RU2487889C2 - Anti-5t4 antibodies and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed are anti-5T4 antibodies, nucleic acids which encode variable regions of such antibodies, antibody/drug conjugates, a method of delivering a drug using such a conjugate, as well as a method of treating a subject with cancer which is characterised by 5T4 antibody expression, by administering the disclosed conjugate.

EFFECT: present invention can further be used in therapy of 5T4-associated diseases.

42 cl, 8 ex, 15 tbl, 15 dwg

Description

RELATED APPLICATIONS

This application claims priority according to provisional application for US patent No. 60/891,248, filed February 23, 2007 and provisional application for US patent No. 60 / 781,346, filed March 10, 2006, each of which is fully incorporated into this description by reference.

FIELD OF THE INVENTION

The present invention relates mainly to antibodies against 5T4 and conjugates of antibodies with a drug (i.e. immunoconjugates) for the diagnosis and / or treatment of neoplasms and malignant tumors. The present invention also relates to isolated nucleic acids encoding the variable region of an antibody and the polypeptides of said region for preparing anti-5T4 antibodies and drug conjugates.

BACKGROUND OF THE INVENTION

The availability of high-affinity monoclonal antibodies has made possible the development of targeted immunotherapy. In accordance with this approach, the therapeutic agent is combined with an antibody characterized by specificity of binding to a specific population of target cells. Therapeutic agents that conjugate with monoclonal antibodies include cytotoxins, biological modulators, enzymes (e.g. ribonuclease), apoptotic proteins and peptides, and radioisotopes. Conjugates of an antibody with cytotoxin are commonly called immunocytoxins. Antibodies associated with low molecular weight drugs, such as methotrexate, are commonly referred to as drug chemoantibody conjugates. Conjugates presented as immunomodulators contain biological response modifiers such as lymphokines (humoral intercellular peptides), growth factors, and complement-activating snake venom factor (CVF). Antibodies labeled with radioactive isotopes include radioactive isotopes that can be used for both radiotherapy and contrast purposes.

The delivery of a drug to tumor cells using antibodies increases the effectiveness of the drug by reducing its penetration into healthy tissues. See, for example: Reffet al. (2002) Cancer Control 9: 152-66; Sievers (2000) Cancer Chemother. Pharmacol 46 Suppl: S18-22; Goldenberg (2001) Cut. Rev. Oncol. Hematol. 39: 195-201. MY-LOTARG® (gemtuzumab ozogamicin) is a commercially available targeted immunotherapy that operates according to the principle described above and which is approved for use in the treatment of acute myeloid leukemia in elderly patients. See Sievers et al. (1999) Blood 93: 3678-84. In this case, the target molecule is a monoclonal antibody against CD33, which is associated with calicheamicin.

However, the use of targeted immunotherapy in humans has been limited, in part due to the side effects associated with monoclonal antibodies obtained from other animals. Early clinical attempts to use antibodies obtained from rodents revealed the response by the human body of antibodies to antibodies obtained from mice (MAMA) and antibodies obtained from rats (HARA), which caused rapid clearance of antibodies. Subsequently, less immunogenic antibodies were developed which include hybrid antibodies, humanized antibodies, PRIMATIZED® antibodies, and human antibodies obtained through the use of transgenic libraries of mice or bacteriophages. See: Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-5; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-33; Newman et al. (1992) Biotechnology (NY) 10: 1455-60; Green et al. (1994) Nat. Genet. 7: 13-21; Marks et al. (1991) J. Mol. Biol. 222: 581-97. The exclusion of the NAMA response allows the use of high and repeated doses to achieve the therapeutic effect.

Selected antibodies for targeted drug administration include antibodies that recognize carcinoembryonic antigens, i.e., antigens that are present in embryonic cells and tumor cells, and which are almost absent in normal adult cells. See, for example, Magdelenat (1992) J. Immunol. Methods 150: 133-43. 5E4 carcinoembryonic antigen is a highly glycosylated transmembrane glycoprotein with a molecular weight of 72 kDa, which includes a 42 kDa non-glycolized nucleus. (Hole et al. (1988) Br. J. Cancer 57: 239-46, Hole et al. (1990) Int. J. Cancer 45: 179-84; international application WO89 / 07947; US Patent No. 5,869,053). 5T4 contains an extracellular domain that is characterized by two leucine-rich repeats (LRRs) and an intermediate hydrophilic region, which is an accessible site for targeted therapy (Myers et al. (1994) J. Biol. Chem. 269: 9319-24).

Human 5T4 is expressed in numerous types of cancer, including carcinomas of the bladder, breast, cervix, endometrium, lung, esophagus, ovary, pancreas, stomach, testes, and is mainly absent in normal tissues, with the exception of syncytotrophoblasts in the placenta (see e.g. Southall et al. (1990) Br. J. Cancer 61: 89-95 (immunohistological distribution of 5T4 antigen in normal and malignant tissues); Mieke et al. (1997) Clin. Cancer Res. 3: 1923-1930 ( low intercellular adhesion of molecule 1 and high expression of 5T4 in tumor cells to correlates with disease-free survival of patients with intestinal cancer); Starzynska et al. (1994) Br. J. Cancer 69: 899-902 (prognostic significance of 5T4 expression of carcinoembryonic antigen in intestinal cancer; Starzynska et al. (1992) Br. J. Cancer 66: 867-869 (expression of 5T4 antigen for bowel and stomach cancer); Jones et al. (1990) Br. J. Cancer 61: 96-100 (expression of 5T4 antigen for cervical cancer); Connor and Stern (199) Int. J. Cancer 46: 1029-1034 (loss of MHC (major histocompatibility complex, HCH) expression of class I in cervical cancer), Ali et al. (2001) Oral Oncology 37: 57-64 (5T4 carcinoembryonic antigen expression model in normal, dysplasia, and malignantly damaged oral mucosa); WO 89/07947; U.S. Patent No. 5,869,053). For example, tissues that are described as lacking 5T4 expression include the liver, skin, spleen, thymus, central nervous system (CNS), adrenal glands and ovaries. Tissues that are described as having focal or low 5T4 expression include: liver, skin, spleen, lymph nodes, tonsils, prostate, and seminal vesicles. Mild and moderate 5T4 expression has been described for the kidney, lung, pancreas, pharynx, and gastrointestinal tract. Syncytiotrophoblast is the only tissue reported to have high 5T4 expression; 5T4 is also absent in normal blood serum or blood serum of pregnant women (i.e., levels less than 10 ng / ml). Overexpression of 5T4 in tumor tissues was made dependent on the development of the disease, and an assessment of 5T4 expression was proposed as a method suitable for identifying patients with short-term prognoses. (Mulder et al. (1997) Clin. Cancer Res. 3: 1923-30, Naganuma et al. (2002) Anticancer Res. 22: 1033-1038, Starzynska et al. (1994) Br. J. Cancer 69: 899 902, Starzynska et al. (1998) Eur. J. Gastroenterol. Hepatol. 10: 479-484; Wrigley et al. (1995) Int. J. Gynecol. Cancer 5: 269-274).

Several anti-5T4 antibodies have been described, including mAb5T4, which is also called the H8 antibody, which distinguishes the conformational epitope of the 5T4 antigen (Shaw et al. (2002) Biochem. J. 363: 137-45, WO 98/55607), rat monoclonal antibody (Woods et al. (2002) Biochem. J. 366: 353-65) and a monoclonal mouse antibody called 5T4 (US Patent No. 5,869,053). Antibodies against 5T4 having a single chain are also described, as well as fusion proteins, which include sequences of antibodies against 5T4 linked to a therapeutic molecule. For example, anti-5T4 antibody sequences linked to the human IgG1 constant domain or mouse extracellular B7.1 domain cause cytolysis of 5T4-expressing tumor cell lines. (Myers et al. (2002) Cancer Gene Ther. 9: 884-896, Shaw et al (2000) Biochim. Biophys. Acta. 1524: 238-246; US Patent Application No. 2003/0018004). Similarly, a single chain anti-5T4 antibody crosslinked with a superantigen can stimulate T cell-dependent cytolysis in in vitro pulmonary carcinoma cells. (Forsberg et al. (2001) Br. J. Cancer 85: 129-136). The first phase of clinical trials using PNU-214936, a Fab fragment of a 5T4 monoclonal antibody crosslinked with a mutated superantigen (sharply enhanced immunogenicity) of staphylococcal enterotoxin A (SEA), revealed limited toxicity and a definite antitumor response (Cheng et al. (2004) J. Clin. Oncol. 22 (4) 602-9). Recombinant 5T4 vaccines are also proposed as a treatment option for the treatment of cancer (Mulryan et al. (2002) Mol. Cancer Ther. 1: 1129-37; US Pat. Nos. 2,370,571 and 2,378,704; EP 1,160,323 and 1,152,060).

The present invention is the creation of new antibodies against 5T4, conjugates of antibodies against 5T4 with the drug, methods for producing the proposed antibodies and conjugates of antibodies with the drug and methods for their use in diagnosis and therapy.

SUMMARY OF THE INVENTION

The present invention provides new antibodies against 5T4, conjugates based on them and methods of using the above objects. Isolated anti-5T4 polypeptides and isolated nucleic acid sequences encoding them are also provided.

Antibodies against 5T4 according to the present invention include antibodies that specifically bind human 5T4 antigen, wherein antibody (a) contains the antigen binding domain of mouse antibodies A1, A2, A3; (b) competes for the binding of 5T4 with mouse antibodies A1, A2, A3; (c) binds the 5T4 epitope to which mouse antibodies A1, A2, A3 bind; or (g) contains a 5T4-binding fragment of antibodies (a) - (c). Antibodies against 5T4 according to the present invention can be hybrid, humanized, single chain, Fab fragment, F (ab) 2 fragment, Fv fragment, four-dimensional, tetravalent, multispecific, domain-specific, single domain antibody, recombinant protein or mouse monoclonal bodies. For example, humanized anti-5T4 antibodies of the present invention include antibodies comprising at least one high molecular weight variable region and at least one low molecular weight variable region in which the humanized antibody or antibody fragment: (a) contains the antigen binding domain of mouse antibodies A1 , A2, A3; (b) competes for the binding of 5T4 with murine antibodies A1, A2, A3; (c) binds to the 5T4 epitope that binds antibodies A1, A2, A3; or (d) a 5T4 binding fragment of antibodies (a) - (c).

Antibodies against 5T4 according to the present invention have an affinity of binding of human 5T4 antigen from at least about 1 × 10 ^-7 mol to about 1 × 10 ^-12 mol. The claimed anti-5T4 antibodies and their conjugates may also exhibit specific affinity for 5T4-expressing cells in vivo.

Representative anti-5T4 antibodies of the present invention include antibodies that comprise a heavy chain variable region comprising (a) a sequence of amino acid residues 20-138 of SEQ ID NO: 2; (b) an amino acid sequence that is at least 85% identical to residues 20-138 of the sequence SEQ ID No: 2; (c) the sequence of amino acid residues 19-135 of the sequence SEQ ID No: 6; (g) an amino acid sequence that is at least 86% identical to residues 19-135 of the sequence SEQ ID No: 6; (e) a sequence of amino acid residues 20-141 of the sequence SEQ ID No: 10; (f) an amino acid sequence that is at least 91% identical to residues 20-141 of the sequence SEQ ID No: 10; (g) the amino acid sequence of any of the sequences of SEQ ID No: 49, 51, 52, 54, 56, 77, 78, 81, or 82; (h) an amino acid sequence that is at least 91% identical to the sequence of SEQ ID No: 51; (i) an amino acid sequence that is at least 78% identical to the sequence of SEQ ID No: 54; (k) an amino acid sequence that is at least 91% identical to the sequence of SEQ ID No: 77; (k) an amino acid sequence that is at least 79% identical to the sequence of SEQ ID No: 78; (m) an amino acid sequence that is at least 80% identical to the sequence of SEQ ID No: 81; or (n) an amino acid sequence that is at least 78% identical to the sequence of SEQ ID No: 82.

Representative anti-5T4 antibodies of the present invention include antibodies that comprise a variable region of a light chain comprising (a) an amino acid residue sequence 21-127 of SEQ ID NO: 4; (b) an amino acid sequence that is at least 94% identical to residues 21-127 of the sequence SEQ ID No: 4; (c) a sequence of amino acid residues 23-130 of the sequence SEQ ID No: 8; (g) an amino acid sequence that is at least 96% identical to residues 23-130 of the sequence SEQ ID No: 8; (e) a sequence of amino acid residues 21-127 of the sequence SEQ ID No: 12; (e) an amino acid sequence that is at least 98% identical to residues 21-127 of the sequence SEQ ID No: 12; (g) the amino acid sequence of any of the sequences of SEQ ID No: 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 80, 83, or 84; (h) an amino acid sequence that is at least 83% identical to the sequence of SEQ ID No: 60; (i) an amino acid sequence that is at least 93% identical to the sequence of SEQ ID No: 70; (k) an amino acid sequence that is at least 85% identical to the sequence of SEQ ID No: 76; (k) an amino acid sequence that is at least 85% identical to the sequence of SEQ ID No: 76; (m) an amino acid sequence that is at least 88% identical to the sequence of SEQ ID No: 79; (n) an amino acid sequence that is at least 84% identical to the sequence of SEQ ID No: 80; (o) an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 83; or (p) an amino acid sequence that is at least 91% identical to the sequence of SEQ ID No: 84.

For example, an anti-5T4 antibody may include (a) a heavy chain variable region comprising a sequence of amino acid residues 20-138 of the sequence SEQ ID No: 2; and a variable region of the light chain containing the sequence of amino acid residues 21-127 of the sequence SEQ ID No: 4; (b) the variable region of the heavy chain containing the amino acid sequence obtained from residues 19-135 of the sequence SEQ ID No: 6; and a variable region of the light chain containing the amino acid sequence obtained from groups 23-130 of the sequence SEQ ID No: 8; or (c) the variable region of the heavy chain containing the amino acid sequence obtained from groups 20-141 of the sequence SEQ ID No: 10; and a variable region of the light chain containing the amino acid sequence obtained from groups 21-127 of the sequence SEQ ID No: 12.

Hybrid and humanized anti-5T4 antibodies of the present invention may comprise constant regions derived from human constant regions, such as, for example, a human antibody light chain constant region derived from a human kappa light chain constant region and a human antibody heavy chain constant region derived from a constant heavy chain regions of human IgG1 or IgG4.

Representative anti-5T4 antibodies of the present invention include antibodies that contain (a) framework regions including residues of the framework region of human antibodies; and (b) one or more hypervariable region of the variable region of the light chain with the sequence of SEQ ID No: 4, 8, or 12; or one or more hypervariable region of the variable region of the heavy chain with the sequence SEQ ID No: 2, 6, or 10. For example, the remains of the frame region of human antibodies may include (a) the frame region of the light chain of a human antibody from clone DPK24 subgroup IV germline, clone the germline of the VHIII subgroup (DPK23, DPK22, DPK20, DPK21) or the VHI subgroup (DPK9, DPK1, O2, DPK7); (b) the frame region of the heavy chain of a human antibody selected from the group consisting of DP-21 (VH7), DP-54 (VH3-07), DP-47 (VH3-23), DP-53 (VH-74), DP-49 (VH3-30), DP-48 (VH3-13), DP-75, DP-8 (VH1-2), DP-25, VI-2b and VI-3 (VH1-03), DP- 15 and V1-8 (VH1-08), DP-14 and V1-18 (VH1-18), DP-5 and V1-24P (VH1-24), DP-4 (VH1-45), DP-7 ( VH1-46), DP-10, DA-6 and YAC-7 (VH1-69), DP-88 (VH1-e), DP-3 and DA-8 (VH1-f); (c) a consensus sequence of framework regions of the heavy chain (b); or (d) a frame region that is at least 63% identical to the frame region (a) to (c).

Representative humanized anti-5T4 antibodies of the present invention may also include two or more hypervariable regions with the sequence: SEQ ID No: 2, 4, 6, 8, 10, or 12, such as two or three hypervariable regions of the light chain variable region, which have the sequence: SEQ ID no: 4, 8 or 12; or two or three hypervariable region of the variable region of the heavy chain, which have the sequence: SEQ ID no: 2, 4 or 10; or one or more hypervariable region of the variable region of the light chain, which has the sequence: SEQ ID No.: 4, 8 or 12, and one or more hypervariable region of the variable region of the heavy chain, which has the sequence: SEQ ID No.: 2, 6 or 12

Typical representatives of hybrid and humanized antibodies against 5T4 include antibodies that contain a sequence of the variable region of the heavy chain, including (a) a sequence of amino acid residues 20-138 of the sequence SEQ ID No: 2; (b) an amino acid sequence that is at least 85% identical to amino acid residues 20-138 of the sequence SEQ ID No: 2; (c) the sequence of amino acid residues 19-135 of the sequence SEQ ID No: 6; (g) an amino acid sequence that is at least 86% identical to amino acid residues 19-135 of the sequence SEQ ID No: 6; (e) a sequence of amino acid residues 20-141 of the sequence SEQ ID No: 10; (e) an amino acid sequence that is at least 91% identical to amino acid residues 20-141 of the sequence SEQ ID No: 10; (g) the sequence of amino acid residues 1-119 of the sequence SEQ ID No: 49; (h) an amino acid sequence that is at least 90% identical to amino acid residues 1-119 of the sequence SEQ ID No: 49; or the amino acid sequence of the humanized variable region of the heavy chain shown in Figures 9A-9C.

Additional hybrid and humanized anti-5T4 antibodies of the present invention include antibodies comprising a variable region of a heavy chain encoded by a nucleic acid that contains: (a) a nucleotide sequence of 58-414 of the sequence SEQ ID No: 1; (b) a nucleotide sequence of 55-405 of the sequence of SEQ ID No: 5; (c) a nucleotide sequence of 58-423 of the sequence of SEQ ID No: 9; (g) the sequence of nucleotides 1-358 of the sequence of SEQ ID no: 48; (e) the nucleotide sequence encoding the variable regions of the humanized antibodies A1, A2 or A3 shown in Figure 9A-9C; (e) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of any of the sequences (a) to (e); or (g) a nucleic acid that specifically hybridizes with the complement of any of (a) to (e) under stringent hybridization conditions.

Typical hybrid and humanized anti-5T4 antibodies include antibodies comprising a light chain variable region sequence that contains: (a) an amino acid residue sequence 21-127 of SEQ ID NO: 4; (b) an amino acid sequence that is at least 94% identical to amino acid residues 21-127 of the sequence SEQ ID No: 4; (c) the sequence of amino acid residues 23-130 of the sequence SEQ ID No: 8; (d) an amino acid sequence that is at least 96% identical to amino acid residues 23-130 of the sequence SEQ ID No: 8; (e) a sequence of amino acid residues 21-127 of the sequence SEQ ID No: 12; (e) an amino acid sequence that is at least 98% identical to amino acid residues 21-127 of the sequence SEQ ID No: 12; or (g) the amino acid sequence of the variable region of the light chain of the humanized antibodies A1, A2, and A3 shown in Figures 9A-9C.

Also provided are drug conjugate antibody conjugates comprising (a) a hybrid or humanized anti-5T4 antibody or antibody fragment of the present invention; and (b) a drug that is directly or indirectly associated with the specified antibody. Typical drugs include therapeutic agents, such as cytotoxins, radioisotopes, immunomodulators, anti-angiogenic agents, antiprofile agents, proapoptotic agents, chemotherapeutic agents, and nucleic acids used in therapeutic agents. The cytotoxin, for example, may be an antibiotic, a tubiline polymerization inhibitor, an alkylating agent, a protein synthesis inhibitor, a protein kinase inhibitor, a phosphatase inhibitor, a topoisomerization inhibitor, or an enzyme. Antibiotic cytotoxins, such as calicheamicin, N-acetyl-γ-calicheamicin or its derivatives, such as N-acetyl-γ-calicheamicin dimethyl hydrazide, are especially effective in anti-cancer therapy.

Proposed conjugates of an anti-5T4 antibody to a drug may include a linker for binding the antibody to the drug. Typical linkers include 4- (4'acetylphenoxy) butyric acid (AcBut), 3-acetylphenylphenylacetic acid (AcPac), and 4-mercapto-4-methylvaleric acid (Amide). Antibody drug conjugates also include polyethylene glycol and other substances used to increase drug incorporation.

To deliver a drug to 5T4-expressing cells, the present invention provides methods for contacting a cell with a conjugate of an antibody with a drug, comprising (i) a hybrid or humanized anti-5T4 antibody; and (ii) a drug that is directly or indirectly associated with humanized anti-5T4 antibody. In accordance with the proposed methods, the drug is internalized into the target cell. The present application also provides methods of therapy that include administering to a subject having a 5T4-positive form of cancer a therapeutically effective amount of an anti-5T4 antibody conjugate with a drug, comprising (i) a hybrid or humanized anti-5T4 antibody or antibody fragment, and (ii) a therapeutic agent which is directly or indirectly associated with a humanized anti-5T4 antibody or antibody fragment. In order to enhance efficacy, anti-5T4 therapy according to the present invention can be combined with any other known therapeutic methods. The second therapeutic agent can be used in combination with the anti-5T4 antibody conjugate with the drug in parallel or sequentially in any order.

Also proposed are isolated nucleic acids encoding the humanized variable regions of anti-5T4 antibodies that can be used to produce the proposed humanized anti-5T4 antibodies. Typical nucleic acids encoding the humanized variable region of the heavy chain of antibodies against 5T4 include: (a) a nucleotide sequence of 58-414 of the sequence SEQ ID No: 1; (b) a nucleotide sequence of 55-405 of the sequence of SEQ ID No: 5; (c) a nucleotide sequence of 58-423 of SEQ ID No: 9; (d) a sequence encoding one of the sequences of SEQ ID No: 48, 50, 53, or 55; (e) a nucleotide sequence that is 89% identical to the sequence of SEQ ID No: 50, if the coverage of the request is 100%; (e) a nucleotide sequence that is 82% identical to the sequence of SEQ ID No: 53, if the coverage of the request is 100%; or (g) a nucleic acid that specifically hybridizes with the complement of any of the sequences from (a) to (g) under stringent hybridization conditions. Typical nucleic acids encoding the humanized variable region of the light chain of an anti-5T4 antibody include (a) a nucleotide sequence of 61-381 of the sequence SEQ ID No: 3; (b) the sequence of nucleotides 67-390 of the sequence SEQ ID no: 7; (c) the nucleotide sequence of nucleotides 61-381. sequences with identification number SEQ ID no: 11; (d) a sequence encoding the humanized variable regions of the light chain of antibodies A1, A2, or A3 of any of the sequences of SEQ ID No: 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75; (e) a nucleotide sequence that is 84% identical to the sequence of SEQ ID No: 59, if the coverage of the request is 100%; (e) a nucleotide sequence that is 86% identical to the sequence of SEQ ID No: 69, if the coverage of the request is 100%; (g) a nucleotide sequence that is 85% identical to the sequence of SEQ ID No: 75, if the coverage of the request is 100%; or (h) nucleic acid that specifically hybridizes to the complement of any of sequences (a) to (g) under stringent hybridization conditions.

BRIEF DESCRIPTION OF ILLUSTRATIONS

On figa-1C shows the sequence of nucleotides and amino acids of the variable regions of the heavy chain and light chain of murine antibodies A1, A2, and A3 against 5T4. The amino acid sequences are provided with comments for recognition of additional hypervariable regions (CDRs) by underlining and the main sequence by double underlining.

Figure 2 shows a Western blot prepared using CT26 / 5T4 cell lysates and tested with the indicated antibodies.

3A-3B are line graphs showing the response curve and binding kinetics for two independent H8 and A1 antibody preparations.

Figures 4A-4C are line graphs that show modulation of the activity of H8, A1, A2, and A3 antibodies by MDAMB435 / 5T4 cells. Antibody levels on the cell surface decrease over time (Figures 4A, 4C (filled dots)), while the antibody level in the supernatant remains constant (Figures 4B, 4C (unfilled dots). MCF stands for cell fluorescence; supt is the supernatant ( supernatant).

Figure 5 shows a schematic representation of the construct of the human ectodomain 5T4 Fc, the construct of the ectodomain 5T4 Fc of the mouse and the construct of the hybrid human / mouse ectodomains. These structures were used to map antigenic determinants as described in Example 4.

6A-6B show graphical results of competing binding of H8 and each of the designated antibodies to the 5T4 Fc ectodomain of the bound human protein. HuH8, a humanized H8 antibody; ChiA1, a hybrid A1 antibody; ChiA1 + C67F, a hybrid A1 antibody carrying a C67F mutation; ChiA2, a hybrid A2 antibody; muA2, A2 mouse antibody; ChiA3, a hybrid A3 antibody; ChiA3 + C91Y, a hybrid A3 antibody carrying the C91Y mutation, muA3, A3 mouse antibody; No Ab - lack of antibody (control).

