TWI406871B - Anti-tat226 antibodies and immunoconjugates - Google Patents

Abstract

Anti-TAT226 antibodies and immunoconjugates thereof are provided. Methods of using anti-TAT226 antibodies and immunoconjugates thereof are provided.

Description

Anti-TAT226 Antibody and Immunoconjugates

The present invention relates to an anti-TAT226 antibody and an immunoconjugate thereof. The invention further relates to methods of using anti-TAT226 antibodies and immunoconjugates thereof.

Antibodies that bind to polypeptides that are expressed on the surface of cancer cells have been shown to be effective anti-cancer therapies. Such anti-systems function via a variety of mechanisms including, for example, activation of antibody-dependent cell-mediated cytotoxicity (ADCC); induction by antibodies with complement-dependent cytotoxicity (CDC); enhancement of cytokine release and induction of cells Apoptosis. See, for example, Cardarelli et al. (2002) Cancer Immunol. Immunother . 51: 15-24. For example, HERCEPTIN And RITUXAN (both from Genentech Inc., South San Francisco, California) are antibodies that have been successfully used to treat breast cancer and non-Hodgkin's lymphoma, respectively. HERCEPTIN A recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. Overexpression of HER2 protein was observed in 25-30% of primary breast cancers. RITUXAN A chimeric murine/human monoclonal antibody is engineered to counteract the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both antibodies were recombinantly produced in CHO cells. HERCEPTIN It seems to work (at least in part) by inhibiting angiogenesis (Izumi et al. (2002) Nature 416: 279-280) and RITUXAN It appears that it acts (at least in part) by inducing apoptosis (Cardarelli et al. (2002) Cancer Immunol. Immunother . 51: 15-24).

Immunoconjugates or "antibody-drug conjugates" are suitable for the local delivery of cytotoxic agents in the treatment of cancer. See, for example, Syrigos et al. (1999) Anticancer Research 19: 605-614; Niculescu-Duvaz et al. (1997) Adv. Drug Deliv. Rev. 26: 151-172; U.S. Patent No. 4,975,278. The immunoconjugate allows targeted delivery of the drug moiety to the tumor, while systemic administration of the unconjugated cytotoxic agent can produce an unacceptable degree of toxicity to normal cells as well as tumor cells to be eliminated. See Baldwin et al. (March 15, 1986) Lancet 603-05; Thorpe (1985) Monoclonal Antibodies '84 : Biological and Clinical Applications (A. Pinchera et al.) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," pp. 475-506. Immunoconjugates targeting cell surface polypeptides have been developed and will continue to be developed for the treatment of cancer. For a discussion, see, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103.

Clearly, there is a continuing need for agents that target cell surface polypeptides for diagnostic and/or therapeutic purposes. The invention described herein satisfies this need and provides other benefits.

All documents cited herein, including patent applications and publications, are hereby incorporated by reference.

The invention provides anti-TAT226 antibodies and methods of use thereof.

In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises at least one, two, three, four, five or six HVRs selected from the group consisting of: (1) comprising SEQ ID NO: HVR-H1 of the amino acid sequence; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (3) HVR- comprising the amino acid sequence corresponding to the sequence identical to SEQ ID NO: H4; (4) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (6) comprising and SEQ ID NO: HVR-L3 of the amino acid sequence of a consistent sequence of 19.

In another aspect, the antibody that binds to TAT226 comprises (a) HVR-H3 comprising at least one amino acid sequence corresponding to the sequence of SEQ ID NO: 11 and (b) at least one, two, three, four Or five HVRs selected from the group consisting of: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; a HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (6) comprising a sequence identical to SEQ ID NO: 19. Consistent amino acid sequence of HVR-L3.

In one embodiment, the antibody comprises HVR-L3 having an amino acid sequence consistent with the sequence of SEQ ID NO: 19. In one embodiment, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4 and HVR-H2 having the amino acid sequence of SEQ ID NO: 5. In one embodiment, the antibody further comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13.

In one embodiment, the antibody comprises HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10. In one embodiment, the antibody further comprises HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-18. In one embodiment, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9, and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 10 and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18. In one embodiment, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4 and HVR-H2 having the amino acid sequence of SEQ ID NO: 5. In one embodiment, the antibody further comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13.

In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises at least one, two, three, four, five or six HVRs selected from the group consisting of: (1) comprising SEQ ID NO: HVR-H1 of the amino acid sequence; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (4) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (6) amino acid sequence comprising SEQ ID NO: 14. HVR-L3.

In another aspect, the antibody that binds to TAT226 comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and (b) at least one, two, three, four or five selected From the following HVRs: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (3) comprising SEQ ID NO HVR-L1 of the amino acid sequence of 12; (4) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (5) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. .

In one embodiment, the antibody comprises HVR-L3 having the amino acid sequence of SEQ ID NO: 14. In one embodiment, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 1 and HVR-H2 having the amino acid sequence of SEQ ID NO: 2. In one embodiment, the antibody further comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 12 and HVR-L2 having the amino acid sequence of SEQ ID NO: 13.

In certain embodiments, any of the above antibodies additionally comprises at least one framework selected from the group consisting of a VH subgroup III consensus framework and a VL subgroup I consensus framework.

In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises a heavy chain variable domain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-25. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31. In one embodiment, the antibody comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:29. In one embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 24 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 29. In one embodiment, the antibody comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:30. In one embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 25 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:30.

In one aspect, an antibody that binds to TAT226 is provided, wherein the antibody comprises a heavy chain variable domain that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody further comprises a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 26. In one embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 20 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 26.

In certain embodiments, a polynucleotide encoding any of the above antibodies is provided. In one embodiment, a vector comprising a polynucleotide is provided. In one embodiment, a host cell comprising a vector is provided. In one embodiment, the host cell is eukaryotic. In one embodiment, the host cell is a CHO cell. In one embodiment, a method of making an anti-TAT226 antibody is provided, wherein the method comprises culturing a host cell under conditions suitable to represent a polynucleotide encoding the antibody and isolating the antibody.

In one aspect, an antibody that binds to TAT226 that is expressed on the cell surface is provided. In one embodiment, the antibody binds to an epitope in the TAT226 region from amino acid 21-115 of SEQ ID NO:75. In one embodiment, the cell is a cancer cell. In one embodiment, the cancer cell is an ovarian cancer cell, a brain tumor cell, or a Wilm's tumor cell.

In certain embodiments, any of the above antibodies are monoclonal antibodies. In one embodiment, the antibody is an antibody fragment selected from the group consisting of a Fab, Fab'-SH, Fv, scFv or (Fab') ₂ fragment. In one embodiment, the antibody is humanized. In one embodiment, the antibody is human. In one embodiment, the antibody binds to the same antigen as the antibody selected from the group consisting of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6. base.

In one aspect, a method of detecting the presence of TAT226 in a biological sample comprising contacting the biological sample with any of the above antibodies and detecting formation between the antibody and TAT226 is provided under conditions permitting binding of the antibody to TAT226 Complex. In one embodiment, the biological sample comprises ovarian tumor cells, brain tumor cells, or Wilm's tumor cells.

In one aspect, a method of diagnosing a cell proliferative disorder associated with an increase in TAT226 expression is provided, the method comprising contacting a test cell with any of the above antibodies; determining the degree of performance of the TAT226 by detecting binding of the antibody to TAT226 And comparing the degree of performance of the test cells to TAT226 to the extent of performance of the control cells to TAT226, wherein the higher degree of performance of the test cells to TAT226 compared to the control cells indicates the presence of a cell proliferative disorder associated with increased performance of TAT226. In one embodiment, the test cell is a cell from a patient suspected of having a cell proliferative disorder. In one embodiment, the cell proliferative disorder is selected from the group consisting of ovarian cancer and Wilm's tumor. In one embodiment, the method comprises determining the extent of performance of TAT226 on the surface of the test cells and comparing the extent of expression of TAT226 on the surface of the test cells to the extent of expression of TAT226 on the surface of the control cells.

The invention further provides immunoconjugates and methods of use thereof.

In one aspect, the immunoconjugate comprises any of the above anti-TAT226 antibodies covalently linked to a cytotoxic agent. In one embodiment, the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope, and a nuclear degrading enzyme.

In one aspect, an immunoconjugate having the formula Ab-(L-D)p is provided, wherein: (a) Ab is any of the above anti-TAT226 antibodies, (b) L is a linker; (c) D is formula D _E or D _F drug And wherein R ² and R ⁶ are each a methyl group, R ³ and R ⁴ are each an isopropyl group, and R ⁷ is a second butyl group, and each R ⁸ group is independently selected from the group consisting of CH ₃ , O-CH ₃ , OH, and H. ; R ⁹ is H; R ¹⁰ is aryl; Z is -O- or -NH-; R ¹¹ is H, C ₁ -C ₈ alkyl or -(CH ₂ ) ₂ -O-(CH ₂ ) ₂ - O-(CH ₂ ) ₂ -O-CH ₃ ; and R ¹⁸ is -C(R ⁸ ) ₂ -C(R ⁸ ) ₂ -aryl; and (d)p is in the range of about 1 to 8.

In one embodiment, the antibody (Ab) comprises 1) HVR-H3 comprising an amino acid sequence consistent with the sequence of SEQ ID NO: 11 and 2) at least one, two, three, four or five An HVR selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (iii) comprising SEQ ID NO: 12 HVR-L1 of the amino acid sequence; (iv) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (v) an amino group comprising the sequence identical to SEQ ID NO: 19. Acid sequence HVR-L3.

In one embodiment, the antibody comprises HVR-L3 having an amino acid sequence consistent with the sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 9 and HVR-L3 having the amino acid sequence of SEQ ID NO: 17. In one embodiment, the antibody comprises HVR-H3 having the amino acid sequence of SEQ ID NO: 10 and HVR-L3 having the amino acid sequence of SEQ ID NO: 18. In one embodiment, the antibody further comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 4; HVR-H2 having the amino acid sequence of SEQ ID NO: 5; having the amino group of SEQ ID NO: HVR-L1 of the acid sequence and HVR-L2 having the amino acid sequence of SEQ ID NO: 13. In one embodiment, the antibody comprises a heavy chain variable region having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-25 and an amine group selected from the group consisting of SEQ ID NOs: 26-31 The acid sequence has a light chain variable region with at least 90% sequence identity. In one embodiment, the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 and at least 90% identical to the amino acid sequence of SEQ ID NO: 29. Sexual light chain variable region. In one embodiment, the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 29. In one embodiment, the antibody comprises a heavy chain variable region having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 25 and at least 90% identical to the amino acid sequence of SEQ ID NO: 30. Sexual light chain variable region. In one embodiment, the antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30.

Additional examples of any of the above immunoconjugates are provided. In one embodiment, the immunoconjugate has cell killing activity in vitro or in vivo. In one embodiment, the linker is linked to the antibody via a thiol group on the antibody. In one embodiment, the linker can be cleaved by a protease. In one embodiment, the linker comprises a val-cit dipeptide. In one embodiment, the linker comprises a p-aminobenzyl unit. In one embodiment, the aminobenzyl unit is placed between the protease and the protease cleavage site in the linker. In one embodiment, the p-aminobenzyl unit is p-aminobenzyloxycarbonyl (PAB). In one embodiment, the linker comprises 6-maleimide hexamethylene. In one embodiment, the 6-maleimide hexyl group is placed between the antibody and the protease cleavage site in the linker. The above embodiments may occur singly or in any combination with each other.

In one embodiment, the drug is selected from the group consisting of MMAE and MMAF. In one embodiment, the immunoconjugate has the following formula Wherein Ab is any of the above anti-TAT226 antibodies, S is a sulfur atom and p is in the range of 2 to 5.

In one embodiment, the immunoconjugate has the following formula Wherein Ab is any of the above anti-TAT226 antibodies, S is a sulfur atom and p is in the range of 2 to 5.

In one aspect, a pharmaceutical composition comprising any of the above immunoconjugates and a pharmaceutically acceptable carrier is provided. In one aspect, a method of treating a cell proliferative disorder, wherein the method comprises administering to a subject a pharmaceutical composition. In one embodiment, the cell proliferative disorder is selected from the group consisting of ovarian cancer, uterine cancer, brain tumors, and Wilm's tumors. In one embodiment, the cell proliferative disorder is associated with increased expression of TAT226 on the cell surface.

In one aspect, a method of inhibiting cell proliferation is provided, wherein the method comprises exposing the cell to any of the above immunoconjugates under conditions that permit binding of the immunoconjugate to TAT226. In one embodiment, the cells are tumor cells. In one embodiment, the tumor cells are ovarian tumor cells, uterine tumor cells, brain tumor cells, or Wilm's tumor cells. In one embodiment, the cells are xenografts. In one embodiment, the exposure occurs outside the body. In one embodiment, the exposure occurs in a living body.

An isolated antibody that binds to TAT226 is provided. An immunoconjugate comprising an anti-TAT226 antibody is also provided. For example, the antibodies and immunoconjugates of the invention are useful for the diagnosis or treatment of conditions associated with altered performance of TAT226, such as increased performance. In certain embodiments, an antibody or immunoconjugate of the invention is useful for the diagnosis or treatment of a cell proliferative disorder, such as a tumor or cancer. In certain embodiments, an antibody or immunoconjugate of the invention is suitable for detecting TAT226, such as TAT226, which is expressed on the surface of a cell.

A polynucleotide encoding an anti-TAT226 antibody is provided. A vector comprising a polynucleotide encoding an anti-TAT226 antibody is provided and a host cell comprising such a vector is provided. Compositions comprising any one or more of the polynucleotides of the invention, an anti-TAT226 antibody or an immunoconjugate, including pharmaceutical formulations, are also provided.

I. General technology

The techniques and procedures described or referenced herein are generally well known to those skilled in the art and are generally employed using conventional methods such as those described in the following documents: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel et al., (2003)); the series Methods in Enzymology (Academic Press, Inc.): Pcr 2 : A Practical Approach (MJMacPherson, BDHames and GRTaylor eds (1995)), Harlow and Lane eds (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RIFreshney ed (1987)); Oligonucleotide Synthesis ( MJGait ed, 1984); Methods In Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), eds. (1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, ed. JB Griffiths and DG Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, ed., 1987); PCR: The Polymerase Chain Reaction , (Mullis et al., 1994); Current Protocols In Immunology (JEColigan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach ( D. Catty. ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra eds, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds. JBLippincott Company, 1993).

II. Definitions and abbreviations A. Definition

An "isolated" antibody is an antibody that is identified and isolated and/or recovered from components of its natural environment. The contaminant component of its natural environment is a substance that interferes with the research, diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody (1) is purified to greater than 95% by weight of the antibody as determined, for example, by the Lowry method and, in some embodiments, greater than 99% by weight; (2) purified to Using, for example, a rotary cup sequencer sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues; or (3) by using, for example, Coomassie blue or under reduced or non-reducing conditions Silver stained SDS-PAGE was purified to homogeneity. An isolated antibody includes an antibody in situ in a recombinant cell, as at least one component of the antibody's natural environment will not be present. However, the isolated antibody will typically be prepared by at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from at least one other nucleic acid molecule that is normally associated with it, for example, in its natural environment. The isolated nucleic acid molecule additionally includes a nucleic acid molecule contained in a cell that normally represents the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

By "purified" is meant that the molecule is present in the sample at a concentration of at least 95% by weight or at least 98% by weight in the sample containing it.

The terms "substantially similar" or "substantially identical" as used herein mean a sufficiently high degree of similarity between two values (eg, one associated with an antibody of the invention and the other associated with a reference/comparative antibody). To the extent that the skilled artisan believes that the difference between the two values is substantially free or biologically and/or statistically significant in the case of biometrics measured by the equivalent (e.g., Kd value). The difference between the two values is a function of the reference/comparison value, such as less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10%.

As used herein, the phrase "substantially lower" or "substantially different" means a sufficiently high degree of difference between two values (generally one associated with a molecule and the other associated with a reference/comparative molecule), To allow those skilled in the art to recognize that the difference between the two values is statistically significant in the case of biometrics measured by the equivalent (eg, Kd value). The difference between the two values is a function of the value of the reference/comparative molecule, such as greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50%.

The term "vector" as used herein is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plastid" which refers to a circular double stranded DNA that can engage additional DNA segments. Another type of vector is a phage vector. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced, such as a bacterial vector having a bacterial origin of replication and a free mammalian vector. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the performance of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply as "expression vectors." In general, expression vectors for use in recombinant DNA techniques are typically in plastid form. In the present specification, since the plastid is the most commonly used carrier form, the "plastid" and the "carrier" can be used interchangeably.

"Polynucleotide" or "nucleic acid" as used interchangeably herein, refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or a base, and/or an analog thereof, or may be incorporated into the polymer by DNA or RNA polymerase or by a synthesis reaction. Any of the qualities. Polynucleotides can comprise modified nucleotides, such as methylated nucleotides and analogs thereof. If present, the nucleotide structure can be modified before or after assembly of the polymer. The nucleotide sequence can be interrupted by a non-nucleotide component. The polynucleotide may comprise modifications that are made after synthesis, such as binding to a label. Other types of modifications include, for example, "hats"; substitution of one or more nucleotides naturally occurring with analogs; internucleotide modifications, such as those having uncharged linkages (eg, methyl phosphonate, phosphotriester, phosphine) Phtharate, urethane, etc.) and those having a charge linkage (eg, phosphorothioate, phosphorodithioate, etc.); such as proteins (eg, nucleases, toxins, antibodies) , signal peptides, ply-L-isoamine, etc.); those with intercalating agents (such as acridine, psoralen, etc.); those who contain chelating agents (such as metals, radioactive metals, boron, oxidizing metals) And the like; those which contain an alkylating agent; those having a modified linkage (for example, a α-helical isomeric nucleic acid, etc.) and an unmodified form of the polynucleotide. In addition, any hydroxyl group typically present in the sugar can be replaced, for example, by a phosphonate group, a phosphate group; an additional linkage that is protected or activated by a standard protecting group to make additional nucleotides, or can be bound to a solid Or a semi-solid support. The 5' and 3' terminal OH groups may be substituted by phosphorylation or by an amine or an organic capping group having 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. The polynucleotide may also contain ribose or deoxyribose sugars, generally in a similar form known in the art, including, for example, 2'-O-methyl-;2'-O-allyl-;2'-fluoro- Or 2'-azido-ribose; carbocyclic sugar analog; α-raceomeric sugar; epimeric sugar, such as arabinose, xylose or lyxose; palladium; furanose; Sedoheptulose; an acyclic analog and a basic nucleoside analog such as a methyl riboside. One or more phosphodiester linkages may be replaced by an alternative linkage group. Such alternative linking groups include, but are not limited to, wherein the phosphate ester is derived from P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR ₂ ( "Amides ester"), P (O) R , P (O) oR ', CO or CH ₂ ( "methylal") replacing the embodiment in which each R or R' is independently H or optionally A substituted or unsubstituted alkyl group (1-20 C), an aryl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group or an araldyl group containing an ether (-O-) bond. All of the bonds in the polynucleotide need not be the same. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" generally refers to a short, generally single-stranded, generally synthetic polynucleotide of generally less than, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.

The "amino acid sequence identity percent (%)" with respect to a reference polypeptide sequence is defined as the amine in the candidate sequence and the reference polypeptide sequence after the alignment sequence is introduced and, if necessary, introduced into the gap to achieve a maximum sequence identity percentage. The percentage of amino acid residues with the same base acid residues, and no conservative substitutions are considered part of the sequence identity. The alignment for the purpose of determining the percent identity of the amino acid sequence can be achieved in various ways in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for the alignment sequence, including any algorithms needed to achieve maximum alignment of the full length of the sequences being compared. However, for the purposes of this document, the amino acid sequence identity % value was generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been filed in U.S. Copyright Office, Washington D.C., 20559 as a user file, which is registered under U.S. Copyright registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Franci Sco, California or can be compiled from source code. The ALIGN-2 program should be compiled for use in a UNIX operating system, preferably a digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In the case where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence sequence of the amino acid sequence A and the designated amino acid sequence B is specified to be % identical (alternatively expressed as a specified amino acid sequence) The designated amino acid sequence A of B having or containing % of a particular amino acid sequence identity is calculated as follows: 100 x fraction X/Y where X is in the alignment of A and B, by sequence alignment program ALIGN -2 is labeled as the number of identically matched amino acid residues, and wherein Y is the total number of amino acid residues in B. It will be appreciated that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the % identity of the amino acid sequence of A and B will not be equal to the % identity of the amino acid sequence of B and A. Unless otherwise specifically stated, all amino acid sequence identity % values used herein were obtained using the ALIGN-2 computer program as described in the previous paragraph.

The term "TAT226" as used herein, unless otherwise indicated, refers to any native source from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats). TAT226. The term encompasses "full length" unprocessed TAT226 and any form of TAT226 produced by processed cells. The term also encompasses naturally occurring TAT226 variants, such as splice variants or dual gene variants. The "mature form" of TAT226 is in the form of a TAT226 that has been processed, for example, in the form of TAT226 that has been cleaved by the N-terminus (eg, signal sequence) and/or C-terminus and/or modified by a GPI binding agent. In one embodiment, the "mature form" of TAT226 is expressed on the cell surface.

"Antibody" (Ab) and "immunoglobulin" (Ig) are glycoproteins having similar structural features. Although antibodies exhibit binding specificity for specific antigens, immunoglobulins include antibodies and other antibody-like molecules that are generally lacking in antigen specificity. The latter polypeptide is produced, for example, by the lymphatic system at low levels and by myeloma at increased levels.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (eg full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (eg bispecific) An antibody, as long as it exhibits the desired biological activity, and may also include certain antibody fragments (as described in more detail herein). The antibody may be a chimeric antibody, a human antibody, a humanized antibody, and/or an affinity matured antibody.

The term "anti-TAT226 antibody" or "antibody that binds to TAT226" refers to an antibody that is capable of binding TAT226 with sufficient affinity to render the antibody suitable for use as a diagnostic and/or therapeutic agent for targeting TAT226. Preferably, the degree of binding of the anti-TAT226 antibody to the unrelated, non-TAT226 protein is less than about 10% of the binding of the antibody to TAT226 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to TAT226 has 1 μM, 100 nM, 10 nM, 1 nM or Dissociation constant (Kd) of 0.1 nM. In certain embodiments, the anti-TAT226 antibody binds to a TAT226 epitope that is conserved in TAT226 from a different species.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal end domain of an antibody heavy or light chain. The heavy chain variable domain can be referred to as "VH." The light chain variable domain can be referred to as "VL." These domains are generally the most variable part of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that the sequences of particular portions of the variable domains between antibodies are generally different and are used for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. It is concentrated in three segments, each of which is referred to as a complementarity determining region (CDR) or a hypervariable region (HVR), in the light and heavy chain variable domains. The higher degree of conservation of the variable domain is referred to as the framework region (FR). The variable domains of the native heavy and light chains each comprise a plurality of four FR regions joined by three CDRs in a beta-sheet configuration that form a loop junction and in some cases form part of a beta sheet structure. The CDRs in each chain are held together in close proximity by the FR region and, together with the CDRs of the other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest , Fifth Edition, National Institute of Health, Betheada, MD (1991)). The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as antibodies involved in antibody-dependent cytotoxicity.

The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be designated as an amino acid sequence based on its constant domain. And one of two completely different types of λ.

An antibody (immunoglobulin) can be assigned to a different species depending on the amino acid sequence of its heavy chain constant domain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of which may be further divided into e.g. _{2, IgG 3, IgG 4,} IgA 1 and IgA subclasses (isotypes IgG _1, IgG ₂ of ). The heavy chain constant domains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al. Cellular and Mol. Immunology , 4th Edition (2000). An antibody can be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as defined below. These terms especially refer to antibodies having a heavy chain comprising an Fc region.

An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion retains at least one of those typically associated with the presence of the portion in the intact antibody, and is as much or all of the functionality as possible. In one embodiment, the antibody fragment comprises the antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment (eg, an antibody fragment comprising an Fc region) retains at least one biological function normally associated with the presence of an Fc region in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC Function and complement combination. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to the intact antibody. For example, such antibody fragments can comprise an antigen binding arm linked to an Fc sequence that confers in vivo stability to the fragment.

Papaya digestion of antibodies produces two identical antigen-binding fragments, each of which has a single antigen-binding site, called a "Fab" fragment, and a residual "Fc" fragment whose name reflects its ability to crystallize readily. Pepsin treatment yields F(ab') ₂ fragments that have two antigen combining sites and are still crosslinkable.

