripheral vein of the arm. Injection termination was considered t = 0.

Samples planned 10 minutes and 6 weeks after injection were omitted in Part B of this test. Instead, samples were collected 240 and 366 hours after injection according to Calibration 1.

Urine was collected for 48 hours after ^99m Tc-labeled hMAb BIWA 4 injection and for 96 hours after ¹⁸⁶ Re-labeled hMAb BIWA 4 injection. Urine was collected at 0-4 hours, 4-8 hours, 8-12 hours and 12-24 hours for the first 24 hours after infusion and at 24-hour intervals for the remainder of the time. The radioactivity of the urine samples was counted to determine radioactivity emissions.

Blood samples were centrifuged to produce serum. Prior to treatment, radioactivity in whole blood was measured. Radioactivity was also measured in serum. Refrigerated storage of blood samples is mandatory, but the samples should not be frozen. Using aliquots preserved from conjugate preparation, gravimetric dilutions of injected patient doses were prepared as standards. According to topical SOPs, patient samples and standards were counted. The results were recorded in the appropriate section of the CRF. Radionuclide content was reported as the percentage of dose injected (expressed as% ID / kg blood or serum). Apart from radioactivity counts, all samples were analyzed for the presence of immune complexes via HPLC analysis.

All sample tubes label the following information: test number, patient number, sample identity confirmation (ie serum, plasma or blood), infusion time, actual time, actual data and radioisotope (ie ^99m Tc or ¹⁸⁶ Re) It became. Measure and record each urine sample volume and label all urine samples with the following information: test number, patient number, total sample volume, collection interval, actual time, data and radioisotope (ie ^99m Tc or ¹⁸⁶ Re) It became.

Data, time and radioactivity measurements of all pharmacokinetic samples were recorded in the CRF.

Plasma samples for ELISA measurement were placed in a cold bath and carefully labeled to identify the original identity, stored at -20 ° C until radioactivity was reduced ( ¹⁸⁶ Re: 4 weeks, ^99m Tc: 3 days) and placed Every four weeks, they were sent to the next department (BI Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany). The plasma sample was sent on dry ice.

3.5.4.2 Analytical Determination

Plasma samples were measured by a validated ELISA method in the following department (Boehringer Ingelheim Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany). Radioactivity counts of the samples were performed by the researcher in whole blood, serum and urine.

3.5.4.3 Parameters and Evaluation

The following pharmacokinetic parameters using WinNonlin 3.1 Professional (Pharsight Corporation, Mountain View, Calif.) Were determined from whole blood, plasma, serum levels and imaging data as described below.

The primary pharmacokinetic parameters are the time point when maximum drug concentration is observed (T _max ), the maximum drug concentration observed (C _max ), area under the concentration-time curve (AUC) _{0 → ∞} , termination elimination half-life (t _1/2 ) , Volume of distribution (termination phase and expected stationary phase), total body clearance (CL) and mean residence time (MRT) _{0 → ∞} .

The primary purpose of pharmacokinetic analysis is to:

To determine and compare the disposition of immunoreactive hMAb BIWA 4 and total radioactivity after a single dose administration of ^99m Tc-labeled (Part A) and ¹⁸⁶ Re-labeled hMAb (Part B) BIWA 4;

From imaging data and blood predisposition data, the fraction (rate) of injected radioactivity that reaches the liver and spleen during this study is to be estimated. To achieve this goal, WinNonlin® software was used to perform non-compartmental pharmacokinetic analysis of plasma concentration versus time profile of immunoreactive hMAb BIWA 4 and total blood radioactivity levels as a function of time after intravenous administration. .

The results are reported as summary statistics.

3.5.5 Pharmacokinetic / Pharmacodynamic Relationships

Compartmental pharmacokinetic models were initially designed to describe the biodistribution and metabolism of ¹⁸⁶ Re-labeled BIWA 4 in humans. These include BIWA 4 plasma levels, data from whole blood and serum radioactivity assessments, systemic imaging and radioactivity from specific areas of interest, administered doses of ¹⁸⁶ Re-labeled BIWA 4, doses of unlabeled BIWA 4, soluble CD44v6 levels, and It is intended to combine the estimates of radiolabeled antibodies prior to injection.

The results should be reported in summary statistics. However, the variable was too large for the rational model.

3.5.6 Primary safety variables

Determination of the innermost tolerated dose and safety assessment were the primary endpoints in Part B of this test. The measurement performed is described in section 3.5.1.6.

3.5.6.1 Dose Limiting Toxicity and Maximum Tolerated Dose

DLT has been defined as drug-related CTC grade 3 non-hematologic toxicity or drug-related CTC grade 4 hematologic toxicity, which excludes vomiting and nausea without proper antiemetic treatment.

MTD was defined as the dose level at which drug-related DLT occurred in less than 2 of 6 patients.

3.6 Data Quality Guarantee

The trial was generally performed in accordance with the principles of good clinical practice criteria as specified in the appropriate enforcement rules and as specified in the company's standard operating procedures that reflect those enforcement rules.

Throughout the study, representatives of Boehringer Ingelheim The Netherlands contacted and / or visited the Institute to monitor the progress of the study. Frequent contact with the researchers and site visits for data accounting audits, including comparison of raw documents with case report forms and drug accounting accountability checks. The researcher or their designee had to be on standby for the Dutch representative Boehringer Ingelheim the Netherlands during these site visits.

Sufficient CRFs were collected regularly. The data was double-data entered into this test. The researchers were asked if this data was considered and difficult to accept.

Serious adverse events were reported in accordance with the guidelines set forth in this protocol and CRF and in accordance with the Boehringer Ingelheim SOPs.

3.7 Determination of statistical methods and sample sizes planned for this protocol

3.7.1 Statistical and Analytical Planning

3.7.1.1 Statistical Design / Model

Designs used in this test are commonly used in Phase I oncology studies.

Part A

Part A of this Phase I trial increased in an uncontrolled manner to determine the safety, tolerability, biodistribution and pharmacokinetics of a single infusion of ^99m Tc-labeled hMAb BIWA 4 in patients with squamous cell carcinoma of the head and neck. Dose was a sequential group study.

Three hMAb BIWA 4 dose levels were used in three patients for each dose level in this part of the study. All patients were found to have tumors in the head and neck and had to undergo surgery.

Part B

Part B of this Phase I trial determines the safety, tolerability, MTD, pharmacokinetics, and preliminary treatment effects of a single infusion of ¹⁸⁶ Re-labeled hMAb BIWA 4 in patients with squamous cell carcinoma of the head and neck who currently have no treatment. For the uncontrolled open dose sequential increase group.

In this part the radiation dose increased sequentially. Two patients were treated at a dose level of which CTC toxicity below grade 2 was observed. If grade 2 toxicity abnormalities were observed, three or more patients per dose group were treated.

Safety and Tolerance Assessment (Part A + B)

The safety and tolerability of this study were evaluated in terms of changes in laboratory parameters, signs of life, occurrence of HAHA and the occurrence of adverse events. The results were recorded separately for each dose level and also as an overall average.

Biodistribution Assessment (Part A)

Biodistribution of ^99m Tc-labeled hMAb BIWA 4 in solid tissue was assessed by radioimmunoassay (see section 3.5.1.1) and radiometric measurements of biopsy specimens (see sections 3.5.1.1 and 3.5.1.2). Since only up to three patients could be involved at each dose level, only descriptive statistics were possible.

Immunoscopy Imaging Assessment (Part A + B)

At some time after the administration of the radiolabeled antibody, an immunofluorescence image was obtained (see section 9.5.1.1). Also, only descriptive statistics were applicable.

Pharmacokinetic Evaluation (Part A + B)

The following pharmacokinetic parameters were determined from plasma or serum, urine levels and imaging data as described below:

Primary parameters (non-compartmental)

T _max , C _max , AUC (0-infinite time point), termination elimination half-life, biodistribution volume (final phase V _z and expected stationary phase V _ss ), CL and MRT (0-infinite time point). Cumulative urine output of radioactivity over time was determined from total urine output.

Secondary Parameters (Segmentability)

Compartmental pharmacokinetic models are planned to be developed to define the biodistribution and metabolism of ¹⁸⁶ Re-labeled hMAb BIWA 4 in humans. The model includes data from serum and urine radioactivity measurements, systemic imaging and radioactivity from specific areas of interest, dose doses of ¹⁸⁶ Re-labeled hMAb BIWA 4, cold-loaded doses of hMAb BIWA 4, soluble CD44v6 levels and infusions. It was matched with the evaluation of all radiolabeled antibodies.

The results were planned to be reported separately and through summary statistics for each dose level.

Therapeutic Efficacy Assessment (Part B)

Tumor response was a parameter for therapeutic efficacy. Tumor response was assessed according to the WHO guidelines. The sum of the products of the largest right-angle diameter of all tumor lesions measured was the primary parameter for tumor response. There are separate evaluation criteria for the response of bone lesions.

3.7.1.2 null and alternative hypotheses

All analyzes in this test were originally descriptive and preliminary. All statistical tests were conducted to provide a statistical framework only to observe the results and to plan further studies. No formal statistical inference was foreseen, so no statistical test was performed.

3.7.1.3 Planned analysis

Two groups were distinguished:

Treatment modalities for the treatment of the subset consisted of all patients with radiolabeled hMAb BIWA 4 injected and baseline data available.