7 is a linear diagram showing the binding of human 5T4 epitopes to H8, A1, A2 and A3. The cited residues are 5T4 antigen residues described by Mayer et al. Myers et al. (1994) J. Biol. Chem. 269 (12): 9319-9324, which are also available at the GenBank Accession Gene Bank No. Z29083 (sequence with identification number SEQ ID No.: 87). LRR, leucine-rich duplication.

Figure 8 shows the results of spherical analyzes carried out as described in Example 6. Conjugates of anti-5T4 antibodies with calicheamicin obtained using antibodies A1 and A3 significantly inhibit the growth of 5T4 expression cells (MDAMB435 / 5T4) compared to control cells (MDAMB435 / neo). CMA-676, anti-CD33 antibody conjugate with calicheamicino; huH8-AcBut-CalichDMH, a humanized H8 antibody conjugated to calicheamicin with 4- (4'-acetylphenoxy) butyric acid (AcBut); CalichDMH, unconjugated calicheamicin; A1-AcBut-CalichDMH, antibody A1 conjugated to calicheamicin with 4- (4'-acetylphenoxy) butyric acid (AcBut); A3-AcBut-CalichDMH, antibody A3 conjugated to calicheamicin with 4- (4'-acetylphenoxy) butyric acid (AcBut).

On figa-9H shows the nucleotide and amino acid sequences of the variable region of the heavy chain of the humanized antibody A1 (huA1 VH) version 1 (sequence with identification number SEQ ID no: 48-49); amino acid sequences of the variable region of the heavy chain of a humanized antibody A1 V version 1.2 and 2.0, amino acid sequences of the variable region of the heavy chain of a humanized antibody A1 (huA1 VL) version 1.0, 2.0 and 3.0; amino acid sequences of the heavy chain variable region of the humanized antibody A2 (huA2 VH) versions 1.0 and 2.0; amino acid sequences of the variable region of the light chain of a humanized antibody A2 (huA2 VL) versions 1.0 and 2.0; amino acid sequences of the heavy chain variable region of the humanized antibody A3 (huA3 VH) versions 1.0 and 2.0; and amino acid sequences of the light chain variable region of the humanized antibody A3 (huA32 VL) versions 1.0 and 2.0. CDRs (hypervariable regions) are underlined.

10A-10B show typical framework sequences of the variable region of the heavy chain that can be used to obtain humanized antibodies against 5T4. Figure 10A shows the correct relative arrangement of the sequences of the Variable region of the heavy chain of human subgroup 1 (sequences with identification numbers SEQ ID No: 14-24) and the consensus framework sequences derived here (sequences with identification numbers SEQ ID No: 25-27). Figure 10B shows the human germline genes of the subgroups VH 7 and VH 3 (sequences with identification numbers SEQ ID No: 88-93).

Figure 11 shows the correct relative arrangement of the sequences of the variable region of the light chain of a person of subgroup VkIII (sequences with identification numbers SEQ ID No: 29-34). The framed sequences are CDRs (hypervariable regions).

Figure 12 shows the correct relative arrangement of the sequences of the variable region of the human light chain of the subgroup VkI (sequences with identification numbers SEQ ID No: 35-44). The framed sequences are CDRs (hypervariable regions).

13 shows additional germline sequences of the subgroups Vk 1 and Vk IV, which have frame regions that can be used to obtain humanized anti-5T4 immunoglobulins (sequences with identification numbers SEQ ID No: 94-99).

On Fig shows the amino acid sequences of typical human C-segments, which can be used for the preparation of hybrid and humanized antibodies against 5T4 (sequences with identification numbers SEQ ID No: 45-47).

15 shows the amino acid sequences of the full 5T4 cynomolgus monkey antigen (cynomolgus monkey - Masas fascicularis) and the 5T4 partial antigen of the black-tailed monkey. Underlined sequences, main sequences. For each 5T4 sequence, the ectodomain contains amino acids 30-356.

DETAILED DESCRIPTION OF THE INVENTION

I. Antibodies against 5T4

The present invention provides new mouse antibodies that bind to the human 5T4 antigen and which are useful for targeted immunotherapy. The human antigen is a 72 kDa non-glycosylated phosphoprotein found on the surface of troboplast cells and a number of types of cancer cells. See See Hole et al. (1988) Br. J. Cancer 57: 239-46, Hole et al. (1990) Int. J. Cancer 45: 179-184; PCT International Publication No. WO 89/07947; U.S. Patent No. 5,869,053.

The mouse antibodies of the present invention are designated A1, A2 and A3 and are prepared as described in Example 1. Anti-5T4 antibodies derived from A1, A2 and A3 are also provided that specifically bind to the human 5T4 antigen. For example, antibodies against 5T4 according to the present invention include antibodies containing antigen binding residues from A1, A2 and A3 antibodies; antibodies that compete for the binding of 5T4 antigen to A1, A2 and A3 antibodies; and antibodies that bind to the aforementioned 5T4 epitope as A1, A2 and A3 antibodies.

In particular, the proposed A1, A2 and A3 antibodies each contain an antigen-binding site that distinguishes a specific epitope of the human 5T4 antigen. Each of these antibodies also binds to an epitope, different from the epitope associated with H8, and each A1, A2 and A3 does not compete with the H8 antibody for binding to human 5T4. See Examples 4-5 and 6-7. Thus, the present invention provides antibodies that specifically bind to residues 30-168 of human 5T4 (e.g., A3), antibodies that specifically bind to residues 224-276 of human 5T4 (e.g., A1), and antibodies that specifically bind to residues 224-355 of human 5T4 (e.g., A2). Also proposed are human 5T4 antigens including epitopes to which antibodies A1, A2 and A3 bind. For example, the invention provides anti-5T4 antigen fragments comprising residues 30-163, 224-276 and 224-355 of a native or full-length 5T4 antigen.

The specific binding of the proposed anti-5T4 antibodies refers to the preferred binding of the antibody to the 5T4 human antigen in a heterogeneous sample that contains various antigens with a complex structure. Typically, specific binding occurs if the binding affinity is at least about 10 ⁻⁷ M or more, for example, at least about 10 ^-8 M or more, including at least about 10 ^-9 M or more, at least about 10 ^-11 M or more, or at least about 10 ^-12 M or more. For example, specific binding of an antibody of the present invention to a 5T4 human antigen includes binding in the range of at least about 10 ^-7 M to about 1 × 10 ^-12 M, as well as in the range of about 1 × 10 ^-8 M to about 1 × 10 ^-12 M, or in the range of about 1 × 10 ^-8 M to about 1 × 10 ^-11 M, or in the range of about 1 × 10 ^-8 M to about 1 × 10 ^-10 M, or in the range of about 1 × 10 ^-9 M to about 1 × 10 ^-10 M. Specific binding also refers to selective targeting to 5T4 antibodies against 5T4-expressing cells following administration of an Ithel subject.

Antibodies against 5T4 according to the present invention can have a tetrameric structure (for example, similar to naturally occurring antibodies), or they can include any other structure having at least one light chain immunoglobulin variable region or at least one heavy chain immunoglobulin V region, or 5T4 binding fragments of the latter (e.g., Fab fragments, modified Fab, F (ab ') _2, or Fv fragments). Single domain antibodies are also included in which one or more hypervariable regions (CDRs), but less than all six hypervariable regions (CDRs), comprise an antigen binding region. Hybrid antibodies, humanized antibodies, superhumanized antibodies, diabodies, single chain antibodies, tetravalent antibodies, and / or multispecific antibodies (e.g., bispecific antibodies) are also provided. These antibody descriptors are not mutually exclusive.

Natural antibodies are tetrameric (H ₂ L ₂ ) glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Two heavy chains are linked to each other via disulfide bonds, and each heavy chain is linked by a disulfide bond to the light chain. Each of the light and heavy chains is further defined by an N-terminal variable region and a C-segment. Variable regions include sequences that differ significantly among antibodies and determine substantially the binding affinity and specificity of a single antibody for a single antigen. The variable regions of the light and heavy chains line up with the formation of an antigen-binding domain.

Hybrid antibodies contain sequences of at least two kinds. As one example, to include variable regions that contain antigen binding sites, recombinant cloning technology from a non-human antibody (i.e., an antibody made from non-human individuals immunized with an antigen) and C-segments derived from human immunoglobulin can be used.

Hybrid antibodies against 5T4 according to the present invention include antibodies containing the variable regions of the heavy chain and light chain of antibodies A1, A2 and A3; those. (a) a variable region of the heavy chain that has a sequence of amino acid residues 20-138 of the sequence SEQ ID NO: 2 and a variable region of the light chain having a sequence of amino acid residues 21-127 of the sequence SEQ ID No: 4; (b) the amino acid sequence of the heavy chain corresponding to amino acid residues 19-135 of the sequence SEQ ID No: 6 and the amino acid sequence of the light chain corresponding to amino acid residues 23-130 of the sequence SEQ ID No: 8; and (c) the amino acid sequence of the heavy chain corresponding to amino acid residues 20-141 of the sequence SEQ ID No: 10 and the amino acid sequence of the light chain corresponding to amino acid residues 21-127 of the sequence SEQ ID No: 12. Typical humanized anti-5T4 antibodies may include a heavy chain variable region corresponding to amino acids 1-119 of the sequence SEQ ID No: 49; or one of the humanized variable regions of the heavy chain shown in figa-9C, and the humanized variable region of the light chain, also shown in figa-9C. The preparation of typical hybrid and humanized antibodies against 5T4 according to the present invention is described in Example 7.

Antibodies against 5T4 according to the present invention may also contain a variable region of the heavy and / or light chain, including an amino acid sequence that is derived or essentially similar to the variable regions of antibodies A1, A2 or A3, or essentially similar to the variable regions of humanized antibodies A1, A2 or A3 . With respect to the substantially identical variable regions of the heavy and light chains, the substantially identical sequences are at least 90% identical to the sequences of the variable regions of any of the sequences of SEQ ID No: 1-12 or the variable regions of the humanized antibodies A1, A2 or A3 displayed on figa-9C, for example, at least 92% identical, or at least 93% identical, or at least 94% identical, or at least 95% identical, or, at least 96% identical, or at least n and 97% identical, or at least 98% identical, or at least 99% identical.

Typical anti-5T4 hybrid antibodies of the present invention, i.e. antibodies that specifically bind the 5T4 antigen also include those that contain (a) the amino acid sequence of the variable region of the heavy chain corresponding to residues 20-138 of the sequence SEQ ID No: 2; residues 19-135 of the sequence of SEQ ID No: 6; residues 20-141 of the sequence SEQ ID No: 10; or any variable region of the heavy chain of humanized antibodies A1, A2 or A3, shown in figa-9C; (b) the amino acid sequence of the variable region of the heavy chain, which is at least 85% identical to residues 20-138 of the sequence SEQ ID No: 2; (c) the amino acid sequence of the variable region of the heavy chain, which is at least 86% identical to residues 19-135 of the sequence SEQ ID No: 6; (d) the amino acid sequence of the variable region of the heavy chain, which is at least 91% identical to residues 20-141 of the sequence SEQ ID No: 10; (d) the amino acid sequence of the variable region of the heavy chain, which is at least 90% identical to residues 1-119 of the sequence SEQ ID No: 49; or (e) the amino acid sequence of the variable region of the heavy chain, which is a derivative of any variable region of the humanized A1, A2 or A3, shown in figa-9C.

The variable region of the heavy chain of a hybrid or humanized anti-5T4 antibody that specifically binds to the 5T4 antigen can encode (a) a nucleic acid containing a nucleotide sequence 58-414 of the sequence SEQ ID No: 1, nucleotides 55-405 of the sequence SEQ ID No: 5, nucleotides 58-423 of the sequence SEQ ID No: 9, nucleotides 1-358 of the sequence SEQ ID No: 48, or a nucleic acid encoding the variable regions of the heavy chain of the humanized antibodies A1, A2 or A3 shown in figa-9C; (b) a nucleic acid containing a nucleotide sequence that is at least 90% identical to a nucleic acid containing a nucleotide sequence 58-414 of the sequence SEQ ID No: 1, nucleotides 55-405 of the sequence SEQ ID No: 5, nucleotides 58- 423 of the sequence SEQ ID No: 9. For example, the variable region of the heavy chain of a hybrid anti-5T4 antibody can be encoded by a nucleic acid that is at least 98% identical to nucleotides 58-414 of SEQ ID NO: 1, a nucleic acid containing a nucleotide sequence that is at least 98% identical to nucleotides 55-405 of the sequence SEQ ID No: 5, or nucleic acid, which is at least 89% identical to nucleotides 1-358 of the sequence SEQ ID No: 48. The variable region of the heavy chain of a hybrid anti-5T4 antibody can also be encoded by a nucleic acid that specifically hybridizes with a nucleic acid complement containing nucleotide sequence 58-414 of SEQ ID No: 1, nucleotides 55-405 of SEQ ID No: 5, nucleotides 58-423 SEQ ID no: 9, or nucleotides 1-358 of SEQ ID no: 48, under stringent hybridization conditions, for example, under conditions of final washing with 0.1X SSC (0.1 fold sodium chloride / sodium citrate buffer) at 65 ° C.

Typical anti-5T4 fusion antibodies of the present invention further include those that contain (a) the amino acid sequence of a light chain variable region corresponding to residues 21-127 of the sequence SEQ ID No: 4; residues 23-130 of the sequence SEQ ID No: 8; residues 21-127 of the sequence SEQ ID No: 12; or residues of the variable region of the light chain of the humanized antibody A1, A2 or A3 shown in figa-9C; or (b) the amino acid sequence of the variable region of the light chain, which is at least 90% identical to residues 21-127 of the sequence SEQ ID No: 12. For example, the amino acid sequence of the variable region of the light chain may include (a) the amino acid sequence of the variable region of the light chain, which is at least 94% identical to residues 21-127 of the sequence SEQ ID No: 4; (b) the amino acid sequence of the variable region of the light chain, which is at least 96% identical to residues 23-130 of the sequence SEQ ID No: 8; (c) the amino acid sequence of the variable region of the light chain, which is at least 98% identical to residues 21-127 of the sequence SEQ ID No: 12; (d) or the amino acid sequence of the variable region of the light chain, which is a derivative of any variable region of the humanized antibodies A1, A2 or A3 shown in figa-9C.

The variable region of the light chain of a hybrid anti-5T4 antibody that specifically binds to the 5T4 antigen can be encoded by (a) a nucleic acid containing a nucleotide sequence 61-381 of the sequence SEQ ID No: 3, nucleotides 67-390 of the sequence SEQ ID No: 7, nucleotides 61 -381 sequences of SEQ ID No: 11, or nucleotides encoding any variable region of the light chain of the humanized antibodies A1, A2 or A3 shown in figa-9C; or (b) a nucleic acid containing a nucleotide sequence that is at least 90% identical to nucleotides 61-381 of SEQ ID No: 3, nucleotides 87-390 of SEQ ID No: 7, or nucleotides 61-381 of SEQ ID No :eleven. For example, the variable region of the light chain of a hybrid anti-5T4 antibody can be encoded by a nucleic acid containing (a) a nucleotide sequence that is at least 97% identical to nucleotides 61-381 of the sequence SEQ ID No: 3; (b) a nucleotide sequence that is at least 98% identical to nucleotides 67-390 of the sequence SEQ ID No: 7; or (c) a nucleotide sequence that is at least 99% identical to nucleotides 61-381 of the sequence SEQ ID No: 11. The variable region of the light chain of a hybrid anti-5T4 antibody that specifically binds to the 5T4 antigen can also be encoded by a nucleic acid that specifically hybridizes to a complement of a nucleic acid containing the nucleotide sequence of nucleotides 61-381 of SEQ ID NO: 3, nucleotides 67-390 of SEQ ID No: 7, or nucleotides 61-381 of the sequence SEQ ID No: 11, under stringent hybridization conditions, for example, under conditions of final washing with 0.1X SSC (0.1 times sodium chloride / sodium citrate buffer ) At 65 ° C.

Humanized antibodies are a type of hybrid antibody in which the residues of the variable region responsible for antigen binding (i.e., the remnants of the hypervariable region, the truncated hypervariable region, or any other residues that are involved in antigen binding) are derived from non-human animals, while other variable region residues (i.e., framework region residues) and constant regions originate, at least in part, from human antibody sequences ESA. A certain number of framework region residues and constant region residues of a humanized antibody may occur from non-human animals. The variable regions of a humanized antibody are also described as Humanized (i.e. humanized variable regions of a light or heavy chain) Non-human animals are typically species used for immunization with antigenic species such as a mouse, rat, rabbit, non-human monkey, or other non-human mammals. Humanized antibodies are usually less immunogenic than traditional hybrid antibodies and exhibit enhanced stability when used in humans. See Benincosa et al. (2000) J. Pharmacol. Exp. Ther. 292: 810-6; Kalofnos et al. (1994) Eur. J. Cancer 30A: 1842-50; Subramanian et al. (1998) Pediatr. Infect. Dis. J. 17: 110-5.

Hypervariable regions (CDRs) are residues of the variable region of an antibody that are involved in antigen binding. In practice, several numbering systems are used to identify hypervariable sections. The definition of Kabat is based on sequence variability; the definition of Chothia is based on the position of closed-loop segments. The definition of AbM is a compromise between the approaches of Kabat and Hotia. According to the Kabat, Hotia, or AbM algorithm, the hypervariable regions (CDRs) of the light chain variable region are limited to residues at positions 24 and 34 (CDR1-L), 50 and 56 (CDR2-L), and 89 and 97 (CDR3-L) . According to the definition of Kabat, the hypervariable regions (CDRs) of the variable region of the heavy chain are limited by residues at positions 31 and 35 V (CDR1-H), 50 and 65 (CDR2-H), and 95 and 102 (CDR3-H) (numbering according to Kabat) . According to Hotia's definition, the hypervariable regions (CDRs) of the variable region of the heavy chain are bounded by residues at positions 26 and 32 (CDR1-H), 52 and 56 (CDR2-H), and 95 and 102 (CDR3-H) (numbering according to Hotia). According to the definition of AbM, the hypervariable regions (CDRs) of the variable region of the heavy chain are bounded by residues at positions 26 and 35 B (CDR1-H), 50 and 58 (CDR2-H), 95 and 102 (CDR3-H) (Kabat numbering). See Martin et al. (1989) Proc. Natl. Acad. Sci. USA 86: 9268-9272; Martin et al. (1991) Methods Enzymol 203: 121-153; Pedersen et al. (1992) Immu№methods 1: 126; and Rees et al. (1996) In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, pp., 141-172.

Specificity Determining Segments (SDRs) are residues within the hypervariable regions that directly interact with the antigen. Specificity-determining segments correspond to hypervariable residues. See Padlan et al. (1995) FASEB J 9: 133-139.

Frame sections are residues of the variable regions of an antibody other than hypervariable or CDR residues. Frame residues can be obtained from natural human antibodies, such as regions of the frame of human antibodies, which are largely similar to the frame regions of antibodies A1, A2 or A3. Artificial framework sequences representing the consensus of the original sequences may also be used. When selecting a framework region for humanization, sequences that are more represented in the human body may be preferred relative to less represented sequences. In order to restore the residues of amino acids of the mouse sequence, which are believed to be involved in the interaction of the antigen and / or residues involved in the structural integration of the antigen binding site, or to increase the expression of the antibody, additional mutations in the acceptor sequences of the framework regions of human antibodies can be used. When studying humanized sequences of variable regions of heavy and light chains to identify and eliminate sites of post-translational modification of proteins introduced during humanization, it is possible to use the prediction of the structure of peptides.

Humanized antibodies can be obtained using any of a variety of methods, which include venation, grafting of hypervariable segments, grafting of truncated hypervariable segments, grafting of specific defining segments, and genetic assembly (Frankenstein assembly), as described below. Humanized antibodies also include hyperhumanized antibodies in which one or more changes have been introduced into the hypervariable segment. For example, amino acid residues of a human sequence can be replaced by residues derived from other animals in the hypervariable segments. To obtain antibodies against 5T4 with any desired sequence, these general approaches can be combined with standard technologies for mutagenesis and synthesis.

Veniation is based on the principle of reducing potentially immunogenic amino acid sequences in the antibodies of rodents or other animals by replacing the sequences of the solvent-accessible outer side of the antibody molecule with human amino acid sequences. Thus, venous antibodies are less alien to human cells than unmodified non-human antibodies. See Padlan (1991) Mol. Immunol. 28: 489-98. An antibody that does not originate from a person is venerated by identifying protruding residues of the outer part of the framework regions in such an antibody that differ from similar residues located in the same places in the framework regions of the human antibody, and by replacing the identified residues with amino acids that typically occupy the same position in human antibodies.

The grafting of hypervariable segments is accomplished by replacing one or more hypervariable segments of an acceptor antibody (e.g., a human antibody or any antibody containing predetermined framework residues) with hypervariable segments of a donor antibody (e.g., an antibody originating from another animal). Acceptor antibodies can be selected based on the similarity of scaffold residues between a possible acceptor antibody and a donor antibody. For example, according to the method of genetic assembly (Frankenstein's method), fragments of human antibodies are defined as having significant sequence homology with each fragments of the corresponding antibodies of another animal, and hypervariable segments of non-human antibodies are grafted onto a set of different fragments of human antibodies. A related method, which is also suitable for the preparation of antibodies according to the present invention, is proposed in application for US patent No. 2003/0040606.

Inoculation of truncated hypervariable segments is a similar approach. Shortened hypervariable regions contain specificity-determining residues and neighboring amino acids, including residues and amino acids at positions 27d-34, 50-55 and 89-96 of the light chain, and at positions 31-35b, 50-58, and 95-101 of the heavy chain (conditional number designations according to Kabat et al. (1987)). See Padlan et al. (1995) FASEB J. 9: 133-9. The grafting of specificity-determining segments is based on the understanding that the binding specificity and paratope affinity are determined by the most highly variable residues within each of the hypervariable segments. The study of the three-dimensional structures of the antibody / antigen complex, in combination with the analysis of the available information on amino acid sequences, can be used to model the variability of the sequence based on the structural heterogeneity of amino acid residues that occur at each position within the hypervariable segment. Specificity determining segments are identified as minimally immunogenic polypeptide sequences consisting of residues of the contact area. See Padlan et al. (1995) FASEB J. 9: 133-139.

Typically, human acceptor framework regions of human antibodies are selected based on the fact that they are substantially similar to the framework regions of donor antibodies, or from those regions that are most similar to the consensus sequence for the subfamily of variable regions. In order to achieve the highest antibody binding efficiency, functionality, use of codons, expression levels, etc. following inoculation, additional changes can be made that include the introduction of non-human residues in the region of the scaffold. See, for example: PCT International Publication No. WO91 / 09967.

For grafting hypervariable segments into framework regions of the heavy chain variable region, suitable framework sequences can be obtained from a DP-21 (VH7), DP-54 (VH3-07), DP-47 (VH3-23), DP-53 (VH- 74), DP-49 (VH3-30), DP-48 (VH3-13), DP-75, DP-8 (VH1-2), DP-25, VI-2b and VI-3 (VH1-03) , DP-15 and V1-8 (VH1-08), DP-14 and V1-18 (VH1-18), DP-5 and V1-24P (VH1-24), DP-4 (VH1-45), DP -7 (VH1-46), DP-10, DA-6 and YAC-7 (VH1-69), DP-88 (VH1-e), DP-3 and DA-8 (VH1-f). Typical variable regions of the heavy chain containing amino acids of the fragments of the framework that are suitable for humanization correspond to the sequences of SEQ ID No: 13-24 and 88-93. Typical fragments of the framework that represent the consensus framework residues of VH1 correspond to the sequences of SEQ ID No: 25-27. See also: figa-10B.

For grafting hypervariable segments into the framework regions of the variable region of the light chain, suitable framework sequences can be obtained from DPK24 subgroup IV of the germline cell clone, subgroup VkIII (DPK23, DPK22, DPK20, DPK21), or subgroup VkI of the germline cell clone (DPK9, DPK1, O2, DPK7). Typical variable regions of the light chain containing frame residues suitable for humanization correspond to the sequences SEQ ID no: 28-34, 35-44 and 94-99. See FIGS. 11-14.

Typical humanized anti-5T4 antibodies of the present invention include antibodies that have one or more hypervariable segments of a non-human anti-5T4 antibody selected from hypervariable regions of the heavy chain variable region of any of sequences with SEQ ID NO: 2, 6, or 10, or the variable region of the light chain of any of the sequences of SEQ ID No: 4, 8, or 12. For example, humanized anti-5T4 antibodies may include two or more hypervariable segments selected from hypervariables sections of the variable region of the heavy chain of any of the sequences of SEQ ID No: 2, 6, or 10, or the variable region of the light chain of any of the sequences of SEQ ID No: 4, 8, or 12. Humanized anti-5T4 antibodies may also contain a heavy chain containing a variable region that has 2 or 3 hypervariable regions of any of the sequences of SEQ ID No: 2, 6, or 10, and a light chain comprising a variable region that has 2 or 3 hypervariable regions of any of the sequences of SEQ ID No: 4, 8, or 12.