"Fv" is the smallest antibody fragment that contains a complete antigen binding site. In one embodiment, the double-stranded Fv species consists of a dimer of one heavy chain that is tightly non-covalently associated with one light chain variable domain. In a single-chain Fv (scFv) species, one heavy chain and one light chain variable domain can be covalently linked by an elastin linker such that the light and heavy chains can be similar to the "dimerization" in a double-stranded Fv species. Structural association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In summary, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of the Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind antigen, although it has a lower affinity than the full binding site.

Fab fragments contain heavy and light chain variable domains and also contain a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxy terminus comprising the heavy chain CH1 domain from one or more of the antibody hinge regions. Fab'-SH is herein referred to as Fab', wherein the cysteine residue of the constant domain carries a free thiol group. The F(ab') ₂ antibody fragment is initially produced in the form of a Fab' fragment pair with hinged cysteine. Other compounding couplings of antibody fragments are also known.

A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein the domains are present in a single polypeptide chain. In general, the scFv polypeptide additionally comprises a polypeptide linker between the VH and VL domains which allows the scFv to form the desired structure for antigen binding. A discussion of the scFV see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "bifunctional antibody" refers to a small antibody fragment having two antigen binding sites comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain. (VH-VL). By using a linker that is too short to be paired between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Bifunctional antibodies can be bivalent or bispecific. Bifunctional anti-systems are more fully described, for example, in EP 404,097; WO 93/11161; Hudson et al. (2003) Nat. Med. 9: 129-134; and Hollinger et al., Proc. Natl. Acad. Sci. USA , 90: 6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al. (2003) Nat . Med. 9: 129-134.

The term "monoclonal antibody" as used herein refers to a population of antibodies that are substantially homogeneous (ie, identical to the individual resistance systems comprising the population, except for possible mutations that may be present in trace amounts (eg, naturally occurring mutations)) Antibody. Thus, the modifier "single plant" refers to an antibody characteristic that is not a mixture of discrete antibodies. In certain embodiments, the monoclonal antibody typically comprises an antibody comprising a polypeptide sequence that binds to a target, wherein the target binding polypeptide sequence is obtained by a method comprising selecting a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method can be to select a unique pure line from a plurality of pure lines (such as the sum of a fusion tumor pure line, a phage pure line, or a recombinant DNA pure line). It will be appreciated that the selected target binding sequence may be further altered, for example to improve affinity for the target; to humanize the target binding sequence; to improve its production in cell culture; to reduce its in vivo immunogenicity; to produce multispecific An antibody or the like, and an antibody comprising a target binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody against the individual antibody preparation is directed against a single determinant on the antigen. In addition to its specificity, the advantage of a monoclonal antibody preparation is that it is generally not contaminated by other immunoglobulins.

The modifier "single plant" refers to the characteristics of an antibody obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques, including, for example, fusion knob methods (e.g., Kohler et al, Nature , 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), recombinant DNA method (see, for example, US patents) No. 4,816,567), phage display technology (see, for example, Clackson et al, Nature , 352: 624-628 (1991); Marks et al, J. Mol . Biol . 222 : 581-597 (1992); Sidhu et al, J .Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol . Biol . 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132 (2004)) and in some or all humans having human immunoglobulin sequences encoding Techniques for producing human or human-like antibodies in immunoglobulin loci or genes in animals (see, for example, WO 98/24893; WO 96/34096; WO 96/33735; WO 91/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al, Nature 362:255-258 (1993); Bruggemann et al, Year in Immunol . 7:33 (1993); U.S. Patent No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al, Bio . Technology 10:779-783 (1992) Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al, Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol . 826 (1996) and Lonberg and Huszar, Intern . Rev. Immunol. 13: 65-93 (1995)).

Monoclonal antibodies herein specifically include "chimeric" antibodies in which portions of the heavy and/or light chains are identical or homologous to corresponding sequences derived from a particular species or antibodies belonging to a particular antibody class or subclass, and the remainder of the strand Partially identical or homologous to a corresponding sequence derived from another species or antibody belonging to another antibody class or subclass; and fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; And Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

A "humanized" form of a non-human (eg, murine) antibody is a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a non-human species (donor antibody) (such as a mouse, rat, rabbit, or rabbit) in which the residue of the hypervariable region of the receptor has the desired specificity, affinity, and/or capacity. Human immunoglobulin (receptor antibody) substituted with residues in the hypervariable region of a non-human primate. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to their loops of non-human immunoglobulins, and all or substantially All FRs are their FRs of human immunoglobulin sequences. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin (typically human immunoglobulin) constant region (Fc). For further elaboration, see Jones et al, Nature 321: 522-525 (1986) ; Riechmann et al, Nature 332: 323-329 (1988) ; and Presta, Curr.Op.Struct.Biol .2: 593-596 (1992). See also the following review article and references cited therein: Vaswani and Hamilton, Ann . Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem . Soc. Transactions 23: 1035-1038 (1995); Hurle And Gross, Curr . Op. Biotech. 5: 428-433 (1994).

A "human antibody" is an amino acid sequence having an amino acid sequence corresponding to an antibody produced by a human and/or an antibody which has been produced using any of the techniques for producing a human antibody as disclosed herein. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of a sequence that is hypervariable and/or forms an antibody variable domain of a structurally defined loop. In general, an antibody comprises six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among the native antibodies, H3 and L3 exhibit the six hypervariable regions with the greatest diversity, and in particular, the H3 has a unique effect of conferring excellent specificity to the antibody. Xu et al. (2000) Immunity 13: 37-45; Johnson and Wu (2003) in Methods in Molecular Biology 248: 1-25 (Lo, Human Press, Totowa, NJ). In fact, a naturally occurring camelid antibody consisting only of heavy chains is functional and stable in the absence of light chains. Hamers-Casterman et al. (1993) Nature 363: 446-448; Sheriff et al. (1996) Nature Struct. Biol. 3: 733-736.

The definitions of various hypervariable regions are used herein and encompassed. Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest , 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). In contrast, Chothia refers to the position of the structural ring (Chothia and Lesk J. Mol. Biol . 196:901-917 (1987)). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structuring loop and is used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable region is based on the analysis of available composite crystal structures. The residues from each of the hypervariable regions are as follows.

The hypervariable region may comprise the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) and VH in VL. Medium 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3). Variable domain residues are numbered according to Kabat et al. (supra) for their respective definitions.

"Framework" or "FR" residues are those variable domain residues that differ from the hypervariable region residues defined herein.

The term "such as the variable domain residue number in Kabat" or "such as the amino acid position number in Kabat" and its variants refer to the Kabat et al. Sequences of Proteins of Immunological Interest , 5th Edition Public Health Service , Numbering System for Antibody Compiled Heavy Chain Variable Domains or Light Chain Variable Domains, National Institutes of Health, Bethesda, MD (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the FR or HVR that shortens or inserts the variable domain. For example, a heavy chain variable domain can include a single amino acid inserted after residue 52 of H2 (residue 52a according to Kabat) and a residue inserted after heavy chain FR residue 82 (eg, according to Kabat Residues 82a, 82b, 82c, etc.). The Kabat numbering of the residues of the indicated antibodies can be determined by alignment of the sequence homology region of the antibody with the "standard" Kabat numbering sequence.

An "affinity mature" antibody is one that has one or more changes in one or more of its HVRs that result in improved affinity of the antibody for the antigen as compared to a parent antibody that does not have such a change. In one embodiment, the affinity matured antibody has a affinity for the target antigen for narm or even picomoles. Affinity mature anti-systems are produced by procedures known in the art. Affinity maturation by VH and VL domain shuffling is described by Marks et al. Bio/Technology 10:779-783 (1992). Random mutations in HVR and/or framework residues are described by Barbas et al. Proc Nat. Acad. Sci. USA 91: 3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton Et al . J. Immunol. 155: 1994-2004 (1995); Jackson et al, J. Immunol. 154(7): 3310-9 (1995); and Hawkins et al. J. Mol. Biol. 226: 889-896 (1992).

A "blocking" antibody or "antagonizing" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonizing antibodies substantially or completely inhibit the biological activity of the antigen.

A "promoting antibody" as used herein is an antibody that mimics at least one functional activity of a polypeptide of interest.

Antibody "effector function" refers to the biological activity attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region) and differs depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors) B cell activation.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is a receptor that binds to an IgG antibody (gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including dual gene variants of such receptors, and alternative spliced forms. Fc[gamma]RII receptors include FcyRIIA ("Activated Receptor") and FcyRIIB ("Inhibition Receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domain. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See Da Ron, Annu . Rev. Immunol. 15:203-234 (1997)). FcR is in Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab . Clin . Med. : 330-41 (1995). Other FcR lines including those that are intended to be recognized in the future are covered by the term "FcR".

The term "Fc receptor" or "FcR" also encompasses the transfer of maternal IgG to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)). And a neonatal receptor that regulates the stability of immunoglobulins, FcRn. Methods for measuring binding to FcRn are known (see, for example, Ghetie 1997, Hinton 2004). In vivo binding and serum half-life of human FcRn high affinity binding polypeptide to human FcRn can be assayed, for example, in a transgenic mouse expressing human FcRn or a transfected human cell strain or a primate Fc variant polypeptide. .

WO 00/42072 (Presta) describes antibody variants with improved or reduced binding to FcRs. The contents of these patent publications are incorporated herein by reference in their entirety. See also Shields et al . J. Biol. Chem. 9(2): 6591-6604 (2001).

A "human effector cell" is a white blood cell that exhibits one or more FcRs and performs effector functions. In certain embodiments, the cell exhibits at least FcyRIII and performs an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killing (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from, for example, the native source of blood.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the binding of secreted Ig to specific cytotoxic cells (eg, natural killing (NK) cells, neutrophils, and macrophages). The Fc receptor (FcR) allows these cytotoxic effector cells to specifically bind to the target cell carrying the antigen and subsequently kill the target cell in a cytotoxic form. Primary cells that mediate ADCC, NK cells only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The FcR expression lines on hematopoietic cells are summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991). In order to evaluate the ADCC activity of the molecule of interest, an in vitro ADCC assay such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, or to the disclosure of U.S. Patent No. 6,737,056. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural kill (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998).

"Complement dependent cytotoxicity" or "CDC" refers to the dissolution of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding the first component of the complement system (Clq) to an antibody that binds to its cognate antigen (an antibody of a suitable subclass). To assess complement activation, a CDC assay such as that described in Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996) can be performed.

Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99/51642. The contents of their patent publications are incorporated herein by reference in their entirety. See also Idusogie et al . J. Immunol. 164 : 4178-4184 (2000).

The term "polypeptide comprising an Fc region" refers to a polypeptide comprising an Fc region, such as an antibody or immunoadhesin. The C-terminal amide acid of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during polypeptide purification or by recombinant engineering of the nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region of the invention may comprise a polypeptide having K447; all K447 removed polypeptides or a mixture of polypeptides having no K447 residues.

The "receptor human framework" for the purposes herein is a framework comprising amino acid sequences derived from the human immunoglobulin framework or the VL or VH framework of the human consensus framework. A receptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise its identical amino acid sequence, or it may contain a pre-existing amino acid sequence change. In some embodiments, the number of previously occurring amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less. When a previously present amino acid change is present in VH, such changes preferably occur only at three, two or one of positions 71H, 73H and 78H; for example, amino acid residues at positions It can be 71A, 73T and/or 78A. In one embodiment, the sequence of the VL receptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

"Human consensus framework" is a framework that represents the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. In general, subgroups of sequences are subgroups as in Kabat et al., Sequences of Proteins of Immunological Interest , 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991). In an embodiment, for VL, the subgroup is a subgroup of Kabat et al. I. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. above.

The "VH subgroup III consensus framework" comprises the consensus sequence obtained from the amino acid sequence in variable weight subgroup III of Kabat et al. (supra). In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 50)-H1-WVRQAPGKGLEWV (SEQ ID NO: 51)-H2-RFTISADTSKNTAYLQMNSLRAEDTAVYYC ( SEQ ID NO: 59) - H3-WGQGTLVTVSS (SEQ ID NO: 35).

"VL Subgroup I Consistent Framework" contains variable lightness by Kabat et al. (ibid.) Consistent sequence obtained from the amino acid sequence in subgroup I. In one embodiment, the VH subgroup I consensus framework amino acid sequence comprises at least a portion or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 60) - L1-WYQQKPGKAPKLLIY (SEQ ID NO: 61) - L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC ( SEQ ID NO: 62) - L3-FGQGTKVEIK (SEQ ID NO: 63).

"Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between a binding member (eg, an antibody and an antigen), unless otherwise indicated. The affinity of the molecule X for its conjugate Y is generally represented by the dissociation constant (Kd). Affinity can be measured by general methods known in the art, including those described herein. Low affinity antibodies generally bind slowly to the antigen and tend to dissociate readily, while high affinity antibodies generally bind more rapidly to the antigen and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present invention. Specific illustrative embodiments are described below.

In one embodiment, the "Kd" or "Kd value" according to the present invention is by a radiolabeled antigen binding assay (RIA) performed by the following Fab-type antibody and its antigen by the following assay. Measure. Fab-to-antigen is measured by balancing the Fab with a minimal concentration of ( ^125I )-labeled antigen in the presence of a continuous titration of unlabeled antigen followed by capture of the antigen bound to the anti-Fab antibody coated plate Solution binding affinity (Chen et al. (1999) J. Mol Biol 293: 865-881). To establish assay conditions, a microtiter plate (Dynex) was coated overnight with anti-Fab antibody (Cappel Labs) captured at 5 μg/ml in 50 mM sodium carbonate (pH 9.6) and then at room temperature (approximately 23 Blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours. In a non-sorbent plate (Nunc #269620), mix 100 pM or 26 pM [ ¹²⁵ I] antigen with serial dilutions of Fab of interest (eg, consistent with anti-VEGF antibody, Fab-12 evaluation, Presta et al. (1997) Cancer Res. 57: 4593-4599). The Fab of interest is then incubated overnight; however, the incubation can last longer (e.g., about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture tray at room temperature for incubation (eg, one hour). The solution was then removed and the dish was washed eight times with 0.1% Tween-20 in PBS. When the disk was dry, 150 μl of scintillant (MicroScint-20; Packard) was added to each well and the plate was counted on a Topcount gamma counter (Packard) for 10 minutes. Each Fab that produces a maximum binding concentration of less than or equal to 20% is selected for use in a competitive binding assay.

According to another embodiment, by using the BIAcore ^TM -2000 or a ^{BIAcore TM -3000 (BIAcore, Inc.,} Piscataway, NJ) at 25 ℃, using a surface plasmon resonance assay at approximately 10 response units (RU) of immobilized antigen The CM5 wafer measures the Kd or Kd value. Briefly, according to the supplier's instructions, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxy amber imine (NHS) Activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc.). The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate, pH 4.8, prior to injection at a flow rate of 5 μl per minute to achieve about 10 reaction units (RU) of the coupled protein. After the antigen was injected, 1 M ethanolamine was injected to block the unreacted groups. For the purpose of kinetic measurement, two-fold serial dilutions (0.78 nM to 500 nM) of Fab were injected in PBS containing 0.05% Tween 20 (PBST) at a flow rate of about 25 μl/min at 25 °C. The association rate ( _kon ) and dissociation rate ( _koff ) were calculated by simultaneously fitting the association and dissociation biosensing curves using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). . The equilibrium dissociation to k _off k _on the calculated ratio / dissociation constant (Kd). See, for example, Chen, Y. et al. (1999) J. Mol Biol. 293:865-881. If the association rate by the above surface plasma resonance test is greater than 10 ⁶ M ^-1 s ^-1 , it may be in a 8000 series such as a spectrophotometer equipped with a stagnant flow (Aviv Instruments) or with a stirred phototube Measurement of 20 nM anti-antigen antibody (Fab format) in PBS at pH 7.2 at 25 ° C using an increased concentration of antigen measured in a spectrophotometer of a SLM-Aminco spectrophotometer (ThermoSpectronic) The fluorescence quenching technique (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) with increased or decreased fluorescence intensity was used to determine the association rate.

"Association rate" of the present invention or "k _on" as described above may also be determined using the BIA _core ^TM -2000 or a BIAcore ^TM -3000 system (BIAcore, Inc., Piscataway, NJ ).

A "condition" is any condition or disease that benefits from treatment with a substance/molecule or method of the invention. It includes chronic or acute conditions, including the pathological conditions in which the mammal is predisposed to the condition in question. Non-limiting examples of conditions to be treated herein include cancers, such as tumors, such as carcinomas (epithelial tumors) and blastomas (embryonic tissue-derived tumors), and in some embodiments ovarian cancer, uterine cancer ( Including endometrial cancer), brain tumors (such as astrocytoma and glioma) and kidney cancer, including nephroblastoma (such as Wilm's tumor).

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a certain degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

As used herein, "tumor" refers to the growth and proliferation of all neoplastic cells (whether malignant or benign) and all precancerous and cancerous cells and tissues. The terms "cancer", "cancer", "cell proliferative disorder", "proliferative disorder" and "tumor" as referred to herein are not mutually exclusive.

The terms "cancer" and "cancer" refer to or describe a mammalian physiological condition that is generally characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (such as Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, intestinal cancer, pancreatic cancer, glioma, and son. Cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, genital cancer, thyroid cancer, liver cancer , leukemia and other lymphoproliferative disorders and various types of head and neck cancer.

"Treatment," as used herein, refers to a clinical intervention that attempts to alter the natural course of the individual or cell being treated, and may be performed for prophylactic purposes or during a clinical pathological process. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease; alleviating the symptoms; reducing any direct or indirect pathological outcome of the disease; preventing cancer metastasis; reducing the rate of disease progression; improving or reducing the disease condition and ameliorating or improving the prognosis. In some embodiments, the anti-system of the invention is used to delay the development of a disease or condition or to slow the progression of a disease or condition.

"Individual" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, livestock (such as cattle), competitive animals, pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal is a human.

"Effective amount" means the amount of dosage and time necessary to effectively achieve the desired therapeutic or prophylactic result.

The "therapeutically effective amount" of a substance/molecule of the invention may vary depending on factors such as the disease condition, the age, sex and weight of the individual, and the ability of the substance/molecule to elicit a desired response in the individual. A therapeutically effective amount also encompasses an amount that treats a beneficial effect over any toxic or detrimental effect of the substance/molecule. "Prophylactically effective amount" means the amount of dosage and time necessary to effectively achieve the desired prophylactic result. Usually, but not necessarily, since the prophylactic dose is administered to the subject before or at the early stage of the disease, the prophylactically effective amount should be less than the therapeutically effective amount.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or blocks cellular function and/or causes cell death or destruction. The term is intended to include radioisotopes (eg, radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu); chemotherapeutic agents such as methotrexate呤, adriamicin, vinblastine (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, benzene Butyric acid mustard, daunorubicin; or other intercalating agents, enzymes and fragments thereof, such as nucleolytic enzymes, antibiotics and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin ( Included are fragments and/or variants thereof and various anti-tumor or anti-cancer agents disclosed below. Other cytotoxic agents are described below. Tumor killing agents cause destruction of tumor cells.

A "toxin" is any substance that has a detrimental effect on cell growth or proliferation.

A "chemotherapeutic agent" is a compound suitable for treating cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN Cyclophosphamide; alkyl sulfonate such as busulfan, improsulfan and piposulfan; aziridine such as benzodopa, carbopol ( Carboquone), meturedopa and uredopa; methyl iminoamine and methylamelamine, including hexamethylene melamine, tri-ethyl melamine, tri-ethyl phosphamide, Tri-ethyl thiophosphonamide and trimethylol melamine; acetogenin (especially bulbatacin and bulbatacinone); δ-9-tetrahydroxyl Cannabinol (dronabinol, MARINOl) ; β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogue topotecan ( Topotecan)(HYCAMTIN ), CPT-11 (irinotecan, CAMPTOSAR) ), acetylcamptothecin, scopolectin and 9-aminocamptothecin; bryostatin; callistatin; CC-1065 ( Including its adozelesin, carzelesin and bizelesin synthetic analogues; podophyllotoxin; podophyllinic acid; teniposide (teniposide); cryptophycin (especially Candida cyclic peptide 1 and Nostoccal cyclic peptide 8); dolastatin; doocarmycin (including synthetic analogues, KW-2189) And CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; mustard nitrogen, such as chlorambucil, naphthyl nitrogen Mustard, cholestyramine, estramustine, ifosfamide, mechlorethamine, nitrous oxide mustard, melphalan, neombibichin, phenesterine ), prednimustine, trofosfamide, uracil mustard; Base ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and lenimostatin Ranimnustine); antibiotics, such as enediyne antibiotics (such as calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, eg, Agnew, Chem Intl. Ed. Engl., 33: 183-) 186 (1994)); dynemicin, including dinamycin A; esperamicin; and new tumor suppressor protein chromophore and related pigment protein enediyne antibiotic chromophore ), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, actinomycin C, carabis ( Carabicin), carminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo- 5-sided oxy-L-posite leucine, ADRIAMYCIN Doxorubicin (including morpholinyl-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolyl-doxorubicin and deoxydoxantine), epirubicin (epirubicin) ), esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, Olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, chain black Streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as Methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, pyrimidine analogs, such as Ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, deoxygenation Doxifluridine, enocitabine, floxuridine; androgens, such as calustronone, dromostanolone propionate, epithiostanol ), mepitiostane, testolactone; anti-adrenalin, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as aldehyde folate (frolinic acid); aceglatone; aldophosphamide glycoside; alanine acetonitrile; eniluracil; amsacrine; betrixine (bestrabucil); bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; Elliptinium acetate; epothilone; etog Lucid); gallium nitrate; hydroxy urea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoxantrone Mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin ); losoxantrone; 2-acetamidine; procarbazine; PSK Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid Triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, rod) Roridin A and anguidine; urethan; vindesine (ELDISINE) FILDESIN ); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; guacittosine; Arabinoside ("Ara-C");thiotepa; paclitaxel, such as TAXOL Paclitaxel (Bristol-Myers Squibb Oncology, Princeton , NJ), ABRAXANE TM episomal Cetyl alcohol polyoxyethylene ethers, for processing works for albumin nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois ) , and TAXOTERE Docetaxel (Rh ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR) 6-thioguanine; guanidine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine (VELBAN) ); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN) ); oxaliplatin; leucovovin; vinorelbine (NAVELBINE) ;;novantrone; edatrexate; daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoride Methyl ornithine (DMFO); a visual pigment such as retinoic acid; capecitabine (XELODA) Any of the above pharmaceutically acceptable salts, acids or derivatives; and combinations of two or more of the above, such as CHOP (cyclophosphamide, doxorubicin, vincristine and pour nylon) the abbreviation) and FOLFOX (Orsay force to platinum (ELOXATIN ^TM) Abbreviation treatment regimen with 5-FU and new combinations of chlorine buckle Wo).

Also included in the definition are anti-hormonal agents that modulate, reduce, block or inhibit the hormonal effects that promote cancer growth and are typically systemic or holistic therapeutic forms. It can be a hormone itself. Examples include anti-estrogen and selective estrogen receptor modulators (SERM) including, for example, tamoxifen (including NOLVADEX) Tamoxifen), EVISTA Raloxifene (EVISTA) Raloxifene), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON Toremifen (FARESTON) Toremifene); anti-progesterone; estrogen receptor down-regulator (ERD); an agent used to inhibit or stop the ovaries, such as luteinizing hormone releasing hormone (LHRH) agonists, such as LUPRON And ELIGARD Illuminated acetonitrile (ELIGARD) Leuprolide acetate), goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide, nilutamide And bicalutamide; and aromatase inhibitors that inhibit the regulation of estrogen production by the estrogen in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, MEGASE Megestrol acetate, AROMASIN Exemestane (AROMASIN) Exemestane), formestanie, fadrozole, RIVISOR Fluconazole (RIVISOR Vorozole), FEMARA Retrazole (FEMARA) Letrozole) and ARIMIDEX Amida Ingot (ARIMIDEX Anastrozole). In addition, this definition of chemotherapeutic agent includes bisphosphonates such as clodronate (eg BONEFOS) Or OSTAC ), DIDROCAL Etidronate (DIDROCAL Etidronate), NE-58095, ZOMETA Zoledronic acid/zoledronate, FOSAMAX Alendronate, AREDIA Pamidronate, SKELID Tiludronate or ACTONEL Risedronate; and troxacitabine (1,3-dioxolan nucleoside cytosine analog); antisense oligonucleotides, especially for their inhibition of abnormal cell proliferation Oligonucleotides involved in the expression of genes in the signal pathway, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE Vaccine and gene therapy vaccines such as ALLOVECTIN Vaccine, LEUVECTIN Vaccine and VAXID Vaccine; LURTOTECAN Type 1 topoisomerase inhibitor; ABARELIX rmRH; lapatinib ditosylate (also known as erbB-2 and EGFR tyrosinate kinase small molecule inhibitors of GW572016) and any of the above pharmaceutically acceptable salts, acids or derivatives Things.