The subset according to the protocol consists of evaluable patients. Patients could be assessed if the following criteria were met:

Part A:

1. Meet the criteria for a treatment modality to treat a subset.

2. Side effects, vital signs and safety Post-treatment in terms of blood and urine samples was adequate.

3. The immunoreactive fraction in at least one of the assays performed was greater than 60%.

4. Appropriate biopsies and scintillation images were available to assess biodistribution.

Part B:

1. Meet the criteria for a treatment modality to treat a subset.

2. Side effects, vital signs and safety Post-treatment in terms of blood and urine samples was adequate.

3. The immunoreactive fraction in at least one of the amount assays performed is greater than 60%.

4. Response: If appropriate tumor measurements were obtained at baseline and the patient was followed over 6 weeks, the patient was evaluable for response. If disease progression was observed within the 6 week period, the patient could also assess the response.

Primary analysis

Primary analysis was performed on a subset according to the protocol.

No evaluable patients were replaced. Thus, for Part A, the evaluable subset was planned to consist of nine patients, and the treatment modality to treat the subset consisted of nine or more patients. For Part B, the number of patients depends on the toxicity encountered. Patients who could be assessed for toxicity but not for response were not replaced.

Pharmacokinetic Data:

Analysis of pharmacokinetic data was performed by the following researchers (Dr. Thomas R. MacGregor, Boehringer Ingelheim Pharmaceuticals Inc. Ridgefield CT, USA).

The primary purpose of pharmacokinetic analysis is to:

1. After administration of a single dose of ^99m Tc-labeled hMAb BIWA 4 and ¹⁸⁶ Re-labeled hMAb BIWA 4, to determine and compare the disposition of immunoreactive hMAb BIWA 4 and total radioactivity.

2. hMAb BIWA 4 is to identify the appropriate cooling-load capacity.

3. To assess the predisposition of the second dose of ¹⁸⁶ Re-labeled hMAb BIWA 4.

4. From the imaging data and blood predisposition data, to estimate the fraction (rate) of injected radiation reaching the liver and spleen during this study. To achieve this goal, the WinNonlin® software package, after intravenous administration, allows non-compartmental pharmacokinetic analysis of plasma concentration versus time profile of immunoreactive hMAb BIWA 4 and total blood radioactivity levels to be performed as a function of time. Was used. All measurements associated with radioactivity were normalized to fractions of protein-bound and immunoreactive radiolabels prior to infusion. Specific pharmacokinetic parameters generated include the following: T _max , C _max , AUC (0-infinite time point), termination elimination half-life, biodistribution volume (termination and expected stationary phase), total body cleaning rate, and average residence time ( 0-infinite time point). Cumulative urine output of radioactivity over time was determined from total urine output.

The secondary purpose of pharmacokinetic analysis to develop a pharmacokinetic model for describing biodistribution and metabolism of ¹⁸⁶ Re-labeled hMAb BIWA 4 in humans was not achieved due to the large data (especially radioscopy data). It was.

Secondary analysis

Secondary analyzes were performed on treatment modalities to treat a subset of patients participating in Part B of this study.

Secondary analysis was limited to major endpoints, such as reaction rate, duration of reaction, and time to progress.

Intermediate analysis

No intermediate analysis was performed.

3.7.1.4 Manipulation of omitted data

In this Phase I study, it was expected that most of the data could be used for analysis. In the case of omitted data, it was assumed that the reason for omission was not related to the result. It is not due to the omitted data.

3.7.2 Determining Sample Size

In Part A, three patients per dose level provide information on the safety of hMAb BIWA 4 and the expected exact dose of 50 mg of unlabeled hMAb BIWA 4.

In Part B, six patients were considered sufficient to determine DLT. Table 3.7.2.1 indicates that more than two out of six patients are likely to be observed to have DLTs for some estimated baseline rates of DLTs in all patient populations.

Estimated population rate of DLT 0.40 0.42 0.45 Probability of DLT observed in 2 or more of 6 patients 0.77 0.80 0.84

Source: Probability was estimated from the cumulative distribution function of the binomial biodistribution plot with n equal to 6 and p varying

According to the incremental scheme in this study, when the basic individual probability of reaching a DLT was 42% or more, the probability of two or more patients exhibiting DLT was 80% or more.

3.8 Changes in the performance of this study or planned analysis

The following changes from the protocol were implemented through calibration:

The timing of blood sample collection was adjusted by the implementation of calibration 1 of September 1, 1999. Samples were dropped 10 minutes and 6 weeks after injection, while additional samples were collected at 240 and 336 hours after termination of injection in Part B of this test. The original blood sampling schedule was found to be insufficient to adequately cover the 50 hour estimated half-life observed in Part A of this test. A total of 3.5 ml of blood was collected in potassium EDTA tubes and 3.5 ml were collected in coagulation tubes.

The protocol states that after determining the MTD, a second dose of ¹⁸⁶ Re-BIWA 4 can be administered to the patient. The process was modified by calibration 2. If a patient responds to the first dose of ¹⁸⁶ Re-BIWA 4 and has no side effects or recovered from these side effects and no HAHA antibody, they are considered eligible for the second treatment cycle. Response was defined as stable disease (ie, no change), partial recovery or complete recovery. The second dose administered is always 50 mCi / m 2. Due to changes in the linker, this test was performed prior to study of the second dose using the dosage regimen described in this protocol. To somehow treat the patients who responded, correction 2 was implemented.

This test was completed and terminated after reaching the MTD due to linker changes. MAG3 will be replaced by MAG2GABA-TFP by the future development of the BIWA 4 project.

Samples could be used to determine sCD44v6 for more time points than originally planned.

The following changes were made after the data evaluation and after discussion within the testing team:

The ratio of% ID / kg tumor to% ID / kg non-tumor tissue was performed only on the bone marrow because of the variable (diversity) for the results.

The same applies to assessing tumor size. The measurements are incomplete and thus threaten reasonable evaluation. Therefore, no formal analysis was done.

4. Study subject

4.1 Postmark of the Subject

Of the 33 patients screened, three patients withdrew from the study because of side effects before even injecting the test drug (Table 4.1: 1). These three patients were not included in this analysis. A total of 30 patients participated in this trial and were treated.

Part A

Of the 30 patients treated, three each received 25 mg and 100 mg of ^99m Tc-BIWA 4, respectively, while four patients contained 10 patients in 50 mg of ^99m Tc-BIWA 4, which were included in Part A. It became. All patients in Part A completed this trial.

Part B

Of the 20 patients treated in Part B, 2 had discontinued treatment due to side effects (2 patients died). Three patients received a second dose of 50 mCi / m 2.

The stigma of the patient Part A Part B gun ^99m Tc-BIWA 4 ¹⁸⁶ Re-BIWA 4 [mCi / ㎡] 25 mg BIWA 4 50 mg BIWA 4 100 mg BIWA 4 20 30 40 50 60 screen 33 Participation 3 4 3 2 4 3 6 5 30 cure 3 4 3 2 4 3 6 5 30 complete 3 4 3 2 3 2 6 5 28 stop 0 0 0 0 One One 0 0 2 AE origin 0 0 0 0 One One 0 0 2 Lack of efficacy 0 0 0 0 0 0 0 0 0 Due to other reasons 0 0 0 0 0 0 0 0 0

Source: Appendix 16.1.9.2 Table 1.1

4.2 Protocol Evaluation

The majority of protocol violations are deviations (> ± 10%) from the time window provided in the protocol for blood and / or urine sampling. The affected patients were not excluded from this study, but the actual collection time of blood or urine was used to determine blood, plasma, serum and urine concentrations.

5. Efficacy / Clinical Pharmacological Evaluation

The results from Part A confirm that the dose of 50 mg BIWA 4 is the optimal dose for treatment. Data from Part B indicates that ¹⁸⁶ Re-BIWA 4 therapy may be clinically beneficial to patients at doses below the dose at which DLT is observed.

The measured concentration of BIWA 4 is proportional to the dose and the appropriate amount of dose administered is excreted through the kidneys.

5.1 Analyzed Data Set

All patients who received one or more doses of the test drug were included in the intensive care analysis. Seven patients from Part A were included in the analysis according to the protocol. For Part B, no analysis was performed according to the protocol.

5.2 Demographics and Other Baseline Features

The average age of all patients included was 56 years (range 37 to 78 years). Nineteen patients were male and 11 were female (Table 5.2: 1).

Three patients treated in Part A had no smoking history, while seven were still smokers (pack age range 19 to 47 years). Frequent alcohol intake was reported in eight patients, while two did not drink alcohol at all (Table 5.2: 1).

All patients in Part B were past smokers or current smokers with an average pack age of 35 years (10 to 86 years) (Table 5.2: 1). Six patients did not drink at all, while the other patients frequently drank (Table 5.2: 1).

The disease stage was locally operable for all patients included in Part A, while patients from Part B had disease recurrence (15 patients) and / or metastases (3 patients). One patient was incapable of local surgery. Information about one patient is omitted.

Concurrent disease or related medical history has been reported in 28 patients (Table 5.2: 1). Simultaneous treatment was desired for all patients. The most frequently used medications were analgesics, sedatives, lactulose, H ₂ -blockers, antiemetics and antimicrobial agents.