The humanized anti-5T4 antibodies of the present invention can be designed such that the variable region of the first chain (i.e., the variable region of the light chain or the variable region of the heavy chain) is humanized, and the variable region of the second chain is not humanized (i.e., the variable region of the antibody, obtained in non-human species). These antibodies are a type of humanized antibody that is called a semi-humanized antibody. Non-human anti-5T4 antibodies that can be used to produce semi-humanized antibodies include antibodies A1, A2 and A3 described herein, as well as the H8 antibody described in PCT International Publication No. WO 98/55607 and Forsberg et al . (1997) J. Biol. Chem. 272 (19): 124430-12436, or rat monoclonal antibody described in Woods et al. (2002) Biochem. J. 366: 353-65. For example, a semi-humanized anti-5T4 antibody may comprise a heavy chain variable region corresponding to amino acids 1-119 of the sequence SEQ ID No: 49 or amino acids of the humanized variable heavy chain region of antibodies A1, A2 or A3 shown in FIGS. 9A-9C of the light chain variable region any of the sequences of SEQ ID No: 4, 8, or 12.

The constant regions of hybrid and humanized anti-5T4 antibodies can be obtained from the constant regions of any of IgA, IgD, IgE, IgG, IgM, any of their isotypes (e.g., IgG1, IgG2, IgG3, or IgG4 IgG isotypes), as well as mutated versions of these immunoglobulins . The choice of a human isotype or a modification of individual amino acids in this isotype can increase or eliminate the activation of immunological defense mechanisms and change the biodistribution of the antibody. See Reff et al. (2002) Cancer Control 9: 152-66. Typical constant regions suitable for the preparation of hybrid and humanized antibodies according to the present invention correspond to the sequences of SEQ ID No. s: 45-47. It is also possible to use okstantnyh areas of the light chain of the lambda of human antibodies, including variants and mutated versions. When cloning sequences encoding the constant region of an immunoglobulin, deletion of intron sequences is permissible.

Hybrid and humanized anti-5T4 antibodies can be constructed using standard techniques known in the art to which this invention pertains. For example, variable regions can be obtained by hybridizing overlapping oligonucleotides encoding the variable regions and ligating them into an expression vector containing a constant region of a human antibody. See, for example, Harlow & Lane (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and US Patent Nos. 4,196,265; 4,946,778; 5,091,513; 5,132,405; 5,260,203; 5,677,427; 5,892,019; 5,985,279; 6,054,561. Tetravalent antibodies (H ₄ L ₄ ) containing two intact tetrameric antibodies, including homodimers and heterodimers, can be prepared, for example, as described in PCT International Application No. WO 02/096948. Antibody dimers can also be prepared by introducing cysteine residue (s) into the constant region of the antibody, which promotes the formation of a disulfide bond between the chains through heterobifunctional cross-linkers (Wolff et al. (1993) Cancer Res. 53: 2560-5), or by recombinant preparation to include a double constant region (Stevenson et al. (1989) Anticancer Drug Des. 3: 219-30). Antigen-binding antibody fragments of the present invention can be prepared, for example, by expression of truncated sequences encoding antibodies, or by post-translational cleavage of full-length antibodies.

Variants of antibodies against 5T4 according to the present invention, i.e. antibodies A1, A2 and A3, as well as hybrid and humanized modifications based on them, can easily be prepared so that they contain a number of changes, substitutions, insertions and deletions. For example, antibody sequences can be optimized for codon use in a particular type of cell used to express the antibody. In order to increase the serum half-life of an antibody in blood serum, it may be, if not already present, that an antigenic determinant (epitope) of binding to the salvage receptor is introduced in heavy. antibody chain. See US Patent No. 5,739,277. Additional modifications to enhance antibody stability include IgG4 modification to replace serine 241 with a proline residue. See Angal et al. (1993) Mol. Immunol. 30: 105-108. Other useful changes include substitutions that are required to optimize the effectiveness of the conjugation of the antibody with the drug. For example, an antibody can be modified at the carboxyl end to include amino acids for drug addition, for example, one or more cysteine residues can be added. Constant regions can be modified to introduce binding sites with carbohydrates or other groups.

Variants of antibodies against 5T4 according to the present invention can be obtained using standard recombinant technologies, including targeted mutagenesis or recombination cloning. By linking genes and converting genes in transgenic non-human animals (US Patent Publication No. 2003/0017534), an extensive spectrum of anti-5T4 antibodies can be prepared, which are then tested by functional assay for the manifestation of the corresponding activities. In particular embodiments of the present invention, anti-5T4 antibody variants are prepared using affinity maturation protocols for mutating the hypervariable segments (Yang et al. (1995) J. Mol. Biol. 254: 392-403), chain shuffling (Marks et al. (1992 ) Biotechnology (NY) 10: 779-783), the use of mutator strains of E. coli (Low et al. (1996) J. Mol. Biol. 260: 359-368), DNA shuffling (Patten et al. (1997) Curr. Opin. Biotechnol. 8: 724-733), phage display (Thompson et al. (1996) J. Mol. Biol. 256: 77-88), and evolving PCR ("sexual PCR" Crameri et al. (1998 ) Nature 391: 288-291). For immunotherapeutic use, appropriate functional assay methods include specific binding to the human 5T4 antigen, internalization of the antibody, and focus on localization (localization) of the neoplasm when administered to an animal with a neoplasm, as described herein below.

The present invention further provides cells and cell lines expressing anti-5T4 antibodies of the present invention. Typical host cells include mammalian and human cells, such as CHO cells, HEK-293 cells, HeLa cells, CV-1 cells, and COS cells. Methods for forming a stable cell line following the transformation of a heterologous structure into a host cell are known in the art. Typical non-mammalian host cells include insect cells (Potter et al. (1993) Int. Rev. Immunol. 10 (2-3): 103-112). Antibodies can also be obtained in transgenic animals (Houdebine (2002) Curr. Opin. Biotechnol. 13 (6): 625-629) and in transgenic plants (Schillberg et al. (2003) Cell Mol. Life Sci. 60 (3) : 433-45).

II. Anti-5T4 antibody polypeptides and nucleic acids encoding them

The present invention also provides isolated nucleic acids encoding the variable regions of the heavy and light chains of anti-5T4 antibodies and isolated polypeptides encoded by the proposed nucleic acids. Nucleic acids and polypeptides according to the present invention include the nucleotide and amino acid sequences of the variable regions of antibodies A1, A2 and A3 and modifications based on them. Isolated nucleic acids and polypeptides can be used to produce hybrid and humanized anti-5T4 antibodies.

II.A Nucleic acids encoding antibodies against 5T4.

Nucleic acids are deoxyribonucleotides or ribonucleotides and their polymers in single-stranded, double-stranded or triplex forms. Unless specifically indicated, nucleic acids may contain known analogs of natural nucleotides that have similar properties with respect to the corresponding natural nucleic acid. Nucleic acids include genes, complementary DNA, messenger RNAs, and complementary RNAs (cDNA, mRNA and cRNA). Nucleic acids can be synthesized, or obtained from any biological source, including any organism. Typical methods for cloning nucleic acids that encode anti-5T4 antibodies are described in Examples 1 and 7.

Typical nucleic acids according to the present invention contain any nucleotide sequence selected from SEQ ID No: 1, 3, 5, 7, 9, 11 and 48. In particular, nucleic acids encoding the variable regions of the heavy chain of antibodies A1, A2 and A3 contain nucleotides 58-41 of SEQ ID NO: 1, nucleotides 55-405 of SEQ ID NO: 5, and nucleotides 58-423 of SEQ ID NO: 9, respectively, which encode variable regions of the heavy chain that have amino acid sequences corresponding to residues amino acids 20-138 The sequence SEQ ID №: 2, residues 19-135 sequence SEQ ID №: 6 and residues 20-141 sequence SEQ ID №: 10, respectively. The nucleic acid encoding the variable region of the heavy chain of the humanized antibody A1, contains nucleotides 1-358 of the sequence SEQ ID no: 48. Nucleic acids encoding the variable regions of the light chains of humanized antibodies A1, A2 and A3 contain nucleotides 61-381 of the sequence SEQ ID No: 3, nucleotides 67-390 of the sequence SEQ ID No: 7 and nucleotides 61-381 of the sequence SEQ ID No: 11, respectively; encoding the variable regions of the heavy chain containing amino acid sequences corresponding to residues 21-127 of the sequence SEQ ID No: 4, residues 23-130 of the sequence SEQ ID No: 8 and residues 21-127 of the sequence SEQ ID No: 12, respectively. Additional nucleic acids according to the present invention contain nucleotides encoding the variable regions of the humanized antibodies A1, A2 and A3 shown in figa-9C.

Nucleic acids according to the present invention may also include nucleotide sequences that are substantially identical to any one of the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, and 48, including nucleotide sequences that are at least 90 % identical to sequences encoding variable regions with any of the sequences of SEQ ID NO: 1, 3, 5, 7, 9 and 11, such as at least about 91% identical or such as at least 92% identical, such at least 93% identical, silt at least 94% identical, or at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least at least 99% identical. For example, the nucleic acids of the present invention may comprise (a) a nucleotide sequence that is at least 98% identical to the sequence of SEQ ID NO: 1 encoding the variable region; (b) a nucleotide sequence that is at least 97% identical to the sequence of SEQ ID No: 3 encoding the variable region; (c) a nucleotide sequence that is at least 98% identical to the sequence of SEQ ID No: 5 encoding the variable region; (d) a nucleotide sequence that is at least 98% identical to the sequence of SEQ ID No: 7 encoding the variable region; (e) a nucleotide sequence that is at least 99% identical to the sequence of SEQ ID No: 11 encoding the variable region; or (e) a nucleotide sequence that is at least 89% identical to the sequence of SEQ ID No: 48 encoding the variable region. The sequences are compared for maximum compliance using a sequence comparison algorithm using a full-length sequence encoding a variable region that has any of the nucleotide sequences of SEQ ID No: 1, 3, 5, 7, 9, 11, and 48, or a nucleotide sequence encoding a variable the region of humanized antibodies A1, A2 and A3, which has the nucleotide sequence shown in Fig.9A-9H as a comparison sequence, as described below, or by visual la. See also Example 1 and Table 1, and Example 7 and Table 11.

The substantially identical sequences may be polymorphic sequences, i.e. alternative sequences or alleles in a population. The difference between alleles can be only one base pair. Substantially identical sequences may also include mutagenized sequences that include silenced mutations (mutations in the non-structural gene). A mutation may include a change in one or more residues, a deletion of one or more residues, or an insertion of one or more additional residues.

Substantially identical nucleic acids, which are further defined as nucleic acids that specifically hybridize or hybridize to substantially full length with any of the sequences of SEQ ID No: 1, 3, 5, 7, 9, 11, or 48; the full length of the sequence encoding the variable region that has any of the sequences of SEQ ID No: 1, 3, 5, 7, 9, 11, or 48, or with the nucleotide sequence encoding the variable region sequence of the humanized antibodies A1, A2 and A3, which has the sequence shown in FIGS. 9A-9H under stringent hybridization conditions. In the context of nucleic acid hybridization, two sequences of compared nucleic acids can be designated as a probe and a target. The probe is a reference nucleic acid molecule, and the target is a test nucleic acid molecule, which is often found within a heterogeneous population of nucleic acid molecules. The target sequence is similar to the test sequence.

Suitable samples for hybridization studies are complementary or correspond to a sequence of at least about 14 to 40 nucleotides in length of the nucleic acid molecule of the present invention. Preferably, the probes contain from 14 to 20 nucleotides, or if desired longer, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides, or up to the full length of any of the sequences SEQ ID NO: 1, 3 5, 7, 9, 11, or 48; the full length of the sequence encoding the variable region, which has any of the sequences SEQ ID no: 1, 3, 5, 7, 9, 11, or 48; or nucleotide sequences encoding the variable region sequences of the humanized antibodies A1, A2, and A3 shown in FIGS. 9A-9C. Such fragments can be easily prepared, for example, by chemical synthesis of the fragment, through nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors to obtain recombinants.

Specific hybridization refers to the binding, duplexing, or hybridization of a molecule with only a specific nucleotide sequence under stringent conditions if that sequence is present in a complex mixture of nucleic acids (e.g., total cellular DNA and RNA). In specific hybridization, erroneous pairings between probe and target sequences may be present depending on the stringency of the hybridization conditions.

Stringent hybridization conditions and stringent washing conditions during hybridization in the context of experimental studies of nucleic acid hybridization, such as, for example, southern blot analysis (for DNA) and norsen-blot analysis (for RNA), depend on both the sequence and the environment. Longer sequences hybridize at higher temperatures. Detailed guidance on nucleic acid hybridization is given in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, Elsevier, New York, New York. Typically, very stringent hybridization and washing conditions are chosen such that the temperature is 5 ° C lower than the melting point temperature (T _m ) of an individual sequence at a given ionic strength and pH. Usually, under strict conditions, the probe will specifically hybridize only with its target subsequence, but not with other sequences.

T _m represents the temperature (at a given ionic strength and pH) at which 50% of the target sequences hybridize with the exact probe. Very stringent conditions are chosen so that they correspond to T _m for a particular probe. An example of stringent hybridization conditions for southern blot or norsen blot analyzes of complementary nucleic acids having more than 100 complementary residues is hybridization overnight in 50% formamide with 1 mg of heparin at 42 ° C. 15 min in 0.1X SSC (0.1 times sodium chloride / sodium citrate buffer) at 65 ° C are examples of very stringent washing conditions. 15 min in 0.2X SSC buffer at 65 ° C are an example of very stringent washing conditions. See Sambrook et al., Eds (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York for a description of the SSC buffer. Often, washing under very severe conditions precedes washing under less stringent conditions to eliminate the background signal of the probe. 15 min in 1X SSC at 45 ° C are an example of medium stringency washing conditions for a duplex of more than 100 nucleotides. 15 min at 4X to 6X SSC at 40 ° C are an example of reduced stringency washing conditions for a duplex of more than 100 nucleotides. For short probes (e.g., 10-50 nucleotides), stringent conditions typically include salt concentrations of less than 1 M Na ⁺ ion, typically about 0.01 M concentrations of Na ⁺ (or other salts) at pH 7 0-8.3, and a temperature of about 30 ° C. Strict conditions can also be achieved by the addition of destabilizing agents such as formamide. In general, a twofold or more difference in the signal-to-noise ratio, in comparison with a random probe in specific hybridization analyzes, indicates the presence of specific hybridization.

The following are examples of hybridization and washing conditions that can be used to identify sequences that are substantially identical to the reference nucleotide sequences of the present invention: the probe nucleotide sequence preferably hybridizes to the target nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA at 50 ° C followed by washing with 2X SSC, 0.1% SDS at 50 ° C; more preferably, probe and target hybridization in 7% sodium dodecyl sulfonate (SDS), 0.5M NaPO ₄ , 1 mM EDTA at 50 ° C followed by washing with 1X SSC, 0.1% SDS at 50 ° C; preferably, probe and target hybridization in 7% sodium dodecyl sulfonate (SDS), 0.5M NaPO ₄ , 1mM EDTA at 50 ° C followed by washing with 0.5X SSC, 0.1% SDS at 50 ° C, probe and target hybridization in 7% dodecyl sulfonate sodium (SDS), 0.5M NaPO ₄ , 1mM EDTA at 50 ° C followed by washing with 0.1X SSC, 0.1% SDS at 50 ° C; more preferably, probe and target hybridization in 7% sodium dodecyl sulfonate (SDS), 0.5M NaPO ₄ , 1mM EDTA at 50 ° C followed by washing with 0.1X SSC, 0.1% SDS at 65 ° C.

A further indicator that the two nucleic acid sequences are substantially identical is that the proteins that encode these nucleic acids are substantially identical have a common three-dimensional structure, or biological functional equivalents. The definition of these terms is given below. Nucleic acid molecules that do not hybridize to each other under stringent conditions are, however, substantially identical if their respective proteins are substantially identical. This may be the case, for example, if two nucleotide sequences contain conservatively substituted variants, like this allowed by genetic code.

Conservatively substituted variants are nucleic acid sequences that are replaced by degenerate codons, in which, in the third position of one or more selected (or all) codons, mixed base residues and / or deoxyinosine (deoxyriboxine) residues are present. See See Batzer et al. (1991) Nucleic Acids Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608; and Rossolini et al. (1994) Mol. Cell Probes 8: 91-98.

Nucleic acids according to the present invention also include nucleic acids complementary to any of the sequences of SEQ ID No: 1, 3, 5, 7, 9, 11, 48, or nucleotide sequences encoding the variable region sequences of the humanized antibodies A1, A2 and A3 shown in figa-9C; or partial or elongated variants of the sequences of SEQ ID No: 1, 3, 5, 7, 9, 11, 48, or nucleotide sequences encoding the variable region sequences of the humanized antibodies A1, A2 and A3 shown in FIGS. 9A-9C and complementary. Complementary sequences are two nucleotide sequences that contain antiparallel nucleotide sequences that can pair with each other through the formation of hydrogen bonds between base pairs. When used in this application, the term "complementary sequences" means nucleotide sequences that are largely complementary, which can be evaluated using methods for comparing identical nucleotides described below, or such sequences are defined as capable of hybridization with the considered nucleic acid segment under relatively stringent conditions hybridization such as the conditions described below. An antisense oligonucleotide is a particular example of a complementary nucleic acid segment.

A partial sequence is a nucleic acid sequence that contains a portion of a longer nucleic acid sequence. A typical partial sequence is a probe as described hereinabove or a primer. As used herein, the term primer refers to a continuous sequence containing 8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20 nucleotides, and more preferably 20-30 nucleotides of a selected nucleic acid molecule. The primers of the present invention encompass oligonucleotides of sufficient length and corresponding sequences such that polymerization is initiated on the nucleic acid molecule of the present invention.

An extended sequence contains additional nucleotides (or other similar molecules) embedded in the nucleic acid. For example, polymerase (e.g. DNA polymerase) can add sequences at the 3'-end of a nucleic acid molecule. In addition, nucleotide sequences can be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, intron sequences, additional restriction enzyme sites, multiple cloning sites, and other coding segments. Thus, the invention also provides vectors that contain the nucleic acids of the present invention, including recombinant expression vectors in which the nucleic acid of the present invention is operably linked to a functioning promoter. Being operably linked to a nucleic acid, the promoter is in functional combination with the nucleic acid such that transcription of the nucleic acid is monitored and regulated by the promoter region. The term “vectors” refers to nucleic acids capable of replication in a host cell, such as plasmids, cosmids, and viral vectors.

The nucleic acids of the present invention may be cloned, synthesized, altered, mutagenized, or a combination of such nucleic acids. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known in the art to which the present invention relates. Site-specific mutagenesis leading to base pair changes, deletions and small insertions are also known in the art. See for example: Sambrook et al. (eds.) (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Silhavy et al. (1984) Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover & Hames (1995) DNA Cloning: A Practical Approach. 2nd ed. IRL Press at Oxford University Press, Oxford / New York; Ausubel (ed.) (1995) Short Protocols in Molecular Biology. 3rd ed. Wiley, New York.

II.V. Anti-5T4 Antibody Polypeptides

The present invention also provides isolated anti-5T4 antibody polypeptides. The terms "polypeptide" and "protein" refer to a compound consisting of a single chain of amino acids linked by peptide bonds. Examples of heavy chain variable region polypeptides are residues 20-138 of the sequence SEQ ID No: 2, residues 19-135 of the sequence SEQ ID No: 6, residues 20-141 of the sequence SEQ ID No: 10, and residues 1-119 of the sequence with SEQ ID No: 49. Typical representatives of the light chain variable region polypeptides are residues 21-127 of the sequence SEQ ID No: 4, residues 23-130 of the sequence SEQ ID No: 8, and residues 21-127 of the sequence SEQ ID No: 12. Additional polypeptides according to the present invention contain amino acids of the humanized variable regions of antibodies A1, A2 and A3 shown in figa-9C.

Additional polypeptides according to the present invention include polypeptides of the variable regions of the heavy chain and light chain, which are substantially similar to the proposed anti-5T4 antibody polypeptides, such as at least about 90% identical to the variable region, which has a sequence selected from SEQ ID Nos: 2, 4, 6, 8, 10, 12, and 49, for example, are at least about 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% dentichny, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical. The sequences are compared for maximum compliance using the sequence comparison algorithm, using any full-sized sequence selected from SEQ ID No: 2, 4, 6, 8, 10, 12, 49, or from the variable region sequences of the humanized antibodies A1, A2 or A3 shown in figa-9C as the sequence of the request, or the sequence of the variable region of these antibodies, or through visual control. The invention further encompasses polypeptides encoded by any of the nucleic acids provided herein.

For example, representative polypeptides of the present invention include: (a) polypeptides having an amino acid sequence that is at least 85% identical to residues 20-138 of the sequence SEQ ID No: 2; (b) polypeptides having an amino acid sequence that is at least 94% similar to residues 21-127 of the sequence SEQ ID No: 4; (c) polypeptides having an amino acid sequence that is at least 86% similar to residues 19-135 of the sequence SEQ ID No: 6; (g) polypeptides having an amino acid sequence that is at least 96% similar to residues 23-130 of the sequence SEQ ID No: 8; (e) polypeptides having an amino acid sequence that is at least 91% similar to residues 20-141 of the sequence SEQ ID No: 10; (e) polypeptides having an amino acid sequence that is at least 98% similar to residues 21-127 of the sequence SEQ ID No: 12; and (g) polypeptides having an amino acid sequence that is at least 90% similar to residues 1-119 of the sequence SEQ ID No: 49. See Example 1 and Table 2, and Example 7 and Table 11.

The polypeptides of the present invention may contain naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof. Polypeptides may include amino acids in both the L-form and the D-form.

Typical non-genetically encoded amino acids include, but are not limited to, 2-aminoadipic acid, 3-aminoadipic acid, (3-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid (piperidic acid); 6-aminocaproic acid, 2-aminoheptane; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosin; 2,2'-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N-ethylsparagin; hydroxyl hydrolysine; ; 3-hydro iprolin; 4-hydroxyproline, isodesmosine, alloisoleucine; N-metilgitsin (sarcosine); N-metilizoleytsin; N-methylvaline, norvaline, norleucine, and ornithine.

Typical amino acid derivatives include, for example, molecules in which the three amino groups have been modified to form amine hydrochlorides, p-toluenesulfonate groups, carbobenzoxy groups, t-butyl carboxyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups can be modified to form salts, methyl or ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be modified to form O-acyl or O-alkyl derivatives. The imidazole nitrogen in histidine can be modified to form N-benzylhistidine.

The present invention also provides fragments of anti-5T4 antibody polypeptides of the present invention, for example, fragments making up the 5T4 antigen binding site. Also provided are polypeptide sequences that are longer than the disclosed sequences. For example, one or more amino acids may be added at the N-terminus or C-terminus of an antibody polypeptide. Such additional amino acids can be used in a number of applications, including, but not limited to, purification purposes. Methods for producing elongated proteins are known in the art.

The anti-5T4 antibody polypeptides of the present invention include amino acid-containing polypeptides that are conservatively substituted variants of any of the sequences of SEQ ID No: 2, 4, 6, 8, 10, 12, or 49. The term “conservatively substituted version” refers to polypeptides, in which one or more amino acid residues has been conservatively substituted with a functionally similar residue.

Examples of conservative substituents include the substitution of one of the non-polar (hydrophobic) residues, such as, for example, isoleucine, valine, leucine or methionine with another; substitution of one polar (hydrophilic) substituent with another, such as arginine with lysine, glutamine with asparagine, glycine with serine; substitution of one basic substituent such as, for example, lysine, arginine or histidine with another; or substitution of one acidic residue, such as aspartic acid or glutamic acid, with another acid.

The isolated polypeptides of the present invention can be purified and characterized by a number of standard methods known to the skilled person. See, for example: Schroder & Lubke (1965) The Peptides. Academic Press, New York; Bodanszky (1993) Principles of Peptide Synthesis. 2nd rev. ed. Springer-Verlag, Berlin / New York; Ausubel (ed.) (1995) Short Protocols in Molecular Biology, 3rd ed. Wiley, New York.

II.C. Comparison of nucleotide amino acid sequences

The terms "identical" and "percent identity" in the context of two or more nucleotide or protein sequences, refer to two or more sequences or partial sequences that are the same or have a certain percentage of amino acid or nucleotide residues that are the same when compared and overlapped with the maximum match, as for example, measured according to one of the sequence comparison algorithms described in this application or visually.

The term "substantially identical" with respect to the nucleotide or protein sequence means that the particular sequence differs from the natural sequence in one or more deletions, substitutions, additions, the combined effect of which does not affect the biological function of the anti-5T4 antibody polypeptide or corresponding nucleic acid.