As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells in vitro or in vivo, such as cells expressing TAT226. Thus, the growth inhibitor can be a growth inhibitor that significantly reduces the percentage of S phase cells, such as cells that express TAT226. Examples of growth inhibitors include agents that block cell cycle progression (except for the S phase), such as agents that induce G1 arrest and M phase arrest. Classic M-phase blockers include vinca (vincristine and vinblastine), taxanes, and type II topoisomerase inhibitors, such as doxorubicin, epirubicin, and Noromycin, etoposide and bleomycin. The agents that stagnate G1 also overflow into the S phase stagnation, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5- Fluorouracil and cytarabine (ara-C). Additional information can be found in Murakami et al., The Molecular Basis of Cancer , edited by Mendelsohn and Israel, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), especially for the 13th. page. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from yew trees. Docetaxel derived from European yew (TAXOTERE) , Rhone-Poulenc Rorer) is paclitaxel (TAXOL) , semi-synthetic analog of Bristol-Myers Squibb). Paclitaxel and docetaxel promote assembly of microtubules by tubulin dimers and stabilize microtubules by preventing depolymerization, which results in inhibition of mitosis in cells.

The term "intracellular metabolite" refers to a compound produced by a metabolic process or reaction within a cell of an antibody-drug conjugate (ADC). The metabolic process or reaction can be an enzymatic process, such as proteolytic cleavage of a peptide linker of an ADC or hydrolysis of a functional group such as a hydrazine, an ester or a guanamine. Intracellular metabolites include, but are not limited to, antibodies that undergo intracellular lysis after entry, diffusion, absorption, or transport into cells, and free drugs.

The term "intracellular cleavage" refers to a metabolic process or reaction within an antibody-drug conjugate (ADC) cell, thereby disrupting the covalent linkage, ie, the linker between the drug moiety (D) and the antibody (Ab), resulting in The free drug dissociates from the antibodies in the cell. Thus the cleavage portion of the ADC is an intracellular metabolite.

The term "bioavailability" refers to the systemic effectiveness (ie, blood/plasma content) of a given amount of a drug administered to a patient. Bioavailability is the absolute form of the measurement that indicates the time (rate) and total amount (degree) of the drug from the dosage form to the total circulation.

The term "cytotoxic activity" refers to the cell killing, cell growth inhibition or growth inhibitory effects of an intracellular metabolite of an antibody-drug conjugate or antibody-drug conjugate. Cytotoxic activity may be expressed as the IC ₅₀ value, the concentration per unit volume when a half cell survival (or molar mass).

"Alkyl" containing normal, second, third or cyclic carbon atoms C ₁ -C ₁₈ hydrocarbon. Examples are methyl (Me, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, isopropyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propene Base (i-Bu, isobutyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, second butyl, -CH(CH ₃ )CH ₂ CH ₃ ), 2-A Benzyl-2-propyl (t-Bu, tert-butyl, -C(CH ₃ ) ₃ ), 1-pentyl (n-pentyl, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (CH(CH ₃ )CH ₂ CH ₂ CH ₃ ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ). 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH(CH ₃ )CH (CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH) ₃ ) (CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl ( -C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ ).

As used herein the term "C ₁ -C ₈ alkyl" means a straight-chain having 1 to 8 carbon atoms or branched, saturated or unsaturated hydrocarbon. Representative "C ₁ -C ₈ alkyl" includes, but is not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, - N-octyl, -n-decyl and -n-decyl; and branched C ₁ -C ₈ alkyl includes, but is not limited to, -isopropyl, -t-butyl, -isobutyl, -third Base, -isopentyl, 2-methylbutyl; unsaturated C ₁ -C ₈ alkyl including, but not limited to, -vinyl, -allyl, 1-butenyl, 2-butene Base, -isobutenyl, 1-pentenyl, 2-pentenyl, -3-methyl-1-butenyl, 2-methyl-2-butenyl, -2,3-di Methyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -ethynyl, -propynyl,-1-butynyl,-2-butynyl,-1-pentynyl ,-2-pentynyl,-3-methyl-1-butynyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, second butyl, tert-butyl , n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2, 3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl Base, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl and isooctyl. The C ₁ -C ₈ alkyl group may be unsubstituted or substituted with one or more groups including, but not limited to, the following groups: -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkane Base, -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C( O) N(R') ₂ -NHC(O)R', -SO ₃ R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ ,- NH ₂ , -NH(R'), -N(R') ₂ and -CN; wherein each R' is independently selected from the group consisting of H, -C ₁ -C ₈ alkyl and aryl.

"Alkenyl" having at least one unsaturated (i.e. a carbon - carbon, sp ² double bond) containing the site of normal, second, third or cyclic carbon atoms of the C ₂ -C ₁₈ hydrocarbon. Examples include, but are not limited to, ethyl or vinyl (-CH=CH ₂ ), allyl (-CH ₂ CH=CH ₂ ), cyclopentenyl (-C ₅ H ₇ ), and 5-hexyl Alkenyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH=CH ₂ ).

"Alkynyl" having at least one unsaturated (i.e. a carbon - carbon, sp triple bond) containing the site of normal, second, third or cyclic carbon atoms of the C ₂ -C ₁₈ hydrocarbon. Examples include, but are not limited to, ethynyl (-C≡CH) and propargyl (-CH ₂ C≡CH).

"Alkyl" means a saturated, branched or straight having from 1 to 18 carbon atoms and having two monovalent groups derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Chain or cyclic hydrocarbon group. Typical alkylene groups include, but are not limited to, methylene (-CH ₂ -), 1,2-ethyl (-CH ₂ CH ₂ -), 1,3-propyl (-CH ₂ CH ₂ CH ₂ -), 1,4-butyl (-CH ₂ CH ₂ CH ₂ CH ₂ -) and the like.

"C ₁ -C ₁₀ alkylene" is a linear saturated hydrocarbon group of the formula -(CH ₂ ) _1-10 -. Examples of the C ₁ -C ₁₀ alkylene group include a methylene group, an exoethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, a decyl group, a decyl group, and a fluorenyl group.

"En stretched alkenyl" means an unsaturated, branched or branched chain having from 2 to 18 carbon atoms and having two monovalent groups derived by the removal of two hydrogen atoms from the same or two different carbon atoms of the parent olefin. Linear or cyclic hydrocarbon group. Typical alkenyl groups include, but are not limited to, 1,2-extended ethyl (-CH=CH-).

"Extend alkynyl" means an unsaturated, branched chain having from 2 to 18 carbon atoms and having two monovalent groups derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Or a linear or cyclic hydrocarbon group. Typical alkynyl groups include, but are not limited to, ethynyl (-C≡C-), propargyl (-CH ₂ C≡C-), and 4-pentynyl (-CH ₂ CH ₂ CH ₂ C≡C) -).

"Aryl" means a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. The carbocyclic aromatic or heterocyclic aromatic group may be unsubstituted or substituted with one or more groups including, but not limited to, the following groups: -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR' , -C(O)N(R') ₂ -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ And -NH(R'), -N(R') ₂ and -CN; wherein each R' is independently selected from the group consisting of H, -C ₁ -C ₈ alkyl and aryl.

"Exoaryl" is an aryl group having two covalent bonds and which may be in the ortho, meta or para configuration as shown in the structure below: Wherein the phenyl group may be unsubstituted or substituted with up to four groups including, but not limited to, the following groups: -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -aryl Base, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R ') ₂ -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH(R'), - N(R') ₂ and -CN; wherein each R' is independently selected from the group consisting of H, -C ₁ -C ₈ alkyl and aryl.

"Aralkyl" means an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (usually a terminal or sp ³ carbon atom) is replaced with an aryl group. Typical aralkyl groups include, but are not limited to, benzyl, 2-phenylethane-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylvinyl-1-yl, naphthoquinonebenzyl, 2-naphthoquinonephenylethane-1-yl and the like. The aralkyl group contains 6 to 20 carbon atoms, for example, the alkyl moiety (including an alkyl group, an alkenyl group or an alkynyl group) of the aralkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.

"Heteroaralkyl" means an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (usually a terminal or sp ³ carbon atom) is substituted with a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkyl group contains 6 to 20 carbon atoms, for example, the alkyl moiety (including an alkyl group, an alkenyl group or an alkynyl group) of the heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms. Atom and 1 to 3 hetero atoms selected from N, O, P and S. The heteroaryl portion of the heteroarylalkyl group may be a monocyclic ring having 2 to 7 ring members (2 to 6 carbon atoms) or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3) One hetero atom selected from N, O, P and S), for example: bicyclo[4,5], [5,5], [5,6] or [6,6] systems.

"Substituted alkyl group", "substituted aryl group" and "substituted aralkyl group" mean an alkyl group, an aryl group and an aralkyl group in which one or more hydrogen atoms are each independently substituted with a substituent. base. Typical substituents include, but are not limited to, -X, -R, -O-, -OR, -SR, -S ^- , -NR ₂ , -NR ₃ , =NR, -CX ₃ , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO ₂ , =N ₂ , -N ₃ , NC(=O)R, -C(=O)R, -C(=O)NR ₂ , -SO ₃ ^- , -SO ₃ H, -S(=O) ₂ R, -OS(=O) ₂ OR, -S(=O) ₂ NR, -S(=O)R, -OP( =O)(OR) ₂ , -P(=O)(OR) ₂ , -PO ₃ -, -PO ₃ H ₂ , -C(=O)R, -C(=O)X, -C(= S) R, -CO ₂ R, -CO ₂ ^- , -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR ₂ , -C( =S)NR ₂ , -C(=NR)NR ₂ , wherein each X is independently halogen: F, Cl, Br or I; and each R is independently -H, C ₂ -C ₁₈ alkyl, C ₆ a -C ₂₀ aryl, C ₃ -C ₁₄ heterocyclic ring, protecting group or prodrug moiety. The alkyl, alkenyl and alkynyl groups as described above may also be similarly substituted.

"Heteroaryl" and "heterocycle" refer to a ring system in which one or more ring atoms are heteroatoms (eg, nitrogen, oxygen, and sulfur). The heterocyclic group contains 1 to 20 carbon atoms and 1 to 3 hetero atoms selected from N, O, P and S. The heterocyclic ring may be a single ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 hetero atoms selected from N, O, P and S) or a bicyclic ring having 7 to 10 ring members ( 4 to 9 carbon atoms and 1 to 3 hetero atoms selected from N, O, P and S), for example: bicyclo[4,5], [5,5], [5,6] or [6,6 ]system.

Heterocyclic is described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7 and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19 and 28; and J. Am . Chem . Soc . (1960) 82: 5566.

Examples of heterocycles include, by way of example and not limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidinyl), thiazolyl, tetrahydrothiophenyl, sulfur-oxidized tetrahydrothiophenyl, pyrimidinyl, Furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thionaphthyl, anthracenyl, porphyrinyl, quinolyl, isoquinolinyl, benzoimidazole , piperidinyl, 4-piperidinone, pyrrolidinyl, 2-pyrrolidone, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetra Hydroquinolinyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolinyl, anthracycline, triazinyl, 6H-1,2,5-thiadiazinyl, 2H, 6H -1,5,2-dithiazinyl, thienyl, thiol, piperidyl, isobenzofuranyl, Alkenyl, , morphine, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridazinyl, isodecyl, 3H-indenyl, 1H-carbazolyl, Sulfhydryl, 4H-quinolizinyl, pyridazinyl, Pyridyl, quinoxalinyl, quinazolinyl, Porphyrin, acridinyl, 4aH-carbazolyl, oxazolyl, β-carboline, phenanthryl, acridinyl, pyrimidinyl, morpholinyl, cyanozinyl, phenothiazine, furazan Base, phenoxazine, different base, Base, imidazolidinyl, imidazolinyl, pyrazolyl, pyrazolinyl, piperazinyl, porphyrinyl, isoindolyl, Pyridyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzoxazole, hydroxyindenyl, benzoxazolinyl and isatinoyl.

By way of example and not limitation, a carbon-bonded heterocyclic ring is bonded at a position 2, 3, 4, 5 or 6 of the pyridine; 3, 4, 5 or 6 of the pyridazine; position 2 of the pyrimidine , 4, 5 or 6; pyrazine position 2, 3, 5 or 6; furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole position 2, 3, 4 or 5; oxazole, imidazole or thiazole Position 2, 4 or 5; isoxazole, pyrazole or isothiazole position 3, 4 or 5; aziridine position 2 or 3; azetidine position 2, 3 or 4; quinoline position 2 Positions 3, 4, 5, 6, 7 or 8 or isoquinolines 1, 3, 4, 5, 6, 7, or 8. More generally, the carbon-bonded heterocyclic ring includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5- Pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6- Pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

By way of example and not limitation, the nitrogen-bonded heterocyclic ring is bonded at the following positions: aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolium, 2 -Imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, hydrazine, porphyrin, 1H-carbazole, position 1; Position 2 of hydrazine or isoindoline; position 4 of morpholine and position of carbazole or β-carboline 9. More typically, the nitrogen-bonded heterocyclic ring includes 1-aziridine, 1-azetyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl and 1-piperidinyl.

"C ₃ -C ₈ heterocyclic ring" means an aromatic or non-aromatic C ₃ -C ₈ carbocyclic ring in which one to four of the ring carbon atoms are independently replaced by a hetero atom selected from the group consisting of O, S and N. . Representative examples of C ₃ -C ₈ heterocycle to include (but not limited to) phenyl Bing furanyl, Bing thiophene, indolyl, pyrazolyl benzene Bing, coumarinyl, isoquinolinyl, pyrrolyl, thienyl , furyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and Tetrazolyl. The C ₃ -C ₈ heterocyclic ring may be unsubstituted or substituted with up to seven groups including, but not limited to, the following groups: -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl ), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O N(R') ₂ , -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH( R'), -N(R') ₂ and -CN; wherein each R' is independently selected from the group consisting of H, -C ₁ -C ₈ alkyl and aryl.

"C ₃ -C ₈ heterocyclic group" means a heterocyclyl group wherein a hydrogen atom of one of the above-defined permuted key of a C ₃ -C ₈ heterocyclyl. The C ₃ -C ₈ heterocyclyl can be unsubstituted or substituted with up to six groups including, but not limited to, the following groups: -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkane Base, -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C( O) N(R') ₂ , -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH (R'), -N(R') ₂ and -CN; wherein each R' is independently selected from the group consisting of H, -C ₁ -C ₈ alkyl and aryl.

"Carbocycle" means a saturated or unsaturated ring having a single ring form of 3 to 7 carbon atoms or a bicyclic form of 7 to 12 carbon atoms. Monocyclic carbocycles have from 3 to 6 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have, for example, 7 to 12 ring atoms arranged in the form of a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or in a bicyclo[5,6] or [6,6] The system is arranged with 9 or 10 ring atoms. Examples of the monocyclic carbocyclic ring include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a 1-cyclopent-1-enyl group, a 1-cyclopent-2-enyl group, a 1-cyclopent-3-enyl group, and a ring. Hexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl and cyclooctyl.

The "C ₃ -C ₈ carbocyclic ring" is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative C ₃ -C ₈ carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1, 3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl,-1,3,5-cycloheptatrienyl, -cyclo Octyl and cyclooctadienyl. The C ₃ -C ₈ carbocyclic group may be unsubstituted or substituted with one or more groups including, but not limited to, the following groups: -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ Alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C (O)N(R') ₂ -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH (R'), -N(R') ₂ and -CN; wherein each R' is independently selected from the group consisting of H, -C ₁ -C ₈ alkyl and aryl.

The "C ₃ -C ₈ carbocyclic group" means a C ₃ -C ₈ carbocyclic group as defined above in which one of the hydrogen atoms of the carbocyclic group is substituted by a bond.

"Linker" refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In various embodiments, linkers include a divalent radical such as an alkyl group, an aryl group, a heteroaryl-diyl; as part of the following _{:-( CR 2) n O (CR} 2) n -, alkoxy groups repeating units (e.g., polyethylene stretch ethoxy, PEG, poly methyleneoxy) and alkylamino group (e.g. ethylamino stretched polyethylene, Jeffamine ^TM); and two acyl esters, and amines including succinate, succinamide Amides , glyoxylic acid ester, malonate and hexylamine.

The term "pivot" refers to a molecule that has a non-overlapping property of a mirror image, and the term "non-palphape" refers to a molecule that overlaps its mirror image.

The term "stereoisomer" refers to a compound that has the same chemical composition but differs in the arrangement of the atoms or groups in space.

"Diastereomer" refers to a stereoisomer that has two or more pairs of palmar centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties such as melting point, boiling point, spectral properties and reactivity. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.

"Enantiomer" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

The stereochemical definitions and conventions used herein are generally based on the McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994). ) John Wiley & Sons, Inc., New York. Many organic compounds exist in a photoactive form, that is, they have the ability to rotate a plane of plane polarized light. In describing photoactive compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to its palm center. The prefixes d and l or (+) and (-) are used to indicate the labeling of a plane-polarized light with a compound, where (-) or l means that the compound is left-handed. Compounds prefixed with (+) or d are dextrorotatory. For a given chemical structure, the stereoisomers are identical except for being mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer and a mixture of such isomers is often referred to as a mixture of enantiomers. The 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate which can be produced in the absence of stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species that are photoactive.

"Debonded group" refers to a functional group which may be substituted with another functional group. Certain detachments are well known in the art and include, but are not limited to, halides (e.g., chloride, bromide, iodide), mesyl, tosyl, three Fluoromethylsulfonyl (triflate) and trifluoromethylsulfonate groups.

B. Abbreviation

Linker component: MC=6-maleimide hexamethylene Val-Cit or "vc" = valine-citrulline (exemplary dipeptide in a linker cleavable by protease) citrulline =2-amino-5-ureidovaleric acid PAB=p-aminobenzyloxycarbonyl (example of "self immolative" linker component) Me-Val-Cit=N-methyl-decylamine Acid-citrulline (where the linker peptide bond is modified to prevent it from being cleaved by cathepsin B) MC(PEG)6-OH=maleimide hexyl-polyethylene glycol (can be attached to antibody half) Cysteine

Cytotoxic drug: MMAE = monomethyl auristatin E (MW 718) MMAF = auristatin E (MMAE) variant with phenylalanine at the C-terminus of the drug (MW 731.5) MMAF-DMAEA = indoleamine with C-terminal phenylalanine MMAF (MW 801.5) MMAF-TEG with DMAEA (dimethylaminoethylamine) in the bond = MMAF MMAF-NtBu with tetraethylene glycol esterified to phenylalanine = as a guanamine attached to the C-terminus of MMAF N-t-butyl

Other abbreviations are as follows: AE is auristatin E; Boc is N-(third butoxycarbonyl); cit is citrulline; dap is dolaproine; DCC is 1,3-dicyclohexylcarbodiimide Imine; DCM is dichloromethane; DEA is diethylamine; DEAD is diethyl azodicarboxylate; DEPC is diethylphosphonium cyanate; DIAD is diisopropyl azodicarboxylate; DIEA is N , N -diisopropylethylamine; dil is doramamic acid; DMA is dimethylacetamide; DMAP is 4-dimethylaminopyridine; DME is ethylene glycol dimethyl ether (or 1, 2-Dimethoxyethane); DMF is N,N-dimethylformamide; DMSO is dimethyl hydrazine; doe is dolaphenine; dov is N,N -dimethylhydrazine Amino acid; DTNB is 5,5'-dithiobis(2-nitrobenzoic acid); DTPA is diethylenetriamine pentaacetic acid; DTT is dithiothreitol; EDCI is 1-(3-dimethylamine) Propyl)-3-ethylcarbodiimide hydrochloride; EEDQ is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; ES-MS is analyzed by electrospray mass spectrometry; EtOAc is ethyl acetate; Fmoc is N- (9-fluorenylmethoxycarbonyl); gly is glycine; HATU is O- (7-aza) Benzotriazole-1-yl)-N,N,N',N'-four Hexafluorophosphate; HOBt is 1-hydroxybenzotriazole; HPLC is high pressure liquid chromatography; ile is isoleucine; lys is lysine; MeCN (CH ₃ CN) is acetonitrile; MeOH is methanol; Is 4-anisyldiphenylmethyl (or 4-methoxytrityl); nor is ( 1S,2R )-(+)-norphedrine; PBS is phosphate buffered saline (pH 7.4); PEG is polyethylene glycol; Ph is phenyl; Pnp is p-nitrophenyl; MC is 6-maleimide hexyl; phe is L-phenylalanine; PyBrop is bromo Pyrrolidinyl hexafluorophosphate; SEC is size exclusion chromatography; Su is amber imine; TFA is trifluoroacetic acid; TLC is thin layer chromatography; UV is ultraviolet; and val is valine.

III. Composition and method of producing the same

An antibody that binds to TAT226 is provided. An immunoconjugate comprising an anti-TAT226 antibody is provided. The antibodies and immunoconjugates of the invention are useful, for example, in the diagnosis or treatment of conditions associated with altered performance (e.g., increased performance) of TAT226. In certain embodiments, an antibody or immunoconjugate of the invention is useful for the diagnosis or treatment of a cell proliferative disorder, such as cancer.

A.Anti-TAT226 antibody

TAT226 ("Tumor Associated Antigen Target No. 226") is a protein that is processed and expressed on the surface of specific cell types, including tumor cells. In particular, human TAT226 has previously been reported to be overexpressed in certain types of tumors, including ovaries, uterus, endometrium, kidney, lung, pancreas, adrenal gland, and hepatocyte tumors. See, for example, U.S. Patent Application Publication Nos. US 2003/0148408 A1, US 2004/0229277 A1 and US 2003/0100712 A1 (TAT 226 is referred to as "PRO9917"); and U.S. Patent No. 6,710,170 B2 (SEQ ID NO: 215). Other database entries and disclosures related to TAT226 are as follows: NCBI registration number AY358628_1 (referred to as "PSCA Hlog" for human TAT226); NCBI registration number AAQ88991.1 and "gi" No. 37182378 (referred to human TAT226 as "PSCA") Hlog"); RIKEN cDNA 2700050C12; US 2003/0096961 A1 (SEQ ID NO: 16); US 2003/0129192 A1 (SEQ ID NO: 215); US 2003/0206918 A1 (Example 5; SEQ ID NO: 215); US 2003/0232056 A1 (SEQ ID NO: 215); US 2004/0044179 A1 (SEQ ID NO: 16); US 2004/0044180 A1 (SEQ ID NO: 16); US 2005/0238649 A1 (referred to as human TAT226) PSCA Hlog"); WO 2003/025148 (SEQ ID NO: 292); WO 2003/105758 (SEQ ID NO: 14); and EP 1347046 (SEQ ID NO: 2640).

Full length TAT226 is processed in cells to produce mature form proteins that are expressed on the cell surface. For example, full length human TAT226 as set forth in SEQ ID NO: 75 contains a predicted signal peptide sequence from amino acid 1-20 or 1-22 that is predicted to cleave from the protein. It is predicted that the C-terminus of the amino acid 116-141 will be cleaved from the protein and the GPI moiety will be linked to the protein at the amino acid 115. The mature form of human TAT226 from amino acid 21-115 or 23-115 of SEQ ID NO: 75 is predicted to be fixed to the cell surface via the GPI moiety. Monkey and rodent TAT226 (see, eg, SEQ ID NO: 76-78) are highly similar to human TAT226 and are therefore susceptible to cleavage and modification at equivalent amino acid positions. See Figure 1. The resulting human, monkey and rodent TAT226 (shown in Figure 1) from the mature form of the amino acid 21-115 or 23-115 are 100% identical.