Part A Part B gun ^99m Tc-BIWA 4 ¹⁸⁶ Re-BIWA 4 [mCi / ㎡] 25 mg BIWA 4 50 mg BIWA 4 100 mg BIWA 4 20 30 40 50 60 Number 4 4 3 2 4 3 6 5 30 Age [average] 45.7 58.5 58.7 57.0 61.0 53.0 57.0 60.4 56.4 Male / female 2/1 2/2 1/2 1/1 3/1 2/1 5/1 3/2 19/11 Concurrent disease 3 3 2 2 4 3 6 5 28 Concurrent therapy 3 4 3 2 4 3 6 5 30 smoker 2 4 One 0 One 0 3 One 12 Past smokers 0 0 0 2 3 3 3 4 15 Drinking alcohol 3 3 2 0 3 3 6 2 22

Demographics and Baseline Features. The mean for age is given. All other numbers refer to the number of patients

Initial diagnosis of the disease is known to patients in Part A approximately one month before participation, whereas patients participating in Part B were ill up to 2 years on average (range 0.1 to 17.5 years; Table 5.2: 2). The Karovskysky score ranged from 70 to 100 for patients from Part A. The Karnovsky score of the patients from Part B was 70 to 90 (Table 5.2: 2).

Metastasis status at diagnosis was reported for one patient in Part B of this trial.

All patients in Part B had prior radiotherapy and / or chemotherapy, whereas patients in Part A had no radiotherapy or chemotherapy precedence reported (Table 5.2: 2). Cisplatin, methotrexate and fluorouracil were the most commonly used systemic anticancer agents.

Prior surgery was basic for 11 of 13 patients in Part B who had undergone surgery. Only one patient from Part A had prior surgery (Table 5.2: 2).

Tumor lesion size (including metastases) is 14-10304 mm 2. Primary tumor sites were appropriately distinguished in the majority of patients. Lymph nodes were affected in 13 patients (Table 5.2: 2).

Part A Part B ^99m Tc-BIWA 4 ¹⁸⁶ Re-BIWA 4 [mCi / ㎡] 25 mg BIWA 4 50 mg BIWA 4 100 mg BIWA 4 20 30 40 50 60 Number 3 4 3 2 4 3 6 5 Years Recognized by Disease 0.11 0.08 0.07 9.30 2.13 1.17 0.08 1.16 Karnovsky score 90 90 100 80 75 76.7 85 80 Basic surgery 0 0 0 2 2 One 2 4 Affected lymph nodes 2 2 0 2 3 2 One One Previous Chemotherapy 0 0 0 One 3 One 4 One Previous radiotherapy 0 0 0 2 4 3 6 5

Demographics and Baseline Features. Means for known disease age and Karnovsky scores are shown. All other numbers refer to the number of patients

5.3 Evaluation of Treatment Compliance

All drugs were administered under the supervision of this researcher or its agents. Blood was collected to determine the pharmacokinetics of BIWA 4. All patients have detectable blood levels of BIWA 4.

5.4 Efficacy / Clinical Pharmacological Results

Three of the six patients experienced stable disease at the innermost dose of 50 mCi / m 2. A dose of 50 mg BIWA 4 was found to be the optimal dose in terms of blood concentration and selective tumor uptake in Part A of this test. Plasma concentrations of BIWA 4 were dose-proportional in the range of 25 mg to 100 mg BIWA 4 in Part A, peaking at 0.9 hours with termination elimination half-lives of 54 to 74 and 94 hours for Parts A and B, respectively.

5.4.1 Efficacy / Pharmacodynamic Analysis

5.4.1.1 Primary Endpoint

The primary endpoint for efficacy was the biodistribution of ^99m Tc-BIWA 4 in Part A. An additional primary endpoint was the analysis of radioimmunoensimetric images and pharmacokinetics (as described in section 5.4.2) for each of parts A and B of this test. Dose determination was performed only for Part B.

In part A ^99m Biodistribution Diagram of Tc-BIWA 4

Tissue samples were obtained during surgery and the uptake rate of ^99m Tc-BIWA 4 was expressed as% injection dose (ID) / kg tissue.

Intensive Care (ITT) Subsets : The relative biodistributions of ^99m Tc-BIWA 4 were Patient 1 (25 mg BIWA 4), Patient 5 (50 mg BIWA 4, no tumor cells) and Patient 9 (100 mg BIWA 4), respectively. Except for this, it was the largest in the tumors of all three dosing groups. Tumor uptake rates for dose groups 25 mg, 50 mg and 100 mg respectively were 6-17% ID / kg, 5-28% ID / kg and 13-17% ID / kg. Mean estimates of tumor uptake versus bone marrow uptake were 1.7, 2.6 and 2 in the 25 mg, 50 mg and 100 mg dose groups, respectively (Tables 7.2: 1 and 7.2: 2).

Subset according to protocol (PP): The relative biodistribution of ^99m Tc-BIWA 4 was greatest in the tumors of all three dosing groups except patient 1 (25 mg BIWA 4). Tumor uptake rates for dose groups 25 mg, 50 mg and 100 mg respectively are 6-17% ID / kg, 23-28% ID / kg and 16% ID / kg. Mean estimates of tumor uptake versus bone marrow uptake were 1.7, 3.2 and 2.5 in the 25 mg, 50 mg and 100 mg dose groups, respectively (Tables 7.2: 1 and 7.2: 3).

A trend toward higher uptake with smaller tumor sizes was considered to exist for the 25 mg BIWA 4 dose group. Similar evaluations were not possible due to the limited number of patients for the 50 mg and 100 mg BIWA 4 dose groups (Table 7.2: 4).

High% ID / kg uptake was observed in bone marrow supernatant (17% ID / kg or less) and plasma or blood (13% ID / kg or less), indicating tumors in the population following protocol except for patient 1 (25 mg BIWA 4). It was always lower than the absorption rate. Skin and mucosal uptake was always lower than primary tumor uptake in the subset according to the protocol.

In part A ^99m Radioimmunoassay scintillation imaging of Tc-BIWA 4

Immediately after injection no radioactivity uptake was recorded in the tumor, whereas a median uptake of 1.9 was recorded 21 hours after injection. The absorption rate of radioimmunoconjugate was low in bone marrow and kidney both immediately after injection and 21 hours after injection. Absorption rates are low or medium in the lungs but higher in the liver.

Similar patterns were observed for the population according to the protocol. The only difference was higher absorption in the kidneys and lower absorption in the lungs compared to intensive care.

In part B ¹⁸⁶ Radioimmune scintillation imaging of Re-BIWA 4

Radioimmune scintillation images were made immediately after injection and at 21, 48, 72, 144 and 336 hours post injection. Immediately after injection, little tumor absorption was observed. Relative tumor uptake is believed to be dose-dependent and increases with time, reaching medium to high uptake after 72-144 hours and descending after 336 hours.

The biodistribution of radioactivity was similar in bone marrow, lung, liver, kidney and intestine and did not show the same dose dependent effect (except intestine). Moreover, the relative radioactivity uptake appears to be consistently or moderately decreasing over time and was similar for all doses of radioactivity. Bone marrow uptake was the lowest for most treatment groups.

5.4.1.2 Secondary Endpoint (s)

Secondary endpoints of efficacy were tumor response to therapy in Part B, respectively, and CD44v6 expression levels in tumors in Part A. Soluble CD44v6 expression was determined for both Part A and B of this test.

CD44v6 Expression in Tumors in Part A

CD44v6 antigen expression determination data is available for 7 patients. CD44v6 antigen expression was observed in at least 90% and at least 80% of tumor cells in 5 patients and 2 patients, respectively, whereas CD44v6 antigen expression was observed in 90 of lymph node transferase cells in 4 patients who were available for evaluation lymph nodes. More than% was detected. Homogeneous CD44v6 antigen expression was observed in the mucosa of all patients.

Tumor Response in Part B

Patients treated with ¹⁸⁶ Re-BIWA 4 at doses up to 40 mCi / m 2 did not experience clinical tumor response. All patients experienced progressive disease. One patient died in the middle and could not be evaluated.

Three of six patients treated with 50 mCi / m 2 ¹⁸⁶ Re-BIWA 4 developed stable disease or no change after the first cycle. In another cycle, two of the three patients treated with 50 mCi / m 2 ¹⁸⁶ Re-BIWA 4 developed progressive disease after the second cycle. Progressive disease was observed after a total of 148 days (patient 109) and 127 days (patient 116) of the first administration of the test drug.

One patient treated with 60 mCi / m 2 ¹⁸⁶ Re-BIWA 4 experienced stable disease after the first cycle. This patient entered progressive disease after the second cycle with 50 mCi / m 2 ¹⁸⁶ Re-BIWA 4 (173 days after the first dose of the test drug).

The total time for progression of patients who did not respond to therapy ranges from 0 to 55 days, with an average of about 5 to 6 weeks regardless of treatment group.

Availability in Parts A and B CD44v6

Soluble CD44v6 was determined in all patients treated with BIWA 4 in this trial.

The amount of soluble CD44v6 detected tended to increase for all patients treated with ¹⁸⁶ Re-BIWA 4 during Part B of this trial (Table 7.2: 6), but this trend was observed for patients treated with ^99m Tc in Part A. Not observed at all.