When comparing two or more sequences, usually one sequence serves as a reference sequence with which one or more of the analyzed sequences is compared. When using the sequence comparison algorithm, the analyzed and reference sequences are introduced into the computer program, if necessary, the coordinates of the partial sequence are indicated and program parameters are selected for the sequence comparison algorithm. The sequence comparison algorithm then calculates the percent sequence identity based on the selected program parameters for the designated analyzed sequence (s) relative to the reference sequence.

Optimal alignment of sequences for comparison can be carried out, for example, by the Smith & Waterman (1981) Adv local homology algorithm according to Smith and Waterman. Appl. Math 2: 482-489, Niedermann and Wünsch homology alignment algorithm Needleman & Wunsch (1970) J. Mol. Biol. 48: 443-453, by the method of finding the likeness of Pearson and Lipman Pearson & Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444-2448, by computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wisconsin), or visually. See Ausubel (ed.) (1995) Short Protocols in Molecular Biology. 3rd ed. Wiley, New York.

The preferred algorithm for determining percent sequence identity is the BLAST algorithm described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Programs for conducting BLAST analyzes are publicly available through the National Center for Biotechnological Information (website address www.ncbi.nlm.nih.gov/).

The parameters of the BLAST algorithm determine the sensitivity and speed of alignment. When comparing two nucleotide sequences, the default BLAST parameters are set to W = 11 (word length) and E = 10 (mathematical expectation) and also include a low complexity filter to mask residues in the test sequence that have low compositional complexity. When comparing two amino acid sequences, the default BLAST parameters are set to W = 3 (word length) and E = 10 (mathematical expectation), use the BLOSUM62 quantification matrix, the price of the gap (gift) = 11 and the increase in the gap = 1, and use the filter low complexity for masking residues in the test sequence that have low compositional complexity. See Example 1.

III. Anti-5T4 antibody conjugates with drug

The present invention further provides drug antibody conjugates comprising an anti-5T4 antibody of the present invention. Methods for preparing conjugates of an antibody with a drug are also provided, according to which the drug is directly or indirectly associated with the antibody. Antibody drug conjugates have the general formula 5T4Ab (-XW) _m .

Wherein:

5T4Ab is a fragment of an anti-5T4 antibody as described herein;

X is a linker that includes the product of any reactive group that can react with an anti-5T4 antibody or fragment of said antibody.

W is a drug;

m represents the average load for the purified conjugation product (for example, such that the drug contains about 3-10% conjugate by weight);

(-XW) _m - is a derivative of the drug.

Methods for preparing conjugates of an antibody with a drug of the present invention are also provided. As one example, an antibody-drug conjugate of the formula 5T4Ab (-XW) _m can be prepared by: (a) adding a derivative drug to an anti-5T4 antibody, wherein the ratio of drug to anti-5T4 antibody is 3-10 weight .%; (b) thermostating the drug derivative and anti-5T4 antibody in a non-nucleophilic, protein-compatible, buffer solution having a pH value of 7-9 to obtain an antibody-drug conjugate, the solution further comprising (i) a suitable organic cosolvent, and (ii) and one or more additives containing at least one bile acid or a salt of this acid, while holding in a thermostat is carried out at temperatures from 30 ° C to 35 ° C for a period of time from 15 minutes to 24 hours owls; and (c) chromatographic treatment of the conjugate obtained in step (b) to separate the antibody conjugates from the drug from the unconjugated anti-5T4 antibody, drug derivative and aggregated conjugates when loaded within 3-10 wt.% of the drug and low conjugated fraction.

III.A. Medicines

A medicament is any compound that has biological activity or detectable activity, for example, therapeutic agents, detectable labels, binding agents, etc., and prodrugs that are metabolized to the active agent in vivo. The drug may also be a derivative of the drug in which functional substituents are introduced into the drug to conjugate with the antibody of the present invention. Typically, these types of conjugates are called immunoconjugates.

Therapeutic agents are compositions that can be used if necessary for the treatment or prevention of a subject's conditions. The therapeutic agents used in the invention include anticancer agents, those. substances exhibiting anticancer activity in 5T4-expressing cells, such as squamous / adenomatous lung carcinoma (non-small cell lung carcinoma), invasive breast carcinoma, colorectal carcinoma, gastric cancer, squamous carcinoma of the uterus carcinoma, invasive carcinoma , ovarian cancer, squamous cancer of the bladder, and choriocarcinoma.

Typical therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulatory agents, antivascular agents, anti-prophylactic agents, proapoptotic agents, and cytostatic and cytolytic enzymes (e.g. ribonuclease). A medicament may also include a therapeutic nucleic acid, such as a gene encoding an immunomodulatory agent, an antivascular agent, an anti-proliferative agent, or a pro-apoptotic agent. These drug descriptions are not mutually exclusive, and thus a therapeutic agent can be described by one or more of the above definitions. For example, selected radioisotopes are also cytotoxins. Therapeutic agents can be used in the form of pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Generally, conjugates having radioisotopes as a medicine are called radioimmunoconjugates, and conjugates comprising a chemotherapeutic agent as a drug are called chemoimmunoconjugates.

Examples of suitable drugs for use in immunoconjugates include taxanes, maytansines, SS-1065 and diocarmycins, calicheamicins and other enedins, and auristatins. Other examples include antifolates, vinca alkaloids (Vinca L), and anthracyclines. Plant oxins, other biologically active proteins, enzymes (i.e. ADEPT), radioisotopes, photosensitizers (i.e. for photodynamic therapy) can also be used in immunoconjugates. Additionally, conjugates can be prepared using secondary carriers as a cytotoxic agent, such as, for example, liposomes or polymers.

The term cytotoxin usually refers to an agent that inhibits or prevents the function of cells and / or leads to cell destruction. Typical cytotoxins include antibiotics, tubilin polymerization inhibitors, alkylating agents that bind to DNA and destroy it, and agents that interfere with protein synthesis or the function of essential cell proteins such as protein kinases, phosphatases, topoisomerases, enzymes and cyclins. Typical cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaryl, pitarubicin, valrubicin, cytarabine, gemcitabine, trinfinincyrindincin, trinfinincinidin, trinfinincinidin, trinfinrincytinurin, dacinfinridin, dacinfinridin, dacinfinridin, , kepecitabine, cladribine, decitabine, phloxuridine, fludarabine, goherotin, pyromycin, tegafur, thiazofurin, adriamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, mecloricin min, prednisone, procarbazine, methotrexate, fluorouracils, etoposide, taxol, taxol analogs, platinum such as cis-platinum and carboplatin, mitomycin, thiotepa, taxanes, vincrristine, daunorubicin, epirubicin, actinomycin azinomycin, autramycin, autramycin azinomycin, autramycin, autramycin , idarubicins, dolastatins / auristatins, hemiasterlins, esperamycins and maytansinoids.

In particular embodiments, the cytotoxin is an antibiotic such as calicheamicin, also called LL-E33288 complex, for example, gamma-calicheamicin (γ ₁ ) or N-acetyl gamma-calicheamicin. See US Patent No. 4,970,198. Additional examples of calicheamicins suitable for the preparation of conjugates of antibodies with the drug according to the present invention are proposed in US Patent Nos. 4,671,958; 5,053,394; 5,037,651; 5,079,233; and 5,108,912, which are fully incorporated into this description by reference. These compounds contain methyl trisulfide, which can be reacted with the corresponding thiols to form disulfides, while simultaneously introducing a functional group such as. for example, hydrazide or another functional group that is used to conjugate calicheamicin with an anti-5T4 antibody. You can also use disulfide analogues of calicheamicin, for example, analogues shown in US Patent Nos. 5,606,040 and 5,770,710, which are combined in this document in its entirety.

For use in radiotherapy, an anti-5T4 antibody of the present invention may contain high activity radioisotopes. The isotope can be directly linked to the antibody, for example, through a cysteine residue present in the antibody, or a chelator can be used as an intermediate in the binding of the antibody and the radioisotope. Radioisotopes suitable for radiotherapy include, but are not limited to, α emitters, β emitters, and Auger electrons. Suitable radioisotopes for use in diagnostics include positron emitters and gamma emitters. The anti-5T4 antibody of the present invention can further be iodinated, for example, at the tyrosine residue of the antibody in order to improve detection or for the therapeutic effect of the antibody.

Typical radioisotopes that can be conjugated with an anti-5T4 antibody include ¹⁸ fluorine, ⁶⁴ copper, ⁶⁵ copper, ⁶⁷ gallium, ⁶⁸ gallium, ⁷⁷ bromine, ^80m bromine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ^99m technetium, ¹⁰⁷ mercury, ²⁰³ mercury, ¹²³ mercury, ¹²⁴ mercury, ¹²⁵ mercury, ¹²⁶ mercury, ¹³¹ mercury, ¹³³ mercury, ¹¹¹ indium, ¹¹³ indium, ^99m rhenium, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ^121m tellurium, ⁹⁹ technetium, ^122m tellurium, ^125m tellurium, ¹⁶⁵ thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium, ⁹⁰ yttrium, and their nitride and oxide derivatives. Other suitable radioisotopes include alpha emitters, such as ²¹³ bismuth, ²¹³ lead, and ²²⁵ actinium.

Antibody drug conjugates of the present invention may include immunomodulators, i.e. agents that elicit an immune response, including humoral immune responses (e.g., production of antigen-specific antibodies) and cell-mediated immune responses (e.g., lymphocyte proliferation). Typical immunomodulatory agents include cytokines, xanthines, interleukins, interferons, and growth factors (e.g. TNF, CSF, GM-CSF and G-CSF), and hormones such as estrogen (diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN® ( fluoxymesterone)), progestins (MEGACE® (megesterol acetate), PROVERA® (medroxyprogesterone acetate)), and corticosteroids (prednisone, dexamethasone, hydrocortisone).

The immunomodulatory agents used in the present invention also include anti-hormones that block the action of hormones on tumors, and immunosuppressive agents that provide cytokine formation, reduce the expression of autoantigen, or mask histocompatible antigens. Typical anti-hormones include antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazoles, 4-hydroxy tamoxifen, trioxifene, keoxifen, LY 117018, onapston, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolin, and goserelin; and the adrenal cortex antogormones. Typical immunosuppressants include 2-amino-6-aryl-5-substituted pyrimidines, azathiopurin, cyclophosphamide, bromocriptine, danazole, dapzone, glutaraldehyde, anti-idiopathic antibodies for histocompatible antigens and histocompatible fragments, cyclosporidin, such as cyclosporin, cytosporin and receptors (e.g., anti-interferon antibodies, anti-IL10 antibodies, anti-TNFα antibodies, anti-IL2 antibodies), steptokinase, TGFβ, rapamycin, T-cell receptor, T-cell receptor fragments, and receptor antibodies ora T cells.

Additional drugs used in the invention include anti-angiogenic agents that inhibit blood vessel formation, e.g., farnesyl transferase inhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroid sulfatase inhibitors (e.g., 2-methoxyestradiol bissulfamate), 2-methoxyestradiol bissulfamate) (2) interleukin-24, thrombospondin, metallospondin proteins, interferon class I, interleukin 12, protamine, angiostatin, laminin, endostatin, and prolactin fragments.

Anti-proliferative agents and proapoptotic agents include PPAR-gamma activators (e.g., cyclopentenone prostoglandins (cyPGs)), retinoids, triterpinoids (e.g., cycloartan, lupane, urzan, oleanan, Fridelan, dammaran, cucurbitin, and limonoid triteritenoids) epidermal growth (e.g., HER4), rampamycin, CALCITRIOL® (1,25-dihydroxycholecalciferol (Vitamin D)), aromatase inhibitors (FEMARA® (letrozone)), telomerase inhibitors, chelate complexes of iron (e.g., theosemicarbazone 3-aminopyridine carboxaldehyde ida (Triapin)), apoptin (viral protein 3 - VP3 of chicken anemia virus), Bcl-2 and Bcl-X (L) inhibitors, TNF-alpha, FAS ligand, TNF-dependent apoptosis inducing ligand (TRAIL / Apo2L), activators TNF-alpha / FAS ligand / TNF-dependent apoptosis inducing ligand (TRAIL / Apo2L) signaling, and PI3K-Akt inhibitors of the survival signal pathway (e.g., UCN-01 and geldanamycin).

Typical chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates, for example, bisulfan, improsulfan and piposulfan; azithidines, for example, benzodopa, carboxwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triaethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trithiomelamine; nitrogen mustards, for example, chlorambucil, chlorofazine, chlorophosphamide, estramustine, ifosfamide, mechioretamine, mechioretamine oxides hydrochloride, melphalan, novembicin, phenesterol, prednimustine, trophostarnide, uracil mustard oil; nitrosoureas, for example, carmustine, chlorozotoxin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, for example, aclacinomusines, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazoin-oxorubicoriboricin-5-oxoriboricin-5-oxoriboricin-5-oxoriboricin-5-oxoriboricin-5-oxoriboricin-oxiboricin-oxiboricin-oxiboricin-5-oxiboricin-oxiboricin idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, zinostatin, zorubicin; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU); folic acid analogues, for example, denopterin, methotrexate, pteropterin, trimexate; purine analogues, for example, fludarabine, 6-mercaptopurine, triampirin, thioguanidine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyuridine, enocytabine, phloxuridine, 5-EU; androgens, for example, calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; anti-adrenal agents, for example, arginoglutetimidine, mitotan, trilostane, folic acid supplementers, for example, frolinic acid; Aceglaton; aldophospharnide glycoside; arninolevulinic acid; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcin, diaziquon; elfornithine; elliptinum acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophyllic acid; 2-ethyl hydrazide; procarbazine; razoxane; sisofiran; spiro germanium; tenuazonic acid; triaziquion; 2,2 ', 2 ”trichlorotriethylamine; turretan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; galatosin; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, for example, paclitaxel (TAXOL®, Bristol-Mayer Squibb Oncology from Princeton, NJ) and doxetaxel (TAXOTERE®, Ron-Pulenck Rohrer from Anthony, France); chiorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, for example, cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitocin C; mitoxantrone; vincristine; vinorelbine; Navelbin; novantron; teniposide; daunomycin; Ainopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinolic acid; esperamycins; and capecitabine.

Additional therapeutic agents that can be conjugated to anti-5T4 antibodies and which can be used in accordance with the therapeutic methods of the present invention include photosensitizing agents (US Patent Application No. 2002/0197262 and US Patent No. 5,952,329) for photodynamic therapy; magnetic particles for thermotherapy (US Patent Application No. 2003/0032995); binding agents, for example, proteins, ligands, cell adhesion ligands, etc., and prodrugs, such as phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, containing substituted phenoxyacetamide prodrugs, or 5-fluorocytosine and other 5-fluoruridine prodrugs that can be converted into a more active drug free of cytotoxicity

For diagnostic methods using anti-5T4 antibodies, the drug may contain a detectable label used to determine the presence of 5T4-expressing cells in vitro or in vivo. Radioisotopes that are detectable in vivo, such as labels that can be detected using scintigraphy, nuclear magnetic tomography, or ultrasound, can be used in clinical diagnostic applications. Suitable scintigraphic labels include positron emitters and gamma emitters. Typical contrast agents for nuclear magnetic resonance imaging are paramagnetic or superparamagnetic ions (e.g., iron, copper, manganese, chromium, erbium, europium, dysprosium, holmium and gadolinium), iron oxide particles, and water-soluble contrast agents. For ultrasonic detection, gases or liquids can be introduced into the pores of the inorganic particles, which are then released as microbubble contrast agents. For in vitro detection, suitable detectable labels include fluorophores, detectable epitopes or binding agents, and radioactive labels.

III.V. Linker molecules.

The drug is conjugated to the hybrid or humanized anti-5T4 antibodies of the present invention, either directly or indirectly through a linker molecule. The linker molecule may be stable or capable of hydrolysis, whereby it is released upon entry into the cell. The main mechanisms by which a drug is cleaved from an antibody include hydrolysis at acidic pHs of liposomes (hydrazone, acetals, and cis-aconitate-like amides), peptide cleavage by liposomal enzymes (cathepsins and other liposomal enzymes), and reduction of disulfides. As a result of the various cleavage mechanisms listed above, the binding mechanisms of the drug to the antibody also vary widely and any suitable linker can be used. Preferably, the conjugation method results in a sample with a minimal fraction with a low degree of conjugation (a fraction of a substantially unconjugated antibody), i.e. less than 10%.

One example of a suitable conjugation technique is based on the conjugation of hydrazides and other nucleophiles with aldehydes generated by oxidation of carbohydrates, which naturally occur in antibodies. Hydrazone-containing conjugates can be obtained by introducing carbonyl groups, this provides the desired properties in the release of the drug substance. Conjugates can also be prepared with a linker that has a disulfide group at one end of the molecule, an alkyl chain at the center, and a hydrazine derivative at the other end of the molecule. Anthracyclines are one example of cytotoxins that can be conjugated with antibodies using this technology.

Linkers containing functional groups other than hydrazones exhibit the ability to cleave lysosomes in an acidic environment. For example, conjugates can be obtained from thiol-reactive linkers that contain a group other than hydrazone that is capable of cleaving within the cell, for example, esters, amides and ketals / acetals. Camptothecin is one of the cytotoxic agents that can be conjugated using these linkers. You can also use ketals obtained from 5-7-membered cyclic ketones, in which one of the oxygen atoms is connected to a cytotoxic agent and the other with a linker for attaching antibodies. Anthracyclines are also an example of a suitable cytotoxin for use with these linkers.

Cis aconitates, which contain a carboxylic acid residue adjacent to the amide group, are another example of a pH class of sensitive linkers. Carboxylic acid accelerates the hydrolysis of amide in the acidic environment of lysosomes. Linkers that accelerate the rate of hydrolysis in a similar manner using some other types of structures can also be used. Maytansinoids are an example of a cytotoxin that can be conjugated to linkers attached at C-9.

Another possible release method for drug conjugates is enzymatic hydrolysis of peptides with liposome enzymes. As an example, a peptide is coupled via an amide bond to a para-aminobenzyl alcohol and then a carbamate or carbonate bond is created between the benzyl alcohol and the cytotoxic agent. Cleavage of the peptides causes the aminobenzyl carbamate or carbonate to decompose or self-decompose. Cytoxic agents illustrating this strategy include anthracyclines, taxanes, mitomycin C, and auristatins. In one example, phenol can also be released during carbamate disruption of the linker. In another embodiment, disulfide reduction is used to initiate the decomposition of para-mercaptobenzyl carbamate or carbonate.

Many of the cytotoxic antibodies conjugated to antibodies have little, if any, solubility in water, and this may limit the loading of the drug onto the conjugate due to aggregation of the conjugate. One approach to avoid this difficulty is to introduce solubility enhancing groups into the linker. You can use conjugates with a linker containing PEG (polyethylene glycol), and you can use a dipeptide comprising PEG (polyethylene glycol), diacid thiol acid, or maleimide acid coupled to an antibody, a dipeptide spacer region, and an amide bond with an amine in anthracycline or duoc analogues. Another example is a conjugate prepared with a PEG-containing linker that is linked via a disulfide bond to a cytotoxic agent and linked via an amide bond to an antibody. Approaches that include PEG groups may be useful in overcoming aggregation and drug loading restrictions.

Typical linkers that are preferred for the preparation of antibody-drug conjugates of the present invention include linkers of the formula:

(CO-Alk ¹ -Sp ¹ -Ar-Sp ² -Alk ² -C (Z ¹ ) = Q-Sp)

wherein,

Alk ¹ and Alk ² independently represent a bond or a branched or unbranched (C ₁ -C ₁₀ ) olefin chain;

Sp ¹ is a bond, -S-, -O-, -CONH-, -NHCO-, -NR'-, -N (CH ₂ CH ₂ ) ₂ N-, or -X-Ar'-Y- (CH ₂ ) _n -Z in which X, Y, and Z independently represent a bond, -NR'-, -S-, or -O-, with the restriction that if n = 0, then at least one Y and Z must be a bond and Ar 'is 1,2-, 1,3-, or 1,4-phenylene, which may be substituted by one, two or three groups of (C ₁ -C ₅ ) alkyl, (C ₁ -C ₄ ) alkoxy, (C ₁ -C ₄ ) thioalkyl, halogen, nitro group, -COOR ', -CONHR', - (CH ₂ ) _n COOR ', -S (CH ₂ ) _n COOR', -O (CH ₂ ) _n CONHR ', or -S (CH ₂ ) _n CONHR', with the restriction that if Alk ¹ is a bond, then Sp ¹ is a bond;

n is an integer from 0 to 5;

R 'represents a branched or unbranched (C ₁ -C ₅ ) chain, possibly substituted by one or two groups-OH, (C ₁ -C ₄ ) alkoxy, (C ₁ -C ₄ ) thioalkoxy, halogen, nitro-group, ( C ₁ -C ₃ ) dialkylamino, or (C ₁ -C ₃ ) trialkylammonium-A ^- , in which A ^- is a pharmaceutically acceptable anion, an addition salt;

Ar represents 1,2-, 1,3-, or 1,4-phenylene, which as substituents may have one, two or three groups (C ₁ -C ₆ ) alkyl, (C ₁ -C ₅ ) alkoxy, (C ₁ -C ₄ ) thioalkoxy, halogen, nitro group, -COOR ', -CONHR', -O (CH ₂ ) _n COOR ', -S (CH ₂ ) _n COOR', -O (CH ₂ ) _n CONHR ' , or —S (CH ₂ ) _n CONHR ′ wherein n and R ′ are as defined above or 1,2-, 1,3-, 1,4-, 1,5-, 1,6- , 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthalene or

,

,

each naphthalene or phenothiazine may have one, two, three or four substituents selected from: (C ₁ -C ₆ ) alkyl, (C ₁ -C ₅ ) alkoxy, (C ₁ -C ₄ ) thioalkoxy, halogen, nitro a group, -COOR ', -CONHR', -O (CH ₂ ) _n COOR ', -S (CH ₂ ) _n COOR', or -S (CH ₂ ) _n CONHR ', in which n and R' are such as defined above, provided that if Ar is phenothiazine, Sp ¹ is a bond connected only to nitrogen;

Sp ² is a bond, —S—, or —O—, provided that if Alk ² is a bond, then Sp ² is a bond;

Z ¹ represents H, (C ₁ -C ₅ ) alkyl, phenyl optionally substituted with one, two or three groups (C ₁ -C ₅ ) alkyl (C ₁ -C ₅ ) alkoxy, (C ₁ -C ₄ ) thioalkoxy , halogen, nitro, -COOR ', -ONHR', -O (CH ₂ ) _n COOR ', -S (CH ₂ ) _n COOR', -O (CH ₂ ) _n CONHR ', or -S (CH ₂ ) _n CONH R ′ in which n and R ′ are as defined above;

Sp is a straight or branched chain disubstituted or trisubstituted (C ₁ -C ₁₈ ) radical, or a disubstituted or trisubstituted aryl or heteroaryl radical, or a disubstituted or trisubstituted (C ₃ -C ₁₈ ) cycloalkyl or heterocycloalkyl radical, or a disubstituted (C tri ₂ -C ₁₈₎ unsaturated alkyl radical, wherein heteroaryl is preferably furyl, thienyl, N-metilpirrolilom, pyridinyl, N-metilimidazolilom, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-metilkarbazoilom, aminok marinilla or fenazinilom and wherein Sp is a trivalent radical, Sp can be additionally substituted by lower (C ₁ -C ₅₎ dialkylamino, lower (C ₁ -C ₅₎ alkoxy, hydroxy, or lower (C ₁ -C ₅₎ alkylthio groups ; and

Q is = NHNCO-, = NHNCS-, = NHNCONH-, = NHNCSNH-, or = NHO-.

Preferably, Alk ¹ is a branched or unbranched (C ₁ -C ₁₀ ) olefin chain; Sp 'is a bond, -S-, -O-, -CONH-, -NHCO-, or -NR', in which R 'are as defined above, with the restriction that if Alk ¹ is a bond, then Sp ¹ is a bond;

Ar is 1,2-, 1,3-, or 1,4-phenylene, which may be substituted with one, two, or three groups (C_one-C₆) alkyl, (C_one-C₅) alkoxy, (C_one-C_four) thioalkoxy, halogen, nitro group, —COOR ′, —CONHR ′, —O (CH₂)_nCOOR ', -S (CH₂)_nCOOR ', -O (CH₂)_nCONHR ', or -S (CH₂)_nCONHR 'in which n and R' are as defined above or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3- , 2,6-, or 2,7-naphthalidene, each of which may have one, two, three or four groups as substituents (C_one-C₆) alkyl, (C_one-C₅) alkoxy, (C_one-C_four) thioalkoxy, halogen, nitro group, —COOR ′, CONHR ′, —O (CH₂)_nCOOR ', -S (CH₂)_nCOOR ', -O (CH₂)_nCONHR ', or -S (CH₂)_nCONHR '.

Z ¹ represents (C ₁ -C ₅ ) alkyl or phenyl optionally substituted with one, two or three groups (C ₁ -C ₅ ) alkyl (C ₁ -C ₅ ) alkoxy, (C ₁ -C ₄ ) thioalkoxy, halogen , nitro group, -COOR ', -ONHR', -O (CH ₂ ) _n COOR ', -S (CH ₂ ) _n COOR', -O (CH ₂ ) _n CONHR ', or -S (CH ₂ ) _n CONH R '; Alk ² and Sp ² together represent a bond; and Sp and Q are as defined above.