Other features of human TAT226 include the predicted N-glycosylation site at 45, which has been empirically demonstrated, and the predicted Ly6/u-PAR domain from amino acid 94-107 of SEQ ID NO: 75. Human TAT226 shares approximately 32% amino acid homology with prostate stem cell antigen (PSCA), a prostate cancer-specific tumor antigen that is expressed on the cell surface via a GPI linkage. See Reiter et al. (1998) Proc. Nat'l. Acad. Sci. USA 95: 1735-1740. PSCA is overexpressed in more than 80% of prostate cancers. (ibid.). Like TAT226, it contains a predicted Ly6/u-PAR domain involved in cellular functions such as signal transduction and cell adhesion. (ibid.).

In one aspect, the invention provides an antibody that binds to TAT226. In some embodiments, an antibody that binds to the mature form TAT226 is provided. In one such embodiment, the mature form of TAT226 has the amino acid sequence from amino acid 21-115 or 23-115 of SEQ ID NO:75. In some embodiments, the antibody to TAT226 binds to the mature form of TAT226 expressed on the cell surface. In some embodiments, an antibody that binds to the mature form of TAT226 expressed on the surface of the cell inhibits cell growth. In some embodiments, the anti-TAT226 antibody binds to the mature form of TAT226 expressed on the cell surface and inhibits cell proliferation. In certain embodiments, the anti-TAT226 antibody binds to the mature form of TAT226 expressed on the cell surface and induces cell death. In some embodiments, the anti-TAT226 antibody binds to the mature form of TAT226 expressed on the surface of cancer cells. In some embodiments, the anti-TAT226 antibody binds to a mature form of TAT226 that is overexpressed on the surface of cancer cells relative to normal cells of the same tissue source.

In one aspect, the anti-TAT226 antibody is a monoclonal antibody. In one aspect, the anti-TAT226 antibody is an antibody fragment, such as a Fab, Fab'-SH, Fv, scFv or (Fab') ₂ fragment. In one aspect, the anti-TAT226 antibody is a chimeric, humanized or human antibody. In one aspect, any of the anti-TAT226 antibodies described herein are purified.

As described in Example B, exemplary monoclonal antibodies derived from phage libraries are provided herein. The antigen used in the screening library is a polypeptide having the amino acid 1-115 sequence of SEQ ID NO: 75, corresponding to the absence of the TAT226 form of the amino acid which is the C-terminus of the putative GPI junction site. The antibodies produced by the library screening were named YWO.32 and YWO.49. YWO.49 is affinity matured to produce YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The alignment of the heavy and light chain variable domain sequences of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 are shown in Figure 11, respectively. And 12 in.

In one aspect, a monoclonal antibody that competes with YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, or YWO.49.H6 for binding to TAT226 is provided. Monoclonal antibodies that bind to the same epitopes as YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6 are also provided.

In one aspect of the invention, a polynucleotide encoding an anti-TAT226 antibody is provided. In certain embodiments, a vector comprising a polynucleotide encoding an anti-TAT226 antibody is provided. In certain embodiments, host cells comprising such vectors are provided. In another aspect of the invention, a composition comprising an anti-TAT226 antibody or a polynucleotide encoding an anti-TAT226 antibody is provided. In certain embodiments, the compositions of the invention are pharmaceutical formulations for the treatment of cell proliferative disorders such as those recited herein.

A detailed description of exemplary anti-TAT226 antibodies is as follows: 1. Specific Examples of Anti-TAT226 Antibodies In one aspect, the invention provides at least one, two, three, four, five or six selected from the group consisting of An antibody to HVR: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4; (b) an amino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: HVR-H2; (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; An HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19.

In one aspect, the invention provides an anti-TAT226 antibody comprising at least one, at least two or all three VH HVR sequences selected from: (a) an amino group comprising SEQ ID NO: 1 or SEQ ID NO: HVR-H1 of the acid sequence; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; and (c) an amine comprising SEQ ID NOS: 3 and 6-11 HVR-H3 of the acid sequence. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 6-11. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having the amino acid sequence of SEQ ID NO: 9 or 10.

In one aspect, the invention provides HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11 and an amino acid selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: Sequence of anti-TAT226 antibody of HVR-H1. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-11 and HVR-H1 having SEQ ID NO: 4. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having SEQ ID NO: 9 or 10 and HVR-H1 having SEQ ID NO: 4. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having SEQ ID NO: 3 and HVR-H1 having SEQ ID NO: 1.

In one aspect, the invention provides HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11 and an amino acid selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: Sequence of anti-TAT226 antibody of HVR-H2. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-11 and HVR-H2 having SEQ ID NO: 5. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having SEQ ID NO: 9 or 10 and HVR-H2 having SEQ ID NO: 5. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-H3 having SEQ ID NO: 3 and HVR-H2 having SEQ ID NO: 2.

In one aspect, the invention provides an HVR-H1 comprising an amino acid sequence having SEQ ID NO: 1 or SEQ ID NO: 4; an amino acid sequence having SEQ ID NO: 2 or SEQ ID NO: HVR-H2; and an anti-TAT226 antibody having HVR-H3 selected from the amino acid sequences of SEQ ID NOS: 3 and 6-11. In one embodiment, HVR-H1 comprises the amino acid sequence of SEQ ID NO: 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises a selected from the group consisting of SEQ ID NO: 6-11 Amino acid sequence. In one embodiment, HVR-H1 comprises the amino acid sequence of SEQ ID NO: 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises SEQ ID NO: 9 or 10. In one embodiment, HVR-H1 comprises the amino acid sequence of SEQ ID NO: 1; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 2; and HVR-H3 comprises the amino acid of SEQ ID NO: sequence.

In one aspect, the invention provides an anti-TAT226 antibody comprising at least one, at least two or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-19. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-19. In one aspect, the invention provides an anti-TAT226 antibody comprising HVR-L3 having the amino acid sequence of SEQ ID NO: 17 or 18.

In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 6-11, and (b) comprising a member selected from the group consisting of SEQ ID NO : HVR-L3 of the amino acid sequence of 14-19. In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and (b) an amino acid sequence comprising SEQ ID NO: 14. HVR-L3. In some embodiments, the TAT226 antibody further comprises (a) HVR-H1 comprising SEQ ID NO: 1 and HVR-H2 comprising SEQ ID NO: 2.

In one aspect, the invention provides an anti-TAT226 antibody comprising (a) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-11 and (b) comprising SEQ ID NO: 14 HVR-L3 of the amino acid sequence of -19. In some embodiments, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9 and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 10 and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the TAT226 antibody further comprises HVR-H1 having SEQ ID NO: 4 and HVR-H2 having SEQ ID NO: 5.

In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4; (b) comprising SEQ ID NO: 2 Or HVR-H2 of the amino acid sequence of SEQ ID NO: 5; (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 and 6-11; (d) comprising SEQ ID NO: 12 HVR-L1 of the amino acid sequence; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) HVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19 -L3.

In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) comprising the amino acid sequence of SEQ ID NO: HVR-H2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) comprising SEQ ID NO: HVR-L2 of the amino acid sequence of 13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) comprising the amino acid sequence of SEQ ID NO: HVR-H2; (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) comprising HVR-L2 of the amino acid sequence of SEQ ID NO: 13; and (f) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19.

In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) comprising the amino acid sequence of SEQ ID NO: HVR-H2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) comprising SEQ ID NO: HVR-L2 of the amino acid sequence of 13; and (f) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17.

In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) comprising the amino acid sequence of SEQ ID NO: HVR-H2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) comprising SEQ ID NO: HVR-L2 of the amino acid sequence of 13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the anti-TAT226 antibody is affinity matured to achieve the desired target binding affinity. In some embodiments, any one or more of the amino acids of the antibody are substituted at the HVR position (Kabat numbering): H98, H99, H100, H100B, L90, L92, L93, L96, and L97. For example, in some embodiments, any one or more of the following substitutions can be made in any combination: - in HVR-H3 (SEQ ID NO: 6): V98I; S99T; R100L or I; G100bA, S or P- In HVR-L3 (SEQ ID NO: 14): Q90R, K, H or N; Y92V; T93F, N, G or A; P96F; T97I or A SEQ ID NO: 11 (HVR-H3) and SEQ ID NO: The consensus sequence of 19 (HVR-L3) covers all possible combinations of the above substitutions.

An anti-TAT226 antibody can comprise any suitable framework variable domain sequence, with the proviso that the antibody retains the ability to bind to TAT226. For example, in some embodiments, an anti-TAT226 antibody of the invention comprises a human subgroup III heavy chain framework consensus sequence. In one embodiment of such antibodies, the heavy chain framework consensus sequence comprises substitutions at positions 71, 73 and/or 78. In one embodiment of such antibodies, position 71 is A, position 73 is T, and/or position 78 is A. In one embodiment, the antibodies comprise a heavy chain variable domain framework sequence of huMAb4D5-8, such as SEQ ID NOs: 50, 51, 59, 35 (FR1, 2, 3, 4, respectively). huMAb4D5-8 is commercially known as HERCEPTIN , Genentech, Inc., South San Francisco, CA, USA; also in U.S. Patent Nos. 6,407,213 & 5,821,337 and Lee et al, J. Mol. Biol. (2004), 340(5):1073-93 and. In one such embodiment, the antibodies additionally comprise humans I light chain framework consistent sequence. In one such embodiment, the antibodies comprise a light chain variable domain framework sequence of huMAb4D5-8.

In one embodiment, the anti-TAT226 antibody comprises a heavy chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises a FR1-FR4 sequence selected from the group consisting of Figures 5A and 5B; HVR H1 comprises SEQ ID NO: amino acid sequence of 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-11. In one embodiment of the antibodies, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9 or 10. In one embodiment, the anti-TAT226 antibody comprises a light chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises a FR1-FR4 sequence selected from the group consisting of Figures 6A and 6B; HVR-L1 comprises The amino acid sequence of SEQ ID NO: 12; HVR-L2 comprises the amino acid sequence of SEQ ID NO: 13; and HVR-L3 comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 14-19. In one embodiment of the antibodies, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17 or 18.

In one embodiment, the anti-TAT226 antibody comprises a heavy chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises the FR1-FR4 sequences of SEQ ID NOs: 50, 51, 59 and 35 as shown in FIG. HVR H1 comprises the amino acid sequence of SEQ ID NO: 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-11. In one embodiment of the antibodies, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9 or 10. In one embodiment, the anti-TAT226 antibody comprises a light chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequences comprise FR1 of SEQ ID NOs: 60, 61, 62 and 63 as shown in Figures 6A and 6B FR4 sequence; HVR-L1 comprises the amino acid sequence of SEQ ID NO: 12; HVR-L2 comprises the amino acid sequence of SEQ ID NO: 13; and HVR-L3 comprises an amine group selected from SEQ ID NOS: 14-19 Acid sequence. In one embodiment of the antibodies, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17 or 18.

In one embodiment, the anti-TAT226 antibody comprises a heavy chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises the FR1-FR4 sequences of SEQ ID NOs: 50, 51, 53 and 35 as shown in FIG. HVR H1 comprises the amino acid sequence of SEQ ID NO: 4; HVR-H2 comprises the amino acid sequence of SEQ ID NO: 5; and HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-11. In one embodiment of the antibodies, HVR-H3 comprises the amino acid sequence of SEQ ID NO: 9 or 10. In one embodiment, the anti-TAT226 antibody comprises a light chain variable domain having a framework sequence and a hypervariable region, wherein the framework sequence comprises the FR1-FR4 sequences of SEQ ID NOs: 60, 61, 62 and 74 as shown in FIG. HVR-L1 comprises the amino acid sequence of SEQ ID NO: 12; HVR-L2 comprises the amino acid sequence of SEQ ID NO: 13; and HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-19 . In one embodiment of the antibodies, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 17 or 18.

In some embodiments, the invention provides for comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25 %, 98% or 99% of the sequence-consistent amino acid sequence heavy chain variable domain anti-TAT226 antibody. In some embodiments, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to a reference sequence An antibody containing a substitution, insertion or deletion, but comprising a peramino acid sequence, retains the ability to bind to TAT226. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted or deleted in a sequence selected from the group consisting of SEQ ID NOs: 20-25. In some embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, in the FR). In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25.

In some embodiments, the invention provides an anti-TAT226 antibody (HERCEPTIN) comprising a light chain variable domain of the humanized 4D5 antibody (huMAb4D5-8) as set forth in SEQ ID NO: 31 , Genentech, Inc., South San Francisco, CA, USA) (also referred to in U.S. Patent No. 6,407,213 and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93).

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108 (SEQ ID NO: 31) (HVR residues are underlined)

In some embodiments, the huMAb4D5-8 light chain variable domain sequence is modified at one or more of positions 30, 66, and 91 (Asn, Arg, and His, respectively, as indicated in bold/italic, above) . In one embodiment, the modified huMAb4D5-8 sequence comprises Ser at position 30, Gly at position 66, and/or Ser at position 91. Thus, in one embodiment, the antibody of the invention comprises a light chain variable domain having the sequence set forth in SEQ ID NO: 26: 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108 (SEQ ID NO: 26) HVR residues are underlined)

The substituted residues for huMAb4D5-8 are indicated above in bold/italic.

In one aspect, the invention provides for comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31. An anti-TAT226 antibody of the light chain variable domain of the amino acid sequence of %, 98% or 99% sequence identity. In some embodiments, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to a reference sequence An antibody containing a substitution, addition or deletion, but comprising a peramino acid sequence, retains the ability to bind to TAT226. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted or deleted in a sequence selected from the group consisting of SEQ ID NOs: 26-31. In some embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, in the FR). In some embodiments, the anti-TAT226 antibody comprises a light chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31.

In one aspect, the invention provides an anti-TAT226 antibody (a) comprising at least 90%, 91%, 92%, 93%, 94% of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25 a heavy chain variable domain of a 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence; and (b) comprising an amino acid selected from the group consisting of SEQ ID NO: 26-31 A light chain variable domain of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity is contained relative to the reference sequence Substitutions, additions or deletions, but antibodies comprising a peramino acid sequence retain the ability to bind to TAT226. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted or deleted in the reference sequence. In some embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, in the FR). In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-25 and a light having an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31 Chain variable domain.

In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-TAT226 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30.

In one aspect, the invention provides an anti-TAT226 antibody comprising: (a) one, two or three VH HVRs selected from the group consisting of Figures 2 and 4; and/or (b) selected from the group consisting of One, two or three VL HVRs shown in Figures 3 and 4. In one aspect, the invention provides an anti-TAT226 antibody comprising a heavy chain variable domain selected from the group consisting of those shown in Figures 9 and 11 and a light chain variable domain selected from those shown in Figures 10 and 12 .

2. Antibody Fragments The present invention encompasses antibody fragments. Antibody fragments can be produced by conventional means such as enzymatic digestion or by recombinant techniques. In certain cases, the use of antibody fragments has advantages over whole antibodies. Fragments of smaller size allow for rapid clearance and can result in improved access to solid tumors. For a discussion of specific antibody fragments, see Hudson et al. (2003) Nat . Med. 9: 129-134.

Various techniques for producing antibody fragments have been developed. Traditionally, such fragments have been produced by proteolytic digestion of intact antibodies (see, for example, Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al, Science , 229:81 ( 1985)). However, such fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can both be expressed in and secreted from E. coli, making it easy to produce large amounts of such fragments. Antibody fragments can be isolated from the above-described antibody phage library. Alternatively, the Fab'-SH fragment can be directly recovered from E. coli and chemically coupled to form an F(ab') ₂ fragment (Carter et al, Bio/Technology 10: 163-167 (1992)). According to another approach, the F(ab') ₂ fragment can be isolated directly from recombinant host cell culture. Fab and F(ab') ₂ fragments having an increased in vivo half-life comprising a residue of a salvage receptor in combination with an epitope are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and scFv are the only species with intact combinatorial sites without constant regions; therefore they are suitable for reduced non-specific binding during in vivo use. The scFv fusion protein can be constructed to produce fusion of effector proteins at the amino or carboxy terminus of the scFv. See Body Engineering, edited by Borrebaeck (supra). For example, antibody fragments can also be "linear antibodies" as described in U.S. Patent No. 5,641,870. Such linear antibodies can be monospecific or bispecific.

3. Humanized Antibodies The present invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced from a non-human source. Such non-human amino acid residues are often referred to as "introduced" residues, which are typically obtained from "introduced" variable domains. The humanization can basically be based on Winter and its collaborators (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534- The method of 1536) is carried out by substituting the corresponding human antibody sequence with a hypervariable region sequence. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which a substantially under-integrated human variable domain has been replaced by a corresponding sequence from a non-human species. In fact, humanized antibodies are typically human antibodies in which some of the hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

Selection of human variable domains (light and heavy) to be used to make humanized antibodies can be important to reduce antigenicity. The variable domain sequences of rodent antibodies are screened against a full library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to the rodent sequence is then accepted as the human framework for humanized antibodies (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol . Biol. 196:901). Another approach uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA , 89: 4285; Presta et al. (1993) J. Immunol ., 151: 2623).

It is generally further desirable that the humanized antibody has a high antigen affinity retained and other advantageous biological properties. To achieve this, humanized antibodies are prepared according to one method by analyzing the parental sequence and various conceptual humanized products using a three-dimensional model of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. A computer program that illustrates and presents a possible three-dimensional configuration of the selected candidate immunoglobulin sequence can be used. Examination of such presentations allows analysis of the possible role of residues in the function of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected from the receptor and introduction sequences and combined to achieve desired antibody characteristics, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and to the greatest extent involved in affecting antigen binding.

4. Human Antibodies The human anti-TAT226 antibodies of the present invention can be constructed by combining Fv pure line variable domain sequences selected from human-derived phage display libraries with the above known human constant domain sequences. Alternatively, the human monoclonal anti-TAT226 antibody of the present invention can be produced by the fusion tumor method. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies are described, for example, by Rozbor J. Immunol ., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Pages 51-63 (Marcel Dekker, Inc., New York, 1987) and Boerner et al, J. Immunol ., 147: 86 (1991).

It is currently possible to produce a transgenic animal (e.g., a mouse) capable of producing a full lineage of human antibodies in the absence of endogenous immunoglobulin production upon immunization. For example, it has been described that homozygous ligation of the antibody re-ligated region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al, Proc. Natl. Acad. Sci USA , 90:2551 (1993); Jakobovits et al, Nature , 362: 255 (1993); Bruggermann et al, Year in Immunol ., 7:33 (1993) .

Gene shuffling can also be used to derive human antibodies from non-human antibodies, such as rodents, wherein human antibodies have similar affinities and specificities as the starting non-human antibodies. According to this method (also referred to as "antigenic determinant effect"), a human V domain gene lineage is substituted for a heavy or light chain variable region of a non-human antibody fragment obtained by phage display technology as described herein. A population of non-human chain/human chain scFv or Fab chimeras. Non-human chain/human chain chimeric scFv or Fab is isolated by antigen selection, wherein the human chain recovers the antigen binding site that is damaged when the corresponding non-human chain in the pure line is removed, ie, the antigenic determinant is dominant (affected The choice of human chain collocation. Human antibodies are obtained when this method is repeated to replace the remaining non-human strands (see PCT WO 93/06213, published Apr. 1, 1993). Unlike humanization of traditional non-human antibodies by CDR grafting, this technique provides fully human antibodies that do not have FR or CDR residues of non-human origin.

5. Bispecific Antibodies Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, the bispecific antibody is a human or humanized antibody. In certain embodiments, one of the binding specificities is for TAT226 and the other is for any other antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of TAT226. Bispecific antibodies can also be used to limit cytotoxic agents to cells expressing TAT226. Such antibodies have a TAT226 binding arm and an arm that binds to a cytotoxic agent such as saporin, anti-interferon alpha, vinblastine, ricin A chain, methotrexate or radioisotope hapten. Bispecific antibodies can be prepared in the form of full length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies).

Methods of making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature , 305:537 (1983)). . Due to the random classification of immunoglobulin heavy and light chains, such fusion tumors (hybridomas) produce a possible mixture of 10 different antibody molecules, of which only one has the proper bispecific structure. Purification of the appropriate molecule, usually by affinity chromatography steps, is quite complex and yields are low. A similar procedure is disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al, EMBO J. , 10:3655 (1991).

The antibody variable domain having the desired binding specificity (antibody-antigen combining site) is fused to an immunoglobulin constant domain sequence according to different methods. For example, fusion with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, the CH2 region, and the CH3 region. In certain embodiments, a first heavy chain constant region (CH1) comprising a site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. In embodiments where three polypeptide chains of unequal ratios are used in the construction to provide optimal yield, the modulation of the mutual ratio of the three polypeptide fragments is greater. However, when at least two polypeptide chains are expressed in equal ratios resulting in high yields or when the ratios are not of particular significance, it is possible to insert coding sequences for two or all three polypeptide chains in one expression vector.

In one embodiment of the method, the bispecific anti-system consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (providing a second Binding specificity) composition. This asymmetric structure was found to facilitate the separation of the desired bispecific compound from the undesired immunoglobulin chain combination, as only the immunoglobulin light chain in which half of the bispecific molecule is present provides a means of easy separation. This method is disclosed in WO 94/04690. For additional details on the production of bispecific antibodies, see, for example, Suresh et al, Methods in Enzymology , 121:210 (1986).

According to another approach, the interface between pairs of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The interface comprises at least a portion of the _CH3 domain of the antibody constant domain. In this way, one or more small amino acid side chains from the first antibody molecule interface are replaced with a larger side chain (eg, tyrosine or tryptophan). Compensatory "holes" having the same or similar size to the larger side chains are produced at the interface of the second antibody molecule by replacing the larger amino acid side chain with a smaller one (e.g., alanine or threonine). This provides a mechanism to increase the yield of heterodimers above other undesirable end products such as homodimers.

Bispecific antibodies include cross-linked or "hetero-synthesis" antibodies. For example, one of the heterozygous antibodies can be coupled to avidin and the other antibodies can be coupled to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). Hetero-joined antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, incorporated herein by reference.

Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al, Science , 229: 81 (1985) describe procedures in which intact antibodies are proteolytically cleaved to produce F(ab') ₂ fragments. The fragments can be reduced in the presence of the dithiol complex sodium arsenite to stabilize the o-dithiol and prevent the formation of disulfides between the molecules. The resulting Fab' fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab'-TNB derivatives is then converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with other molar amounts of other Fab'-TNB derivatives to form a bispecific antibody. The bispecific antibody produced can be used as an agent for selectively immobilizing an enzyme.

Recent advances have led to the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J. Exp . Med ., 175: 217-225 (1992) describe the production of fully humanized bispecific antibody F(ab') ₂ molecules. Each Fab' fragment is secreted independently from E. coli and subjected to directed chemical coupling in vitro to form a bispecific antibody. The bispecific antibody thus formed binds to cells overexpressing the HER2 receptor and normal human T cells, as well as triggering the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for directly producing and isolating bispecific antibody fragments from recombinant cell culture have also been described. For example, bispecific antibodies have been made using leucine zippers. Kostelny et al, J. Immunol ., 148(5): 1547-1553 (1992). The leucine zipper peptide from the Fos and Jun proteins was ligated to the Fab' portion with two different antibodies by gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer and then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The "bifunctional antibody" technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) joined to the light chain variable domain (VL) by a linker that is too short to allow pairing of the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol ., 152: 5368 (1994).

Covers antibodies with more than two valences. For example, a trispecific antibody can be prepared. Tutt et al . J. Immunol. 147: 60 (1991).

6. Multivalent antibodies Multivalent antibodies can be internalized (and/or catabolized) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibody of the present invention may be a multivalent antibody (other than an IgM species) having three or more antigen-binding sites (for example, a tetravalent antibody), which can be easily produced by recombinantly expressing a nucleic acid encoding an antibody polypeptide chain. A multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more amino-terminal ends to the antigen binding site of the Fc region. In certain embodiments, the multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, the multivalent antibody comprises (or consists of) four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (eg, two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, and Fc is a polypeptide of the Fc region The chain, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For example, the polypeptide chain can comprise: a VH-CH1-elastic linker-VH-CH1-Fc region chain; or a VH-CH1-VH-CH1-Fc region chain. Multivalent antibodies herein may additionally comprise at least two (eg, four) light chain variable domain polypeptides. For example, a multivalent antibody herein can comprise from about two to about eight light chain variable domain polypeptides. A light chain variable domain polypeptide encompassed herein comprises a light chain variable domain and, as the case may be, further comprises a CL domain.

7. Single Domain Antibodies In some embodiments, the antibodies of the invention are single domain antibodies. A single domain antibody is a single polypeptide chain comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). In one embodiment, the single domain anti-system consists of all or part of the heavy chain variable domain of the antibody.