5.4.2 Relationship with Drug Dose, Drug Concentration, and Response

The pharmacokinetics of BIWA 4 will be presented separately for Parts A and B because the radiolabels are different (Part A: ^99m Tc; Part B: ¹⁸⁶ Re).

5.4.2.1 Analytical Performance for BIWA 4, HAHAs and CD44v6

The ratified assay was used for plasma sample analysis. Analysis of quality control samples was obtained as a result of high accuracy and precision.

Analytical Performance on the Gamma Coefficient of Samples

The signal linearity of the scintillation crystals in the gamma counter was not tested. This can be estimated based on the type of device above the energy range used in the present study. Calibration samples typically exhibited 40000 to 200000 cpm versus less than 50 to 100 cpm in the background sample. The accuracy of the seven measurements per calibration standard is regularly within 2%. This also applies to the accuracy of the individual triple measurements in blood, serum and urine. Blood and serum samples typically exhibited an activity of 2000 to 100000 cpm.

There was no quality control sample for counting radioactivity. The accuracy of repeated measurements of calibration standards and samples derived from the current study indicates that the accuracy of triplicate measurements is at least 2% better.

5.4.2.1 Pharmacokinetics

Part A

Part A consisted of 10 patients who received a single dose of BIWA 4 in doses ranging from 25 to 100 mg with a constant dose of radiolabeled 20 mCi ^99m Tc. The pharmacokinetic parameters in plasma for BIWA 4 measured by ELISA after intravenous infusion within a short period of time are shown in Table 5.4.2.2:1:

Pharmacokinetic Parameters for BIWA 4 in Plasma from Part A of the Study patient BIWA 4 dose (mg) Tmax (minutes) Cmax (ng / mL) Half-life (hours) AUC _inf (ugh / mL) Vz (L) CL (mL / min) MRT (hours) One 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 14900 64.9 1112.3 4.2 0.749 84.8 5 50 121 15500 102.8 1580.2 4.7 0.527 135.2 8 50 5 13900 53.0 831.0 4.6 1.003 71.7 10 50 41 16700 83.8 1461.6 4.1 0.570 110.0 6 100 4 30500 85.0 2548.9 4.8 0.654 112.0 7 100 17 30600 76.0 2709.6 4.0 0.615 110.4 9 100 30 24500 44.6 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 15216 73.8 1208.8 4.4 0.689 97.5 100 mg 13 28383 66.0 2276.4 4.2 0.732 91.1

An increase in C _max BIWA 4 plasma concentration was observed and the area under the curve was proportional to the dose administered (FIGS. 4 and 5).

Serum concentrations of radiolabels ( ^99m Tc) were also determined and pharmacokinetic parameters are shown in Table 5.4.2.2:2:

Pharmacokinetic Parameters for ^99m Tc-BIWA 4 in Serum from Part A of the Study patient ^99m Tc-BIWA 4 Capacity (mCi) Tmax (minutes) Cmax (% ID / kg) Half-life (hours) AUC _inf (% ID · h / kg) Vz (kg) CL (kg / min) MRT (hours) One 19.0 mCi 117 52.66 44.6 3376.9 1.9 0.030 63.8 2 20.0 mCi 10 28.08 55.6 2151.7 3.7 0.048 79.1 3 21.4 mCi 10 39.11 27.5 1445.3 2.7 0.072 41.5 4 19.5 mCi 29 31.84 31.3 1432.8 3.2 0.072 43.8 5 19.1 mCi 121 32.29 39.6 1934.8 3.0 0.054 56.3 8 18.8 mCi 114 28.41 42.8 1620.1 3.8 0.060 60.8 10 19.3 mCi 41 40.3 43.1 2247.1 2.8 0.042 62.3 6 19.3 mCi 120 30.45 45.3 1946.5 3.4 0.054 65.2 7 19.8 mCi 77 35.79 36.0 1628.6 3.2 0.060 51.1 9 18.3 mCi 238 31.94 35.5 1805.6 2.8 0.054 50.6 Harmonic mean 38.7 Geometric mean 57.9 34.5 39.4 1898.0 3.0 0.053 56.5

The geometric mean half-life for BIWA 4 measured by ELISA in plasma after intravenous infusion ranged from 54 to 73.8 hours for the three treatment groups studied in Part A. The geometric mean half-life for ^99m Tc-radiolabeled BIWA 4 for the same sample was 39.4 hours in serum and shorter at 46.4 hours in blood. The discrepancy between the two mean estimates was due to the longer sampling time possible with BIWA 4, due to radioactivity limitations.

Radioactivity was released to urine in the amounts collected in Table 5.4.2.2:3.

Since the collection over 48 hours is insufficient (ie cumulative data cannot always be equal due to changes in the collection period), no further evaluation of the urine release data has been made. For six patients who completed a 48 hour collection, the average amount of radioactivity collected was 10% of the initial dose.

The amount of radioactivity ( ^99m Tc) released to urine in Part A (% of initial dose) patient Collection interval (hours) Cumulative amount (% ID) 0-4 4-8 8-12 12-24 24-48 One 2.80 10.13 5.08 n.a. n.a. 18.01 2 1.94 0.97 0.52 30.9 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 5 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 10 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, data not available

Part B:

Part B consisted of 20 patients receiving a 50 mg dose of BIWA 4 with varying radiolabeled doses of ¹⁸⁶ Re-BIWA 4 in the range of 20-60 mCi / m 2. Three patients received a second intravenous infusion, but the other 17 patients received only one dose during the course of treatment.

Graphically, there is a consistent relationship between plasma BIWA 4 and serum radioactivity for each patient and occurred twice in three patients receiving ¹⁸⁶ Re-BIWA 4. This graphically observed consistency contrasts with longer half-life detection of radioactivity when modeling data (noncompartmental assessment). The geometric mean half-life of 122 hours for the radioactive portion of ¹⁸⁶ Re-BIWA 4 was longer than the 94-hour ELISA-determined plasma half-life for BIWA 4.

Gathering pharmacokinetic parameters as the amount of radioactivity administered (Tables 5.4.2.2.:6 and, Table 5.4.2.2:7), increasing doses of radioactivity administered tended to result in longer exposures, but in each dose group The number of individuals in me was too small to come up with a consistent conclusion.

Geometric mean for plasma BIWA 4 pharmacokinetic parameters collected as presented radioactivity ¹⁸⁶ Re capacity (mCi / m ² ) Number Cmax (ng / mL) Half-life (hours) AUC _inf (% ID · h / kg) Vz (kg) CL (kg / h) MRT (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 9.6 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

* Includes patients who received two 50 mCi / m2 doses.

Geometric Means for Serum ¹⁸⁶ Re-BIWA 4 Pharmacokinetic Parameters Collected with Given Radioactivity ¹⁸⁶ Re capacity (mCi / m ² ) Number Cmax (ng / mL) Half-life (hours) AUC _inf (㎍h / mL) Vz (L) CL (mL / h) MRT (hours) 20 2 30.88 114.4 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 3586 4.73 0.026 172.4 50 9 * 40.22 123.3 4182 4.25 0.024 162.5 60 5 32.10 145.2 3493 6.00 0.029 185.5

* Includes patients who received two 50 mCi / m2 doses.

5.4.2.2 Pharmacokinetic / Pharmacodynamic Analysis

No pharmacokinetic / pharmacodynamic analyzes were performed.

5.4.3 Statistical / analytic issues

The analysis was performed as originally planned. Details are given in Section 3.7.1.3.

5.4.3.1 Primary Endpoint (s)

Not applicable

5.4.3.2 Secondary Endpoint (s)

Not applicable

5.4.4. Drug-drug and drug-disease interactions

No tests on drug-drug or drug-disease interactions were performed.

5.4.5 Sub-Object Display

See sections 6.4.2.2 and 6.4.2.3 for details.

5.5 Efficacy / Clinical Pharmacological Conclusions

Part A:

The results from Part A identified a 50 mg dose of BIWA 4 as the optimal dose for treatment based on blood concentration and tissue uptake. The distribution as assessed by radioscopy and biopsy measurements was highest in tumors in almost all cases compared to other tissues. Radioactivity uptake increased in tumors over time. CD44v6 expression was seen at least 80% and at least 90% in primary tumor and lymph node metastatic cells, respectively, and all mucosal samples were obtained.

The amount of soluble CD44v6 appeared to be constant before and after administration of ^99m Tc-BIWA 4.

The concentration of BIWA 4 measured was dose-proportional in BIWA 4 ranging from 25 mg to 100 mg. The appropriate amount of dose administered was excreted through the kidneys. The% ID released in urine was similar for all dose groups.

Part B:

Data from Part B indicates that the patient may be clinically advantageous from the ¹⁸⁶ Re-BIWA 4 treatment in MTD. One of five patients treated with 60 mCi / m 2 showed stable disease. Three of six patients experienced stable disease at a maximum tolerated dose of 50 mCi / m 2. The time to disease progression took 127 to 173 days in patients administered a second dose of 50 mCi / m 2. Radioimmunoascent scintigraphy directs the absorption of radioactivity in tumor tissues. Biodistribution was equivalent in other tissues regardless of dose. Dosage analysis did not show unexpectedly high uptake doses in tissues other than tumors except the testes. However, the relevance of the observed testicular dose to fertility impairment is currently unclear.