US patent No. 5,773,001, fully included in the description of this application, discloses linkers that can be used with nucleophilic drugs, in particular hydrazides and related nucleophiles made from calicheamicins. These linkers are especially useful in those cases in which the best activity is obtained if the chemical bond formed between the drug and the linker can undergo hydrolysis. These linkers contain two functional groups, including (1) a group for interacting with an antibody (e.g., carboxylic acid), and (2) a carbonyl group (e.g., an aldehyde or ketone) for reacting with the drug. The carbonyl group may react with the hydrazide group of the drug to form a hydrazone bond. This bond is capable of hydrolysis, which allows the therapeutic agent to be released from the conjugate after binding to target cells.

As one example, an anti-5T4 antibody can be conjugated to a cytotoxic drug by: (1) adding a derivative of a cytotoxic drug to an anti-5T4 antibody, wherein the cytotoxic drug is taken in a ratio of 4.5-11 wt.% To the protein carrier; (2) thermostating a derivative of a cytotoxic drug and anti-5T4 antibody in a non-nucleophilic, protein-compatible, buffered solution having a pH in the range of 7 to 9, leading to the formation of a monomeric antibody-drug conjugate, the solution further comprising (a) a suitable organic cosolvent, and (b) an additive containing at least one C ₆ -C ₁₈ carboxylic acid or its salt, wherein said thermostating is carried out at a temperature of from 30 ° C to 35 ° C for a period time, which is selected in the range from 15 minutes to 24 hours; and (3) chromatographic separation of the conjugate obtained in step (2), with the aim of separating monomeric conjugates loaded with a cytotoxic drug in the range from 3 wt.% to 10 wt.% and a low conjugated fraction below 10% of the unconjugated antibody derived from a cytotoxic drug agents, and aggregated conjugates.

Chromatographic separation in step (3) may include processes such as size exclusion chromatography, ultrafiltration / diafiltration, high pressure liquid chromatography (HPLC), rapid liquid chromatography of proteins (FPLC), chromatography on Sephacryl S-200. Chromatographic separation can also be followed by hydrophobic chromatography using phenyl Sepharose 6 Fast Flow chromatography media, butyl Sepharose 6 Fast Flow chromatography media, octyl Sepharose 6 Fast Flow chromatography media, Toyopearl Ether-650M chromatography media, Macro-Prep methyl hydrophobic media chromatography or Macro-Prep t-butyl hydrophobic chromatography media.

Typical methods for preparing anti-5T4 antibody conjugates with a drug include those described for the preparation of CMC-554 according to U.S. Patent Application No. 2004-082764A1 and U.S. Patent Application No. 10 / 699,874, which is fully incorporated herein. Conjugation can be carried out using the following conditions: 10 mg / ml antibody, 8.5 wt.% Of calicheamicin derivative, 37.5 mM sodium decanoate solution, 9 vol.% Ethanol, 50 mM HEPES (N- (2-hydroxyethyl) piperazine-N '- ( 4-butanesulfonic acid)), pH 8.5, 32 ° C, 1 hour. Hydrophobic chromatography can be carried out using FF butyl Sepharose resin, a buffer for loading with 0.65 M potassium phosphate, a buffer for washing with 0.49 M potassium phosphate and an elution buffer with 4 mM potassium phosphate. Buffer exchange may be accompanied by size exclusion chromatography (SEC), ultrafiltration / diafiltration, or other suitable means. The formulation of the conjugate of the antibody with the drug can be compiled in 1.5% Dextran-40, 0.9% sucrose, 0.01% TWEEN®-80, 20 mm Tris / 50 mm NaCl (Tris - hydroxymethylaminomethane, then use the reduction Tris, pH 8.0). It is also possible to use a solution with an alternative formula containing 5% sucrose, 0.01% TWEEN®-80, 20 mM Tris / 50 mM NaCl, pH 8.0. Lyophilization cycles are used based on the developed formulation. The concentration of the formulation may be 0.5 mg conjugate / ml. Each ampoule contains 1 mg of conjugate, i.e. 2 ml filling. If necessary, you can prepare other filling volumes, for example, 5 ml filling.

Other typical methods include the methods described for CMD-193, which are also described in US Patent Application No. 20060002942. Conjugation can be carried out using the following conditions: 10 mg / ml antibody, 7 wt.% Calicheamicin derivative, 10 mM deoxycholate solution, 50 mM HEPES (N- (2-hydroxyethyl) piperazine-N '- (4-butanesulfonic acid)), 9 vol.% ethanol, pH 8.2, 32 ° C, 1 hour. The reaction mixture can be diluted 10 times with a 0.65M potassium phosphate solution with pH 8.56, and hydrophobic chromatography can be performed using butyl sepharose FF resin, 0.60M potassium phosphate buffer for loading and washing, and 20 mM Tris / 50 mM NaCl elution buffer . Buffer exchange may be accompanied by ultrafiltration / diafiltration with an artificial cellulose membrane. The conjugate can be diafiltered against 20 mM Tris / 10 mM NaCl pH 8.0 (10 dia volumes). The formulation of the conjugate of the antibody with the drug can be prepared in 5% sucrose solution, 0.01% TWEEN®-80, 20 mm Tris / 50 mm NaCl, pH 8.0. The concentration of the conjugate mass after composing the mixture may be 1 mg / ml, and the ampoule filling may be 5 mg / ampoule, i.e. 5 ml filling, or if necessary, other filling volumes can be prepared.

In a particular embodiment of the present invention, the linker used is 4- (4-acetylphenoxy) butyric acid (AcBut). Antibody drug conjugates are prepared by reacting β-calicheamicin, γ-calicheamicin or N-acetyl of γ-calicheamicin or derivatives thereof, with 3-mercapto-3-methyl butyric acid hydrazide, AcBut linker, and the anti-5T4 antibody of the present invention . See, for example, US Patent No. 5,773,001. This linker results in conjugates that are substantially stable in the circulatory system, secreting the expected 2% NAc-gamma DMH per day, and which easily release NAc-gamma DMH in acid lysosomes. In another embodiment, antibody-drug conjugates are prepared using 3-acetylphenylacetic acid (AcPac) or 4-mercapto-4-methylvaleric acid (Amide) as linker molecules.

Typical linkers suitable for conjugating radioisotopes include diethylenetriamine pentaacetate (DTPA) -isothiocyanate, succinimidyl 6-hydrazino nicotinate hydrochloride (SHNH), and hexamethylpropyleneamine oxime (HMPAO) (Bakker et al. (0101: 15). Nucl. Al. (1990) J. Nucl. -1509, Chattopadhyay et al. (2001) Nucl. Med. Biol. 28: 741-744, Dewanjee et al. (1994) J. Nucl. Med. 35: 1054-63, Krenning et al. (1989) Lancet 1 : 242-244, Sagiuchi et al. (2001) Ann. Nucl. Med. 15: 267-270); U.S. Patent No. 6,024,938). Alternatively, a derivative of the targeting molecule can be prepared so that the radioisotope can be attached directly to it (Yoo et al. (1997) J. Nucl. Med. 38: 294-300). Methods for introducing iodine are also known in the art, typical methods can be found in the literature, for example, Krenning et al. (1989) Lancet 1: 242-4 and in Bakker et al. (1990) J. Nucl. Med. 31: 1501-9.

To further increase the number of drug molecules per antibody-drug conjugate, the drug can be conjugated to polyethylene glycol (PEG), including linear or branched polymers and polyethylene glycol monomers. The polyethylene glycol monomer has the formula - (CH ₂ CH ₂ O) -. Medicines and / or proteins can be bound directly or indirectly to polyethylene glycol, i.e. through acceptable spatial groupings, for example, Sugars. The PEG / antibody / drug composition may also include additional lipophilic and / or hydrophilic components to promote drug stability and in vivo delivery to the target site. Typical techniques for preparing PEG-containing compositions can be found, along with other sources, in US Patent Nos. 6,461,603; 6,309,633; and 5,648,095.

For example, in order to increase the amount of calicheamicin in the conjugates of an antibody with calicheamicin, the antibody is conjugated before conjugation with calicheamicin with PEG, for example using PEG-SPA, PEG-SBA, or PEG-bismaleiminimide. Antibody conjugates prepared with PEG may show reduced binding affinity for the target antigen, but are still effective due to increased drug loading. Supplements such as deoxycholate and decanoate can be used to produce antibody / calicheamicin conjugates with low levels of unconjugated antibodies and low levels of aggregation.

The hydrophobic nature of many drugs, including calicheamicins, can lead to aggregation of antibody conjugates with the drug. To obtain monomeric antibody-drug conjugates with higher drug delivery rates, yield and reduced aggregation, the conjugation reaction can be carried out in non-nucleophilic, protein-compatible, buffered solutions containing (i) propylene glycol as a co-solvent and (ii) an additive containing at least one C ₆ -C ₁₈ carboxylic acid. Suitable acids include C ₇ to C ₁₂ acids, for example, octanoic or caprylic acid or their salts. You can use other than ethylene glycol, protein compatible organic cosolvents, for example, ethylene glycol, ethanol, DMF, DMSO, etc. Some or all of the organic cosolvents are used to add the drug and the conjugation mixture. Suitable buffers for preparing antibody-drug conjugates using N-hydroxysuccinimide esters (OSu) or other relatively activated esters include saline phosphate buffer (PBS) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer). The buffers used in conjugation reactions should not contain substantially free amines and nucleophiles. In another embodiment, the conjugation reaction can be carried out in a non-nucleophilic, protein-compatible, buffered solution that contains tert-butanol without additional additives. See, for example: US Patent Nos. 5,712,374 and 5,714,586. Additional conjugation techniques for conjugate containing calicheamicin are also described in US Pat. Nos. 5,739,116 and 5,877,296.

The optimal conditions for the formation of monomeric conjugates can be determined empirically by varying the parameters of the reaction, for example, temperature, pH, administration of a calicheamicin derivative, concentration of the additive. Typical amounts of propylene glycol are in the range of 10% to 60%, for example, 10% to 40%, or about 30% of the total solution volume. Typical amounts of additives containing at least one C ₆ -C ₁₈ carboxylic acid are in the range of 20 mM to 100 mM, for example, 40 mM to 90 mM, or about 60 mM to 90 mM. The concentration of C ₆ -C ₁₈ carboxylic acid can be increased to 150-300 mm and reduce the amount of co-solvent to from 1% to 10%. In typical embodiments of the present invention, the carboxylic acid is caprylic (octanoic) acid, capric (decanoic) acid, or their corresponding salts. For example, you can use a 200 mm solution of caprylic acid with 5% propylene glycol or ethanol. The conjugation reaction can be carried out at a slightly elevated temperature (30-35 ° C) and pH (8.2-8.7). The concentration of the antibody can be in the range of 1 to 15 mg / ml, and the concentration of the calicheamicin derivative, for example, N-acetyl gamma-calicheamicin DMH AcBut OSu ester, can be in the range of 4.5% to 11% by weight of the antibody. Acceptable conditions for conjugation for other drugs can be determined by qualified specialists in this field of technology without much additional research.

III.C. Purification of antibody conjugates with drug

After the conjugation process, the monomeric conjugates can be separated from the unreacted reagents and / or aggregated forms of the conjugates by conventional methods, for example, size exclusion chromatography, hydrophobic chromatography, ion exchange chromatography, or chromatofocusing. The purified conjugates are monomeric and usually contain from 3% to 10% by weight of the drug. Antibody drug conjugates can also be purified by hydrophobic chromatography, which provides some advantages relative to size exclusion chromatography, which include (1) the ability to effectively reduce the content of the low conjugated fraction as well as the aggregated; (2) the possibility of using large volumes of reaction mass; and (3) minimal dilution of the product. Large-capacity hydrophobic chromatography medium suitable for use in large scale production includes Sepharose 6 Fast Flow phenyl chromatography medium, Sepharose 4 Fast Flow butyl chromatography medium, Sepharose 4 Fast Flow chromatography medium, Toyopearl Ether-650M chromatography medium, Macro-Prep methyl hydrophobic chromatography medium; or Macro-Prep t-butyl hydrophobic chromatography medium. Utilization / diafiltration can also be used for buffer exchange.

In a typical purification process, multiple operations are performed, including a cell removal step in a centrifuge, a Protein A affinity capture step, followed by one or two orthogonal chromatographic purification steps, a viral filtration step, and a tangential flow filtration step for concentration and formulation. The purification process preferably results in a product with less than 5% aggregation, less than 20 ppm Protein A, less than 50 ppm protein of the host cell, and a total recovery of more than 50%.

A typical anti-5T4 antibody preparation with calicheamicin contains predominantly (about 95%) conjugated antibody containing 5-7 moles of calicheamicin per mole of antibody. The conjugate was reproducibly obtained on a laboratory scale (10-200 mg). The drug load, which is expressed as μg of calicheamicin per mg of monoclonal antibody, is determined by assigning the concentration of calicheamicin (μg / ml) to the concentration of the antibody (mg / ml). These values are determined by measuring the UV absorption of the conjugate solution at 280 nm and 310 nm. It is important to note that this value is the average load and that the actual load is a quasi-Gaussian distribution centered on the average load, i.e. a certain amount of antibody is loaded to a greater extent than the average value of the load and a certain amount of antibody is loaded to a lesser extent than the average value of the load An unconjugated antibody (low-conjugated fraction), which can be determined by high-resolution analytical hydrophobic liquid chromatography, is a population of antibodies that are small degrees conjugated or unconjugated with calicheamicin. This value is a measure of the distribution of calicheamicin on the antibody and generally does not affect the dose of calicheamicin. Non-conjugated calicheamicin, the amount of which can be determined by enzyme-linked immunosorbent assay, is defined as the amount of calicheamicin, which is not conjugated to the antibody and is expressed as a percentage of calicheamicin. The quantitative determination of the drug load does not distinguish between unconjugated and conjugated calicheamicin. When quantifying the drug load, the amount of unconjugated calicheamicin is undetectable or negligible; as a result, these analyzes effectively show the amount of conjugated calicheamicin.

Analytical methods can be used to determine the release and stability of humanized 5T4 conjugates with calicheamicin. Conjugates can be assessed for identity, potency (total protein and total loading of calicheamicin), purity (unconjugated calicheamicin, low conjugated antibody, aggregate content and SDS-PAGE Reduced (polyacrylamide gel electrophoresis assay in the presence of reducing conditions), and immuno enzyme-linked immunosorbent assay for binding to the antibody). Additional analytical studies known to the person skilled in the art can be used. of the methods, it is possible to maintain intergroup correspondence between the produced batches for commercial purposes.

III.D, Pharmacokinetics of Antibody Conjugates

The pharmacokinetics of 5T4-specific immunoconjugates can be determined and compared with the pharmacokinetics of unconjugated calicheamicin in various animals. For example, this can be done after a single intravenous administration of the drug to female nude mice, male Speg-Dawley rats, and female cynomolgus monkeys (cynomolgus monkey). The pharmacokinetics of the anti-5T4 antibody are generally characterized by low clearance, low volume of distribution, and a long observed half-life in various species. It is believed that serum concentrations of unconjugated calicheamicin derivatives are below the limit of quantitation. The toxicity profile for these conjugates in a single dose toxicity study is expected to be similar to the toxicity levels obtained for other antibody / calicheamicin conjugates taken in comparable doses.

IV. Functional analyzes of characterization of antibodies against 5T4 and conjugates of the antibody with the drug

The invention further provides assay methods for characterizing the activities of anti-5T4 antibodies in vitro and in vivo, including 5T4 binding activity, internalization of cells followed by binding to 5T4 antigen present on the cell surface, and specificity for 5T4-expressing cells of the subject. When conjugated to a cytotoxin, the antibodies of the present invention may exhibit anti-cancer activity, including inhibiting the growth of 5T4-expressing cancer cells and / or the death of 5T4-expressing cells. Antibodies against 5T4 according to the present invention can exhibit one or more of the above activities.

Techniques for determining the binding of anti-5T4 antibodies to 5T4 antigen are known in the art, for example, BIACORE® measurements are shown in Example 2. Additional typical techniques include centrifugation, affinity chromatography and other immunochemistry methods. See, for example, Manson (1992) Immunochemical Protocols, Humana Press, Totowa, New Jersey, United States of America; Ishikawa (1999) Ultrasensitive and Rapid Enzyme Immunoassav, Elsevier, Amsterdam / New York. Antigen binding measurements can be performed using an isolated 5T4 antigen or 5T4 expression cells. See Example 2.

The specificity of the binding of antibodies against 5T4 can be further characterized by determining the binding epitope, i.e. identifying residues including non-contiguous residues that are involved in binding to the antigen; and / or determining residues that influence binding to the antigen. See Examples 4-5.

The internalization of anti-5T4 antibodies and drug conjugates of 5T4-expressing cells can be quantified by determining the number of antibodies or conjugates that bind to the surface of 5T4-expressing cells over time. Typical methods for determining the localization value of antibodies and antibody conjugates with a drug on the membrane are shown in Example 3.

Functional analysis methods also include methods for determining the anticancer activity of antibody-drug conjugates, for example, the ability to kill existing cancer cells, or to slow down or inhibit the growth of cancer cells. The types of cancer that are targeted by the drug antibody conjugate therapy of the present invention include both primary and metastasized tumors and carcinomas of any tissue of the subject, including carcinomas and malignant diseases of the blood, such as leukemia and lymphosarcoma.

Anti-5T4 antibodies that have anti-growth activity can eliminate 5T4-expressing cells or inhibit or reduce the proliferation of 5T4-expressing cells. Typical methods for rapidly in vitro determining inhibition of cell growth are described in Jones et al. (2001) J. Immunol. Methods 254: 85-98.

Anti-5T4 antibodies can also be capable of stimulating cell death, for example, programmed cell death characterized by nuclear DNA degradation, nuclear degeneration or condensation, loss of membrane integrity, and phagocytosis Typical biological experiments to evaluate cell activity are described in Hoves et al. (2003) Methods 31: 127-34; Peng et al. (2002) Chin. Med. Sci. J. 17: 17-21; Yasuhara et al. (2003) J. Histochem. Cytochem. 51: 873-885.

For example, to determine the cytotoxicity of antibody-drug conjugates in vitro, MDAMB435 / 5T4 cells (human breast cancer cells overexpressing human 5T4 antigen) and MDAMB435 / neo cells (control cells) are cultured in the presence of drug-conjugate conjugates or free calicheamicin, practically as described in Boghaert et al. (2004), Clin. Cancer Res. 10: 4538-4549. The cytotoxicity of each agent is given in ED50 (ng / ml), which is the amount of calicheamicin taken as a conjugate or as a free drug, which causes 50% inhibition of cell culture compared to the untreated control sample. The number of cells in the culture is determined by vital staining after treatment with the drug. See also Example 6.

The cytotoxicity of conjugates of antibodies with calicheamicin can also be assessed by the use of MDAMB435 / 5T4 and MDAMB435 / neo cells cultured in a manner that is suitable for the formation of spheroids. Cells are cultured in the presence of conjugates of antibodies with calicheamicin or free calicheamicin, and after the exposure of the drug, the sizes of each spheroid are determined. The effectiveness of each agent in suppressing spheroid growth is reported as ED50 (ng / ml), i.e. the amount of calicheamicin taken in the form of a conjugate or in the form of a free drug, which causes 50% inhibition of spheroid growth compared to the untreated control sample. See Example 6.

To evaluate the cytotoxicity of conjugates of anti-5T4 antibodies with calicheamicin in vivo, tumors are prepared in nude mice (nude mouse with nude mutation) by subcutaneous injection of MDAMB435 / 5T4 cells (overexpressing human 5T4 human antigen human breast cancer cells), NCI-H157 (non-small cell cells human lung cancer), PC14RE6 cells (non-small cell lung cancer cells), or N87 (human stomach cancer cells). Conjugates of antibodies with calicheamicin and control compounds are administered to mice having tumor cells, for example, by intraperitoneal administration after a 4-day interval with a total of 3 doses, for example, on days 1, 5 and 9. Measurable therapeutic responses include inhibition of tumor cell growth.

To further evaluate the targeting potential of anti-5T4 antibody conjugates with calicheamicin, an orthoptic model for non-small cell and small cell cancer can be used, essentially as described by Onn et al. (2003) Clin. Cancer Res. 9 (15): 5532-5539. Briefly, human lung adenocarcinoma cells (PC14RE6) are introduced into the tail vessels of “naked” mice, which then migrate to form tumors in the lung. Tumors can occur as solid nodules in the lung parenchyma and cause hemorrhagic pleural effusions containing suspended tumor cells. Control compounds and conjugates of antibodies with calicheamicin are administered to mice having tumors, for example, intraperitoneally, starting from day 6 after injection of tumor cells with a total of 3 doses administered at a 4-day interval, for example, on days 6, 10 and 14. Measurable therapeutic responses include reduced pleural effusions and increased survival.

V. Use of anti-5T4 antibodies and antibody-drug conjugates

Anti-5T4 antibodies and antibody-drug conjugates of the present invention can be used both in vitro and in vivo in applications related to 5T4-expressing cells. Types of cancers expressing 5T4 include squamous / adenomatous pulmonary carcinoma (non-small cell lung carcinoma), invasive cancer breast, colorectal carcinoma, gastric cancer, squamous cervical cancer, invasive endometrial adenocarcinoma, invasive pancreatic cancer, ovarian cancer, squamous cancer of the bladder and choriocarcinoma. 5T4 was found in high levels in carcinomas of the bronchi, breast, colon, rectum, stomach, cervix, endometrium, pancreas, ovary, chorium and seminal vesicles.

V.A. In vitro applications

The present invention provides in vitro technology for the use of antibodies against 5T4. For example, the proposed antibodies can be used either alone or in combination with cytotoxic agents or other drugs for specific binding of 5T4-positive cancer cells to isolate similar cells from a population of cells. Methods are also proposed for inducing apoptosis and / or inhibiting cell proliferation by contacting 5T4-expressing cells conjugates of an antibody to a drug containing an anti-5T4 antibody conjugated to a cytotoxin. Typical in vitro methods are provided herein above under the heading "Functional Assays for Characterization of Anti-5T4 Antibodies and Antibody-Drug Conjugates."

Antibodies against 5T4 according to the present invention can also be used to determine 5T4-positive cells in vitro, which is based on their ability to specifically bind 5T4 antigen. A method for determining 5T4-expressing cells may include: (a) preparing a biological sample containing cells; (b) contacting an anti-5T4 antibody with an in vitro biological sample; and (c) determining binding of the anti-5T4 antibody. To facilitate detection, the antibody may be conjugated to a label.

V.B. In Vivo Detection and Diagnostics

Antibodies against 5T4 according to the present invention can be used in in vivo detection methods, for example, for diagnosis, for providing intraoperative support, or for determining doses. After administration of a labeled anti-5T4 antibody to a subject, after a period of time sufficient for binding, biodistribution of antibody-bound 5T4-expressing cells can be observed. The proposed diagnostic methods can be used in combination with treatment methods. Additionally, antibodies against 5T4 according to the present invention can be used for two purposes: diagnosis and therapy.

Typical non-invasive detection methods include scintigraphy (e.g., SPECT (computed tomography of single-proton emission), PET (positron emission tomography), gamma-ray tomography, and linear scanning), magnetic resonance imaging (e.g., traditional magnetic resonance imaging, tomography with magnetization transfer, proton magnetic resonance spectroscopy, diffusion-weighted tomography and functional magnetic resonance chromatography, and ultrasound.

V.C. Therapeutic application

The present invention further provides methods and compositions suitable for inducing cytolysis of 5T4-expressing cancer cells in a subject. Anti-5T4 antibody conjugates with the drug of the present invention are useful for inhibiting the growth of cancer cells and cells not associated with neoplasms of proliferative disorders, for example, hyperplasia, metaplasia or, in particular, dysplasia (for a review of such abnormal growth conditions, see DeVita, Jr. et a. (2001), Cancer: Principles and Practice, 6 ^th edition, Lippincott Williams & Wilkins.

Cancer species that are sensitive to targeted therapy with drug conjugates include 5T4-expressing primary and metastasized tumors of the breast, colon, rectum, lung, oropharynx (mesopharynx), hypopharynx (hypopharynx), esophagus, stomach, pancreas, liver, gall bladder, bile ducts, small intestine, urinary tract, including kidney, bladder, urethra, female genital tract, cervix, ovaries, male genital tract, prostate, seminal vesicles, testicles, endocrine glands , thyroid gland, adrenal gland, pituitary gland, skin, bone, soft tissues, brain, blood vessels, nerves, eyes, and meninges. Other relevant cancers are 5T4-expressing leukemias and lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), including sluggish, aggressive, low-grade, intermediate-grade, high-grade leukemia or lymphoma.