8. Antibody Variants In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. For example, such modifications include deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construction, with the proviso that the final construction has the desired characteristics. The amino acid change can be introduced into the subject antibody amino acid sequence upon sequence generation.

As described by Cunningham and Wells (1989) Science, 244: 1081-1085, a method suitable for identifying a particular antibody residue or region that is a preferred location for a mutation is referred to as an "alanine scanning mutation." Here, a population of residues or target residues (eg, charged residues (such as arg, asp, his, lys, and glu)) are identified and neutralized or neutralized with a neutral or negatively charged amino acid (eg, alanine or poly Alanine) substitutions affect the interaction of the amino acid with the antigen. These amino acid positions that are functionally sensitive to the substitution performance are then improved by introducing additional or other variants at or at the substitution site. Therefore, although the site of introduction of the amino acid sequence variation is predetermined, the nature of the predetermined mutation itself is not required. For example, to analyze the mutational potency of a given site, an ala scan or random mutation is performed at the target codon or target region and the desired activity indicative of the immunoglobulin is screened.

Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having N-terminal methionine residues. Other insertional variants of the antibody molecule include fusion of the N-terminus or C-terminus of the antibody with an enzyme (eg, ADEPT) or a polypeptide that increases the serum half-life of the antibody.

In certain embodiments, the antibodies of the invention are altered to increase or decrease the extent of antibody glycosylation. Glycosylation of a polypeptide is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartic acid residue. The tripeptide sequence aspartic acid-X-serine and aspartate-X-threonine (where X is any amino acid other than proline) is to partially conjugate the carbohydrate to The recognition sequence of the aspartic acid side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide produces a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetyl galactosamine, galactose or xylose to hydroxylamine, which is most commonly seric acid or threonine, although 5-Hydroxyproline or 5-hydroxy lysine is used.

Addition or deletion of an antibody glycosylation site is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences are generated or removed (for N-linked glycosylation sites). Alterations can also be made by adding, deleting or substituting one or more serine or threonine residues in the initial antibody sequence (for O-linked glycosylation sites).

When the antibody comprises an Fc region, the carbohydrate to which it is attached can be altered. For example, an antibody lacking trehalose in a mature carbohydrate structure linked to the Fc region of an antibody is described in US Patent Application No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). An antibody having an aliquot of N-acetyl galactosamine (GlcNAc) in a carbohydrate linked to the Fc region of an antibody is cited in WO 2003/011878 to Jean-Mairet et al. and U.S. Patent No. 6,602,684 to U.S. An antibody having at least one galactose residue in an oligosaccharide linked to the Fc region of an antibody is reported in WO 1997/30087 to Patel et al. See also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) for antibodies having altered carbohydrates linked to their Fc region. See also US 2005/0123546 (Umana et al.) for antigen binding molecules with modified glycosylation.

In certain embodiments, the glycosylation variant comprises an Fc region, wherein the carbohydrate structure linked to the Fc region lacks trehalose. These variants have improved ADCC function. Optionally, the Fc region additionally comprises a further modification of one or more amino acid substitutions of the ADCC, such as at positions 298, 333 and/or 334 of the Fc region (Eu numbering of residues). Examples of publications relating to "de-alginization" or "trehalose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; Okazaki et al. J. Mol .Biol. 336 :1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell strains which produce de-alcoholized antibodies include Lec13 CHO cells which are deficient in protein seaweed (Ripka et al. Arch. Biochem. Biophys . 249: 533-545 (1986); US Patent Application No. US 2003/0157108 A1 , Presta, L; and Adams et al. WO 2004/056312 A1, especially in Example 11) and gene knockout cell lines, such as the α-1,6-trehalyltransferase gene FUT8 , gene knockout CHO cells (Yamane- Ohnuki et al. Biotech. Bioeng. 87:614 (2004)).

Another variant type is an amino acid substitution variant. The variants have at least one amino acid residue in the antibody molecule displaced by a different residue. Sites of interest for substitutional mutations include hypervariable regions, but also encompass FR changes. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". If such substitutions result in a desired change in biological activity, more substantial changes and screened products designated as "exemplary substitutions" in Table 1 or further described below with reference to the amino acid species can be incorporated.

Substantial modification of the biological properties of an antibody is accomplished by selective substitutions that maintain (a) a backbone structure of the substitution region, such as a folded or helical configuration; (b) charge or hydrophobicity of the molecule at the target site , or (c) the effect of a large number of side chains is significantly different. Amino acids can be grouped according to the similarity of their side chain properties (in Biochem, 2nd Edition, pages 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala (A) ), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M)(2) Polarity without charge: Gly(G), Ser (s), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidity: Asp (D), Glu (E) (4) Alkaline: Lys ( K), Arg(R), His(H)

Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobicity: n-leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: Cys, ser, Thr , Asn, Gln; (3) Acidity: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will cause one of the classes to be exchanged for another species. These substituted residues can also be introduced into a conservative substitution site or a remaining (non-conservative) site.

One type of substitution variant includes one or more hypervariable region residues that replace a parent antibody (eg, a humanized or human antibody). In general, the resulting variants selected for further development will have modified (e.g., improved) biological properties relative to the parent antibody from which they are produced. A convenient way to generate such substituted variants involves the use of phage to exhibit affinity maturation. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each point. The antibody thus produced is presented as a fusion with filamentous phage particles as a fusion with at least a portion of the phage sheath protein (e.g., the gene III product of M13) encapsulated within each particle. Phage-presented variants are then screened for their biological activity (e.g., binding affinity). To identify candidate hypervariable region sites for modification, a scan mutation (eg, alanine scan) can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to recognize the point of contact between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to techniques known in the art. Once such variants are produced, the variant plates are subjected to screening using known techniques in the art, including those described herein, and may select antibodies with superior properties in one or more of the relevant assays. For further research and development.

Nucleic acid molecules encoding antibody amino acid sequence variants are prepared by various methods known in the art. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutations, PCR mutations, and previous preparations. Prepare by mutation of a sequence cassette of an antibody variant or non-variant translation.

It may be desirable to introduce one or more amino acid modifications in the Fc region of an antibody of the invention, thereby producing an Fc region variant. An Fc region variant can be included in a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising one or more amino acid positions of hinge cysteine comprising an amino acid modification (eg, a substitution).

In accordance with this description and teachings of the technology, antibodies encompassing the invention in some embodiments may comprise one or more changes, for example, in the Fc region, as compared to a wild-type counterpart antibody. However, such antibodies will generally retain the same characteristics required for therapeutic use as their wild-type counterparts. For example, as described, for example, in WO 99/51642, it is believed that specific changes in C1q binding and/or complement dependent cytotoxicity (CDC) that produce a change (ie, improved or reduced) can be made in the Fc region. See also, for example, Duncan & Winter Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or reduced binding to FcR. The contents of these patent publications are incorporated herein by reference in their entirety. See also Shields et al . J. Biol. Chem. 9(2): 6591-6604 (2001). An antibody responsible for the transfer of maternal IgG to the fetus with increased half-life and improved binding to the neonatal Fc region receptor (FcRn) (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24: 249 (1994)) is described in US 2005/0014934 A1 (Hinton et al.). The antibodies comprise one or more substituted Fc regions having a modified Fc region that binds to FcRn. A polypeptide variant having a modified Fc region amino acid sequence and an increased or decreased C1q binding ability is described in U.S. Patent No. 6,194,551 B1, WO 99/51642. The contents of their patent publications are hereby incorporated by reference in their entirety. See also Idusogie et al . J. Immunol. 164 : 4178-4184 (2000).

In one aspect, the invention provides a modified antibody comprising an Fc polypeptide interface comprising an Fc region, wherein the modifications promote and/or facilitate heterodimerization. The modifications comprise introducing a protuberance into the first Fc polypeptide and introducing a cavity into the second Fc polypeptide, wherein the protuberance can be localized in the cavity to facilitate recombination of the first and second Fc polypeptides. Methods for producing antibodies having such modifications are known in the art, as described in U.S. Patent No. 5,731,168.

9. Antibody Derivatives The antibodies of the invention can be further modified to contain additional non-protein portions known in the art and readily available. The portion suitable for derivatizing the antibody is preferably a water-soluble polymer. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidine Ketone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and Dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol and mixtures thereof . Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The amount of polymer attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific characteristics or functions of the antibody to be modified, whether the antibody derivative will be used under defined conditions. Therapy, etc.

In another embodiment, a conjugate of an antibody and a non-protein moiety that is selectively heated by exposure to radiation is provided. In one embodiment, the non-protein portion is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, a wavelength that does not damage normal cells but heats the non-protein portion to a temperature that kills cells adjacent to the antibody-non-protein portion.

B. Specific methods for making antibodies

1. Method based on specific fusion tumor The anti-TAT226 monoclonal antibody of the present invention can be produced by the fusion method described first by Kohler et al., Nature , 256:495 (1975), or by recombinant DNA method (USA) Manufactured in Patent No. 4,816,567.

In the fusion tumor method, a mouse or other appropriate host animal such as a hamster is immunized to elicit a lymphocyte that produces or is capable of producing an antibody that specifically binds to a protein for immunization. Antibodies to TAT226 are generally increased in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of TAT226 and adjuvant. TAT226 can be prepared using methods well known in the art, some of which are further described herein. For example, TAT226 can be produced recombinantly. In one embodiment, the animal is immunized with a TAT226 derivative comprising an extracellular portion of TAT226 fused to the Fc portion of the immunoglobulin heavy chain. In one embodiment, the animal is immunized with a TAT226-IgG1 fusion protein. In one embodiment, the animal is immunogenic with TAT226 in a solution with monophosphoryl lipid A (MPL) / trehalose dimylate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT). The derivative is immunized and the solution is injected intradermally at multiple sites. The animals were challenged two weeks later. Animals were bled seven to fourteen days later and serum anti-TAT226 titers were assayed. Inspire the animal until the price reaches a steady state.

Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form a fusion tumor cell (Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)).

The thus prepared fusion tumor cells are seeded and grown in, for example, a suitable medium containing one or more substances that inhibit growth or survival of non-fused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of the fusion tumor will typically include hypoxanthine, aminopurine and thymidine (HAT medium). These substances prevent the growth of cells lacking HGPRT.

In certain embodiments, the myeloma cells are such that they are efficiently fused, by a stable, high level of cell-supporting antibody of the selected antibody-producing antibody, and to cells that are sensitive to a medium such as HAT medium. Exemplary myeloma cell lines include, but are not limited to, mouse myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, And myeloma cell lines of SP-2 or X63-Ag8-653 cells available from American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol ., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Pages 51-63 (Marcel Dekker, Inc., New York, 1987)).

To generate a monoclonal antibody that binds to TAT226, the medium in which the fusion tumor cells are grown is assayed. Preferably, the binding specificity of the monoclonal antibodies produced by the fusion tumor cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of a monoclonal antibody can be determined by Scatchard analysis by Munson et al., Anal . Biochem ., 107: 220 (1980).

After identifying a fusion tumor cell that produces an antibody having the desired specificity, affinity, and/or activity, the pure line can be subcultured by a limiting dilution procedure and grown by standard methods ( Going, Monoclonal Antibodies: Principles and Practice , Pp. 59-103 (Academic Press, 1986)). For example, suitable media for this purpose include D-MEM or RPMI-1640 medium. In addition, the fusion tumor cells can be grown in vivo as an ascites tumor in an animal. Individual antibodies secreted by sub-pure lines can be adapted from culture medium, ascites or serum by conventional immunoglobulin purification procedures such as protein A agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Ground separation.

2. Specific Library Screening The anti-TAT226 antibody of the present invention can be produced by using a combinatorial library to screen for antibodies having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are generally described in Hoogenboom et al. (2001) Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ), and in some embodiments described in Lee et al. Human (2004) J. Mol . Biol . 340: 1073-1093.

In fact, the synthetic antibody pure line is selected by screening a phage library containing phage displaying various fragments of the antibody variable region (Fv) fused to the phage sheath protein. These phage libraries are panned by affinity chromatography against the desired antigen. A pure line that exhibits an Fv fragment that binds to the desired antigen is adsorbed onto the antigen and thereby separated from the unbound pure line in the library. The bound pure line is then detached from the antigen and further enriched by additional antigen adsorption/dissolution cycles. Any of the anti-TAT226 antibodies of the invention can be obtained by the following procedure: a suitable antigen screening procedure designed to select a pure line of the phage of interest, followed by Kabat et al, Sequences of Proteins of Immunological Interest , Fifth Edition, NIH Publication 91- 3242, Bethesda MD (1991), Fv sequence from the phage-pure line of interest and the appropriate constant region (Fc) sequence described in Volumes 1-3 construct a full-length anti-TAT226 antibody line.

In certain embodiments, the antigen binding domain of an antibody is formed by two variable (V) regions of about 110 amino acids, each from a light chain (VL) and a heavy chain (VH), both There are three hypervariable loops (HVRs) or complementary decision regions (CDRs). As described in Winter et al, Ann . Rev. Immunol., 12: 433-455 (1994), the variable domain can be a single chain Fv (scFv) fragment (wherein VH and VL are covalently linked via a short, elastic peptide) Or Fab fragments (wherein each fused to a constant domain and non-covalently interacting) form functionally presented on the phage. The scFv encoding the phage-pure line and the Fab encoding the phage-pure line as used herein are collectively referred to as "Fv phage pure line" or "Fv pure line".

As described in Winter et al., Ann . Rev. Immunol., 12: 433-455 (1994), the lineages of the VH and VL genes can be independently selected by polymerase chain reaction (PCR) and randomized in phage libraries. Recombination, followed by searching for phage libraries for antigen-binding pure lines. A library of immunogenic sources provides high affinity antibodies to the immunogen without the need to construct a fusion cell. Alternatively, as described by Griffiths et al., EMBO J , 12: 725-734 (1993), an alternative natural lineage is provided to provide a wide range of non-autoantigen and autoantigen human antibodies without any immunization. Single source. Finally, as described by Hoogenboom and Winter, J. Mol . Biol ., 227:381-388 (1992), unrearranged V gene segments can also be selected from stem cells and PCR primers containing random sequences can be used. The high-variation CDR3 region is encoded and the in vitro rearrangement is completed to synthesize a natural library.

In certain embodiments, the filamentous phage system is used to present antibody fragments by fusion with a minimal amount of sheath protein pill. Antibody fragments can be presented as single-chain Fv fragments (wherein the VH and VL domains are linked to the same polypeptide chain by an elastic polypeptide spacer, such as Marks et al, J. Mol . Biol ., 222: 581-597 (1991). Or a Fab fragment form (one of which is fused to pIII and the other is secreted into the periplasm of the bacterial host cell, in which the Fab sheath protein structure presented on the surface of the phage is assembled by moving some wild-type sheath proteins, For example, Hoogenboom et al., Nucl. Acids Res ., 19: 4133-4137 (1991)).

In general, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library of anti-TAT226 pure lines is desired, the subject is immunized with TAT226 to generate an antibody response, and spleen cells and/or circulating B cells, and other peripheral blood lymphocytes (PBL) are recovered for use in constructing the library. In a preferred embodiment, the anti-TAT226 antibody response is produced in a transgenic mouse carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that TAT226 is immunized against TAT226 The human antibody producing B cells is obtained to obtain a library of human antibody gene fragments which are biased against the pure TAT226 lineage. The production of a transgenic mouse producing a human antibody is described below.

B cells expressing TAT226-specific membrane-bound antibodies can be isolated by using a suitable screening procedure, for example by cell separation using TAT226 affinity chromatography or by adsorption of cells to fluorescent dye-labeled TAT226, followed by streaming Additional enrichment of the anti-TAT226 reactive cell population was obtained by activated cell sorting (FACS).

Alternatively, use spleen cells and/or B cells from unimmunized donors or other PBLs to provide a better representation of possible antibody lineages, and also allow for the construction of antibodies using any animal (human or non-human) species in which TAT226 is not antigenic. Library. For libraries incorporating in vivo antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding the unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as humans, mice, rats, rabbits, luprines, canines, felines, pigs, cows, horses, and poultry.

Nucleic acids encoding antibody variable gene segments, including VH and VL segments, are recovered and amplified from the cells of interest. In the case of rearrangement of the VH and VL gene pools, as described in Orlandi et al, Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), by genomic DNA or mRNA Isolation from lymphocytes, followed by polymerase chain reaction (PCR) with primers that match the 5' and 3' ends of the rearranged VH and VL genes to obtain the desired DNA, thereby producing different V gene lineages for expression. The V gene can be amplified from cDNA and genomic DNA as described in Orlandi et al. (1989) and Ward et al, Nature , 341: 544-546 (1989), wherein the backward primer encodes a mature V domain. At the 5' end of the neutron and the forward primer is based on the J segment. However, for cDNA amplification, as described in Jones et al, Biotechnol ., 9:88-89 (1991), the backward primer can also be based on the guide exon; and as in Sastry et al, Proc As described in .Natl. Acad. Sci. (USA) , 86: 5728-5732 (1989), the forward primer is located in the constant region. To maximize complementarity, degeneracy can be incorporated into the primers as described by Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, such as described in Methods of Marks et al, J. Mol . Biol ., 222: 581-597 (1991) or Orum et al, Nucleic Acids Res ., 21: 4491-4498 (1993) As described in the methods, all available VH and VL arrangements present in the nucleic acid sample of the immune cell are amplified by maximizing pool diversity using PCR primers that target each V gene family. In order to select the amplified DNA into the expression vector, very few restriction sites can be used as markers for one end as described in Orlandi et al. (1989) or as Clackson et al., Nature , 352: 624-628 (1991). It was introduced into the PCR primer by further PCR amplification with a labeled primer.

The synthetic rearranged V gene lineage can be derived in vitro from the V gene segment. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al, J. Mol . Biol ., 227:776-798 (1992)) and plotted (in Matsuda et al, Nature Genet ., 3 : 88-94 (1993); as described in Hoogenboom and Winter, J. Mol . Biol ., 227:381-388 (1992), these selected colonies (including all of the H1 and H2 rings) The primary configuration can be used to generate different VH gene lineages with PCR primers encoding H3 loops of different sequences and lengths. As described in Barbas et al, Proc. Natl. Acad. Sci. USA , 89: 4457-4461 (1992), it is also possible to produce a VH lineage in which all sequence diversity is concentrated in a single length of long H3 loop. Human V And the Vλ segment has been cloned and sequenced (reported in Williams and Winter, Eur . J. Immunol ., 23: 1456-1461 (1993)) and can be used to make synthetic light chain lineages. Synthetic V gene lineages based on VH and VL folding and L3 and H3 length ranges will encode antibodies with substantial structural diversity. After amplification of the V gene encoding DNA, the germline V gene segment can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol . Biol ., 227:381-388 (1992).

Antibody fragment lineages can be constructed by combining VH and VL gene lineages in a number of ways. Each lineage can be produced in a different vector and recombined in vitro, for example as described in Hogrefe et al, Gene , 128: 119-126 (1993), or recombinantly in vivo by a combination infection, such as Waterhouse et al. The loxP system described in Nucl. Acids Res ., 21: 2265-2266 (1993). The in vivo recombination pathway utilizes the double-stranded nature of the Fab fragment to overcome the limitations imposed by the E. coli transport efficiency on the size of the library. The native VH and VL lines are independently selected, one is selected into the phagemid and the other is selected into the phage vector. The two libraries were then combined by phage infection of bacteria containing phagemids such that each cell contained a different combination and the pool size was limited only by the number of cells present (approximately 10 ¹² pure lines). Both vectors contain an in vivo recombination signal such that the VH and VL genes are recombined on a single replicon and co-packaged in phage virions. Such huge libraries provide a large number of good affinity (about 10 ^-8 M of K _d ^-1) of various antibodies.

Alternatively, the lineage can be serially ligated into the same vector as described, for example, in Barbas et al, Proc. Natl. Acad. Sci. USA , 88: 7978-7982 (1991), or assembled by PCR and then, for example, Colonization is described in Clackson et al., Nature , 352: 624-628 (1991). PCR assembly can also be used to join VH and VL DNA with DNA encoding an elastin spacer to form a single chain Fv (scFv) lineage. In another technique, as described in Embleton et al., Nucl. Acids Res ., 20: 3831-3837 (1992), "intracellular PCR assembly" is used to combine VH and VL genes by PCR in lymphocytes, And then the lineage of the linked gene is selected.

As above, the Winter et al. (1994), by the natural antibody library (natural or synthetic) can be produced with a moderate affinity of (about ¹⁰⁶ to 10 ⁷ M ^-1 of the K _d ^-1), but also by The second library was constructed and re-selected to simulate affinity maturation in vitro. For example, by Hawkins et al, J. Mol . Biol ., 226: 889-896 (1992) and Gram et al, Proc . Natl . Acad . Sci USA , 89: 3576-3580 (1992) In a method, a mutation is randomly introduced in vitro using a misusable polymerase (reported in Leung et al., Technique , 1:11-15 (1989)). In addition, affinity maturation can be performed by randomly mutating one or more CDRs and screening for higher affinity pure lines in selected individual Fv lines, for example, using PCR with primers carrying random sequences spanning the CDRs of interest. WO 9607754 (published on March 14, 1996) describes a method for inducing mutations in the complementarity determining regions of immunoglobulin light chains to produce a light chain gene pool. Another effective route, as described in Marks et al, Biotechnol ., 10:779-783 (1992), is the natural production of selected VH or VL domains from phage and from unimmunized donors. The V domain variant lineage is recombined and screened for higher affinity in several rounds of chain shuffling. This technique allows for the production of antibodies and antibody fragments having an affinity of 10 ^-9 M or less than 10 ^-9 M.

Library screening can be accomplished by a variety of techniques known in the art. For example, TAT226 can be used to coat the pores of the adsorption plate, on host cells attached to the adsorption plate or for cell sorting, or to biotin for coating with streptavidin. The beads are captured or used in any other method for screening phage display libraries.

The phage library sample is contacted with immobilized TAT226 under conditions suitable for binding at least a portion of the phage particles to the adsorbent. Typically, conditions including pH, ionic strength, temperature, and the like are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then lysed as acid as described, for example, by Barbas et al, Proc. Natl. Acad. Sci USA , 88: 7978-7982 (1991); or eg, Marks et al, J. Mol . Biol , 222: 581-597 (1991) is dissolved in an alkali; or, for example, in a procedure similar to the antigen competition method of Clackson et al., Nature , 352: 624-628 (1991), competitively dissociates by TAT226 antigen. Phage can be enriched 20-1,000 times in a single round. In addition, enriched phage can be grown in bacterial cultures and additionally subjected to several rounds of selection.

The efficiency of selection depends on a number of factors, including the dissociation kinetics during washing and whether multiple antibody fragments of a single phage can simultaneously engage the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinity) can be maintained by using short washes, multivalent phage presentation, and high antigen coating density in the solid phase. The high density not only stabilizes the phage via multivalent interactions, but also facilitates recombination of the dissociated phage. Long-term washing and monovalent phage presentation as described in Bass et al, Proteins , 8: 309-314 (1990) and WO 92/09690 and as in Marks et al, Biotechnol ., 10:779-783 (by: The low antigen coating density described in 1992) facilitates the selection of antibodies with slow dissociation kinetics (and superior binding affinity).

It is possible to choose between phage antibodies with different affinities for TAT226, even with slightly different affinities. However, random mutations in selected antibodies (eg, mutations by some affinity maturation techniques) may result in a variety of mutants, most of which bind to the antigen and a few that have a higher affinity. By limiting TAT226, competition eliminates very few high affinity phage. To maintain all of the higher affinity mutants, the phage can be grown in excess of biotinylated TAT226, but wherein the biotinylated TAT226 has a molar concentration that is lower than the constant target molar concentration of TAT226. High affinity binding phage can then be captured by the streptavidin coated paramagnetic beads. This "balance capture" allows for the selection of antibodies based on their binding affinity by allowing the sensitivity of a mutant line having an affinity of as low as twice as high to be separated from a large excess of phage having a lower affinity. Conditions for washing the phage bound to the solid phase can also be manipulated to distinguish based on dissociation kinetics.