The amount of soluble CD44v6 detected tends to increase for ¹⁸⁶ Re-BIWA 4 treated patients.

Plasma concentrations of BIWA 4 peaked at 0.92 hours and antibodies were removed with a 94 hour geometric mean half-life for BIWA 4 determined by ELISA. C _max and AUC values (ELISA measurements) were similar to those obtained in Part A of this test for the 50 mg BIWA 4 dose group.

Dose limiting toxicity occurred at a dose of 60 mCi / m 2, while a dose of 50 mCi / m 2 ¹⁸⁶ Re-BIWA 4 was found to be the maximum tolerated dose in this test. The dose limiting toxicity was side effects from bone marrow, namely thrombocytopenia and leukopenia.

6.1 Exposure Degree

All 10 patients treated in Part A of this study received a single dose of ^99m Tc-BIWA 4. The dose of BIWA 4 was 25 mg, 50 mg or 100 mg, whereas the ^99m Tc was 20 mCi.

In Part B, 20 patients received a single dose of ¹⁸⁶ Re-BIWA 4. Three patients received a second dose of 50 mCi / m 2 due to stable disease (no change). The dose of BIWA 4 remained stable at 50 mg, while the radioactivity of ¹⁸⁶ Re increased by 10 mCi / m 2 per body surface area.

Dose was calculated according to body surface area (Table 6.1: 1).

Average dose administered Part A Part B ^99m Tc-BIWA 4 ¹⁸⁶ Re-BIWA 4 [mCi / m ² ] 25 mg BIWA 4 50 mg BIWA 4 100 mg BIWA4 20 30 40 50 60 Number 3 4 3 2 4 3 6 5 mCi ^99m Tc 20.1 ± 1.2 19.2 ± 0.3 19.1 ± 0.8 n.a. n.a. n.a. n.a. n.a. mCi ¹⁸⁶ Re n.a. n.a. n.a 33.1 ± 1.7 52.4 ± 5.6 70.6 ± 5.2 87.2 ± 8.5 97.2 ± 8.3

n.a. = Not applicable, mean values and standard deviations are given.

The radiochemical purity of the drug is greater than 95% and the immunoreactive fraction is greater than 80%.

Observations were made for at least 6 weeks after administration of this test drug.

6.2 Side Effects (AEs)

Adverse events were recorded in all treated patients. Two patients were discontinued due to AE during the observation period of Part B. After treatment was complete, no action was taken with the test drug.

Part A:

The majority of patients in Part A experienced side effects as a whole (50%), which may be from advanced surgery and consequent pain. No special pattern of adverse events was recorded, and no patients who showed CTC criteria 3 or 4 were judged to be drug-related (Tables 6.2.2: 1 and 2).

Three patients experienced severe side effects as described in section 6.3.1.

Part B:

Side effects of the SOC 'body as a whole' were noted in 65% of patients, and side effects of the systemic organ classification 'platelet, bleeding and coagulation disorders' were noted in 60% of patients. Side effects of 'leukocyte and reticuloendothelial disorder' have been reported in 50% of patients. Drug-related thrombocytopenia and leukopenia were seen in 11 (55%) and 10 (50%) patients on average 21 days after initiation of radioimmunotherapy and were generally reversible after 6 weeks. Dose-response to these side effects was observed (Table 6.2.2: 3).

Hematological dose-limiting toxicity, defined as drug-related CTC grade 4, has been reported in three patients.

Non-hematologic dose-limiting toxicity, defined as drug-related CTC grade 3 or 4 non-hematologic toxicity, was seen in 4 patients (at 30 mCi / m 2, one patient experienced rash and quinque edema, 60 mCi 2 patients experienced fever and 1 patient treated with 60 mCi / m 2).

Six of the nine patients who experienced serious adverse events died. Mortality rates and serious adverse events are described in section 12.3.1.

6.2.2 Side Effects Display

Part A:

The following side effects, categorized by three dose groups: SOC, preferred due date, and BIWA 4, were reported to one or more patients in Part A of this study (Table 6.2.2: 1). Preferred terms as well as more detailed descriptions are given in section 7.3.1.

Patients with adverse events in Part A (preoperative treatment with a single dose of 20 mCi ^99m Tc-BIWA 4) ^99m Tc-BIWA4 25 mg BIWA4 50 mg BIWA4 100 mg BIWA4 gun Number of patients (% of patients treated) 3 (100) 4 (100) 3 (100) 10 (100) The number of patients with any side effects 3 (100) 4 (100) 3 (100) 10 (100) Adaptive position disorder 2 (66.7) 0 (0) 0 (0) 2 (20.0) As a whole 2 (66.7) 2 (50.0) 1 (33.3) 5 (50.0) ache 1 (33.3) 1 (25.0) 1 (33.3) 3 (30.0) Metabolic and nutritional disorders 1 (33.3) 2 (50.0) 1 (33.3) 4 (40.0) Weight loss 1 (33.3) 1 (25.0) 0 (0) 2 (20.0) Resistance system disorder 0 (0) 4 (100) 0 (0) 4 (40.0) infection 0 (0) 3 (75.0) 0 (0) 3 (30.0) Vascular disorders 1 (33.3) 1 (25.0) 1 (33.3) 3 (30.0) Bleeding (not specific) 1 (33.3) 1 (25.0) 0 (0) 2 (20.0)

Preferred deadlines and SOCs have been suggested when reported in more than one patient; The numbers in parentheses refer to the proportion of patients treated.

The side effects that are graded according to the CTC criteria and considered drug-related by the researcher are listed in Table 6.2.2: 2. The side effect is grade 1 according to the CTC.

Severity of drug-related (by investigator judgment) side effects according to CTC criteria reported in Part A (preoperative treatment with a single dose of 20 mCi ^99m Tc-BIWA 4) ^99m Tc-BIWA4 25 mg BIWA4 50 mg BIWA4 100 mg BIWA4 gun Number of patients 3 4 3 10 The number of side effects that are considered drug-related 0 2 0 2 Hepatobiliary disorders 0 2 0 2 SGOT increasing 0 One 0 One SGPT increasing 0 One 0 One

sGOT = serum glutamic oxalacetic acid amine group transferase; sGPT = serum glutamic pyruvate amine group transferase.

Part B:

Table 6.2.2: 3 shows the number of patients exhibiting side effects categorized by SOC, preferred due date, and radiation dose group of ¹⁸⁶ Re-BIWA 4.

¹⁸⁶ Re-BIWA4 (first and second doses) considered ¹⁸⁶ Re-BIWA4 [mCi / m ² ] 20 30 40 50 60 gun Number of patients 2 (100) 4 (100) 3 (100) 6 (100) 5 (100) 20 (100) Number of patients with adverse events 2 (100) 4 (100) 3 (100) 6 (100) 5 (100) 20 (100) As a whole 2 (100) 3 (75.0) 2 (66.7) 2 (33.3) 4 (80.0) 13 (65.0) Allergic reaction 0 (0) 1 (25.0) 0 (0) 0 (0) 1 (20.0) 2 (10.0) fatigue 0 (0) 0 (0) 0 (0) 1 (16.7) 0 (0) 2 (10.0) Fever 0 (0) 0 (0) 2 (66.7) 0 (0) 2 (40.0) 4 (20.0) Oral edema 1 (50.0) 0 (0) 0 (0) 1 (16.7) 0 (0) 2 (10.0) ache 0 (0) 2 (50.0) 0 (0) 1 (16.7) 1 (20.0) 4 (20.0) Central and Peripheral Nervous System Disorders 0 (0) 1 (25.0) 2 (66.7) 1 (16.7) 0 (0) 4 (20.0) Gastrointestinal disorders 0 (0) 1 (25.0) 1 (33.3) 2 (33.3) 3 (60.0) 7 (35.0) Mucositis 0 (0) 0 (0) 0 (0) 1 (16.7) 3 (60.0) 4 (20.0) Stomatitis 0 (0) 0 (0) 0 (0) 1 (16.7) 1 (20.0) 2 (10.0) Hepatobiliary disorders 1 (50.0) 0 (0) 0 (0) 0 (0) 1 (20.0) 2 (10.0) Increased sGPT 1 (50.0) 0 (0) 0 (0) 0 (0) 1 (20.0) 2 (10.0) Metabolic and nutritional disorders 1 (50.0) 0 (0) 1 (33.3) 1 (16.7) 1 (20.0) 4 (20.0) tumor 0 (0) 0 (0) 2 (66.7) 0 0 (0) 2 (40.0) 4 (20.0) Intensified malignancy 0 (0) 0 (0) 1 (33.3) 0 (0) 2 (40.0) 3 (15.0) Platelet, bleeding and coagulation disorders 0 (0.0) 1 (25.0) 1 (33.3) 5 (83.3) 5 (100) 12 (60.0) Thrombocytopenia 0 (0.0) 1 (25.0) 1 (33.3) 4 (66.7) 5 (100) 11 (55.0) Erythrocyte disorder 0 (0) 0 (0) 0 (0) 2 (33.3) 3 (60.0) 5 (25.0) anemia 0 (0) 0 (0) 0 (0) 2 (33.3) 3 (60.0) 5 (25.0) Respiratory system disorders 0 (0) 1 (25.0) 1 (33.3) 0 (0) 2 (40.0) 4 (20.0) bronchitis 0 (0) 1 (25.0) 0 (0) 0 (0) 1 (20.0) 2 (10.0) Dyspnea 0 (0) 0 (0) 0 (0) 0 (0) 2 (40.0) 2 (10.0) Rhinitis 0 (0) 0 (0) 1 (33.3) 0 (0) 1 (20.0) 2 (10.0) Skin and its adnexal disorders 1 (50.0) 1 (25.0) 1 (33.3) 0 (0) 1 (20.0) 4 (20.0) rash 0 (0) 1 (25.0) 0 (0) 0 (0) 1 (20.0) 2 (10.0) Leukocytes and RES Disorders 0 (0) 0 (0) 1 (33.3) 4 (66.7) 5 (100) 10 (50.0) Granulocytopenia 0 (0) 0 (0) 0 (0) 1 (16.7) 2 (40.0) 3 (15.0) Leukopenia 0 (0) 0 (0) 1 (33.3) 4 (66.7) 5 (100) 10 (50.0)

Preferred due dates and SOCs are indicated when reported in one or more patients; sGPT = serum glutamic pyruvate amine group transferase; RES = reticuloendothelial system; The numbers in parentheses refer to the proportion of patients treated.