In particular, it is known that 5T4 is expressed by cells of squamous / adenomatous pulmonary carcinoma (non-small cell lung cancer), invasive breast cancer, colorectal carcinoma, gastric cancer, squamous carcinoma of the cervix uteri, invasive carcinoma of the endometrial carcinoma, invasive carcinoma, carcinoma bladder, and choriocarcinoma. 5T4 is found in high quantities in cancer of the bronchi, breast, colon, rectum, stomach, cervix, endometrium, pancreas, ovary, chorium and seminal vesicles. The 5T4 antigen distribution on the cell surface may be homogeneous or heterogeneous. In colorectal carcinoma, gastric cancer, and ovarian cancer, 5T4 expression is directly related to the development of the disease. In breast cancer, there is an increased intensity of 5T4 staining on metastatic nodules, despite this, 5T4 expression is not dependent on the stage of the disease. Carcinomas can also express Lewis Y carbohydrate antigen, including carcinomas of the breast, colon, stomach, esophagus, pancreas, lung, bladder, and kidney, and gastric and non-benign neuroendocrine tumors. See US Patent No. 6,310,185.

Thus, patients to be treated with conjugates of anti-5T4 antibodies with the drug of the present invention can be determined based on the expression of a biomarker, including but not limited to increased expression of 5T4 antigen, which leads to the selection of a group of patients based on increased expression of the target, rather than based on the location or histology of the tumor. Target expression can be measured as a function of the number of stained cells in combination with cell staining intensity. For example, the classification for high 5T4 expression includes those patients who have more than 30% (i.e. 40%, 50%, or 60%) of cells defined as positive for immunohistochemical staining for 5T4 with a value of 3+ (on a scale of 1 to 4), while moderate 5T4 expression may include those patients who have staining of more than 20% of cells with a value from 1+ to 2+.

It is also permissible to use biomarkers other than 5T4 antigen expression for patient selection, including determining tumor parameters based on, for example, multidrug resistance. About 50% of human cancers are either completely resistant to chemotherapy or sensitive only for a short time, after which they are no longer affected by commonly used anticancer drugs. This phenomenon is called MDR (multidrug resistance) and is initially observed in some types of tumors, although other tumors acquire multidrug resistance after chemotherapy treatment. P-glucoprotein, a drug pump, mediates most types of multidrug resistance associated with cytotoxic chemotherapy. In order to establish the correspondence of a specific mechanism (mechanisms) of multidrug resistance for specific types of tumors, it is possible to carry out a phenotypic and functional analysis of the mechanisms of multidrug resistance existing in tumor samples of patients with cancer patients.

The terms “cancer growth / development” or “pathological proliferation” refer to any of the values of indicators that suggest a change in the cells towards a more advanced form of cancer or stage of the disease. Inhibition of the growth of cancer cells or cells not related to neoplasms of a proliferative disorder can be investigated by methods known in the art, for example, delaying tumor growth or inhibiting metastases. Other indicators for measuring suppression of cancer cell growth include: a decrease in cancer cell survival, a decrease in tumor size or morphology (for example, determined by computed tomography, sonography, or other tomography methods), destruction of the vasculature of the tumor, improvement in the delayed skin hypersensitivity test, increased activity cytolytic T-lymphocytes and a decrease in the levels of tumor-specific antigens.

Not limited to any particular treatment method, both antigen-targeted targeted therapy and passive targeted therapy with anti-5T4 antibody conjugates with the drug can contribute to antitumor efficacy. Antigen-targeted targeted therapy refers to the preferred movement and / or accumulation of a peptide or peptide analog in target tissues (i.e., tissues containing 5T4-expressing cells and a putative site for accumulation of anti-5T4 antibody conjugate with drug) compared to the control tissue (i.e., tissue that is believed to be substantially free of 5T4-expressing cells and lacking the binding and / or accumulation of the anti-5T4 antibody conjugate used with the drug). The preferred localization of the antibody-drug conjugate in the general case is such that the amount of said antibody-drug conjugate in the target tissue is 2 times higher than the amount of the antibody-drug conjugate in the control tissue, for example, 5 or more times higher, or 10 or more times higher.

Passive targeted therapy usually refers to the binding of antibodies or antibody conjugates to a drug in the localization of the tumor, due to local changes in the vascular network. For example, anti-5T4 antibody conjugates with the drug can leave the vasculature at the site of the tumor, which is fenestrated due to increased VEGF formation, bind to 5T4-expressing cells and trigger the internalization mechanism of the anti-5T4 antibody drug conjugate. Low venous and lymphatic drainage of the tumor also leads to sequestration of unbound conjugates of anti-5T4 antibodies with the drug. Antibodies conjugated to drugs via acid-unstable linkers can release the drug upon diffusion into the tumor cells. The antitumor effect of passive targeted therapy is variable or not as significant as the effect caused by antigen-targeted targeted therapy, but can nevertheless contribute to the overall effectiveness of the treatment.

V.D. Recipes

Anti-5T4 antibodies and anti-5T4 antibody conjugates with the drug of the present invention are readily prepared and formulated for reliable and effective clinical use. Suitable formulations for administration to a subject include aqueous and non-aqueous sterile injectable solutions, which may contain anti-oxidants, buffers, bacteriostats, antibacterial and antifungal agents (e.g., parabens, chlorobutyl alcohol, phenol, ascorbic acid, and thimerosal), dissolved substances, leading to to the isotonicity of the formulation with respect to the body fluids of the recipient (for example, sugars, salts and polyols), suspending agents and thickeners. Suitable solvents include water, ethanol, polyhydric alcohols (e.g. glycerol, propylene glycol, and liquid polyethylene glycol), and mixtures thereof. The preparations can be presented in the form of packages containing the drug in single doses or in several doses, for example, sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) state, which requires only the addition of a sterile liquid carrier immediately before use by a patient or for subsequent radioisotope labeling with an isotope that matches the intended use. Antibodies against 5T4 and conjugates of the antibody with the drug according to the present invention are preferably prepared in the form of effective doses described below.

As one example, a typical formulation of an anti-5T4 antibody or anti-5T4 antibody conjugate with a drug includes a multi-dose formulation that contains 40 mg / ml antibody or antibody conjugate with a drug, 25 mM acetate, 150 mM trehalose, 0.9 % benzyl alcohol, 0.02% polysorbate 20 at pH 5.0, and which has a minimum shelf life of 2 years at 2-8 ° C. As another example, the formulation of an anti-5T4 antibody or anti-5T4 antibody drug conjugate may consist of 10 mg / ml antibody or antibody-drug conjugate in 9.0 mg / ml sodium chloride, 7.35 mg / ml sodium citrate dihydrate, 0.7 mg / ml polysorbate 80 and sterile water, pH 6.5. Typical anti-5T4 antibody conjugate formulations for calicheamicin for use in experimental mouse models contain 2 or 4 micrograms of calicheamicin (See Examples 3, 4, and 7), which can be suitably increased for use in humans.

A stable lyophilized formulation of an anti-5T4 antibody or anti-5T4 antibody conjugate with a drug can be prepared by (a) dissolving the antibody conjugate with a drug to a final concentration of 0.5 to 2 mg / ml in a solution containing 1.5% -5 cryoprotectant % by weight, polymer filler in a concentration of 0.5% -1.5% by weight, electrolytes with a concentration of 0.01 M to 0.1 M, solubility facilitating agent in a concentration of from 0.005% to 0.05% by weight, a buffering agent at a concentration of 5-50 mM, thus so that The pH of the resulting solution was 7.8-8.2, and water; (b) moving the solution obtained above into vials at a temperature of + 5 ° C to + 10 ° C; (c) freezing the solution at a refrigerator temperature of from -35 ° C to -50 ° C; (d) initial freezing-drying of the frozen solution at an initial drying pressure of 20 to 80 microns at a shelf temperature of from -10 ° C to -40 ° C for 24 to 78 hours; and (e) secondary drying the product of step (d) at a drying pressure of 20 to 80 microns at a shelf temperature of + 10 ° C to + 35 ° C for 15 to 30 hours.

Typical cryoprotectants suitable for use in lyophilization include aldithiol, mannitol, sorbitol, inositol, polyethylene glycol, aldonic acid, uric acid, aldaric acid, aldoses, ketoses, aminosugar, alditols, inositol, glyceric aldehydes, arabino, xylose, galactose, glucose, hexose, idose, mannose, talose, heptose, glucose, fructose, gluconic acid, sorbitol, lactose, mannitol, methyl α-glucopyranose, maltose, iso-ascorbic acid, ascorbic acid, lactone, lactone, aroic acid, erythrose, threose, arabinose, allose, altrorz, gulose, idose, talose, erythrulose, ribose, xylulose, psicose, tagatose, gluconuric acid, gluconic acid, glucuronic acid, galucanoic acid, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galacturon, galucurin, trialose, neuramic acid, arabinose polymers, fructose polymers, fucans, galactans, galacturans, glucans, mannans, xylans, levans, fucoidans, carrageenin, galactacarolose, pectins, pectic acids, amylose, pullulan, glycogen, cellulose, amylop, pustulan, chitin, aragose, keratin, chondroitin, dermatan, gualuronic acid, alginic acid, xanthan gum, starch, sucrose, glucose, lactose, trehalose, ethylene glycol, polyethylene glycol, polypropylene glycol, glycerol penta.

For example, sucrose can be used as a cryoprotectant in concentrations of 1.5% by weight, polymer filler Dextran 40 or hydroxyethylated starch 40 can be used in a concentration of 0.9% by weight, the electrolyte used in the lyophilization solution is sodium chloride, which is present at concentrations of 0.05 M, and the buffering agent tromethamine can be used at a concentration of 0.02 M. A dissolving agent (for example, a surfactant such as Polysorbate 80) can also be used in about the time of the lyophilization process. Typically, said dissolving agent is a surfactant. Typical steps for preparing a lyophilized formulation include freezing vials at -45 ° C; the frozen solution is subjected to the initial stage of drying in the cold at a primary drying pressure of 60 microns and at a shelf temperature of -30 ° C for 60 hours; then subjected to cold-dried product of the secondary stage of drying at a drying pressure of 60 microns at a temperature of + 25 ° C for 24 hours.

Anti-5T4 antibodies and drug antibody conjugates are prepared in a pharmaceutically acceptable carrier, for example, using slowly metabolizing macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polymeric lactic acids, polymeric amino acids, amino acid copolymers and inactivated viral particles. Pharmaceutically acceptable salts can also be used, for example, salts of mineral acids, such as hydrochlorides, hydrobromides, carbonates, phosphates and sulfates, or salts of organic acids, such as acetates, propionates, malonates and benzoates. The formulations may further contain liquids such as water, saline solutions, glycerin and ethyl alcohol, and / or excipients, such as wetting or emulsifying agents or pH stabilizing buffers, may be present in such a composition. These carriers allow the preparation of compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions and suspensions for oral administration.

V.E. Dosage and Administration

Anti-5T4 antibodies and anti-5T4 antibody conjugates with the drug of the present invention can be administered parenterally, for example, intravascularly. subcutaneously, intraperitoneally or intramuscularly. To deliver the compositions in pulmonary routes, the compositions can be administered in the form of an aerosol or large spray, i.e. transnasally. Intrathecal, intramedullary or intraventicular administration can be used in the treatment of cancers of the central nervous system or central nervous system-related cancers. Antibodies against 5T4 and conjugates of antibodies against 5T4 with a drug can also be prescribed transdermally, transdermally, locally, enteral, intravaginal, sublingual or rectal. Intravenous administration can be used regularly in the clinic. The route of administration is chosen based on such considerations as the conditions and place of treatment, the type of antibody formulation and therapeutic efficacy of the composition.

The present invention provides that the subject is administered an effective amount of an anti-5T4 antibody and anti-5T4 antibody conjugate with a drug, i.e. the amount of anti-5T4 antibody and anti-5T4 antibody conjugate with the drug is sufficient to induce the desired biological response. For example, when administered to a cancer patient, an effective amount includes an amount sufficient to achieve anticancer activity, including cancer cell cytolysis, suppression of cancer cell proliferation, cancer cell apoptosis, reduction of cancer cell antigens, slowing tumor growth, and / or inhibiting metastasis. Reducing the size of the tumor is quite acceptable as a clinical sign, replacing efficiency. Another quite acceptable measure of effectiveness is an increase in free survival. Conjugates of anti-5T4 antibody with calicheamicin generally show at least a 25% increase in key performance parameters, for example, an increase in average survival, an increase in tumor development time, and an overall degree of response.

In the General case, the effective dose is in the range from 0.01 mg / m ² to 50 mg / m ² , for example, from 0.1 mg / m ² to 20 mg / m ² , or up to 15 mg / m ² , each dose is calculated on the basis of the amount of anti-5T4 antibody. The effective dose of the anti-5T4 antibody conjugate with the drug can also be calculated based on the amount of conjugated drug. For example, typical doses of an anti-5T4 antibody conjugate with calicheamicin for administration in an experimental mouse model include from 2 to 4 micrograms of calicheamicin and can be correspondingly increased for use in humans. For example, conjugates of an anti-5T4 antibody with calicheamicin according to the present invention can be administered to patients every 3 weeks for up to 6 cycles. For anti-5T4 antibodies labeled with radioactive isotopes, the effective dose is usually in the range of 1 mCi to 300 mCi, typically 5 mCi to 100 mCi, depending on the radioisotope and the binding activity of the antibody.

To detect 5T4-positive cells using the disclosed anti-5T4 antibodies, a detectable amount of a composition according to the present invention is administered to a subject, i.e. the dose of the anti-5T4 antibody is sufficient so that the presence of the antibody can be determined in vitro or in vivo. For scintigraphy using radioisotopes, typical doses of the radioisotope may include activities from 10 µCi to 50 mCi, or from 100 µCi to 25 mCi, or from 500 µCi to 20 mCi, or from 1 µCi to 10 mCi, or 10 mCi.

The actual dose levels of the active ingredients in the composition according to the present invention can be varied so as to introduce the amount of composition that is effective to achieve the desired diagnostic or therapeutic response. Reception modes can also vary. It is acceptable to use single or multiple injections. The dosage level and regimen chosen will depend on a number of factors, including activity and stability (i.e. half-life) of the therapeutic composition, formulation, route of administration, combination with other drugs and prescriptions, a disease or disorder that needs to be detected and / or treated, and the physical condition and previous medical history of the subject being treated.

For anti-5T4 antibodies or anti-5T4 antibody conjugates of the medicament of the present invention, the therapeutically effective dose can be pre-determined either in a cell culture study or in animal models, for example, rodents, rabbits, pigs, and / or primates. An animal model can also be used to determine acceptable concentration levels and routes of administration. Such information can then be used to determine effective doses and routes of administration in humans. A minimal dose is usually administered and the dose is increased in the absence of a dose limiting cytotoxicity. The determination and correction of an effective amount or dose, as well as the assessment of where and how to make such adjustments, are known to ordinary specialists in the field of medicine.

For combination therapy, anti-5T4 antibodies, anti-5T4 antibody conjugates with the drug and / or additional therapeutic or diagnostic agents are administered over a period of time suitable for the prescribed therapy or diagnosis. So, individual agents can be administered almost simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially in any order. For example, treatment with individual agents can be carried out within 1 year of each other, for example, within 10, 8, 6, 4, or 2 months, or within 4, 3, 2 or 1 week (s), or within 5, 4 3, 2, or 1 day (s).

For additional guidance on formulation, dosage, administration regimens, and measurable therapeutic responses, see Berkow et al. (2000) The Merck Manual of Medical Information, Merck & Co, Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, CRC Press, Boca Raton, Florida; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania; Katzung (2001) Basic & Clinical Pharmacology, Lange Medical Books / McGraw-Hill Medical Pub. Div., New York; Hardman et al. (2001) Goodman & Gilman's the Pharmacological Basis of Therapeutics, The McGraw-Hill Companies, Columbus, Ohio; Speight & Holford (1997) Avery's Drug Treatment: A Guide to the Properties, Choices, Therapeutic Use and Economic Value of Drugs in Disease Management, Lippincott, Williams, & Wilkins, Philadelphia, Pennsylvania.

V.F. Combination therapy

The proposed anti-5T4 antibodies of the present invention and anti-5T4 antibody conjugates with a drug can be used as a primary treatment, or for treating conditions that are immune to conventional methods of treatment. Additionally, anti-5T4 antibodies and anti-5T4 antibody conjugates with the drug can be used in combination with other types of therapy (e.g., surgical excision, radiotherapy, additional anti-cancer agents, etc.), thereby causing an additional or enhanced therapeutic effect and / or reducing the hepatotoxicity of some anti-cancer agents. Anti-5T4 antibodies and anti-5T4 antibody conjugates with the drug of the present invention can be co-administered, or mixed together with additional agents, or formulated for sequential administration with additional agents in any order.

Typical agents suitable for combination therapy include any of the drugs described herein above as suitable for the preparation of anti-5T4 antibody conjugates with a drug. Anti-5T4 antibodies and anti-5T4 antibody conjugates of the drug of the present invention can also be used in combination with other therapeutic antibodies and anti-drug antibody conjugates, including anti-5T4 antibodies other than the anti-5T4 antibodies disclosed herein, as well as antibodies and conjugates directed by another antigen. Typical antibodies that can be used alone or as a drug conjugate of antibodies a include anti-CD19 antibodies, anti-CD20 antibodies (e.g. RITUXAN®, ZEVALIN®, BEXXAR®), anti-CD22 antibodies. anti-CD33 antibodies (e.g. MYLOTARG®), anti-CD33 antibody conjugates with a drug, anti-Lewis Y antigen antibodies (e.g. Hu3S193, Mthu3S193, AGmthu3S193), anti-HER-2 antibodies (e.g. HERCEPTIN® (trastuzumab), MDX-210 , OMNITARG® (pertuzumab, rhuMAb 2C4)), anti-CD52 antibodies (e.g. CAMATH®), anti-EGFR antibodies (e.g. ERBITUX® (cetuximab), ABX-EGF (panitumumab)), anti-VEGF antibodies (e.g. AVASTIN® ( bevacizumate)), anti-DNA / histone complex antibodies (e.g. ch-TNT-1 / b), anti-CEA antibodies (e.g. CEA-Cide, YMB-1003) hLM609, anti-CD47 antibodies (e.g., 6H9), anti-VEG antibodies FR2 (receptor containing the KDR kinase insertion domain) (e.g., 1MS-1C11), anti-Ep-CAM antibodies (e.g., ING-1), anti-FAP antibodies (e.g., sibrotuzumab), anti-DR4 antibodies (e.g., TRAIL-R) , anti-progesterone receptor antibodies (e.g., 2C5), anti-CA19.9 antibodies (e.g., GIVAREX®), and anti-fibrin antibodies (e.g., MH-1).

Anti-5T4 antibody conjugates with the drug can also be administered as part of a treatment regimen in conjunction with a combination of one or more cytotoxic agents. Suitable cytotoxic drugs for these purposes include CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone, and procarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone); COP (cyclophosphamide, vincristine and prednisone); SAR-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine and prednisone); m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and leucovorin); ProMACE-MORP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin, mechloethamine, vincristine, prednisone and procarbazine); RgoMACE-CytaBOM (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin, cytarabine, bleomycin and vincristine); MASOR-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin); MOPR (mechloethamine, vincristine, prednisone and procarbazine); ABVD (adriamycin / doxorubicin, bleomycin, vinblastine and dacarbazine); MOPP (mechloethamine, vincristine, prednisone, and procarbazine) attested with ABV (adriamycin / doxorubicin, bleomycin, vinblastine); MOPP (mechloethamine, vincristine, prednisone and procarbazine) alternating with ABVD (adriamycin / doxorubicin, bleomycin, vinblastine and dacarbazine); ChlVPP (chlorambucil, vinblastine, procarbazine, prednisone); IMVP-16 (ifosfamide, methotrexate, etoposide); MIME (methyl gag, ifosfamide, methotrexate, etoposide); DHAP (dexamethasone, high doses of cytaribine and cisplatin); ESHAP (etoposide, methylpredisolone, high doses of cytarabine, and cisplatin); CEPP (B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin), CAMP (lomustine, mitoxantrone, cytarabine and prednisone); and CVP-1 (cyclophosphamide, vincristine and prednisone); DHAP (cisplatin, high doses of cytarabine and dexamethasone); CAP (cyclophosphamide, doxorubicin, cisplatin); PV (cisplatin, vinblastine or vindesine); CE (carboplatin, etoposide); EP (etoposide, cisplatin); MVP (mitomycin, vinblastine or vindesine, cisplatin); PFL (cisplatin, 5-fluorouracil, leucovorin); IM (ifosfamide, mitomycin); IE (ifosfamide, etoposide); IP (ifosfamide, cisplatin), MIP (mitomycin, ifosfamide, cisplatin); ICE (ifosfamide, carboplatin, etopozod); PIE (cisplatin, ifosfamide, etoposide); Viorelbine and Cisplatin; Carboplatin and paclitachel; CAV (cyclophosphamide, doxorubicin, vincristine); CAE (cyclophosphamide, doxorubicin, etoposide); CAVE (cyclophosphamide, doxorubicin, vincristine, etoposide); EP (etoposide; cisplatin); and CMCcV (cyclophosphamide, methotrexate, lomustine, vincristine).

Antibodies against 5T4 and conjugates of antibodies against 5T4 with calicheamicin can be used in combination with systemic anticancer drugs, for example, epithilones (BMS-247550, EPO-906), reformulation of taxanes (Abraxan, Xiotax), microtubilin inhibitors (MST-997, TTI-237 ), or with targeted cytotoxins, for example, CMD-193 and SGN-15. Additional suitable anti-cancer agents include TAXOTERE®, TARCEVA®, GEMZAR® (gemcitabine), 5-FU, AVASTIN®, ERBITUX®, TROVAX®, anatumomab mafenatox, letrazole, docetaxel, and anthracyclines.

In combination therapy methods, anti-5T4 antibodies and anti-5T4 antibody conjugates with a drug and / or one or more additional therapeutic or diagnostic agents are administered over a period of time suitable for the intended therapy or diagnosis. Thus, individual agents can be administered almost simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially in any order. For example, treatment with individual agents can be carried out within 1 year in relation to each other, for example, within 10, 8, 6, 4, or 2 months or within 4, 3, 2 or 1 week (s), or within 5, 4, 3, 2, or 1 day (s). Administration of an anti-5T4 antibody or conjugate of an anti-5T4 antibody with calicheamicin in combination with a second therapeutic agent preferably has a greater effect than administering either of them individually.

EXAMPLES

The following examples were included to illustrate the methods of the present invention. Some aspects of the examples are described in terms of techniques and technologies, which, as established or assumed by the authors of this work, work well in the implementation of the present invention. These examples illustrate standard laboratory methods of work of the authors. Given the present description and the general level of specialized knowledge, those skilled in the art should understand that the following examples are only samples, and that numerous changes, modifications and variations can be made without departing from the scope of the present invention.

EXAMPLE 1

Mouse antibodies against 5T4

Antibodies against 5T4 were obtained in mice using human 5T4 antigen and standard immunization methods. Hybridoma cell lines producing antibodies A1, A2 and A3 were obtained by hybridization of individual B cells and myeloma cells.

The variable regions of the heavy and light chains of antibodies A1, A2 and A3 against 5T4 were cloned by means of a SMART® cDNA synthesis system (Clontech Laboratories Inc. Moutain View, California, Clontech Laboratories Inc. of Mountain View, California) followed by PCR amplification. cDNA was synthesized from 1 microgram of total RNA isolated from A1, A2 or A3 hybridomas using oligo (dT) and SMART® IIA oligo (Clontech Laboratories Inc.) with reverse transcriptase POWERSCRIPT ™ (Clontech Laboratories Inc.). The cDNA was then amplified by PCR using a primer that hybridizes to the oligotype SMART® IIA sequence and a primer specific for the constant region of mouse antibodies (mouse Kappa light chain, mouse IgG2a for heavy chain A1, mouse IgG2b for heavy chain A2, and mouse IgG1 for heavy chain A3) with VENT® polymerase (New England Biolabs Inc. Ipswich, Mass. New England Biolabs Inc. of Ipswich, Massachusetts). The PCR products of the variable regions of the heavy and light chains were subcloned into the expression vector pED6 and the nucleic acid sequence was determined. This method is advantageous in that no prior knowledge of the DNA sequence is required. In addition, when using degenerate PCR primers, the resulting DNA sequence does not change.