The anti-TAT226 pure line can be selected based on the activity. In certain embodiments, the invention provides an anti-TAT226 antibody that binds to a living cell that naturally expresses TAT226. In one embodiment, the invention provides an anti-TAT226 antibody that blocks binding between a TAT226 ligand and TAT226, but does not block binding between a TAT226 ligand and a second protein. Fv pure lines corresponding to such anti-TAT226 antibodies can be selected by the following methods: (1) separating the anti-TAT226 pure line from the phage library as described above and optionally amplifying by growing a population in a suitable bacterial host. Phage-pure elite population; (2) select TAT226 and second protein that require blocking and non-blocking activity, respectively; (3) adsorb anti-TAT226 phage pure line to fixed TAT226; (4) use excess second protein to identify and dissociate The binding of the diproteins determines any undesired pure lines of the TAT226 binding determinant that overlap or share; and (5) the pure line that remains adsorbed after the dissolving step (4). Depending on the situation, the pure line with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

Easy to isolate and sequence encode the invention using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from a fusion tumor or phage DNA template) The fusion-derived monoclonal antibody or phage exhibits Fv-pure DNA. After isolation, the DNA can be placed in a performance vector and subsequently transfected into host cells (such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) that do not otherwise produce immunoglobulins. The synthesis of the desired monoclonal antibody is obtained in a recombinant host cell. Review articles on recombinant expression of DNA encoding antibodies in bacteria include Skerra et al, Curr. Opinion in Immunol ., 5: 256 (1993) and Pluckthun, Immunol. Revs , 130: 151 (1992).

A DNA encoding an Fv pure line of the invention can be combined with a known DNA sequence encoding a heavy chain and/or a light chain constant region (for example, an appropriate DNA sequence obtainable from Kabat et al. (supra) to form a full length code or Part of the length of the heavy chain and / or light chain. It will be appreciated that any isotype constant region can be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be obtained from any human or animal species. As used herein, a "chimeric" or "hybridization" antibody includes variable domain DNA derived from an animal (such as a human) species and then fused to constant region DNA of another animal species to form for "hybridization". An Fv pure line of the full length heavy and/or light chain coding sequence. In certain embodiments, Fv pure lines derived from human variable DNA are fused to human constant region DNA to form coding sequences for full length or partial length human heavy and/or light chains.

The DNA encoding the anti-TAT226 antibody derived from the fusion cell of the present invention can also be modified, for example, by replacing the homologous mouse sequence derived from the fusion tumor with the coding sequence of the human heavy and light chain constant domains (eg, Morrison et al. , Proc. Natl. Acad. Sci. USA , Method 81: 6851-6855 (1984)). DNA encoding an antibody or fragment derived from a fusion tumor or Fv pure line may be further modified by covalent attachment to an immunoglobulin coding sequence, all or part of a coding sequence of a non-immunoglobulin polypeptide. In this manner, a "chimeric" or "hybridization" antibody having the binding specificity of an antibody derived from an Fv pure line or a fusion line of the present invention is prepared.

3. Vectors, Host Cells, and Recombinant Methods The antibodies of the present invention are produced recombinantly, and the nucleic acid encoding the same is isolated and inserted into a replicable vector for further selection (amplification of DNA) or expression. The DAN encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody). A variety of carriers can be used. The choice of vector depends in part on the host cell to be used. In general, host cells have a prokaryotic or eukaryotic (generally mammalian) source. It will be appreciated that any isotype constant region can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species.

a) Fine production of antibodies using a prokaryotic host: (1) Vector construction A polynucleotide sequence encoding a polypeptide component of an antibody of the present invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from the antibody producing cells, such as fusion tumor cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR technique. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector that can replicate in a prokaryotic host and display a heterologous polynucleotide. A wide variety of vectors that are available and known in the art can be used for the purposes of the present invention. The choice of a suitable vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed by the vector. Each vector contains various components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it is present. Vector components generally include, but are not limited to, an origin of replication, selection of a marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insertion, and a transcription termination sequence.

Generally, a plastid vector containing a replicon and a control sequence derived from a species compatible with the host cell is used in conjunction with the host. Vectors typically carry a replication site, as well as a marker sequence that provides phenotypic selection in transformed cells. For example, E. coli is typically transformed with the plastid pBR322 derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides an easy means of identifying transformed cells. pBR322, its derivatives or other microbial plastids or bacteriophages may also contain or be modified to contain a promoter that can be used by microorganisms to express endogenous proteins. An example of a pBR 322 derivative for the expression of a specific antibody is described in detail in U.S. Patent No. 5,648,237 to Carter et al.

In addition, a phage vector containing a replicon and a control sequence compatible with the host microorganism can be used as a transformation vector in combination with the host. For example, phage such as λGEM.TM.-11 can be used to make recombinant vectors that can be used to transform sensitive host cells such as E. coli LE392.

An expression vector of the invention may comprise two or more promoter-cistronic pairs encoding each polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are usually divided into two categories: inducible and constitutive. An inducible promoter is a promoter that elicits an increased degree of cistronic transcription under the control of changes in its reaction culture conditions (eg, presence or absence of nutrients or temperature changes).

A large number of promoters recognized by various possible host cells are well known. The selected promoter can be operably linked to the coding light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Cistron DNA. Native promoter sequences and many heterologous promoters can be used to directly amplify and/or represent a target gene. In some embodiments, a heterologous promoter is utilized as it generally permits more transcription and higher yield of the expressed target gene as compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, beta-galactosidase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria, such as other known bacteria or bacteriophage promoters, are also suitable. Nucleotide sequences thereof have been disclosed, whereby one skilled in the art can operably bind to a cistron encoding a target light and heavy chain using a linker or adaptor (Siebenlist et al. (1980) Cell 20:269) to supply any desired restriction sites.

In one aspect of the invention, each cistron in the recombinant vector comprises a secretion signal sequence component that directs translocation across the membrane of the expression polypeptide. In general, the signal sequence can be a component of the vector, or it can be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention shall be a signal sequence which is recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from alkaline phosphatase, penicillinase, Ipp or thermostability. A group consisting of enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence used to represent the two cistrons of the system is the STII signal sequence or a variant thereof.

In another aspect, the production of an immunoglobulin according to the invention can occur in the cytoplasm of a host cell, and thus there is no need to have a secretion signal sequence in each cistron. In this regard, the immunoglobulin light and heavy chains are expressed, folded, and assembled to form a functional immunoglobulin within the cytoplasm. A particular host strain (e.g., E. coli trxB-strain) provides cytoplasmic conditions that facilitate disulfide bond formation, thereby permitting proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene , 159: 203 (1995).

Antibodies of the invention can also be produced by the use of expression systems in which the quantitative ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted and appropriately assembled antibodies of the invention. This modulation is accomplished, at least in part, by simultaneously adjusting the translational strength of the polypeptide component.

A technique for modulating the translational strength is disclosed in U.S. Patent No. 5,840,523 to Simmons et al. It utilizes translational initiation region (TIR) variants in the cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be generated within the range of translational strengths, thereby providing a convenient way to adjust this factor to the desired degree of performance of a particular strand. TIR variants can be produced by conventional mutation techniques that result in codon changes that alter the amino acid sequence. In certain embodiments, the nucleotide sequence changes to be static. For example, a TIR change can include a change in the number or spacing of the Shine-Dalgarno sequences and a change in the signal sequence. One method of generating a mutated signal sequence is to generate a "codon set" at the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., changes to quiescent). This can be accomplished by altering the third nucleotide position of each codon; in addition, some amino acids (such as leucine, serine, and arginine) have multiples that increase the complexity of making the group. One and second positions. This mutation method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

In one embodiment, a set of vectors is generated with a range of TIR intensities of each of the cistrons. This restriction set provides a comparison of the extent of performance of each strand and the desired antibody product yield under various combinations of TIR intensities. The TIR intensity can be determined by quantifying the degree of expression of the conductor gene as detailed in U.S. Patent No. 5,840,523, issued to Simmons et al. Based on the comparison of translational intensities, the desired individual TIRs are selected for combination in the construction of the expression vector of the present invention.

Prokaryotic host cells suitable for use in the expression of the antibodies of the invention include primitive bacteria (Archaebacteria) and eubacteria (Eubacteria), such as Gram-negative or Gram-positive organisms. Examples of suitable bacteria include Escherichia (e.g., Escherichia coli), Bacilli (e.g., B. subtilis), Enterobacteria, and Pseudomonas species. (eg P. aeruginosa), attenuated Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as a host of the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, A strain 33D3 having the genotype W3110 ΔfhuA(ΔtonA)ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE)degP41 kanR is included (U.S. Patent No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537) and E. coli RVS08 (ATCC 31,608) are also suitable. The examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above-described bacteria having the defined genotype are known in the art and are described, for example, in Bass et al., Proteins, 8: 309-314 (1990). It is generally necessary to consider the reproducibility of replicons in bacterial cells to select appropriate bacteria. For example, when a well-known plastid such as pBR322, pBR325, pACYC177 or pKN410 is used to supply a replicon, Escherichia coli, Serratia or Salmonella species can be suitably used as a host. Host cells should typically secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can be ideally incorporated into cell culture.

(2) Antibody production The host cell is transformed with the above expression vector, and cultured in a conventional nutrient medium suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence.

Transformation means introducing DNA into a prokaryotic host such that the DNA is reproducible as an extrachromosomal element or by a chromosomal component. Depending on the host cell used, transformation is carried out using standard assays suitable for such cells. Calcium treatment with calcium nitride is generally used for bacterial cells containing substantial cell wall barriers. Another method of transformation uses polyethylene glycol/DMSO. Another technique used is electroporation.

Prokaryotic cell lines used to produce the polypeptides of the invention are grown in a medium known in the art and suitable for use in culturing selected host cells. Examples of suitable media include luria broth (LB) plus the necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to a medium for growing cells expressing an ampicillin resistance gene.

In addition to the carbon, nitrogen and inorganic phosphate sources, any necessary supplement may also be included, either alone or in a mixture with another supplement or medium, such as a complex nitrogen source. The medium may optionally contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, guanidine acetate, dithioerythritol, and dithiothreitol.

Prokaryotic host cells are cultured at a suitable temperature. In certain embodiments, for E. coli growth, the growth temperature is from about 20 °C to about 39 °C; from about 25 °C to about 37 °C, or about 30 °C. The pH of the medium depends primarily on the host organism and can be any pH in the range of from about 5 to about 9. In certain embodiments, for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation. In one aspect of the invention, the PhoA promoter is used to control transcription of the polypeptide. Thus, the transformed host cells are cultured in phosphate-limited medium for induction. In certain embodiments, the phosphate-limiting medium is C.R.A.P medium (see, for example, Simmons et al, J. Immunol. Methods (2002), 263: 133-147). As is known in the art, various other inducers can be used depending on the vector construction employed.

In one embodiment, the expressed polypeptide of the invention is secreted in the host cell periplasm and recovered from the host cell periplasm. Protein recovery typically involves destroying microorganisms generally by means such as osmotic shock, ultrasonic treatment or lysis. After disrupting the cells, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be delivered to and isolated from the culture medium. The cells were removed from the culture and the culture supernatant was filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further separated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

In one aspect of the invention, a large amount of antibody is produced by fermentation. Various large-scale batch feed fermentation procedures can be used to produce recombinant proteins. Large scale fermentation has a capacity of at least 1000 liters, and in some embodiments, a capacity of about 1,000 to 100,000 liters. These fermenters use a stirring impeller to distribute oxygen and nutrients, especially glucose (preferably carbon/energy). Small-scale fermentation generally refers to fermentation in a fermenter having a capacity of no greater than about 100 liters and ranging from about 1 liter to about 100 liters.

In the fermentation process, the induction of protein expression is typically initiated after the cells are grown under suitable conditions to a desired density of, for example, OD550 of about 180-220 (at which stage the cells are in an early growth arrest phase). As is known in the art and as described above, various inducers can be used depending on the vector employed. Cells can grow for a short period of time prior to induction. Although longer or shorter induction times can be used, the cells are typically induced for about 12-50 hours.

In order to improve the production yield and quality of the polypeptide of the present invention, various fermentation conditions can be improved. For example, to improve the proper assembly and folding of the secreted antibody polypeptide, additional vectors that overexpress the accompanying protein, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (with accompanying protein activity) may be used. Peptidyl amidoxime cis-trans isomerase) to co-transform host prokaryotic cells. Accompanying proteins have been shown to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274: 19601-19605; U.S. Patent No. 6,083,715 to Georgiou et al; U.S. Patent No. 6,027,888 to Georgiou et al.; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100 -17105; Ramm and Pluckthun (2000) J. Biol. Chem . 275: 17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.

To minimize proteolysis of the heterologous proteins exhibited (especially for those proteolytically sensitive), specific host strains lacking proteolytic enzymes can be used in the present invention. For example, a host cell strain can be modified to achieve a genetic mutation in a gene encoding a known bacterial protease such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combination. Some E. coli protease deficient strains are available and are described, for example, in Joly et al. (1998) (supra); U.S. Patent No. 5,264,365 to Georgiou et al; U.S. Patent No. 5,508,192 to Georgiou et al; Hara et al., Microbial Drug Resistance , 2: 63-72 (1996).

In one embodiment, an E. coli strain lacking a proteolytic enzyme and exhibiting a plastid transformation of one or more accompanying proteins is used as a host cell in the expression system of the invention.

(3) Antibody Purification In one embodiment, the antibody protein produced herein is further purified to obtain a substantially homogeneous preparation for further assay and use. Standard protein purification methods known in the art can be employed. The following procedure is an exemplary purification procedure: immunoaffinity fractionation or ion exchange column, ethanol precipitation, reverse phase HPLC, ceria chromatography or ion exchange resin chromatography such as DEAE, chromatofocusing, SDS-PAGE, sulfuric acid Ammonium precipitation and gel filtration using, for example, Sephadex G-75.

In one aspect, protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody product of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds to the Fc region of the antibody with high affinity. Lindmark et al. (1983) J. Immunol. Meth. 62: 1-13. The solid phase of immobilized protein A can be a column comprising a glass or cerium oxide surface, or a controlled pore glass column or citrate column. In some applications, the tubing is coated with a reagent such as glycerin in an attempt to prevent non-specific adhesion of contaminants.

As a first step of purification, a formulation derived from a cell culture as described above can be applied to the solid phase of immobilized protein A to allow specific binding of the antibody of interest to protein A. The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by dissolution.

b) Production of antibodies using eukaryotic host cells: Vectors for use in eukaryotic host cells generally comprise one or more of the following non-limiting components: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters and Transcription termination sequence.

(1) Signal sequence component The vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected can be a signal sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signal sequences as well as viral secretory guides, such as the herpes simplex gD signal, can be used. The DNA of the previously driven region is ligated into the DNA encoding the antibody in the reading frame.

(2) Origin of replication Generally, mammalian expression vectors do not require replication of the starting component. For example, the SV40 origin can usually be used simply because it contains an early promoter.

(3) Selection of gene components The expression and selection vectors may contain a selection gene, also referred to as a selectable marker. Typical selection genes encode (a) confer resistance to antibiotics or other toxins (eg, ampicillin, neomycin, methotrexate or tetracycline); (b) supplement auxotrophy (when relevant) or (c) supply is not self-complexing The protein of the standard nutrient obtained in the medium.

One example of a selection mechanism utilizes a drug that stagnates host cell growth. The cells that have been successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus preserves the selection protocol. An example of this advantage is the use of the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a suitable selectable marker for a mammalian cell is such a selectable marker that enables recognition of a cell capable of absorbing the antibody nucleic acid, such as DHFR; thymidine kinase; metallothionein I and II, preferably primates Animal metallothionein gene; adenosine deaminase; ornithine decarboxylase.

For example, in some embodiments, cells transformed with a DHFR selection gene are first identified by culturing all transformants in a medium containing a competitive antagonist of methotrexate (Mtx), DHFR. In some embodiments, when wild-type DHFR is employed, the appropriate host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (eg, ATCC CRL-9096).

Alternatively, the DNA sequence of the encoded antibody can be selected for transformation by cell growth in a medium containing a selectable marker selection agent such as an aminoglycoside antibiotic (eg, kanamycin, neomycin or G418) or Co-transformed host cells (especially wild-type hosts containing endogenous DHFR), wild-type DHFR proteins, and another selectable marker, such as aglycoside 3'-phosphotransferase (APH). See U.S. Patent No. 4,965,199.

(4) Promoter components The expression and selection vectors typically contain a promoter that is recognized by the host organism and operably linked to a nucleic acid encoding a polypeptide of interest (e.g., an antibody). The promoter sequence of eukaryotic cells is known. For example, virtually all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream from the initiation of transcription of many genes is the CNCAAT region where N can be any nucleotide. The 3' end of most eukaryotic genes is the AATAAA sequence, which can be a signal that adds a poly A tail to the 3' end of the coding sequence. In certain embodiments, any or all of such sequences can be suitably inserted into a eukaryotic expression vector.

Vector transcripts from mammalian host cells are controlled, for example, by promoters derived from viral genomes such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), milk Head tumor virus, avian sarcoma virus, giant cell virus, retrovirus, hepatitis B virus and simian virus 40 (SV40); heterologous mammalian promoter, such as actin promoter or immunoglobulin promoter A heat shock promoter whose restriction conditions are compatible with the host cell system for such promoters.

The early and late promoters of the SV40 virus are conveniently obtained in the form of SV40 restriction fragments that also contain the SV40 viral origin of replication. The immediate early promoter of the human cell giant virus is conveniently obtained in the form of a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al, Nature 297: 598-601 (1982), which describes the expression of human beta interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as a promoter.

(5) Enhancer component The DNA encoding the antibody of the present invention by higher order eukaryotic transcription is usually increased by inserting the enhancer sequence into the vector. Many enhancer sequences are known to be derived from mammalian genes (blood globulin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are commonly used. Examples include the SV40 enhancer located at the back of the replication origin (bp 100-270); the cellular giant virus early promoter enhancer; the polyoma enhancer on the posterior side of the replication origin and the adenovirus enhancer. See also Yaniv, Nature 297: 17-18 (1982) describing the enhancer element used to activate eukaryotic promoters. The enhancer can be spliced into the vector at the 5' or 3' position of the sequence encoding the antibody polypeptide, but is typically positioned at the 5' position of the promoter.

(6) Transcription termination component The expression vector for use in a eukaryotic host cell also contains a sequence necessary for terminating transcription and stabilizing mRNA. Such sequences are typically obtained from the 5' and (occasionally) 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding the antibody. One suitable transcription termination component is the bovine growth hormone polyadenosine region. See WO 94/11026 and the expression vectors disclosed therein.

(7) Selection and Transformation of Host Cells Suitable host cells for the selection or expression of DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. The propagation of vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of suitable mammalian host cell strains are SV40 transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (secondarily selected for growth of 293 or 293 cells in suspension culture) , Graham et al., J.Gen Virol .36: 59 (1977 )); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc.Natl.Acad. Sci. USA 77:4216 (1980)); mouse podocytes (TM4, Mather, Biol . Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells ( VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung Cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 ( 1982)); MRC 5 cells; FS4 cells and human liver tumor cell lines (Hep G2).

The host cell is transformed with the above-described expression or selection vector to produce an antibody, and cultured in a conventional nutrient medium modified as needed for inducing a promoter, selecting a transformant, or amplifying a gene encoding the desired sequence.

(8) Culture of host cells Host cells for producing the antibody of the present invention can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma) and Dubecco's Modified Ig's Medium (DMEM), Sigma) is suitable for culturing host cells. Further, Ham et al, Meth. Enz. 58: 44 (1979), Barnes et al, Anal . Biochem . 102: 255 (1980), U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; 4,560,655 No. 5,122,469; any of the media described in WO 90/03430; WO 87/00195 or U.S. Patent No. Re. 30,985 can be used as a medium for host cells. Any such medium may optionally be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor); salts (such as sodium chloride, calcium salts, magnesium salts and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ^(TM) drugs), trace elements (defined as inorganic compounds typically present in the final concentration in the micromolar range), and glucose or equivalent energy. They may also include any other supplements known to those skilled in the art at appropriate concentrations. Culture conditions such as temperature, pH, and the like are those of the host cells previously selected for performance and will be apparent to those of ordinary skill in the art.

(9) Purification of antibodies When recombinant techniques are used, antibodies can be produced intracellularly or directly secreted into a medium. If the antibody is produced intracellularly as a first step, the microparticle fragments (host cells or lysed fragments) are removed, for example, by centrifugation or ultrafiltration. When the antibody is secreted into the culture medium, the supernatant from these performance systems is first concentrated using a commercially available protein concentration filter (eg, Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a convenient technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J. Immunol. Methods 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3 (Guss et al, EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached may be agarose, but other matrices may be used. Mechanically stable matrices such as controlled microporous glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times compared to agarose achievables. Where the antibody comprises a CH3 domain, Bakerbond ABX ^TM resin (JTBaker, Phillipsburg, NJ) is suitable for purification. Depending on the antibody to be recovered by the set may also be used, such as ion-exchange column fractionation, ethanol precipitation, reverse phase HPLC, chromatography on silicon dioxide, heparin SEPHAROSE ^TM chromatography on an anion or cation exchange chromatography resin (such as a polyaspartic amine Acid column), chromatographic focusing, SDS-PAGE and other protein purification techniques for ammonium sulfate precipitation.

After any preliminary purification step, the mixture comprising the antibody of interest and the contaminant can be used, for example, by using a pH of between about 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0-0.25 M salt). The lower pH hydrophobic interaction chromatography of the dissolution buffer was subjected to further purification.

In general, various methods for preparing antibodies for research, testing, and clinical use have been identified in the art, consistent with the above methods and/or as deemed suitable by the skilled artisan for the particular antibody of interest.

C. Immunoconjugate

The invention also provides an immunoconjugate (alternatively referred to as "antibody-drug conjugate" or "ADC") comprising any of the anti-TAT226 antibodies of the invention conjugated to one or more cytotoxic agents, the cytotoxic agent Such as chemotherapeutic agents, drugs, growth inhibitors, toxins (eg, enzymatically active toxins or fragments thereof of bacterial, fungal, plant or animal origin) or radioisotopes (ie, radioactive conjugates).

Immunoconjugates can be used in the treatment of cancer for the local delivery of cytotoxic agents, ie drugs that kill or inhibit the growth or proliferation of tumor cells (Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26: 151-172; U.S. Patent No. 4,975,278). The immunoconjugate can allow for delivery of a drug moiety to the tumor and intracellular accumulation therein, wherein systemic administration of unconjugated drugs can produce an unacceptable degree of toxicity to normal cells and tumor cells to be eliminated (Baldwin et al, Lancet (March 15, 1986, p. 603-05); Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer The rapy: A Review," in Monoclonal Antibodies '84 : Biological And Clinical Applications (A. Pinchera et al. Edited, pp. 475-506. Multiple strains of antibodies and monoclonal antibodies have been reported to be suitable for such strategies (Rowland et al. (1986) Cancer Immunol. Immunother . 21: 183-87). Drugs for use in such methods include daunorubicin, doxorubicin, methotrexate and vindesine (same as Rowland et al., (1986) above). Toxins for antibody-toxin conjugates include bacterial toxins such as diphtheria toxin; phytotoxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al. (2000) J. of the Nat .Cancer Inst .92(19): 1573-1581; Mandler et al. (2000) Bioorganic & MedChem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791); Maytansinoids) (EP 1391213; Liu et al, (1996) Proc. Natl. Acad. Sci. USA 93: 8618-8623) and calicheamicin (Lode et al. (1998) Cancer Res. 58: 2928; Hinman et al. (1993) Cancer Res. 53: 3336-3342). These toxins exert their cytotoxic effects by a mechanism including tubulin binding, DNA binding or topoisomerase inhibition. Some cytotoxic drugs tend to be inert or less active when conjugated to large antibody or protein receptor ligands.