Drug-related side effects have been reported in 75% of patients. Drug-related leukopenia was reported in 50% of patients and thrombocytopenia was reported in 55% of patients, respectively.

Adverse events considered drug-related are graded according to CTC and given in Table 6.2.2: 4.

CTC criteria reported in Part B (first and second doses considered) Side Effect ¹⁸⁶ Re-BIWA4 [mCi / m ² ] 20 30 40 50 60 gun CTC Grade 1 One 0 2 6 5 14 Facial edema One n.a. 0 0 0 One Leukopenia 0 n.a. One 2 0 3 Moniliosis 0 n.a. 0 0 One One Mucositis 0 n.a. 0 0 One One Platelet abnormalities 0 n.a. 0 One 0 One Purpura 0 n.a. 0 0 One One Stomatitis 0 n.a. 0 One 0 One Thrombocytopenia 0 n.a. One 2 2 5 CTC Grade 2 0 One 0 7 7 15 anemia n.a. 0 n.a. One One 2 ventilation n.a. 0 n.a. One 0 One Leukopenia n.a. 0 n.a. One 2 3 Mucositis n.a. 0 n.a. 2 3 5 Stomatitis n.a. 0 n.a. 0 One One Loss of taste n.a. 0 n.a. One 0 One Thrombocytopenia n.a. One n.a. One 0 2 CTC Grade 3 0 2 2 8 12 Allergic reaction n.a. One n.a. 0 0 One fatigue n.a. 0 n.a. 0 One One Fever n.a. 0 n.a. 0 2 2 Leukopenia n.a. 0 n.a. One One 2 rash n.a. One n.a. 0 0 One Thrombocytopenia n.a. 0 n.a. One 4 5 CTC Grade 4 0 0 0 3 5 8 anemia n.a. n.a. n.a. 0 One One Granulocytopenia n.a. n.a. n.a. One 2 3 Leukopenia n.a. n.a. n.a. One 2 3 Thrombocytopenia n.a. n.a. n.a. One 0 One

The number of side effects is shown; n.a. = Not applicable.

6.2.3 Analysis of Side Effects

Part A:

Except for one patient who experienced mild and reversible CTC Grade 1 elevations of AST and ALT, no adverse events developed in Part A of this study were considered drug-related.

Compared to the different BIWA 4 doses, no difference in AE profile was observed.

Part B:

Thrombocytopenia and leukopenia were dose-dependent (Tables 6.2.2: 3 and 6.2.2: 4) and dose-limited. The time course is given in section 6.4.2.1.

Causes dose-limiting toxicity (Tables 6.2.2: 3 and 6.2.2: 4).

Allergic reactions were reported in two patients, one of whom experienced drug-related quinque edema and rash (30 mCi / m2), and the other one experienced an allergic reaction after platelet concentrate perfusion (60 mCi / M 2), which was not considered drug-related. One patient (20 mCi / m 2) showed urticaria that was not considered drug-related and one patient (20 mCi / m 2) experienced drug-related facial edema.

HAHAs were detected in two patients (see section 12.4.3).

Test discontinuation due to side effects occurred in two patients. Both patients died.

Because of the side effects, simultaneous treatment was initiated in 17 patients. These patients are briefly described in section 6.3.1.

6.3 Death, Other Serious Side Effects, and Other Significant Side Effects

Serious adverse events were reported in 12 patients. Six of these patients died during or after the course of this trial. All of the patients who died were treated in Part B of this trial. None of the patients treated in Part A died. All disasters were due to disease progression.

Two patients in Part B were prematurely discontinued due to side effects (both 112 and 105 died).

Most of the side effects that required treatment were thrombocytopenia, leukopenia and fever.

Additions to this text

7.1 Demographic Data

Details of demographics are not included.

7.2 Efficacy / Pharmacological Data

Table 7.2: 1 Tumor to Bone Marrow Absorption Ratio-Part A of this Test

Table 7.2: 2 Tumor to Bone Marrow Absorption Ratio (ITT Subset) —Part A of this Test

Table 7.2: 3 Tumor to Bone Marrow Absorption Ratio (PP Subset) —Part A of This Test

Table 7.2: 4 Tumor Size and Antibody Uptake-Part A of this Test (Depending on Protocol Population)

Table 7.2: 5 CD44v6 Antigen Expression in Tumors and Other Tissues—Part A of This Test

TABLE 7.2: 6 Soluble CD44v6 in Serum. Mean values are given in ng / ml.

Tumor to Bone Marrow Absorption Ratio-Part A of this Test Part A ^99m Tc-BIWA4 Patient No. Tumor uptake rate (% ID / kg; calculated) Bone marrow uptake (% ID / kg; calculated) Tumor / Bone Marrow Ratio (Calculated) 25 mg BIWA4 One* 6.14 5.58 1.10 2* 15.78 7.49 2.11 3 * 16.78 8.63 1.95 50 mg BIWA4 4* 28.10 6.98 4.03 8* 27.76 7.57 3.67 10 * 22.63 11.54 1.96 100 mg BIWA4 6 * 15.89 6.47 2.46 7 16.99 7.70 2.21 9 13.31 1011 1.32

No = number; * = Included according to protocol analysis; % ID =% injection dose; Estimates were taken into account for different sample weights.

Tumor to Bone Marrow Absorption Ratio (ITT Subset) —Part A of this Test Part A ^99m Tc-BIWA4 Tumor / Bone Marrow Ratio (Calculated) 25 mg BIWA4 1.72 50 mg BIWA4 2.57 100 mg BIWA4 1.99

Mean values are presented; ITT = intensive care; Estimates were taken into account for different sample weights.

Tumor to Bone Marrow Absorption Ratio (PP Subset)-Part A of this Test Part A ^99m Tc-BIWA4 Tumor / Bone Marrow Ratio (Calculated) 25 mg BIWA4 1.72 50 mg BIWA4 3.22 100 mg BIWA4 2.46

Mean values are presented; PP = according to the protocol; Estimates were taken into account for different sample weights.

Tumor size and% antibody uptake-Part A of this test (depending on protocol population) Part A ^99m Tc-BIWA4 Patient No. Tumor Size [mm ² ] Tumor uptake rate (% ID / kg; calculated) 25 mg BIWA4 One 1325 6.14 2 900 15.78 3 225 16.78 50 mg BIWA4 4 n.a. 28.10 8 1504 27.76 10 1400 22.63 100 mg BIWA4 6 800 15.89

No = number of patients; n.a. = Not applicable, only length or width is given, not length and width; Estimates were taken into account for different sample weights.

CD44v6 Antigen Expression in Tumors and Other Tissues-Part A of this Test Part A ^99m -Tc BIWA4 Patient No. CD44v6 Antigen Expression in Tumors CD44v6 Expression in the Mucosa CD44v6 Expression in Lymph Node Metastases 25 mg BIWA4 One* n.a. n.a. n.a. 2* ++ / +++; > 80% +++ ++ / +++; > 90% 3 * +++; > 90% +++ +++; > 90% 50 mg BIWA4 4* +++; > 90% +++ +++; > 90% 8* n.a. n.a. n.a. 10 * ++; > 90% +++ +++; > 90% 100 mg BIWA4 6 * +++; > 80% +++ n.a. 7 +++; > 90% +++ n.a. 9 +++; > 90% +++ n.a.

No = number; * = Included according to protocol analysis; n.a. = Not applicable

Soluble CD44v6 in serum (mean values are given in ng / ml) Part A Part B ^99m Tc-BIWA4 ¹⁸⁶ Re-BIWA4 [mCi / m ² ] 25 mgBIWA4 50 mg BIWA4 100 mg BIWA4 20 30 40 50 60 Number 3 4 3 2 4 3 6 5 Before input 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 hours 188 282 182 444 284 516 233 195 240/336 hours n.a. n.a. n.a. 445 337 n.a. n.a. n.a. 6 Weeks 203 310 210 289 220 487 226 162

n.a. = Not applicable, data included second dose.