The nucleotide sequences of the variable regions of the heavy chain of antibodies A1, A2 and A3 correspond to nucleotides 58-414 of the sequence SEQ ID No: 1, nucleotides 55-405 of the sequence SEQ ID No: 5, and nucleotides 58-423 of the sequence SEQ ID No: 9, respectively. The amino acid sequences of the variable regions of the heavy chain of antibodies A1, A2, and A3 correspond to residues 20-138 of the sequence SEQ ID No: 2, residues 19-135 of the sequence SEQ ID No: 6, and residues 20-141 of the sequence SEQ ID No: 10, respectively. The nucleotide sequences of the variable regions of the light chain of antibodies A1, A2, and A3 correspond to nucleotides 61-381 of the sequence SEQ ID No: 3, nucleotides 67-390 of the sequence SEQ ID No: 7, and nucleotides 61-381 of the sequence SEQ ID No: 11, respectively. The amino acid sequences of the variable regions of the light chain of antibodies A1, A2 and A3 correspond to residues 21-127 of the sequence SEQ ID No: 4; residues 23-130 of the sequence SEQ ID No: 8 and residues 21-127 of the sequence SEQ ID No: 12, respectively. See also figa-1C.

In order to evaluate the novelty of the variable region sequences of antibodies A1, A2, and A3 against 5T4, we performed a BLASTp search (for the requested protein sequences) with the default parameters Waiting = 10, Word length = 3, low-complexity filter and BLOSUM62 matrix, introducing a gap ( gap existence) E = 11, and the expansion of the gap (gift extension) W = 1. BLASTn searches (for the requested nucleotide sequences) were performed with the default parameters Waiting = 10, Word Length = 11, and a low complexity filter. BLAST search results are presented as a list of sequences related to the requested sequence in order of the value of E, which is an indicator of statistical significance matches found in the database. The sequences that are most closely related to the variable region sequences used for BLAST assays are shown in Table 1 (BLASTn) and Table 2 (BLASTp).

Table 1 BLASTn analyzes Requested sequence Identity (%), closest sequence Description of the closest sequence A1 VH (SEQ ID No: 1) 97% gi | 31322165 | gb | AY169686.1 | clone Mus musculus VGBC1.13 precursor of the immunoglobulin heavy chain variable region gene, incomplete cds A1 VL (SEQ ID No. 3) 96% gi | 804922 | dbj | D50385.1 | MUSIKCVRJ mPHK Mus musculus for the variable region of the kappa immunoglobulin chain, incomplete sequence, cell line: K3F10 A2 VH (SEQ ID No: 5) 97% gi | 11612012 | gb | AF303853.1 | AF303853 clone Mus musculus J558 22 mPHK immunoglobulin heavy chain variable region, incomplete cds A2 VL (SEQ ID No: 7) 97% gi | 1556423 | emb | X79906.1 | MMMABMST2 Mus musculus mPHK light chain monoclonal antibody MST2 A3 VH (SEQ ID No: 9) 99% gi | 3420272 | gb | AF064445.1 | AF064445 Mus musculus immunoglobulin heavy chain variable region (Vh10.2) gene, Vh10.2a allele, incomplete cds A3 VL (SEQ ID No: 11) 98% gi | 2906107 | gb | AF045512.1] AF045512 Mus musculus 9E10 variable region of the kappa light chain monoclonal antibody, (IgK) mRNA, incomplete cds

table 2 BLASTp analyzes Requested sequence Identity (%) of the closest sequence Description of the closest sequence A1 VH (SEQ ID No: 2) 84% gi | 15865327 | emb | CAC82228.1 | immunoglobulin heavy chain [Mus musculus] A1 VL (SEQ ID No: 4) 93% gi | 644862 | gb | AAA62143.1 | Kappa chain variable region of immunoglobulin against integrin alpha 4 A2 VH (SEQ ID No: 6) 85% gi | 15149453 | gb | AAK85298.1 | single-chain antibody HFN7.1 [synthetic construct] A2 VL (SEQ ID No: 8) 95% gi | 297678 | emb | CAA80086.1 | immunoglobulin variable region [Mus musculus domesticus] A3 VH (SEQ ID No: 10) 90% gi | 2906050 | gb | AAC04511.1 | anti-poly (dC) monoclonal antibody heavy chain [Mus musculus] A3 VL (SEQ ID No: 12) 97% gi | 2906108 | gb | AAC04540.1 | kappa monoclonal antibody light chain [Mus musculus]

EXAMPLE 2

Binding specificity and affinity of murine anti-5T4 antibodies

To evaluate the binding specificity and affinity of antibodies A1, A2, and A3, BIACORE® analysis was performed using human 5T4 antigen immobilized on a CM5 chip. BIACORE® technology uses changes in the refractive index on the layer surface when the antibody binds to the 5T4 antigen immobilized in the layer. Binding is determined by surface plasma resonance of a laser beam reflected from the surface. A study of the kinetics of the signal by the rate of filling and rate of departure (on- and off-rates) allows us to distinguish between non-specific and specific interactions. An H8 anti-5T4 antibody was used as a standard. H8 is hybridoma-formed monoclonal mouse IgG1 antibodies described in PCT International Publication No. WO 98/55607 and in Forsberg et al. (1997) J. Biol. Chem. 272 (19): 124430-12436.

Table 3 BIACORE® Analysis Results Antibody KD (M) KA (1 / M) kd (1 / s) ka (1 / Ms) H8 4.1 × 10 ^-10 2.5 × 10 ⁹ 5.1 × 10 ^-5 1.3 × 10 ⁵ A1 6.4 × 10 ^-10 1.6 × 10 ⁹ 1.3 × 10 ^-4 2.0 × 10 ⁵ A2 1.5 × 10-8 6.5 × 10 ⁷ 8.7 × 10 ^-4 5.6 × 10 ⁴ A3 2.2 × 10 ^-9 4.6 × 10 ⁸ 5.2 × 10 ^-5 2.4 × 10 ⁴

The data obtained by BIACORE® show that antibodies H8 and A1 have a higher affinity for 5T4 compared to antibodies A2 and A3. A2 is an antibody with a relatively small affinity. Unusual cysteine is present at position 67 of the variable region of the heavy chain A1 and at residue 91 of the variable region of the heavy chain A1. Replacing these residues with phenylalanine (A1) or tyrosine (A3) does not affect antibody binding properties or expression levels.

The affinity of binding of antibodies H8, A1, A2 and A3 was also evaluated by Western blotting using cell lysates CT26 / 5T4, which showed strong binding to H8, A1 and A3. See FIG. 2.

The ability of antibodies H8, A1, A2, and A3 to bind cells expressing 5T4 antigen was evaluated by fluorescence activated cell sorting (FACS) of PC14RE6 cells. Antibodies showed specific binding to 5T4-expressing PC14PE6 cells, however, the level of A2 binding was significantly lower than the level observed for H8, A1 and A3. See table 4.

Table 4 FACS Analysis Results Antibody The average value of cell fluorescence Control (Secondary Ab) four Control (mouse IgG) four H8 24 A1 eighteen A2 7 A3 27

To assess the possible variability in the production of antibodies, two independently prepared batches A1 and H8 were studied. When comparing antibodies from different lots, the binding properties and kinetics of each antibody did not differ significantly. See FIGS. 3A-3B.

EXAMPLE 3

Internalization of murine antibodies against 5T4 5T4-expressing cells

To evaluate the internalization of antibodies upon binding to the 5T4 antigen, the amount of H8 and A1 antibodies found on the cell surface compared to the supernatant was measured as a function of time. Non-enzymatically dissociated MDAMB435 / 5T4 cells (human breast cancer cells) were exposed to anti-5T4 antibodies for 1 hour at 4 ° C. Cells were washed and thermostated in a medium at 37 ° C from 4 hours to 24 hours. The amount of antibody bound to the cell membranes with respect to the unbound antibody (i.e., presence in the supernatant) was determined by the FACS method. The disappearance of 5T4 antibodies from the surface of MDAMB435 / 5T4 cells shows modulation of the 5T4-antigen / antibody complex on the cell surface, which possibly means internalization and / or dissociation. See FIGS. 4A-4C.

EXAMPLE 4

Epitope mapping using 5T4 chimeric proteins

To identify the epitopes to which each of the antibodies A1, A2, A3, and H8 binds, an ELISA (enzyme-linked immunosorbent assay) was performed using (1) 5T4 - Fc ectodomain constructs with deleted or mutated sequences, and (2) 5T4 chimeric constructs expressed for a short time in COS-1 cells. The ectodomain contains an amino-terminal segment, two leucine-rich repeats, and a transition hydrophilic segment. Chimeric proteins containing the 5T4 ectodomain and Fc constant regions from human IgG1 were prepared using 5T4 mice (amino acids 1-136), 5T4 rats (amino acids 1-361), 5T4 cynomolgus monkey) (amino acids 1-355), 5T4 chimpanzees (amino acids 1-355), and 5T4 black-tailed monkey (amino acids 1-355). 5T4 hybrid constructs are depicted in FIG. 5. The binding results are summarized in Table 5, the results show specific binding, partial binding or lack of binding of each of the antibodies H8, A1, A2 and A3. Humanized H8 antibody and chimeric antibodies A1, A2 and A3 showed binding properties similar to mouse H8, A1, A2 and A3, respectively.

Based on these results, it was found that humanized H8 antibody binds to human 5T4 between residues 173 and 252. Humanized H8 binds to 5T4 in the presence or absence of N-linked glycosylation at residue 344, which confirms that the binding of humanized H8 to human 5T4 is not membrane proximal. Antibody A1 has a first region of contact with human 5T4 between residues 173 and 252 and a second contact with 5T4 of human between residues 282 and 361. Antibody A2 binds human 5T4 between residues 282 and 361. Antibody A3 binds the first leucine-rich repeat of human 5T4 between residues 83 and 163. The epitopes that bind each antibody are depicted in Fig.7.

Table 5 Epitope mapping results using recombinant 5T4 ectodomain proteins with Fc and 5T4 human / mouse chimeras construct 5T4 antigen Antibody H8 A1 A2 A3 5T4 human ectodomain / Fc + + + + 5T4 mouse / Fc ectodomain - - - - 5T4 rat / Fc ectodomain - +/- - - 5T4 ectodomain of cynomolgus monkey / Fc - + + + ectodomain 5T4 chimpanzee \ Fc + + + + ectodomain 5T4 black-tailed monkey / Fc +/- + + - human / mouse 83-163 + + + - human / mouse 173-361 - - - + human / mouse 173-258 - +/- + +

human / mouse 282-361 (with or without N-bond at position 344) + +/- - + (+) binding; (-) lack of binding; (+/-) partial binding

Based on the different binding observed for human 5T4 ectodomains and cynomolgus macaques, targeted mutagenesis was performed to identify residues that are involved in antibody binding. The binding of the humanized H8 antibody was determined with each of the mutated 5T4 ectodomains shown in Table 6 below, i.e. 5T4 human ectodomains, which include residues from cynomolgus macaque in the marked position. These results indicate that residues 213 and 214 of the human 5T4 antigen are required for the binding of the epitope to the humanized H8 antibody.

Table 6 Epitope Mapping Results Using the Human 5T4 Ectodomain / Fc Hybrid Protein Directed Mutagenesis mutation binding to humanized H8 E189K + V200k + L204V + R213H +/- R213H and R214L - (+) binding; (-) lack of binding; (+/-) partial binding

In addition to the direct determination of binding, competitive binding was measured using a biotinylated humanized H8 antibody and each of A1, A2, and A3 antibodies. Inhibition of binding to human 5T4 was not observed, which confirmed that each of the antibodies A1, A2 and A3 binds to the 5T4 epitope, which is different from the epitope associated with the H8 antibody.

EXAMPLE 6

Epitope mapping with BIACORE®

The epitope of antibodies H8, A1, A2 and A3 was also mapped using BIACORE® using a CM5 chip with human 5T4 associated antigen. The chip was saturated with antibodies H8, A1, A2 and A3, and the first response was measured. Then, the chip was saturated with a second antibody among antibodies H8, A1, A2 and A3, and a second response was measured. For repeated experiments, the chip was regenerated by dissociation of bound antibodies in 10 mM glycine, pH 1.5, followed by washing with a buffer solution. The results are summarized in Table 7 below. The percentages given are units of the response measured by binding the second antibody directly to the CM5 chip, relative to units of the response measured by binding of the second antibody to the CM5 chip saturated with the first antibody. These results indicate that each of the H8, A1, A2, and A3 antibodies binds to a different human 5T4 epitope.

Epitopes associated with antibodies H8 and A3 are sterically close to each other so that the rate of association with the antigen decreases if the binding of H8 is determined in the presence of A3 and vice versa. Similar results were obtained using hybrid and humanized antibodies H8, A1, A2 and A3, which were prepared as described in Example 7 below in this document. See table 8.

Table 7 BIACORE® Competitive Binding Assay Results — Percent Response of the Second Antibody Following Saturation with the First Antibody Second antibody First antibody H8 A1 A2 A3 H8 -------- 114% 102% 85% A1 109% -------- 109% 98% A2 99% 98% -------- 94% A3 73% 104% 106% --------

Table 8 BIACORE® Competitive Binding Assay Results — Percentage of Second Antibody Following Saturation with First Antibody second antibody / first antibody binding time after administration of the second antibody (seconds) 19 37.5 75 150 300 600 humanized H8 / chimeric A3 44.9% 57.0% 69.1% 79.4% 86.6% 91.3% chimeric A3 / humanized H8 46.2% 51.2% 58.4% 67.5% 76.2% 83.6% chimeric A2 / chimeric A1 102.9% 93.5% 90.1% 89 0% 88 9% 89 1% chimeric A1 / chimeric A2 92.5% 90.6% 91.4% 92.7% 93.9% 95 5% chimeric A3 / 82.1% 82.0% 84.5% 87.8% 90 8% 92.8%

chimeric A1 chimeric A1 / chimeric A3 98.8% 96.5% 97.0% 98.0% 98.8% 99.6% chimeric A3 / chimeric A2 92.2% 88.6% 89.5% 91.5% 93.4% 94.6% chimeric A2 / chimeric A3 89.2% 88.4% 89.8% 91.5% 92.9% 94.3% humanized H8 / chimeric A1 93.2% 92.7% 94.2% 95.9% 96.9% 97.3% chimeric A1 / humanized H8 92.7% 92.4% 93.8% 95.8% 97.3% 98.7% humanized H8 / chimeric A2 93.8% 94.0% 96.0% 98.1% 99.8% 101.3% chimeric A2 / humanized H8 86.9% 84.7% 86.9% 90.5% 93.7% 96.7%

The combined results of the study of epitope mapping obtained using hybrid constructs (see Example 4) and BIACORE® are presented in Fig.7.

EXAMPLE 6

The effectiveness of conjugates of antibodies against 5T4 with calicheamicin

Vital dye staining (MTS) was used to determine the number of surviving cells after using different drugs. MTS (non-radioactive kit for determining cell proliferation) was purchased from Promega (Madison, Wisconsin) and used in accordance with the manufacturer's technical description. For each cell line, a calibration curve was constructed (the number of cells depending on optical density after 2 hours) to determine a suitable initial seeding density. Then the cells were seeded in a 96-well plate with a density of 750 to 5000 cells per well. Immediately after seeding, cells were treated with different concentrations (0, 0.01, 0.05, 0.1, 1, 10, 100 and 500 ng of calicheamicin equivalents / ml) of calicheamicin, CMA-676 and calicheamicin conjugates with anti-5T4 antibodies. Following determination of the number of cells surviving after 96 hours of drug exposure based on logistic regression parameters derived from dose-response curves, an ED _{50 was} calculated. ED _{50 was} defined as the concentration of the drug (CalichDMH), which caused a 50% decrease in the number of cells after 96 hours of exposure to the drug. The calicheamicin equivalent (cal. Eq - cal. Equiv.) Is the concentration of calicheamicin given either as a pure compound or as a conjugate. Depending on the amount of calicheamicin binding to the antibody (drug loading of the antibody), different equivalents of calicheamicin may mean different protein concentrations.

The results of the MTS analysis are shown in Table 9. The conjugates of antibodies with calicheamicin prepared using antibodies A1 and A3 against 5T4 significantly reduced the viability of MDAMB435 / 5T4 cells. The selectivity values were calculated by comparing the specific activity of the conjugates with non-specific activity. That is, the multiplicity of CalichDMH for 5T4 expressing cells was related to the multiplicities of CalichDMH for non-expressing cells. If a non-specific antibody is used, for example, hp67.6 (CMA-676), the multiplicities of the CalichDMH values are approximately the same, which means that the selectivity is 1.

Table 9 MTS analysis results Treatment Cell line MDAMB435 / neo ED50 (ng / ml) MDAMB435 / 5T4 ED50 (ng / ml) Calichddh 3.3-5.0 5.0-8.0 huHS-ActBut-CalichDMH 0.4-0.8 0.08-0.1 SMA-676 34-60 50-100 A1-ActBut-CalichDMH 22-34 0.4-0.6 A2-ActBut-CalichDMH 60 40 A3-ActBut-CalichDMH 20-20 0.3-20 treatment Cell line MDAMB435 / neo multiplicity CalichDMH MDAMB435 / 5T4 multiplicity CalichDMH Calichddh 1.0-1.0 1.0-1.0 huH8-ActBut-CalichDMH 4.0-12.5 50-100 SMA-676 0.08-0.1 008-0.1 AI-ActBut-CalichDMH 0.14-0.15 13-13 A2-ActBut-CalichDMH 0.06 0.13 A3-ActBut-CalichDMH 0.17-0.25 04-17

Selectivity: H8 = 8; hP67.6 = 1; A1 = 93; A3 = 1.6

CalichDMH, unconjugated calicheamicin

huHS-AcBut-CalichDMH, a humanized H8 antibody conjugated to calicheamicin by 4- (4'-acetylphenoxy) butyric acid (AcBut)

SMA-676, anti-CD33 antibody conjugate with calicheamicin

A1-AcBut-CalichDMH, A1 antibody conjugated to calicheamicin via 4- (4'-acetylphenoxy) butyric acid (AcBut)

A2-AcBut-CalichDMH, A2 antibody conjugated to calicheamicin via 4- (4'-acetylphenoxy) butyric acid (AcBut)

A3-AcBut-CalichDMH, A3 antibody conjugated to calicheamicin via 4- (4'-acetylphenoxy) butyric acid (AcBut)

The cytotoxicity of anti-5T4 antibody conjugates with calicheamicin was also measured using a three-dimensional spheroid cell culture that more closely approximates the cell environment in vivo. Spheroids were prepared essentially according to the method of Yuhas et al. (1977) Cancer Res. 37: 3639-3643. Briefly, 10 ⁵ cells in 5 ml of culture medium were plated on 60 mm cell culture polystyrene plates that were previously coated with 5 ml of 0.65% agar. for cell tissues in a culture medium (Sigma St. Lois, Missouri). The cups were thermostated for 5-6 days at 37 ° C and with a CO ₂ content of 5% in air. Spheroids with a diameter of 0.2 mm were selected and placed in a 24-well plate. Each well contained 0.5 ml of agar support, 1 spheroid, and 1 ml of the top layer of the culture. The spheroids were then exposed to various concentrations (0, 0.091, 0.365, 1.46, 5.86, 23.44, 93.75 and 375 ng calicheamicin equiv / ml) calicheamicin, CMA-676 and anti-5T4 antibody conjugates with calicheamicin prepared using anti-5T4 antibodies A1 and A3 and AcBut linker. Both conjugates of antibodies against 5T4 with calicheamicin significantly inhibited the growth of MDAMB435 / 5T4 cells. See FIG.

EXAMPLE 7

Preparation and study of the binding properties of hybrid and humanized antibodies against 5T4

Hybrid antibodies H8, A1, A2 and A3 were obtained using sequences of the variable regions of the light and heavy chains of mouse H8 and the constant regions of the heavy chain of human IgG4 and the constant regions of the human kappa light chain. The cysteine present at position 67 of the variable region of the heavy chain of antibody A1 was optionally replaced with phenylalanine, and the cysteine present at position 91 of the variable region of the heavy chain of A3 was arbitrarily replaced with tyrosine. These options correspond to the sequence of SEQ ID No: 2 (A1 VH) and the sequence of SEQ ID No: 10 (A3 VH). The presence or absence of intron sequences and the substitution of cysteine residues did not affect antibody expression. To clone sequences encoding IgG constant regions, intron sequences were deleted.

Humanized H8 antibodies were prepared as described in PCT Application No. WO 2006/031653. Humanized antibodies A1 were prepared by transferring hypervariable sites as described below in this document. Hypervariable regions of murine antibodies A1, A2, and A3 were identified using the AbM definition, which is based on sequence variability as well as localization of closed structure segments. In general, human acceptor scaffolds were selected on the basis that they were substantially similar to the framework regions of murine antibodies, or were most similar to the consensus sequence of the subfamily of the variable region. The frequency of occurrence of framework loci in humans was also taken into account, with the proviso that more widely represented sequences were generally preferable to less common sequences. To restore murine residues that are believed to be involved in the interaction of the antigen and / or residues involved in the structural integrity of the antigen-binding site, additional mutations of acceptor sequences of frame regions of human antibodies were introduced. Amino acid sequences can also be optimized taking into account the preference of codons in CHO cells and to remove restriction enzyme sites. To analyze the humanized sequences of the variable regions of the heavy and light chains in order to identify and avoid the sites of post-translational protein modifications introduced during humanization, a protein structure prediction program can be used.

The variable regions of the heavy chain of the humanized antibody A1 (A1 VH version 1.0) were designed to include the hypervariable regions of the mouse antibody A1 that were grafted into the human DP-21 frame region (VH7 subgroup, access code No..CAA43346, sequence number SEQ ID No.: 88), which contains a mutation in the framework (S82A) and one reverse mutation (E46K). Options were prepared by removing the reverse mutation (A1VH versions 1.1 and 1.2). The second variable region of the heavy chain of humanized A1 was prepared by grafting the hypervariable regions of A1 into the framework region of the DP-54 human germline (A1 VH version 2.0). Six (6) reverse mutations were performed to obtain A1 VH version 2.1. As described below, both variable regions of the A1 heavy chain retained 5T4 binding properties. The frame regions of DP-21 and DP-54 showed 63% identity of the amino acid sequence along their length, this shows that multiple amino acid changes can be made while maintaining the binding specificity of the antibody, including the ability to bind to a specific epitope. The similarity of the variable regions of the heavy chain of humanized A1 is shown in Table 10. Typical nucleotide sequences encoding the variable regions of the heavy chain of humanized A1 correspond to the sequences SEQ ID Nos. 48, 50, 53, and 55. Typical amino acid sequences of the variable regions of the heavy chain of humanized antibodies correspond to SEQ sequences ID No.: 49, 51, 52, 54 and 56. See also FIGS. 9A-9B.

Humanized variable regions of the light chain were designed to include mouse hypervariable regions A1 grafted onto the framework regions of DPK24 (VKIV subgroup), DPK9 (VKI subgroup) and DPK23 (VKIII subgroup) of the human germline. After the introduction of S67Y reverse mutation into the framework regions of the variable region of the light chain of humanized A, antibodies prepared with such framework regions showed binding to 5T4. See below, including Table 13. The DPK24 framework regions show 74% and 73% amino acid sequence identity with DPK9 and DPK23, respectively. The framework region of DPK9 shows 74% amino acid sequence identity with DPK23. The similarity of the humanized A1 light chain variable regions is shown in Table 10. Numerous variants of the humanized framework regions of the light chain variable regions show that multiple amino acid changes can be made while maintaining the binding specificity of the antibody, including the ability to bind to a particular epitope. Typical nucleotide sequences encoding the variable regions of the light chain of a humanized antibody A1 correspond to sequences SEQ ID NOS: 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75. Typical amino acid sequences of the variable regions of the light chain of a humanized A1 correspond to SEQ ID: No. 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76. See also Figs. 9C-9F.

Table 10 The binding of humanized A1 antibodies The first variable region / The second variable region Percent identity HuA1 VL v1.1 (SEQ ID No. 60) / HuA1 VL v2.4 protein (SEQ ID No. 70) 86% HuA1 VL v1.1 (SEQ ID No: 60) / HuA1 VL v3.1 Protein (SEQ ID No: 75) 86% HuA1 VL v2.4 protein (SEQ ID No: 70) / HuA1 VL v3.1 protein (SEQ ID No: 75) 85% HuA1 VH v1.1 protein (SEQ ID No: 52) / HuA1 VH v1.0 protein (SEQ ID No: 54) 78%

Humanized A2 and A3 antibodies were constructed using a similar strategy. Typical amino acid sequences of the variable regions of the heavy chain of a humanized antibody A2 and the variable regions of the light chain of a humanized antibody A2 correspond to sequences SEQ ID Nos. 77-78 and SEQ ID Nos. 79-80, respectively. See also Fig. 9G. Typical amino acid sequences of the variable regions of the heavy chain of a humanized antibody and the variable regions of the light chain of a humanized antibody A3 correspond to SEQ ID No. 81-82 and SEQ ID No. 83-84, respectively. See also Fig. 9H.

To assess the novelty of the variable regions of the heavy and light chains of the humanized antibodies A1, A2 and A3, BLASTn and BLASTp analyzes were performed as described in Example 1. The results are presented in Table 11.