ZEVALIN (Ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate that is a murine IgG1 against CD20 antigen found on the surface of normal and malignant B lymphocytes. Monoclonal antibodies and ¹¹¹ In or ⁹⁰ Y radioisotope compositions bound by a thiourea linker-chelating agent (Wiseman et al., (2000) Eur. Jour. Nucl. Med. 27(7): 766-77; Wiseman et al. (2002) Blood 99(12): 4336-42; Witzig et al., (2002) J. Clin. Oncol. 20(10): 2453-63; Witzig et al., (2002) J. Clin. Oncol. (15): 3262-69). Although ZEVALIN is active against B-cell non-Hodgkin's lymphoma (NHL), administration in both patients results in severe and long-term cytopenia. It was approved in 2000 one kind MYLOTARG ^TM (gemtuzumab (gemtuzumab ozogamicin), Wyeth Pharmaceuticals) by the treatment of acute myeloid leukemia by injection (Drugs of the Future engaged by the connection of calicheamicin to an antibody composition of hu CD3 antibody drug (2000) 25(7): 686; U.S. Patent No. 4,970, 198; No. 5, 907, 533; No. 5,558, 089; No. 5,560, 040; No. 5, 769, 372; No. 5, 739, </ RTI> No. 5, 767, 285; The antibody drug conjugate Cantuzumab mertansine (Immunogen, Inc.) consisting of the huC242 antibody linked to the maytansine drug moiety DM1 via the disulfide linker SPP is advanced to treat cancers that exhibit CanAg (such as colon cancer, Phase II trial of pancreatic cancer, gastric cancer and others. An antibody therapeutic conjugate MLN-2740 (Millennium Pharm., BZL Biologics, Immunogen Inc.) consisting of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the mayonoid drug moiety DM1 is in the potential therapeutic development of prostate tumors in. The synthetic analogs of dolastatin (alastatin), auristatin E (AE) and monomethyl auristatin (MMAE) are ligated to the chimeric monoclonal antibody cBR96 (specific for Lewis Y on cancer) And cAC10 (specific for CD30 on hematological malignancies) (Doronina et al. (2003) Nature Biotechnol. 21(7): 778-784) and in therapeutic development.

In certain embodiments, the immunoconjugate comprises an anti-TAT226 antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents suitable for use in generating an immunoconjugate are described herein (eg, above). Enzymatically active toxins and fragments thereof can also be used and are described herein.

In certain embodiments, the immunoconjugate comprises an anti-TAT226 antibody and one or more small molecule toxins including, but not limited to, small molecule drugs such as calicheamicin, maytansine, dolastatin, auristatin, single ended Trichothecene and CC1065; and derivatives of such drugs having cytotoxic activity. Examples of such immunoconjugates are described in further detail below.

1. An exemplary immunoconjugate The immunoconjugate (or "antibody-drug conjugate"("ADC")) of the invention can have the formula I below, wherein the anti-TAT226 antibody is joined via an optional linker (L) (ie Covalently linked) to one or more drug moieties (D). Ab-(L-D) _p I Thus, an anti-TAT226 antibody can be conjugated to a drug either directly or via a linker. In Formula I, p is the average number of drug moieties per antibody, which may range, for example, from about 1 to about 20 drug moieties per antibody, and in certain embodiments from 1 to about 8 drug moieties per antibody. Within the scope.

a) Exemplary Linkers A linker can comprise one or more linker components. Exemplary linker components include 6-maleimide hexamethylene ("MC"), maleimide propyl sulfonate ("MP"), lysine- citrulline ("val-cit" Or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimide 4-(2-pyridylthio) valerate ("SPP"), N-succinimide 4-(N-maleimidomethyl)cyclohexane-1carboxylate ("SMCC") and N-succinimide (4- Iodo-ethenylaminobenzoate ("SIAB"). Various linker components are known in the art, some of which are described below.

A linker can be a "cleavable linker" that promotes drug release in a cell. For example, an acid labile linker (eg, purine), a protease sensitive (eg, peptidase sensitive) linker, a photolabile linker, a dimethyl linker, or a linker containing a disulfide bond can be used (Chari et al. , Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

In some embodiments, a linker component can comprise a "stretch unit" that attaches an antibody to another linker moiety or to a drug moiety. Exemplary stretch units are shown below (where the wavy line indicates a covalent attachment site to the antibody):

In some embodiments, the linker component can comprise an amino acid unit. In one such embodiment, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating the release of the drug from the immunoconjugate when exposed to an intracellular protease such as a lysosomal enzyme. See, for example, Doronina et al. (2003) Nat . Biotechnol. 21:778-784. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit); alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); or N -Methyl-proline- citrulline (Me-val-cit). Exemplary tripeptides include: glycine-proline-glycine (gly-val-cit) and glycine-glycine-glycine. The amino acid unit can comprise naturally occurring amino acid residues, as well as trace amounts of amino acids and non-naturally occurring amino acid analogs such as citrulline. Amino acid units can be designed and optimized for their enzymatic cleavage by specific enzymes such as tumor associated proteases, cathepsins B, C and D or plasmin proteases.

In some embodiments, a linker component can comprise a "spacer" unit that attaches the antibody to the drug moiety, either directly or via a stretching unit and/or an amino acid unit. The spacer unit can be "self-destructive" or "non-destructive". A "non-destructive" spacer unit is a spacer unit that retains binding to a drug moiety when part or all of the spacer units are cleaved (eg, proteolytically) by the ADC. Examples of non-self-destructive spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Other combinations of peptide spacers susceptible to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease will result in the release of the glycine-glycine drug moiety from the remainder of the ADC. In one such embodiment, the glycine-glycine drug moiety is then subjected to an independent hydrolysis step in the tumor cells to partially cleave the glycine-glycine spacer unit from the drug.

The "self-destructive" spacer unit allows the drug moiety to be released without the need for an independent hydrolysis step. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, the aminobenzyl alcohol is attached to the amino acid unit via a guanamine linkage and a urethane, methyl carbamate or carbonate is formed between the benzyl alcohol and the cytotoxic agent. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the pendant phenyl moiety of the aminobenzyl unit is substituted with Qm, wherein Q is -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -halogen, -nitro or -cyano; and m is an integer in the range 0-4. Examples of self-destructive spacer units additionally include, but are not limited to, aromatic compounds that are electrically similar to aminobenzyl alcohol (see, for example, US 2005/0256030 A1), such as 2-aminoimidazole-5-methanol derivatives (Hay Et al. (1999) Bioorg. Med. Chem. Lett. 9: 2237) and o- or p-aminobenzyl acetal. A spacer which undergoes cyclization upon hydrolysis of the indole bond, such as substituted and unsubstituted 4-aminobutyric acid decylamine (Rodrigues et al, Chemistry Biology , 1995, 2, 223); appropriately substituted bicyclo[ 2.2.1] and bicyclo[2.2.2] ring systems (Storm et al, J. Amer . Chem . Soc ., 1972, 94, 5815); and 2-aminophenyl phenyl decanoate (Amsberry et al, J. Org . Chem ., 1990, 55, 5867). The elimination reaction of amine-containing drugs substituted at the a position of glycine (Kingsbury et al, J. Med . Chem ., 1984, 27, 1447) is also an example of a self-destructive spacer suitable for ADC.

In one embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) as described below, which can be used to incorporate and release a plurality of drugs.

Wherein Q is -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -halogen, -nitro or -cyano; m is an integer in the range 0-4; n is 0 or 1; and p is in the range of 1 to about 20.

A linker can comprise any one or more of the above linker components. In certain embodiments, the linker is shown as a scaffold in ADC Formula II below: Wherein A is a stretching unit and a is an integer from 0 to 1; W is an amino acid unit and w is an integer from 0 to 12; Y is a spacer unit; and y is 0, 1 or 2; and Ab, D and p As defined above for Formula I. Illustrative embodiments of such a linker are described in US 2005-0238649 A1, which is expressly incorporated herein by reference.

Exemplary linker components and combinations thereof are shown in the ADC content of Formula II below:

Linking subcomponents, including stretching units, spacer units, and amino acid units, can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1.

b) Exemplary Drugs (1) Maytansine and Maytans In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more maytansine molecules. Maytansine is a mitotic inhibitor that acts by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansines such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed in, for example, U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268 No. 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663 and No. 4, 371, 533.

The mayaconine drug moiety is an absorbent drug moiety in the antibody-drug conjugate because it is: (i) relatively easy to prepare by fermentation or chemical modification or derivatization of the fermentation product, (ii) susceptible to Suitable for the effect of functional group derivatization by attachment to a non-disulfide linker, (iii) stable in plasma; and (iv) effective against various tumor cell lines.

The maytansin compounds suitable for use as a portion of the maytansine drug are well known in the art and can be isolated from natural sources or produced using genetic engineering techniques according to known methods (see Yu et al. (2002) PNAS 99: 7968-7973). . Maytanol and maytansinol analogs can also be prepared synthetically according to known methods.

Exemplary mayaconine drug moieties include those having a modified aromatic ring, such as: C-19-dechlorination (U.S. Patent No. 4,256,746) (Resination of lithium aluminum hydride by ansamycin P2 (ansamytocin P2) Prepared); C-20-hydroxy (or C-20-desmethyl) +/- C-19-dechlorinated (US Patent Nos. 4,361,650 and 4,307,016) (by using Streptomyces or Preparation of actinomyces by demethylation or dechlorination using LAH); and C-20-demethoxy, C-20-decyloxy (-OCOR) +/- dechlorination (US patent) No. 4,294,757) (prepared by the use of mercapto chloride) and which have been modified by other positions.

Exemplary maytansinoid drug moieties also include their having such as the following modified by: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by maytansinol with H ₂ S or P ₂ S ₅ reactions); C -14-alkoxymethyl (demethoxy/CH ₂ OR) (US 4,331,598); C-14-hydroxymethyl or decyloxymethyl (CH ₂ OH or CH ₂ OAc) (US Pat. No. 4,450,254) No.) (prepared by Nocardia); C-15-hydroxy/decyloxy (US 4364866) (prepared by conversion of maytansinol with streptomycin); C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); C-18-N-demethylation (U.S. Patent Nos. 4,362,663 and 4,322,348) (by virtue of streptomycin Prepared by methylation) and 4,5-deoxy (US 4,371,533) (prepared by the reduction of titanium trichloride/LAH of maytansinol).

Illustrative examples of the maytansine drug moiety include: DM1, DM3, and DM4 having the following structure: The wavy line indicates the covalent attachment of the sulfur atom of the drug to the linker (L) of the antibody-drug conjugate. HERCEPTIN connected to DM1 by SMCC has been reported (trastuzumab) (WO 2005/037992; US 2005/0276812 A1).

Other exemplary maytan base antibody-drug conjugates have the following structures and abbreviations (where Ab is an antibody and p is from 1 to about 8):

An exemplary antibody-drug conjugate in which DM1 is linked to a thiol group of an antibody via a BMPEO linker has the following structure and abbreviations: Wherein Ab is an antibody; n is 0, 1 or 2; and p is 1, 2, 3 or 4.

An immunoconjugate comprising a meringine base, a method of making the same, and a therapeutic use thereof are disclosed in, for example, U.S. Patent Nos. 5,208,020, 5,416,064, US 2005/0276812 A1, and European Patent EP 0 425 235 B1, which are incorporated herein by reference. The disclosure is expressly incorporated herein by reference. Liu et al . Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describes an immunoconjugate comprising a mersalamine named DM1 linked to a monoclonal antibody C242 against human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and exhibited anti-tumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52: 127-131 (1992) describe a murine antibody A7 in which maytansine is bound to an antigen on a human colon cancer cell line via a disulfide linker or conjugated to a carcinogen with HER-2/neu An immunoconjugate of another murine monoclonal antibody TA.1 that binds to the gene. The cytotoxicity of the TA.1-Medang base conjugate to the human breast cancer cell line SK-BR-3 expressing 3 x 10 ⁵ HER-2 surface antigen in each cell was tested in vitro. The drug conjugate achieves a degree of cytotoxicity similar to that of the free maytan base drug, which can be increased by increasing the number of mannitol molecules per antibody molecule. The A7 mayenamine conjugate exhibits low systemic cytotoxicity in mice.

The antibody maytansine conjugate is prepared by chemically linking the antibody to the maytansine molecule without significantly reducing the biological activity of the antibody or the maytansine molecule. See, for example, U.S. Patent No. 5,208,020, the disclosure of which is expressly incorporated by reference. On average, 3-4 meridian base molecules per antibody molecule have been shown to enhance the cytotoxicity of target cells without adversely affecting the function or solubility of the antibody, although even if only one toxin/antibody molecule is expected to be enhanced during use of the naked antibody Cytotoxicity. Maytansine is well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansines are disclosed, for example, in U.S. Patent No. 5,208,020 and other patents and non-patent publications mentioned above. Preferably, the maytansine is a maytansinol and a meta-alcohol analog, such as various maytansinol, which is modified at the aromatic ring or other position of the maytansinol molecule.

A number of linking groups are known in the art for the manufacture of antibody-Medogen base conjugates, including, for example, U.S. Patent No. 5,208,020 or EP Patent No. 0 425 235 B1; Chari et al. Cancer Research 52: 127-131 (1992) They are disclosed in US 2005/016993 A1, the disclosures of which are expressly incorporated herein by reference. The antibody-meta base conjugate comprising the linker component SMCC can be prepared as disclosed in US 2005/0276812 A1, "Antibody-drug conjugates and Methods". As disclosed in the above patents, the linker comprises a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group or an esterase labile group. Other linkers are described and exemplified herein.

The conjugate of the antibody with the maytansine can be made using various bifunctional protein coupling agents, such as N-succinimido-3-(2-pyridyldithio)propionate (SPDP), amber imine. Bifunctionality of -4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), quinone imido ester Derivatives (such as hexamethyleneimine dimethyl ester HCl), active esters (such as dinonyl succinimide), aldehydes (such as glutaraldehyde), bis- azide compounds (such as double (pairs) Nitrobenzylbenzyl) hexamethylenediamine), double nitrogen salt derivatives (such as bis(p-diazepin)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In certain embodiments, the coupling agent is N-succinimido-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al, Biochem. J. 173: 723-737 (1978) ) or N-succinimide-4-(2-pyridylthio) valerate (SPP) to provide a disulfide bond.

The linker visual connection type is linked to the mayon base at various locations. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position with a hydroxyl group, at the C-14 position modified with a hydroxymethyl group, at the C-15 position modified with a hydroxy group, and at the C-20 position having a hydroxy group. In one embodiment, a linkage is formed at the C-3 position of the maytansinol or maytansinol analog.

(2) Auristatin and dolastatin In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to a dolastatin or a hare toxic peptide analog or derivative (e.g., auristatin) (U.S. Pat. No. 5,635, 548; 5780588) number). Haitoxin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anticancer (US Patent No. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). The dolastatin or auristatin drug moiety can be linked to the antibody via the N (amino) or C (carboxy) terminus of the peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF, which are disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Digest No., published March 28, 2004. In 623, the entire disclosure of the disclosure is expressly incorporated by reference.

The peptide drug moiety can be selected from the following formulas D _E and D _F :

Wherein the wavy line of D _E and D _F represents a covalent attachment site to the antibody or antibody-linker component and is independent at each position: R ² is selected from H and C ₁ -C ₈ alkyl; R ³ is Selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocyclic, aryl, C ₁ -C ₈ alkyl-aryl, C ₁ -C ₈ alkyl-(C ₃ -C ₈ carbocycle , C ₃ -C ₈ heterocyclic ring and C ₁ -C ₈ alkyl-(C ₃ -C ₈ heterocyclic ring); R ⁴ is selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocyclic ring , aryl, C ₁ -C ₈ alkyl-aryl, C ₁ -C ₈ alkyl-(C ₃ -C ₈ carbocyclic), C ₃ -C ₈ heterocyclic ring and C ₁ -C ₈ alkyl-( C ₃ -C ₈ heterocycle); R ⁵ is selected from H and methyl; or R ⁴ and R ^{5 are} joined to form a carbocyclic ring and have the formula -(CR ^a R ^b ) _n - wherein R ^a and R ^b independently Selected from H, C ₁ -C ₈ alkyl and C ₃ -C ₈ carbocycles and n is selected from 2, 3, 4, 5 and 6; R ⁶ is selected from H and C ₁ -C ₈ alkyl; ⁷ is selected from the group consisting of H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocyclic, aryl, C ₁ -C ₈ alkyl-aryl, C ₁ -C ₈ alkyl-(C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl - (C ₃ -C ₈ heterocycle); each R ⁸ are independently selected H, OH, C ₁ -C ₈ alkyl C ₃ -C ₈ carbocycle and 0- (C ₁ -C ₈ alkyl); R ⁹ is selected from H and C ₁ -C ₈ alkyl; R ¹⁰ is selected from aryl or C ₃ -C ₈ heterocycle Z is O, S, NH or NR ¹² , wherein R ¹² is C ₁ -C ₈ alkyl; R ¹¹ is selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, -(R ¹³ O) _m -R ¹⁴ or -(R ¹³ O) _m -CH(R ¹⁵ ) ₂ ; m is an integer in the range from 1 to 1000; R ¹³ is C ₂ -C ₈ alkyl; R ¹⁴ is H or C ₁ -C ₈ alkyl; each occurrence of R ^{15 is} independently H, COOH, -(CH ₂ ) _n -N(R ¹⁶ ) ₂ , -(CH ₂ ) _n -SO ₃ H or -( CH ₂ ) _n -SO ₃ -C ₁ -C ₈ alkyl; each occurrence of R ^{16 is} independently H, C ₁ -C ₈ alkyl or -(CH ₂ ) _n -COOH; R ¹⁸ is selected from - C(R ⁸ ) ₂ -C(R ⁸ ) ₂ -aryl, -C(R ⁸ ) ₂ -C(R ⁸ ) ₂ -(C ₃ -C ₈ heterocycle) and -C(R ⁸ ) ₂ - C(R ⁸ ) ₂ -(C ₃ -C ₈ carbocycle); and n is an integer in the range of 0 to 6.

In one embodiment, R ³ , R ⁴ and R ^{7 are} independently isopropyl or a second butyl group and R ⁵ is -H or methyl. In an exemplary embodiment, R ³ and R ⁴ are each isopropyl, R ⁵ is -H and R ⁷ is a second butyl group.

In still another embodiment, each of R ² and R ⁶ is methyl and R ⁹ is —H.

In yet another embodiment, each occurrence of R ⁸ is -OCH ₃ .

In an exemplary embodiment, R ³ and R ⁴ are each isopropyl, R ² and R ⁶ are each methyl, R ⁵ is -H, and R ⁷ is a second butyl, each occurrence of R ⁸ is -OCH ₃ and R ⁹ is -H.

In one embodiment, Z is -O- or -NH-.

In one embodiment, R ¹⁰ is an aryl group.

In an exemplary embodiment, R ¹⁰ is -phenyl.

In an exemplary embodiment, when Z is -O-, R ¹¹ is -H, methyl or a tert-butyl group.

In one embodiment, when Z is -NH, R ¹¹ is -CH(R ¹⁵ ) ₂ , wherein R ¹⁵ is -(CH ₂ ) _n -N(R ¹⁶ ) ₂ and R ¹⁶ is -C ₁ - C ₈ alkyl or -(CH ₂ ) _n -COOH.

In another embodiment, when Z is -NH, R ¹¹ is -CH (R ¹⁵⁾ _2, wherein R ¹⁵ is _{- (CH 2) n -SO 3} H.

Examples of formula D _E exemplary auristatin embodiment is MMAE, wherein the wavy line indicates the covalent attachment to the antibody - linker joining the pharmaceutical composition of (L):

An exemplary auristatin embodiment of Formula _DF is MMAF, wherein the wavy line indicates a linker (L) covalently linked to the antibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006) Bioconjugate Chem. 17:114 -124):

Other drug moieties include the following MMAF derivatives, wherein the wavy line indicates a covalent linkage to the linker (L) of the antibody-drug conjugate:

In one aspect, as described above may include (but are not limited to) triethylene glycol (TEG) of the hydrophilic group is attached to the drug moiety at R ¹¹ shown in FIG. Without being bound by any particular theory, the hydrophilic group contributes to the internalization and non-coagulation of the drug moiety.

Illustrative examples of ADCs of Formula I comprising auristatin/sea rabbit toxin or a derivative thereof are described in US 2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114, which is expressly incorporated herein by reference. -124. Illustrative examples of ADCs of Formula I comprising MMAE or MMAF and various linker components have the following structures and abbreviations (wherein "Ab" is an antibody; p is from 1 to about 8; "Val-Cit" is a proline- Citrate dipeptide and "S" is a sulfur atom):

Illustrative examples of ADCs of Formula I comprising MMAF and various linker components additionally include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Interestingly, immunoconjugates comprising MMAF linked to an antibody by a non-proteolytically cleavable linker have been shown to have activity comparable to immunoconjugates comprising MMAF linked to the antibody by proteolytic cleavage of the linker . See Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. In such cases, the release of the drug is achieved by degradation of the antibody in the cell. Same as above.

Peptide-based drug moieties can generally be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments. Such peptide bonds may, for example, be based on liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schr Der and KL Bke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). The Auristatin/Hytotoxin drug moiety can be prepared according to the methods in the following documents: US 2005-0238649 A1, U.S. Patent No. 5,635, 548; U.S. Patent No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. : 5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, GR et al. Synthesis , 1996, 719-725; Pettit et al. (1996) J. Chem . Soc . Perkin Trans . 1 5: 859-863; and Doronina (2003) Nat. Biotechnol. 21(7): 778-784.

In particular, the auristatin/conne toxin drug moiety of formula D _F , such as MMAF and its derivatives, can be prepared using the methods described in US 2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. preparation. The Auristatin/Hytotoxin drug moiety of Formula D _E , such as MMAE and its derivatives, can be prepared using the method described in Doronina et al. (2003) Nat. Biotech. 21:778-784. The drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE are available, for example, by Doronina et al. (2003) Nat. Biotech. 21: 778-784 and patent applications. The conventional method described in the publication US 2005/0238649 A1 is conveniently synthesized and then ligated to the antibody of interest.

(3) Kazimycin In other embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The antibiotic calicheamicin family is capable of producing double-stranded DNA breaks at concentrations below the picomole. For the preparation of conjugates of the calicheamicin family, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (both Belongs to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-ethylindolyl-γ ₁ ^I , PSAG, and θ ^I ₁ (Hinman et al., Cancer Research 53: 3336-3342 (1993), Lode et al, Cancer Research 58: 2925-2928 (1998), and the aforementioned U.S. patent to American Cyanamid. Another anti-tumor drug that can bind antibodies is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and are not easily traversing the plasma membrane. Thus, the uptake of such agents via antibody-mediated internalization greatly enhances their cytotoxic effects.

c) Other cytotoxic agents Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozocin, vincristine, and 5-fluorouracil, a family of agents collectively referred to as the LL-E33288 complex ( It is described in U.S. Patent Nos. 5,053,394, 5,770,710, and esperamicin (U.S. Patent No. 5,877,296).

Useful enzyme-active toxins and fragments thereof include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, azithro A chain, Modi Modecin A chain, alpha-sarcin, oil ketone (Aleurites fordii) protein, carnation protein, foreign commercial protein (PAPI, PAPII and PAP-S), bitter melon inhibitor ( Momordica charantia inhibitor), curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogen, restrictocin, phenolic acid (phenomycin), enomycin and mycotoxins. See, for example, WO 93/21232, published October 28, 1993.

The invention further encompasses immunoconjugates (e.g., ribonucleases or DNA endonucleases, such as deoxyribonuclease; DNase) formed between the antibody and a compound having nuclear degrading activity.

In certain embodiments, the immunoconjugate can comprise a highly radioactive atom. Various radioisotopes can be used to generate radioconjugated antibodies. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When an immunoconjugate is used for detection, it may comprise a radioactive atom for scintillation scanning studies, such as tc ^99m or I ¹²³ , or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri). Spin labeling such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, cesium, manganese or iron.

Radiolabels or other labels can be incorporated into the immunoconjugate in a known manner. For example, the peptide can be synthesized biosynthesis or can be synthesized by chemical amino acid synthesis using a suitable amino acid precursor (eg, including the replacement of hydrogen with fluorine-19). Labels such as tc ^99m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ¹¹¹ can be linked via a cysteine residue in the peptide.钇-90 can be linked via an amine acid residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

In certain embodiments, an immunoconjugate can comprise an anti-TAT226 of the invention conjugated to a prodrug activating enzyme that converts a prodrug (eg, a peptidyl chemotherapeutic agent, see WO 81/01145) to an active drug (such as an anti-cancer drug). antibody. These immunoconjugates are suitable for antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that can be conjugated to an anti-TAT226 antibody of the invention include, but are not limited to, alkaline phosphatase, which is suitable for converting a phosphate-containing prodrug to a free drug; an arylsulfatase enzyme suitable for containing a sulfate The prodrug is converted to a free drug; cytosine deaminase, which is suitable for converting non-toxic 5-fluorocytosine to anticancer drug 5-fluorouracil; proteases such as Serratia protease, thermolysin, Bacillus subtilis Proteases, carboxypeptidases and cathepsins (such as cathepsins B and L), which are suitable for the conversion of peptide-containing prodrugs to free drugs; D-alaninyl carboxypeptidase, which is suitable for conversion containing D-amino acid substitutions a prodrug; a carbohydrate-lyase, such as beta-galactosidase and a neuraminidase, which is suitable for converting a glycosylated prodrug into a free drug; beta-endoaminase, which is suitable for Conversion of a drug derived from β-indoleamine to a free drug; and penicillin amidase, such as penicillin V glutaminase and penicillin G glutaminase, which are suitable for use as phenoxyethyl phenyl or benzene Base B An amine-based drug-derived nitrogen at respectively into free drugs. The enzyme can be covalently bound to the anti-TAT226 antibody of the invention by recombinant DNA techniques well known in the art. See, for example, Neuberger et al, Nature 312:604-608 (1984).

d) Drug loading rate The drug loading rate is expressed in p, which is the average number of drug portions per antibody in the molecule of formula I. The drug loading rate can be in the range of 1 to 20 drug portions (D) per antibody. The ADC of Formula I includes a collection of antibodies conjugated to a bulk of drug moiety (1 to 20). The average number of drug moieties per antibody prepared by the ligation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the ADC can also be determined from p. In some cases, separation, purification, and characterization of a homogeneous ADC (where p is a particular value) can be achieved from an ADC having other drug loading rates, such as by reverse phase HPLC or electrophoresis.