Efficacy Result:

Part A:

The results from Part A identified a 50 mg dose of BIWA 4 as the optimal dose for treatment based on blood concentration and tissue uptake. The distribution as assessed by radioscopy and biopsy measurements was highest in tumors in almost all cases compared to other tissues. Over time, radioactivity uptake increased in tumors. CD44v6 expression was at least 80% and at least 90%, respectively, in primary tumor and lymph node metastatic cells, and all mucosal samples were obtained.

No correlation between tumor size and radioactivity uptake was observed in the 50 mg BIWA 4 dose group. The amount of soluble CD44v6 is believed to be constant before and after administration of ^99m Tc-BIWA 4.

The measured concentration of BIWA 4 is dose-proportional in BIWA 4 ranging from 25 mg to 100 mg. Appropriate amount of dose administered was excreted through the kidneys. The% ID released in urine was similar for all dose groups.

Part B:

Data from Part B indicates that the patient may be clinically advantageous from ¹⁸⁶ Re-BIWA 4 treatments under the maximum tolerated dose (MTD). One of five patients treated with 60 mCi / m 2 showed stable disease. Three of six patients experienced stable disease at a maximum tolerated dose of 50 mCi / m 2. The time to disease progression took 127 to 173 days in patients administered a second dose of 50 mCi / m 2. Radioimmunoascent scintigraphy directs the absorption of radioactivity in tumor tissues. Biodistribution was equivalent in other tissues regardless of dose. Dosage analysis did not show unexpectedly high uptake doses in tissues other than tumors except the testes. The amount of soluble CD44v6 detected tends to increase for ¹⁸⁶ Re-BIWA 4 treated patients.

Plasma concentrations of BIWA 4 peaked at 0.92 hours and antibodies were removed with a 94 hour geometric mean half-life for BIWA 4 determined by enzyme-linked immunosorbent assay (ELISA) assay. C _max and AUC values (ELISA) were similar to those obtained in Part A of this test for the 50 mg BIWA 4 dose group.

The tolerance of the single dose BIWA 4 coupled with the low radiation dose of technetium-99 was acceptable. Two of the three serious side effects were due to surgical complications.

Part B:

Maximum tolerated capacity (MTD) is 50 mCi / m 2 ¹⁸⁶ Re-BIWA 4.

Dose-limiting side effects involved fever in individual patients and were dose dependent and reversible decreases in platelet and leukocyte levels. The clinical sign of thrombocytopenia is mild point bleeding.

Mucositis was observed in patients treated with higher radiation doses, but this was not dose-limiting.

During the course of this test, no relevant changes in thyroid stimulating hormone (TSH) levels were observed.

Twelve patients experienced serious side effects. Of these, six patients died mainly due to disease progression during Part B of this trial.

Allergic reactions were rarely observed, with one person having severe drug-related quinque edema. No allergic reactions occurred during or immediately after this drug infusion.

Two patients showed HAHA. Repeated administration did not induce HAHA expression.

conclusion:

The results indicate clinical benefit and uptake of ¹⁸⁶ Re-BIWA 4 in tumor tissues in patients with head and neck squamous cell carcinoma. The safety profile is considered acceptable. BIWA 4 showed dose-proportional pharmacokinetics and tumor uptake did not change with respect to 50 mg to 100 mg BIWA 4 doses.

Example 4

Introduction

Patients with advanced head and neck squamous cell carcinoma (HNSCC) are at high risk of developing a mobile local recurring tumor and / or distant metastasis. For these patients, development of effective adjuvant systemic therapies is desired. Since HNSCC is inherently radiation sensitive, the selective targeting of radionuclides to HNSCC by using monoclonal antibodies as a form of radioimmunotherapy may contribute to more effective therapies.

purpose

To determine the safety, maximum tolerated dose (MTD), immunogenicity and efficacy of radioimmunotherapy with rhenium- ¹⁸⁶ ( ¹⁸⁶ Re) -labeled human adapted monoclonal antibody BIWA 4 in HNSCC patients.

Patient and method

Currently, phase I dose escalation studies have been performed in HNSCC patients without treatment. A total of 20 patients received ¹⁸⁶ Re-labeled BIWA 4 intravenously at 20, 30, 40, 50 or 60 mCi / m 2 doses. At least three months after the 50 or 60 mCi / m 2 dose was administered, three patients received a second dose of 50 mCi / m 2.

result

All single doses as well as repeated doses were widely tolerated and no acute side effects were observed. The only real signs of toxicity seen at higher doses are oral mucositis and dose-limited myeloid toxicity consisting of thrombocytopenia and leukopenia. MTD was set at 50 mCi / m 2. One patient showed a human-anti-human response after a single dose. In five patients treated at the highest dose level, stable disease was observed to last for 4 to 19 weeks.

conclusion

Radioimmunotherapy with ¹⁸⁶ Re-labeled BIWA 4 is considered safe for HNSCC patients and can reach a carcinoma dose. Moreover, it appears that repeated administrations at low rates of immunogenicity are possible. The results of the Phase I study further promote the further development of radioimmunotherapy with rhenium- ¹⁸⁶ ( ¹⁸⁶ Re) -labeled human adapted monoclonal antibody BIWA 4 for the development of adjuvant therapy for head and neck cancer patients. I let you.

Claims (80)