Table 11 BLASTn and BLASTp analyzes Requested follower
nost Identity (%) of the closest sequence Description of the closest sequence Humanized
new A1. Vl
DNA version 1.1 (SEQ ID No: 59) 83% * DETERMINATION partial mRNA of the variable region of the kappa chain of the homo sapiens immunoglobulin (IGKV2 gene). CODE AM040532 Humanized
new A1. Vl
protein version 1.1 (SEQ ID No: 60) 82% DEFINITION segment B17 of the kappa precursor chain V-IV Ig. CODE P06314 Humanized
new A1. Vl
option 24 DNA 85% * DEFINITION clone SC4064 mRNA of the variable region of the light chain of the immunoglobulin Homo sapiens

(SEQ ID No. 69) against rabies virus, full cds. CODE AY942044 Humanized
new A1. Vl
option 24 protein
(SEQ ID No: 70) 92% DEFINITION The variable region of the kappa chain of a humanized immunoglobulin against integrin alpha 4 CODE AAA62146 Humanized
new A1. Vl
DNA variant 3.1
(SEQ ID No: 75) 84% * DEFINITION clone 136e06 (IGL @) mPHK of the variable region of the light chain of Homo sapiens immunoglobulin against tetanus toxin, partial cds. CODE AY867377 Humanized
new A1. Vl
version 3.1 protein
(SEQ ID No: 76) 84% DEFINITION scFv antibody against Burkholderia mallei [synthetic construct]. CODE AB197018 Humanized
new A1. Vh
DNA version 1.1
(SEQ ID No: 50) 88% * DEFINITION ID CLL097 mPHK of the IgA heavy chain variable region of Homo sapiens, partial cds. CODE AF021940 Humanized
new A1. Vh
protein variant 1.1
(SEQ ID No: 51) 90% DEFINITION IgA heavy chain variable region [Homo sapiens] CODE AAC09074 Humanized
new A1. Vh
option 20 DNA
(SEQ ID No: 53) 81% * DEFINITION clone 23u-45 mRNA of the variable region of the heavy chain of the immunoglobulin Homo sapiens (IGH), partial cds. CODE AF062241 Humanized
new A1. Vh
option 2.0 77% DEFINITION Circuit D, Insights in Erbb signal transmission from the structure

squirrel
(SEQ ID No: 54) Erbb2-Pertuzumab complex CODE 1S78_D Humanized
New A2 VL
version 1.0 protein
(SEQ ID No: 79) 87% DEFINITION Chain A, Crystal structure of the Fab region of human cold monoclonal Igm agglutinin. CODE 1QLR_A Humanized
A2. Vl
protein option 2.0
(SEQ ID No: 80) 83% DEFINITION immunoglobulin kappa 1 light chain [Homo sapiens] CODE AAD29608 Humanized
A2. Vh
protein option 1.0
(SEQ ID No: 77) 88% DEFINITION Chain A, Crystal structure of a humanized Fv 528 antibody. CODE 1WT5_A Humanized
A2. Vh
protein option 2.0
(SEQ ID No: 78) 78% DEFINITION Circuit D, Insights into Erbb signal transmission from the structure of the Erbb2-Pertuzumab complex. CODE 1S78_D Humanized
new A3. Vl
version 1.0 protein
(SEQ ID No: 83) 85% DEFINITION VLJ segment of the kappa immunoglobulin light chain [Homo sapiens]. CODE BAC01708 Humanized
new A3. Vl
protein option 2.0
(SEQ ID No: 84) 90% DEFINITION HerMel [synthetic construct]. CODE CAL47329 Humanized
New A3 VH
protein option 1.0
(SEQ ID No: 81) 79% DEFINITION of Igh-1a protein [Mus musculus]. CODE AAN80671

Humanized
new A3. Vh
protein option 2.0
(SEQ ID No: 82) 77% DEFINITION of Igh-1a protein [Mus musculus]. CODE AAH80671 * If request coverage = 100%

On figa-10B shows additional sequences of the variable region of the heavy chain that can be used as scaffolds for the preparation of humanized antibodies A1, A2 and A3 against 5T4. 11-13 show additional sequences of the variable region of the light chain that can be used as a framework for the production of humanized antibodies A1, A2 and A3 against 5T4. On Fig shows a typical cosntant region that can be used to obtain hybrid and humanized antibodies A1, A2 and A3 against 5T4.

To assess the binding ability and affinity of the hybrid and humanized antibodies H8, A1, A2 and A3, BIACORE® analysis was performed using 5T4 antigen immobilized on a CM5 chip. See Example 2. The results obtained for hybrid antibodies A1, A2 and A3 are shown in Table 12 below.

Table 12 BIACORE® Analysis Data Antibody KD (M) KA (1 / M) kd (1 / s) ka (1 / Mc) Humanized H8 1.5 × 10 ^-10 6.5 × 10 ⁹ 4.0 × 10 ^-5 26 × 10 ⁵ Chimeric A1 4.4 × 10 ^-10 2.3 × 10 ⁹ 6.7 × 10 ^-5 1.5 × 10 ⁵ Chimeric A2 1.8 × 10 ^-9 5.7 × 10 ⁸ 2.5 × 10 ^-4 1.4 × 10 ⁵ Chimeric A3 ~ 1.8 × 10 ^-10 ~ 5.4 × 10 ⁹ ~ 1.0 × 10 ^-5 ~ 5.4 × 10 ⁴ Chimeric A1 + 3.8 × 10 ^-10 2.6 × 10 ⁹ 6.3 × 10 ^-5 1.7 × 10 ⁵

C67f Chimeric A3 + C91Y 5.9 × 10 ^-9 1.7 × 10 ⁹ 1.6 × 10 ^-5 2.7 × 10 ⁴

In general, hybridization / humanization increases the affinity of H8, A1, A2, and A3 antibodies to human 5T4. Compare with Table 3. The increase in binding activity seems to occur mainly as a result of a slower dissociation of the antibody from the antigen compared to faster association. Hybrid antibodies A2 and A3 showed the best binding properties after obtaining their chimeras.

All of the variable regions of the heavy chain of the humanized antibody A1 retain 5T4 binding properties. In addition, the removal of the K46 reverse mutation from the variable regions of the heavy chain of humanized A1 antibodies does not affect the parameters of their binding to 5T4. Humanized variable regions of the light chain of humanized A1 exhibit moderate 5T4 binding properties. The humanized variable regions of the light chain of antibody A1 obtained using the DPK9 and DPK23 framework regions bind 5T4 with greater affinity than the humanized variable regions of the light chain of humanized A1 obtained using the DPK24 framework. Inverse mutations were introduced to restore and / or optimize 5T4 binding. Substitution of the serine residue at position 67 with the tyrosine residue, as seen in the mouse skeleton A1, completely restores the binding properties to 5T4. See table 13.

Table 13 Inhibition of binding of a biotinylated hybrid antibody A1 to 5T4 ELISA Version A1 antibodies IC ₅₀ Chimeric A1 16-20 nm huA1 V _H v2.0 + huA1 V _L v1.1 > 1 µM huA1 V _H v2.0 + huA1 V _L v1.1 28 nM huA1 V _H v2.0 + huA1 V _L v2.0 > 1 µM huA1 V _H v2.0 + huA1 V _L v2.4 16 nM huA1 V _H v2.0 + huA1 V _L v v.0 > 1 µM huA1 V _H v2.0 + huA1 V _L v3.1 27 nM huA1, humanized A1 v version

EXAMPLE 8

Cross-reactivity of antibodies against 5T4

To detect interspecific in vivo efficacy and toxicological analysis, the reactivity of the anti-5T4 antibodies proposed in this document in various animal species was determined. To further characterize the epitope that binds each antibody, the dependence of binding activity and the affinity of various 5T4 ectodomains were also used. Binding was determined using 5T4 ectodomains from various species fused to human IgG1 Fc. The percent identity of each ectodomain site with 5T4 person is shown in Table 14.

Table 14 5T4 affinity from various species Types The percentage of identity with human 5T4 Ectodomain Amino Acids Person one hundred 1-355 Mouse 84.0 1-361 ^a Rat 83.1 1-361 ^a Chimpanzee (partial sequence - 396/420 amino acids) ~ 99.5 1-355 Javanese macaque 96.7 1-355 Black-tailed monkey (partial sequence 367/420 amino acids) ~ 94.6 1-355 Dog 87.9 1-355 Cow 86.9 1-355 ^a Contains a direct repeat of 6 amino acids in the hydrophilic domain

Complete or partial 5T4 sequences of humans, mice, rats, dogs, or cows have been previously described via GenBank access codes Z29083 (human, SEQ ID NO: 87), AJ012160 (mouse), BC087011 (rat), XM539020 (dog) and XM593502 ( cow). A virtual 5T4 chimpanzee sequence was obtained by aligning mRNA and genome sequences. Nucleic acids encoding 5T4 proteins were isolated from cynomolgus monkey and black-tailed monkey. The amino acid sequences of these additional 5T4 antigens are shown in FIG. 15 and are also described as SEQ ID No. 86 (cynomolgus monkey) and SEQ ID No. 85 (black-tailed monkey).

To evaluate the novelty of the cynomolgus monkey and black-tailed monkey sequences, BLAST analyzes were performed as described in Example 2. When using the full black-tailed monkey sequences as the requested sequence, the closest sequence was identified as 5T4 person (GenBank reference number NP_006661.1) with 94% identity (302/320 amino acids). The sequences also differed in carboxyl ends, with amino acids 1-19 of the sequence with identification number SEQ ID No: 85 not aligned with the closest sequence. Using the full sequences of cynomolgus macaques as the requested sequence, the closest real sequence is identified as the precursor of the troboplast glycoprotein also from cynomolgus macaques (GenBank BAE00432.11) with 99% identity (364/366 amino acids). The sequences also differed in carboxyl ends, and amino acids 1-25 of the sequence with identification number SEQ ID No: 86 did not align with the closest sequence.

To determine the binding of antibodies against 5T4, recombinant 5T4 ectodomain / Fc proteins were transfected temporarily into COS-1 cells, and ELISA analysis was performed. As control samples, irrelevant human IgG4 and IgG1 antibodies were used. The interspecific reactivity of anti-5T4 antibodies is summarized in Table 15.

Table 15 Intertype reactivity of antibodies against 5T4 (ED50 in nM) huH8 γ4 huH8 γ1 ChiA1 γ4 ChiA2 γ4 ChiA3 γ4 person 0.19 0.20 0.28 0.21 0.20 chimpanzees 0.19 0.22 0.27 0.22 0.20 black-tailed monkey +/- +/- 0.24 0.22 - javanese macaque - - 0.18 0.18 0.23 rat - - +/- - - mouse - - - - - huH8 γ4, a humanized H8 antibody having IgG4 constant regions huH8 γ1, a humanized H8 antibody having IgG1 constant regions ChiA1 γ4, a hybrid A1 antibody having IgG4 constant regions ChiA2 γ4, a hybrid A2 antibody having IgG4 constant regions ChiA3 γ4, a hybrid A3 antibody having IgG4 constant regions (+/-), partial binding (-), lack of binding

Claims (42)

1. An isolated antibody or antibody fragment that specifically binds a human 5T4 antigen, said antibody or said specific antibody fragment containing
(a) three hypervariable regions of the variable region of the heavy chain, which has the sequence of SEQ ID NO: 2, and three hypervariable regions of the variable region of the light chain, which has the sequence of SEQ ID No: 4;
(b) three hypervariable regions of the variable region of the heavy chain, which has the sequence of SEQ ID No: 6, and three hypervariable regions of the variable region of the light chain, which has the sequence of SEQ ID No: 8; or
(c) three hypervariable regions of the variable region of the heavy chain, which has the sequence of SEQ ID NO: 10, and three hypervariable regions of the variable region of the light chain, which has the sequence of SEQ ID No: 12. 2. The antibody according to claim 1, characterized in that said antibody is a chimeric antibody, a humanized antibody, a single chain antibody, a Fab fragment, an F (ab) ₂ fragment, an Fv fragment, a tetrameric antibody, a tetravalent antibody, a domain-specific antibody or recombinant protein. 3. The antibody according to claim 1, characterized in that said antibody is a mouse monoclonal antibody. 4. The antibody according to claim 1, characterized in that said antibody has a binding affinity for the 5T4 human antigen of at least about 1 · 10 ^-7 M to about 1 · 10 ^-12 M. 5. The antibody according to claim 1, characterized in that said antibody is specifically directed to 5T4-expressing cells in vivo. 6. The antibody according to claim 1, characterized in that the variable region of the heavy chain of the indicated antibody contains a sequence of amino acid residues 20-138 of the sequence SEQ ID No: 2, residues 19-135 of the sequence SEQ ID No: 6 or residues 20-141 of the SEQ ID sequence No .: 10. 7. The antibody according to claim 1, characterized in that the variable region of the light chain of the indicated antibody contains a sequence of residues 21-127 of the sequence SEQ ID No: 4, residues 23-130 of the sequence SEQ ID No: 8 or residues 21-127 of the sequence SEQ ID No : 12. 8. The antibody according to claim 1, characterized in that the variable region of the heavy chain of the indicated antibody contains the sequence of amino acid residues 20-138 of the sequence SEQ ID No: 2 and in which the variable region of the light chain contains the sequence of amino acid residues 21-127 of the sequence SEQ ID No: four. 9. The antibody according to claim 1, characterized in that the variable region of the heavy chain of the indicated antibody contains an amino acid residue sequence derived from residues 19-135 of the sequence SEQ ID No: 6, and in which the variable region of the light chain contains an amino acid residue sequence obtained from residues 23-130 of the sequence of SEQ ID No: 8. 10. The antibody according to claim 1, characterized in that the variable region of the heavy chain of the indicated antibodies contains an amino acid residue sequence derived from residues 20-141 of the sequence SEQ ID No: 10, and in which the variable region of the light chain contains an amino acid residue sequence derived from residues 21-127 of the sequence of SEQ ID No: 12. 11. The isolated antibody according to claim 1, which specifically binds to the 5T4 human epitope, containing residues 173-252 and 276-355 of the sequence SEQ ID No: 87. 12. The isolated antibody according to claim 1, which specifically binds to the human 5T4 epitope, which contains amino acids 276-355 of the sequence SEQ ID No: 87. 13. The isolated antibody according to claim 1, which specifically binds to the 5T4 human epitope, which contains amino acids 83-163 of the sequence SEQ ID No: 87. 14. The antibody according to claim 2, characterized in that said antibody is a hybrid or humanized anti-5T4 antibody. 15. The antibody of claim 14, wherein said antibody comprises constant regions derived from constant regions of human antibodies. 16. The antibody according to clause 15, wherein the constant region of the human light chain is obtained from the constant region of the human kappa light chain. 17. The antibody or antibody fragment of claim 15, wherein the human heavy chain constant region is derived from the human heavy chain constant region IgGI, IgG2, IgG3, or IgG4. 18. The antibody or antibody fragment according to 17, characterized in that the constant region of the heavy chain of human IgG4 contains proline at position 241. 19. The antibody of claim 14, wherein the variable region of the light chain and / or the variable region of the heavy chain of the specified antibody contains a frame region containing the remains of the frame region of a human antibody. 20. The antibody according to claim 19, characterized in that the residues of human amino acids are amino acid residues of the framework regions of human antibodies selected from the group consisting of
(a) the frame region of the heavy chain of a human antibody selected from the group consisting of: DP-21 (VH7), DP-54 (VH3-07), DP-47 (VH3-23), DP-53 (VH-74), DP-49 (VH3-30), DP-48 (VH3-13), DP-75, DP-8 (VH1-2), DP-25, VI-2b and VI-3 (VH1-03), DP- 15 and V1-8 (VH1-08), DP-14 and V1-18 (VH1-18), DP-5 and V1-24P (VH1-24), DP-4 (VH1-45), DP-7 ( VH1-46), DP-10, DA-6 and YAC-7 (VH1-69), DP-88 (VH1-e), DP-3 and DA-8 (VH1-f);
(b) the framework region of the light chain of a human antibody of a clone of the germline of DPK24 subgroup IV, subgroup VkIII (DPK23, DPK22, DPK20, DPK21), or a clone of the germline of the subgroup VkI (DPK9, DPK1, O2, DPK-7);
(c) the consensus sequence of the heavy chain framework region of claim (a); and
(d) a frame region that is at least 63% identical to the frame region according to (a) to (c). 21. The antibody according to 14, characterized in that the sequence of the variable region of the heavy chain of the specified antibody contains
(a) a sequence of amino acid residues 20-138 of the sequence of SEQ ID No: 2;
(b) an amino acid sequence that is at least 85% identical to residues 20-138 of the sequence SEQ ID No: 2;
(c) the sequence of amino acid residues 19-135 of the sequence SEQ ID No: 6;
(d) an amino acid sequence that is at least 86% identical to residues 19-135 of the sequence SEQ ID No: 6;
(e) the amino acid sequence of residues 20-141 of the sequence of SEQ ID No: 10;
(e) an amino acid sequence that is at least 91% identical to residues 20-141 of the sequence SEQ ID No: 10;
(g) an amino acid residue sequence corresponding to any of the sequences of SEQ ID NO: 49, 51, 52, 54, 56, 77, 78, 81, or 82;
(h) an amino acid sequence that is at least 91% identical to the sequence of SEQ ID No: 51;
(i) an amino acid sequence that is at least 78% identical to the sequence of SEQ ID No: 54;
(k) an amino acid sequence that is at least 89% identical to the sequence of SEQ ID No: 77;
(k) an amino acid sequence that is at least 79% identical to the sequence of SEQ ID No: 78;
(m) an amino acid sequence that is at least 80% identical to the sequence of SEQ ID No: 81;
(n) an amino acid sequence that is at least 78% identical to the sequence of SEQ ID No: 82,
moreover, these percentages of identity are achieved through one or more conservative substitutions of amino acids in the indicated sequences. 22. The antibody according to 14, characterized in that the sequence of the variable region of the light chain of the specified antibody contains
(a) a sequence of amino acid residues 21-127 of the sequence SEQ ID No: 4;
(b) an amino acid sequence that is at least 94% identical to residues 21-127 of the sequence SEQ ID No: 4;
(c) a sequence of amino acid residues 23-130 of the sequence SEQ ID No: 8;
(g) an amino acid sequence that is at least 96% identical to residues 23-130 of the sequence SEQ ID No: 8;
(e) a sequence of amino acid residues 21-127 of the sequence SEQ ID No: 12;
(e) an amino acid sequence that is at least 98% identical to residues 21-127 of the sequence SEQ ID No: 12;
(g) the amino acid residue sequence of any of the sequences of SEQ ID No: 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 80, 83 or 84;
(h) an amino acid sequence that is at least 83% identical to the sequence of SEQ ID No: 60;
(i) an amino acid sequence that is at least 93% identical to the sequence of SEQ ID No: 70;
(k) an amino acid sequence that is at least 85% identical to the sequence of SEQ ID No: 76;
(k) an amino acid sequence that is at least 88% identical to the sequence of SEQ ID No: 79;
(m) an amino acid sequence that is at least 84% identical to the sequence of SEQ ID No: 80;
(n) an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 83; or
(o) an amino acid sequence that is at least 91% identical to the sequence of SEQ ID No: 84,
moreover, these percentages of identity are achieved through one or more conservative substitutions of amino acids in the indicated sequences. 23. The antibody of claim 14, which is hybrid or humanized and includes
(a) a variable region of the light chain that contains an amino acid sequence selected from the group consisting of the sequence of amino acid residues 21-127 of SEQ ID No: 4 and any of SEQ ID No: 58, 60, 62, 64, 66, 68, 70 , 72, 74 or 76; and
(b) a variable region of the heavy chain that contains an amino acid sequence selected from the group consisting of a sequence of amino acid residues 20-138 of SEQ ID No: 2 and any of SEQ ID No: 49, 51, 52, 54 or 56. 24. The antibody of claim 14, which is hybrid or humanized and includes
(a) the variable region of the light chain, which contains the amino acid sequence of SEQ ID No: 70; and
(b) the variable region of the heavy chain, which contains the amino acid sequence of SEQ ID No: 54. 25. The conjugate of the antibody with the drug for drug delivery to cells expressing 5T4, including
(a) an antibody according to any one of claims 1-24; and
(b) a drug that is directly or indirectly associated with the antibody. 26. The antibody-drug conjugate of claim 25, wherein said drug is a therapeutic agent selected from the group consisting of a cytotoxin, a radioisotope, an immunomodulating agent, an anti-angiogenic agent, an anti-prophylactic agent, a pro-apoptotic agent, a chemotherapeutic agent and a therapeutic nucleic acid. 27. The conjugate of the antibody with the drug according to p, characterized in that the therapeutic agent is a cytotoxin. 28. The antibody-drug conjugate of claim 26, wherein the cytotoxin is an antibiotic, tubulin polymerization inhibitor, alkylating agent, protein synthesis inhibitor, protein kinase inhibitor, phosphatase inhibitor, topoisomerase inhibitor or enzyme. 29. The conjugate of the antibody with the drug according to p, characterized in that the cytotoxin is an antibiotic. 30. The conjugate of the antibody with the drug according to clause 29, wherein the antibiotic is calicheamicin. 31. The conjugate of the antibody with the drug according to item 30, wherein the calicheamicin is an N-acetyl derivative or disulfide analogue of calicheamicin. 32. The conjugate of the antibody with the drug according to p, characterized in that calicheamicin is an N-acetyl-γ-calicheamicin. 33. The conjugate of the antibody with the drug according to p, characterized in that the drug is associated with the antibody through a linker. 34. The antibody-drug conjugate according to claim 33, wherein the linker is selected from the group consisting of 4- (4'-acetylphenoxy) butyric acid (AcBut), 3-acetylphenylacetic acid (AcPac), 4-mercapto-4 -methyl-valeric acid (Amide) and their derivatives. 35. A method for delivering a drug to 5T4-expressing cells, comprising bringing the cells into contact with an antibody conjugate with a drug containing (i) an anti-5T4 antibody according to claim 1 and (ii) a drug that is associated with an anti-5T4 antibody directly or indirectly. 36. The method according to clause 35, wherein the drug penetrates into the target cell. 37. A method of treating a subject having a 5T4-positive cancer, which comprises administering to the subject a therapeutically effective amount of an anti-5T4 antibody conjugate with a drug, said conjugate comprising (i) an anti-5T4 antibody according to any one of claims 1 to 24 of the claims and (ii) a therapeutic agent that is directly or indirectly linked to an anti-5T4 antibody. 38. The method according to clause 37, wherein the anti-5T4 antibody conjugate with the drug is an anti-5T4 antibody conjugate with calicheamicin, the method further comprising administering a second therapeutic agent, wherein the anti-5T4 antibody conjugate with calicheamicin and a second drug are administered simultaneously or sequentially in any order. 39. An isolated nucleic acid encoding a variable region of a heavy chain of an anti-5T4 antibody or a conservatively substituted variant thereof, including
(a) a nucleotide sequence of 58-414 of the sequence of SEQ ID No: 1;
(b) a nucleotide sequence of 55-405 of the sequence of SEQ ID No: 5;
(c) a nucleotide sequence of 58-423 of the sequence of SEQ ID No: 9;
(d) a sequence encoding any of the sequences of SEQ ID No: 48, 50, 53 or 55;
(e) a nucleotide sequence that encodes a variable region of the heavy chain corresponding to any of SEQ ID No: 49, 51, 54, 56, 77, 78, 81 and 82;
(f) a sequence that is 89% identical to the sequence of SEQ ID No: 50, if the coverage of the request is 100%;
(g) a sequence that is 82% identical to the sequence with identification number SEQ ID No.; 53 if the coverage of the request is 100%; or
(h) a nucleic acid that specifically hybridizes with the complement of any of sequences (a) to (d) under stringent hybridization conditions. 40. An isolated nucleic acid encoding a variable region of a light chain of an anti-5T4 antibody or a conservatively substituted variant thereof, including
(a) a nucleotide sequence of 61-381 of the sequence of SEQ ID No: 3;
(b) the sequence of nucleotides 67-390 of the sequence SEQ ID no: 7;
(c) a nucleotide sequence of 61-381 of the sequence of SEQ ID No: 11;
(d) a nucleotide sequence encoding the variable region of the light chain of a humanized antibody that has any sequence from SEQ ID No: 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75;
(e) a nucleotide sequence encoding a variable region of the light chain of a humanized antibody that has any sequence from SEQ ID No: 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 80, 83 and 84 ;
(e) a nucleotide sequence that is 84% identical to the sequence of SEQ ID No: 59, if the coverage of the request is 100%;
(g) a nucleotide sequence that is 86% identical to the sequence of SEQ ID No: 69, if the query coverage is 100%;
(h) a nucleotide sequence that is 85% identical to the sequence of SEQ ID No: 75, if the coverage of the request is 100%; or
(i) a nucleic acid that specifically hybridizes with the complement of any of sequences (a) to (d) under stringent hybridization conditions. 41. The use of antibodies according to claim 1 for the production of antibodies with multiple specificity. 42. The use of the antibody according to claim 1 for the production of a single domain antibody that specifically binds to human 5T4 antigen.

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