An antibody molecule comprising a variable region of a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 1, or a fragment thereof, an allelic variant, a functional variant, a glycosylation variant, a fusion molecule or a chemical derivative. An antibody molecule consisting of amino acids wherein the variable region of the heavy chain is characterized by the amino acid sequence of SEQ ID NO: 1. An antibody molecule comprising a variable region of a light chain as characterized by an amino acid sequence as defined in SEQ ID NO: 2, or a fragment thereof, an allelic variant, a functional variant, a glycosylation variant, a fusion molecule or a chemical derivative. An antibody molecule consisting of amino acids wherein the variable region of the light chain is characterized by the amino acid sequence of SEQ ID NO: 2. An antibody molecule comprising a variable region of a light chain as characterized by an amino acid sequence as defined in SEQ ID NO: 3, or a fragment thereof, an allelic variant, a functional variant, a glycosylation variant, a fusion molecule or a chemical derivative. An antibody molecule consisting of amino acids wherein the variable region of the light chain is characterized by the amino acid sequence of SEQ ID NO: 3. 4. The variable region of claim 1 or 3 comprising the variable region of the heavy chain as characterized by the amino acid sequence as defined in SEQ ID NO: 1 and characterized by the amino acid sequence as defined in SEQ ID NO: An antibody molecule comprising a variable region of a light chain as described above, or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or chemical derivative thereof. The variable region of claim 2 or 4, wherein the variable region of the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 1, and the variable region of the light chain is characterized by the amino acid sequence of SEQ ID NO: Antibody molecule consisting of the same amino acid. The variable region of claim 1 or 5 comprising a variable region of a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 1 and characterized by an amino acid sequence as defined in SEQ ID NO: An antibody molecule comprising a variable region of a light chain as described above, or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or chemical derivative thereof. 7. The variable region of claim 2 or 6, wherein the variable region of the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 1, and the variable region of the light chain is characterized by the amino acid sequence of SEQ ID NO: Antibody molecule consisting of the same amino acid. An antibody molecule comprising a variable region of a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, or a fragment thereof, an allelic variant, a functional variant, a variant based on a degenerate nucleic acid code, a fusion molecule or a chemical derivative . An antibody molecule in which the variable region of the heavy chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 4. An antibody molecule comprising a variable region of a light chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 5, or a fragment thereof, an allelic variant, a functional variant, a variant based on a degenerate nucleic acid code, a fusion molecule or a chemical derivative . An antibody molecule in which the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 5. An antibody molecule comprising a variable region of a light chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 6, or a fragment thereof, an allelic variant, a functional variant, a variant based on a degenerate nucleic acid code, a fusion molecule or a chemical derivative . An antibody molecule in which the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 6. The variable region of a light chain according to claim 11 or 13, comprising a variable region of a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, and encoded by a nucleic acid sequence as defined in SEQ ID NO: Antibody molecules comprising, or fragments, allelic variants, functional variants, variants based on degenerate nucleic acid codes, fusion molecules or chemical derivatives thereof. 15. The antibody of claim 12 or 14, wherein the variable region of the heavy chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 4 and the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 5. molecule. The variable region of a light chain according to claim 11 or 15, comprising a variable region of a heavy chain encoded by a nucleic acid sequence as defined in SEQ ID NO: 4, and encoded by a nucleic acid sequence as defined in SEQ ID NO: Antibody molecules comprising, or fragments, allelic variants, functional variants, variants based on degenerate nucleic acid codes, fusion molecules or chemical derivatives thereof. 17. The antibody of claim 12 or 16, wherein the variable region of the heavy chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 4 and the variable region of the light chain is encoded by a nucleic acid sequence as defined in SEQ ID NO: 6. molecule. The antibody molecule according to any one of claims 1 to 20, wherein each of the variable region of the light chain and the variable region of the heavy chain is linked separately from the human constant region. The antibody molecule of claim 21, wherein the human constant region of the light chain is a human kappa constant region. The antibody molecule of claim 21 or 22, wherein the human constant region of the heavy chain is a human IgG1 constant region. 24. A compound according to any one of the preceding claims comprising a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 7 and characterized by an amino acid sequence as defined in SEQ ID NO: An antibody molecule comprising a light chain, or a fragment thereof, an allelic variant, a functional variant, a glycosylation variant, a fusion molecule or a chemical derivative thereof. The amino acid according to any one of claims 1 to 24, wherein the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 7 and the light chain is characterized by the amino acid sequence of SEQ ID NO: 8. An antibody molecule consisting of. 26. A compound according to any one of claims 1 to 25 comprising a heavy chain as characterized by an amino acid sequence as defined in SEQ ID NO: 7 and characterized by an amino acid sequence as defined in SEQ ID NO: 9. An antibody molecule comprising a light chain, or a fragment thereof, an allelic variant, a functional variant, a glycosylation variant, a fusion molecule or a chemical derivative thereof. 27. The amino acid according to any one of claims 1 to 26, wherein the heavy chain consists of amino acids as characterized by the amino acid sequence of SEQ ID NO: 7 and the light chain is characterized by the amino acid sequence of SEQ ID NO: 9. An antibody molecule consisting of. 28. The method according to any one of claims 1 to 27, comprising a heavy chain as encoded by a nucleic acid sequence as defined in SEQ ID NO: 10 and characterized by a nucleic acid sequence as defined in SEQ ID NO: 11. An antibody molecule comprising a light chain as described above, or a fragment thereof, an allelic variant, a functional variant, a variant based on a degenerate nucleic acid code, a fusion molecule or a chemical derivative. The antibody molecule of any one of claims 1-28, wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID NO: 10 and the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 11. 29. 30. The composition of any one of claims 1 to 29, comprising a heavy chain as encoded by a nucleic acid sequence as defined in SEQ ID NO: 10 and characterized by a nucleic acid sequence as defined in SEQ ID NO: 12. An antibody molecule comprising a light chain as described above, or a fragment thereof, an allelic variant, a functional variant, a variant based on a degenerate nucleic acid code, a fusion molecule or a chemical derivative. 31. The antibody molecule of any one of the preceding claims, wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID NO: 10 and the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 12. 32. A compound according to any one of claims 1 to 31 comprising a heavy chain as encoded by a nucleic acid sequence as defined in SEQ ID NO: 13 and characterized by a nucleic acid sequence as defined in SEQ ID NO: 14. An antibody molecule comprising a light chain as described above, or a fragment thereof, an allelic variant, a functional variant, a variant based on a degenerate nucleic acid code, a fusion molecule or a chemical derivative. 33. The antibody molecule of any one of the preceding claims, wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID NO: 13 and the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 14. 34. The composition of any one of claims 1 to 33, comprising a heavy chain as encoded by a nucleic acid sequence as defined in SEQ ID NO: 13 and characterized by a nucleic acid sequence as defined in SEQ ID NO: 15. An antibody molecule comprising a light chain as described above, or a fragment thereof, an allelic variant, a functional variant, a variant based on a degenerate nucleic acid code, a fusion molecule or a chemical derivative. 35. The antibody molecule of any one of the preceding claims, wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID NO: 13 and the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 15. 36. The antibody molecule of any one of claims 1 to 35, comprising a heavy chain and a light chain as encoded by a nucleic acid sequence as defined in SEQ ID NO: 16, or fragments, allelic variants, functionalities thereof. Variants, variants, fusion molecules or chemical derivatives based on degenerate nucleic acid codes. The antibody molecule of any one of claims 1-36, wherein the heavy and light chains are encoded by the nucleic acid sequence of SEQ ID NO: 16. 38. The antibody protein of any one of claims 1-37, wherein the antibody protein is conjugated with a therapeutic agent. The antibody protein according to claim 38, wherein the therapeutic agent is a therapeutic agent selected from the group consisting of radioisotopes, toxins, toxoids, inflammatory agents and chemotherapeutic agents. The antibody protein of claim 39, wherein the therapeutic agent is linked to the antibody protein via a linker selected from the group of MAG-3 GABA, MAG-2 GABA, and N2S2. The antibody protein of claim 40, wherein the therapeutic agent is linked to the antibody protein via MAG-2 GABA. The antibody protein of any one of claims 38-41, wherein the radioisotope is a β-emitting radioisotope. 43. The antibody protein of claim 42, wherein the radioisotope is selected from the group consisting of ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ¹³¹ iodine and ⁹⁰ yttrium. The antibody protein of claim 43, wherein the radioisotope is ¹⁸⁶ rhenium. 45. The method of any one of claims 39 to 44, wherein from about 0.5 to about 15 mCi / mg, or from about 0.5 to about 14 mCi / mg, preferably from about 1 to about 10 mCi / mg, preferably about 1 An antibody protein having a specific activity of from about 5 mCi / mg, and most preferably 2 to 6 mCi / mg or 1 to 3 mCi / mg. 38. The antibody protein according to any one of claims 1 to 37, characterized by labeling. 47. The antibody protein of claim 46, wherein the 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, digoxigenin and biotin. 38. The antibody protein according to any one of claims 1 to 37 conjugated with an imageable agent. The antibody protein of claim 49, wherein the imageable agent is a radioisotope. 51. The antibody protein of claim 50, wherein the radioisotope is a γ-emitting radioisotope. 52. The antibody protein of claim 51, wherein the radioisotope is ¹²⁵ I. A pharmaceutical composition comprising an antibody protein according to any one of claims 1 to 45 and a pharmaceutically acceptable carrier or excipient. The antibody protein of claim 53, wherein the antibody protein is conjugated with a radioisotope according to any one of claims 39-44, and from about 0.5 to about 15 mCi / mg, or from about 0.5 to about 14 mCi / mg, preferably The pharmaceutical composition has a specific activity of 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. The pharmaceutical composition of claim 53 or 54, wherein the amount of radiolabeled antibody in the pharmaceutical composition to be administered to the patient is 10, 20, 30, 40, 50, or 60 mCi / m 2. The pharmaceutical composition of any one of claims 53-55, wherein the amount of radiolabeled antibody in the pharmaceutical composition to be administered to the patient is 50 mCi / m 2. 57. The compound according to any one of claims 53 to 56, wherein ascorbic acid, gentis acid, redux acid, erythorbic acid, p-aminobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2,5-di A pharmaceutical composition further comprising at least one radioprotective agent selected from the group of hydroxy-1,4-benzenedisulfonic acid, povidone, inositol, and / or citrate. 58. The pharmaceutical composition of any one of claims 53-57, wherein the radioprotectant is ascorbic acid. 59. The method of any one of claims 53-58, further comprising a radioprotectant ascorbic acid, wherein the antibody protein is linked to ¹⁸⁶ rhenium through MAG-2 GABA. A pharmaceutical composition comprising an antibody molecule selected from the group of antibody molecules of claims 20, 24, 25, 28, 29, 29, 32, 33, 36 or 37. Use of an antibody protein according to any one of claims 1 to 45 in the manufacture of a medicament for the treatment of cancer. 61. The use of an antibody protein according to claim 60, wherein the 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 cancer of the brain. 62. The antibody of claim 60 or 61, wherein the antibody protein is conjugated with a radioisotope according to any one of claims 39 to 44, and from about 0.5 to about 15 mCi / mg, or from about 0.5 to about 14 mCi /. an antibody with a specific activity of 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 Use of Proteins. The use of an antibody protein according to claim 62, wherein the amount of radiolabeled antibody in the pharmaceutical composition to be administered to the patient is 10, 20, 30, 40, 50 or 60 mCi / m 2. The use of an antibody protein according to claim 62, wherein the amount of radiolabeled antibody in the pharmaceutical composition to be administered to the patient is 50 mCi / m 2. 46. An antibody protein according to any one of claims 1 to 45 is administered once or several times to an individual in need thereof, wherein said antibody protein selectively binds CD44v6 to a tumor cell via a therapeutic agent linked to said antibody protein. Destroying, and therapeutic success is monitored. 66. The method of claim 65, wherein the tumor is a cancer selected from the group of colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic cancer of the brain. A nucleic acid encoding the antibody protein according to any one of claims 1 to 37. A recombinant DNA vector containing a nucleic acid according to claim 67. 69. The recombinant DNA vector of claim 68, wherein the recombinant DNA vector is an expression vector. 70. The recombinant DNA vector of claim 68 or 69, wherein the vector is pAD-CMV or a functional derivative thereof. The recombinant DNA vector according to any one of claims 68 to 70, which is a vector N5KG1Val or a derivative thereof. A host comprising the vector according to any one of claims 68 to 71. 73. The host of claim 72, which is a eukaryotic host cell. 74. The host of claim 72 or 73, which is a mammalian cell. 75. The host of any one of claims 72 to 74, which is a BHK, CHO or COS cell. 73. The host of claim 72, wherein the host is a bacteriophage. 73. The host of claim 72, which is a prokaryotic host cell. Culturing the host under conditions which express the antibody protein according to any one of claims 1 to 37 by a host cell according to any one of claims 72 to 77; The method for producing an antibody protein according to any one of claims 1 to 37, characterized in that it comprises the step of separating the antibody protein. 79. The method of claim 78, wherein the host is a mammalian cell, preferably a CHO or COS cell. 79. The method of claim 78 or 79, wherein the host cell is co-transfected with two plasmids carrying expression units for the light or heavy chain.