For some antibody-drug conjugates, p can be limited by the number of attachment sites on the antibody. For example, as in the above exemplary embodiments, when linked to a cysteine thiol, the antibody may have only one or several cysteine thiol groups, or may have only one or several via It can be attached to a fully reactive thiol group of the linker. In certain embodiments, a higher drug loading rate, such as p > 5, can result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the ADC of the present invention has a drug loading rate in the range of from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In fact, it has been demonstrated that for a particular ADC, the optimal ratio per antibody portion of the antibody can be less than 8 and can range from about 2 to about 5. See US 2005-0238649 A1.

In certain embodiments, a portion of the drug that is less than the theoretical maximum is conjugated to the antibody during the ligation reaction. As described below, the antibody may contain, for example, an lysine residue that is not reactive with the drug-linker intermediate or linker reagent. In general, antibodies do not contain many free and reactive cysteine thiol groups that can be attached to the drug moiety; in fact, most of the cysteine thiol residues in the antibody are present as disulfide bridges. In certain embodiments, the antibody can be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to produce a reactive cysteine thiol group. . In certain embodiments, the antibody is subjected to denaturing conditions to expose a reactive nucleophilic group such as an amine acid or cysteine.

The loading rate of the ADC (drug/antibody ratio) can be controlled in different ways: for example by (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the time or temperature of the ligation reaction; And (iii) partial or limiting reduction conditions for cysteine thiol upgrading.

It will be appreciated that when more than one nucleophilic group is reacted with a drug-linker intermediate or linker reagent, and then with a drug moiety reagent, the resulting product is one in which one or more drug moieties are linked to the antibody. A mixture of ADC compounds. The average number of drugs per antibody can be calculated from the mixture by double ELISA antibody assay, which is specific for the antibody and specific for the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, for example, Hamblett, KJ et al. "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti -CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, SC et al. "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004 ). In certain embodiments, a homogeneous ADC having a single carrier value can be separated from the ligation mixture by electrophoresis or chromatography.

e) Specific Methods for Preparing Immunoconjugates The ADCs of Formula I can be prepared by employing several routes of organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) nucleophilic groups of antibodies The group reacts with the divalent linker reagent to form an Ab-L via a covalent bond, which in turn reacts with the drug moiety D; and (2) the nucleophilic group of the drug moiety reacts with the divalent linker reagent to form via a covalent bond D-L, which in turn reacts with the nucleophilic group of the antibody. An exemplary method of preparing an ADC of Formula I via the latter route is described in US 2005-0238649 A1, which is expressly incorporated herein by reference.

The nucleophilic group of the antibody includes, but is not limited to, (i) an N-terminal amine group; (ii) a side chain amine group such as an amide acid; (iii) a side chain thiol group such as cysteine and (iv) a glycosyl or amine group to which the antibody is glycosylated. The amine, thiol and hydroxyl group are nucleophilic and are capable of reacting to form a covalent bond with an electrophilic group comprising a linker moiety and a linker reagent: (i) an active ester such as an NHS ester, a HOBt ester, Haloformate and acid halide; (ii) alkyl and benzyl halides such as haloacetamide; (iii) aldehydes, ketones, carboxyl groups and maleimine groups. A particular antibody has a reducible interchain disulfide bond, ie a cysteine bridge. The antibody may be reacted with a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) to allow complete or partial reduction of the antibody. Thus, each cysteine bridge will theoretically form two reactive thiol nucleophiles. The amine can be converted by introducing an additional nucleophilic group into the antibody by, for example, reacting an oleic acid residue with a 2-iminothiolane (Traut's reagent) to modify the amine group to introduce an additional nucleophilic group. It is a thiol. By introducing one, two, three, four or more cysteine residues (for example by preparing a variant antibody comprising one or more non-native cysteine amino acid residues) A reactive thiol group is introduced into the antibody.

The antibody-drug conjugate of the present invention can also be produced by reaction between an electrophilic group on the antibody (such as an aldehyde or a ketone carbonyl group) and a linker reagent or a nucleophilic group on the drug. Suitable nucleophilic groups for use on linker reagents include, but are not limited to, hydrazine, hydrazine, amine, hydrazine, amino thiourea, carboxylic acid hydrazine, and aryl hydrazine. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a linker reagent or a nucleophilic substituent on the drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized, for example, by an iodate oxidizing reagent to form an aldehyde or ketone group reactive with the linker reagent or the amine group of the drug moiety. The resulting imine Schiff base can form a stable linkage or can be reduced, for example, via a borohydride reagent to form a stable amine linkage. In one embodiment, the reaction of the carbohydrate moiety of the glycosylated antibody with galactose oxidase or sodium metaperiodate produces a carbonyl (aldehyde and ketone) group in an antibody reactive with a suitable pharmaceutically acceptable group ( Hermanson, Bioconjugate Techniques). In another embodiment, an antibody comprising an N-terminal serine acid or a threonine residue can be reacted with sodium metaperiodate to produce an aldehyde instead of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem . 3: 138-146; US 5,362,852). This aldehyde can be reacted with a drug moiety or a linker nucleophile.

The nucleophilic group on the drug moiety includes, but is not limited to, an amine, a thiol, a hydroxy group, a hydrazine, a hydrazine capable of reacting to form a covalent bond with an electrophilic group on the linker moiety and the linker reagent. Anthracene, amino thiourea, carboxylic acid hydrazine and aryl sulfhydryl groups, such linker moieties and linker reagents include: (i) active esters such as NHS esters, HOBt esters, haloformates and acid halides (ii) an alkyl group and a benzyl halide such as haloacetamide; (iii) an aldehyde, a ketone, a carboxyl group and a maleimine group.

The compounds of the invention expressly encompass, but are not limited to, ADCs prepared from the following crosslinking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo -EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (amber succinimide-(4-vinyl fluorene) benzene Acid esters, which are commercially available (for example, available from Pierce Biotechnology, Inc., Rockford, IL., USA; see, for example, the 2003-2004 application manual and catalogue pages 467-498).

Immunoconjugates comprising antibodies and cytotoxic agents can also be made using various bifunctional protein coupling agents, such as N-succinimido-3-(2-pyridyldithio)propionate (SPDP), amber醯imino-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), imidothiolane (IT), quinone imide Bifunctional derivatives (such as hexamethyleneimine dimethyl ester HCl), active esters (such as dinonyl succinimide), aldehydes (such as glutaraldehyde), diazido compounds (such as double (p-azidobenzylhydrazine) hexamethylenediamine), double nitrogen derivatives (such as bis(p-diabenzamide)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and double activity Fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the attachment of radionucleotides to antibodies. See WO 94/11026.

Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be made, for example, by recombinant techniques or peptide synthesis. The recombinant DNA molecule can comprise a region encoding the antibody and a cytotoxic moiety of the conjugate that is adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

In yet another embodiment, an antibody can be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, wherein the antibody-receptor conjugate is administered to a patient, which in turn will use a scavenger The unbound conjugate is removed from the cycle and then administered to a "ligand" (eg, avidin) that is conjugated to a cytotoxic agent (eg, a radioactive nucleotide).

D. Pharmaceutical formulations

In one aspect, the invention further provides a pharmaceutical formulation comprising at least one anti-TAT226 antibody of the invention and/or at least one immunoconjugate thereof. In some embodiments, a pharmaceutical formulation comprises 1) an anti-TAT226 antibody and/or an immunoconjugate thereof, and 2) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises 1) an anti-TAT226 antibody and/or an immunoconjugate thereof, and optionally 2) at least one additional therapeutic agent. Other therapeutic agents include, but are not limited to, those described in Section E.2 below.

A pharmaceutical formulation comprising an antibody or immunoconjugate of the invention is by using an antibody or immunoconjugate of the desired purity with an optional physiologically acceptable carrier, excipient or stabilizer ( Remington's Pharmaceutical Sciences) The 16th edition, Osol, A. (1980)) is mixed and prepared in the form of an aqueous solution or lyophilized or other dry formulation for storage. Acceptable carriers, excipients or stabilizers in the dosages and concentrations employed are non-toxic to the receptor and include buffering agents such as phosphates, citrates, histidines and other organic acids; Oxidizing agents, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexahydroxy quaternary ammonium chloride, benzalkonium chloride, benzethonium chloride); phenol, butanol Or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol; a molecular weight (less than about 10 residues) polypeptide; a protein such as serum albumin, gelatin or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamic acid, Aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannose Alcohol, trehalose or sorbitol; forming a counter ion of a salt, such as sodium; (E.g. Zn- protein complexes), and / or nonionic surfactant, ^(TM) such as TWEEN, ^(TM) PLURONICS or polyethylene glycol (PEG). Pharmaceutical formulations to be administered for in vivo administration are generally sterile. This is easily accomplished by filtration through a sterile filtration membrane.

In gelled drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, the active ingredient can also be captured, for example, by coacervation techniques or by interfacial polymerization. The microcapsules prepared are, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980).

A sustained release preparation can be prepared. Suitable examples of sustained release formulations include semipermeable matrices containing solid hydrophobic polymers of the antibodies or immunoconjugates of the invention, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters; hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)); polylactide (U.S. Patent No. 3,773,919); - -L- glutamate and γ-ethyl ester of the copolymer bran amine; of non-degradable ethylene - vinyl acetate; the non-degradable lactic acid - glycolic acid copolymers such as the LUPRON DEPOT ^TM (lactic acid - glycolic acid copolymer And injectable microspheres composed of leuprolide acetate; and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow molecules to be released for more than 100 days, some hydrogels still release proteins for a shorter period of time. When the encapsulated antibody or immunoconjugate is maintained in the body for a prolonged period of time, it can denature or agglomerate upon exposure to moisture at 37 °C, resulting in loss of biological activity and possibly immunogenicity. A reasonable strategy for stabilizing the effects can be designed based on the mechanisms involved. For example, if the agglutination mechanism is found to be an intermolecular S-S bond exchange via a sulfur-disulfide bond, the water content can be controlled by lyophilization from an acidic solution by modifying the sulfhydryl residue. Additives and development of specific polymer matrix compositions to achieve stabilization.

E. Method of using anti-TAT226 antibody and immunoconjugate

1. Diagnostic Methods and Detection Methods In one aspect, the anti-TAT226 antibodies and immunoconjugates of the invention are useful for detecting the presence of TAT226 in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as the tissue listed in Figure 13. In certain embodiments, such tissue comprises normal and/or cancerous tissue exhibiting TAT226 to a higher degree relative to other tissues, such as ovary, kidney, brain, endometrium, adrenal gland, bone, lung, skin And soft tissue.

In one aspect, the invention provides a method of detecting the presence of TAT226 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with an anti-TAT226 antibody under conditions that permit binding of the anti-TAT226 antibody to TAT226, and detecting whether a complex is formed between the anti-TAT226 antibody and TAT226.

In one aspect, the invention provides a method of diagnosing a condition associated with increased performance of TAT226. In certain embodiments, the method comprises contacting the test cell with an anti-TAT226 antibody; determining the degree of performance (quantitative or qualitative) of the test cell for TAT226 by detecting binding of the anti-TAT226 antibody to TAT226; and comparing the test cell to TAT226 The degree of performance is compared with the degree of performance of the control cells to TAT226 (the control cells are, for example, normal cells of the same tissue source as the test cells, or cells expressing TAT226 to a degree comparable to the normal cells), wherein the test is compared to the control cells. The higher degree of performance of the cells to TAT226 indicates the presence of conditions associated with increased TAT226 performance. In certain embodiments, the test cells are obtained from an individual suspected of having a condition associated with increased performance of TAT226. In certain embodiments, the condition is a cell proliferative disorder, such as a cancer or a tumor.

Exemplary cell proliferative disorders that can be diagnosed using the antibodies of the invention include cancer conditions, such as tumors, such as carcinomas (epithelial neoplasms) and blastomas (embryonic tissue-derived tumors); and in certain embodiments, ovarian cancers , uterine cancer (including endometrial cancer) and kidney cancer, including nephroblastoma (such as Wilm's tumor). In particular, ovarian cancer encompasses a heterogeneous population of malignant tumors derived from the ovary. Approximately 90% of malignant ovarian tumors are of epithelial origin; the rest are germ cell tumors and stromal tumors. Epithelial ovarian tumors are classified into the following histological subtypes: serous adenocarcinoma (about 50% of epithelial ovarian tumors); endometrioid adenocarcinoma (about 20%); mucinous adenocarcinoma (about 10%); clear cell carcinoma ( About 5-10%); Brenner (transitional cell) tumors (relatively uncommon). The sixth most common form of cancer in women with ovarian cancer is usually poor prognosis, with a five-year survival rate in the range of 5-30%. For a discussion of ovarian cancer, see Fox et al. (2002) "Pathology of epithelial ovarian cancer," in Ovarian Cancer, Chapter 9 (Jacobs et al., Oxford University Press, New York); Morin et al. (2001) "Ovarian Cancer. "In the Encyclopedic Reference of Cancer , pp. 654-656 (edited by Schwab, Springer-Verlag, New York). The invention encompasses methods of diagnosing or treating any of the epithelial ovarian tumor subtypes described above (and in particular, serous adenocarcinoma subtypes).

In certain embodiments, methods of diagnosis or detection such as those described above comprise detecting binding of an anti-TAT226 antibody to TAT226 expressed on a cell surface or expressed in a membrane preparation obtained from cells expressing TAT226 on its surface. In certain embodiments, the method comprises contacting the cell with an anti-TAT226 antibody under conditions that permit binding of the anti-TAT226 antibody to TAT226, and detecting whether a complex is formed between the anti-TAT226 antibody and TAT226 on the cell surface. An exemplary assay for detecting binding of an anti-TAT226 antibody to TAT226 that expresses TAT226 on the cell surface is a "FACS" assay, such as described in Example D below.

Certain other methods can be used to detect binding of the anti-TAT226 antibody to TAT226. Such methods include, but are not limited to, antigen binding assays well known in the art, such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, firefly Photoimmunoassay, protein A immunoassay and immunohistochemistry (IHC).

In certain embodiments, the anti-TAT226 antibody is labeled. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels, and radioactive labels) and indirectly detected, for example, via enzymatic or molecular interactions. Part, such as an enzyme or a ligand. Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I; fluorophores such as rare earth chelating agents or luciferins and derivatives thereof, rhodamine and Its derivatives, tanshinyl, ketone, luciferase (such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin; 2,3-dihydropyridazine II Ketone; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; sugar oxidase (eg glucose oxidase, galactose oxidase and glucose-6-phosphate Salt dehydrogenase; heterocyclic oxidase, such as uricase and xanthine oxidase, coupled to an enzyme that oxidizes a dye precursor, such as HRP, lactoperoxidase or microperoxidase, using hydrogen peroxide. Biotin/avidin; spin labeling, phage labeling, stable free radicals and the like.

In certain embodiments, the anti-TAT226 antibody is immobilized on an insoluble substrate. Immobilization requires the isolation of any TAT226 from the TAT226 antibody that remains free from the solution. It is conventionally known to render the anti-TAT226 antibody insoluble by adsorption to a water insoluble substrate or surface (Bennich et al., US 3,720,760) or by covalent coupling (eg, crosslinking with glutaraldehyde) prior to the assay procedure, or This is accomplished by insolubilizing the anti-TAT226 antibody, eg, by immunoprecipitation, after formation of a complex between the anti-TAT226 antibody and TAT226.

Any of the above diagnostic or detection embodiments can be carried out using an immunoconjugate of the present invention in place of or in addition to the anti-TAT226 antibody.

2. Methods of Treatment For example, the antibodies or immunoconjugates of the invention can be used in in vitro, ex vivo and in vivo therapeutic methods. In one aspect, the invention provides a method of inhibiting cell growth or proliferation in vivo or in vitro, the method comprising: exposing the cell to an anti-TAT226 antibody or immunoconjugate thereof under conditions permitting binding of the immunoconjugate to TAT226 . "Inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and This includes inducing cell death. In certain embodiments, the cell is a tumor cell. In certain embodiments, the cell is an ovarian tumor cell, a uterine tumor cell, a brain tumor cell, or a renal tumor cell. In certain embodiments, such as exemplified herein, the cells are xenografts.

In one aspect, an antibody or immunoconjugate of the invention is used to treat or prevent a cell proliferative disorder. In certain embodiments, the cell proliferative disorder is associated with increased performance and/or activity of TAT226. For example, in certain embodiments, a cell proliferative disorder is associated with increased performance of TAT226 on the cell surface. In certain embodiments, the cell proliferative disorder is a tumor or cancer. Examples of cell proliferative disorders to be treated with the antibodies or immunoconjugates of the invention include, but are not limited to, cancerous conditions such as tumors, such as carcinomas (epithelial neoplasms) and blastomas (tumors derived from embryonic tissue) And in certain embodiments ovarian cancer; uterine cancer, including endometrial cancer; brain tumors (eg, astrocytoma, including advanced glioma, also known as glioblastoma multiforme); and kidney Cancer, including nephroblastoma (such as Wilm's tumor).

In one aspect, the invention provides a method of treating a cell proliferative disorder comprising administering to an individual an effective amount of an anti-TAT226 antibody or immunoconjugate thereof. In certain embodiments, a method of treating a cell proliferative disorder comprises administering to a subject an effective amount of a pharmaceutical formulation comprising an anti-TAT226 antibody and, optionally, at least one other therapeutic agent, such as those provided below . In certain embodiments, a method of treating a cell proliferative disorder comprises administering to a subject an effective amount of a pharmaceutical formulation comprising: 1) an immunoconjugate comprising an anti-TAT226 antibody and a cytotoxic agent; and optionally 2) At least one other therapeutic agent, such as those provided below.

In one aspect, at least some of the antibodies or immunoconjugates of the invention can bind to TAT226 from species other than humans. Thus, an antibody or immunoconjugate of the invention can be used, for example, in a cell culture containing TAT226, in a human or in other mammals having a TAT226 that cross-reacts with an antibody or immunoconjugate of the invention (e.g., chimpanzee, baboon TAR226 is bound in vivo, scorpion, cynomolgus monkey and macaque, pig or mouse. In one embodiment, an anti-TAT226 antibody or immunoconjugate can be used to inhibit TAT226 activity by contacting the antibody or immunoconjugate with TAT226 such that inhibition of TAT226 activity. In an embodiment, the TAT226 is a human TAT226.

In one embodiment, an anti-TAT226 antibody or immunoconjugate can be used in a method of binding TAT226 in an individual having a disorder associated with increased TAT226 expression and/or activity, the method comprising administering to the individual an antibody or immunizing The conjugate is incorporated into the TAT226 in the individual. In one embodiment, the TAT226 is a human TAT226 and the individual is a human individual. Alternatively, the individual can be a mammal that expresses TAT226 in combination with an anti-TAT226 antibody. In addition, the individual can be a mammal into which TAT226 is introduced (eg, by administering TAT226 or by expressing a transgene encoding TAT226).

Anti-TAT226 antibodies or immunoconjugates can be administered to humans for therapeutic purposes. In addition, anti-TAT226 antibodies or immunoconjugates can be administered to non-human mammals (eg, primates, pigs, rats, or mice) that express TAT226 that cross-reacts with antibodies for veterinary purposes or as an animal model of human disease. Things. With regard to the latter, such animal models can be adapted to assess the therapeutic efficacy of the antibodies or immunoconjugates of the invention (e.g., to test the dosage and schedule of administration).

The antibodies or immunoconjugates of the invention may be used alone or in combination with other compositions in therapy. For example, an antibody or immunoconjugate of the invention can be co-administered with at least one other therapeutic agent and/or adjuvant. In certain embodiments, the additional therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, or a growth inhibitory agent. In one of these embodiments, the chemotherapeutic agent is a medicament or combination of agents for treating ovarian cancer, such as a platinum compound (eg, cisplatin or carbopol); a taxane (eg, paclitaxel or docetaxel); topography Anthracycline; anthracycline (eg, doxorubicin (ADRIAMYCIN) ) or lipid doxorubicin (DOXIL) ); gemcitabine; cyclophosphamide; vinorelbine (NAVELBINE) ); hexamethylene melamine; ifosfamide and etoposide. In another of these embodiments, the chemotherapeutic agent is a combination of agents or agents for the treatment of uterine or endometrial cancer, such as cisplatin, carboplatin, doxorubicin, paclitaxel, methaqualin, Fluorouracil and medroxyprogesterone. In another of these embodiments, the chemotherapeutic agent is a medicament or combination of agents for treating a brain tumor, such as a nitrosourea (such as carmustine or lomustine); a cytotoxic agent (such as irinotene) Kang or temozolamide; anti-angiogenic agents (such as thalidomide, TNP-470, platelet factor 4, interferon and endostatin); differentiation agents (eg retinoids, phenylbutyrate, benzene) Base acetate and anti-neoplaston; anti-invasive agents (eg, matrix metalloproteinase inhibitors, such as marimastat); signal transduction regulators (eg, tamoxifen, Bryostatin and O-6 benzylguanine); topoisomerase inhibitors (such as irinotecan or topotecan) and growth factor inhibitors (such as tyrosine kinase inhibitors). In another of these embodiments, the chemotherapeutic agent is a pharmaceutical or pharmaceutical combination for treating kidney cancer (eg, Wilm's tumor), such as vincristine, actinomycin D, adriamycin, Doxorubicin, cyclophosphamide, ifosfamide, etoposide and carboplatin. In certain embodiments, an antibody of the invention can be combined with an anti-inflammatory agent and/or a preservative.

The combination therapies described above encompasses combination administration (wherein two or more therapeutic agents are included in the same or separate formulations) and is administered separately, in which case administration of the antibody or immunoconjugate of the invention may be Occurs before, concurrently with, and/or after administration of other therapeutic agents and/or adjuvants. The antibodies or immunoconjugates of the invention may also be used in combination with radiation therapy.

The antibody or immunoconjugate of the invention (and any other therapeutic or adjuvant) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and if desired, localized Treatment, the lesion is administered. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Furthermore, antibodies or immunoconjugates are suitably administered by pulse infusion, especially with reduced antibody or immunoconjugate doses. Partially depending on the short or long term administration, it can be administered by any suitable route, for example by injection, such as intravenous or subcutaneous injection.

Certain embodiments of the invention provide antibodies or immunoconjugates to traverse the blood brain barrier when the binding target is in the brain. There are several ways known in the art to transport molecules across the blood brain barrier including, but not limited to, physical methods, lipid based methods, stem cell based methods, and receptor based and channel based methods.

Physical methods of transporting antibodies or immunoconjugates across the blood-brain barrier include, but are not limited to, completely surrounding the blood-brain barrier or by creating an opening in the blood-brain barrier. Surrounding methods include, but are not limited to, direct injection into the brain (see, for example, Papanastassiou et al, Gene Therapy 9:398-406 (2002)); interstitial infusion/convection enhanced delivery (see, eg, Bobo et al, Proc. Natl) .Acad.Sci.USA 91: 2076-2080 (1994)) and the transmission device implanted in the brain (see, e.g., Gill et al., Nature Med .9: 589-595 (2003 ); and Gliadel Wafers ^TM, Guildford Pharmaceutical) . Methods of creating openings in the barrier include, but are not limited to, supersonics (see, e.g., U.S. Patent Publication No. 2002/0038086); osmotic pressure (e.g., by administering hypertonic mannitol) (Neuwelt, EA, Implication) Of the Blood-Brain Barrier and its Manipulation , Vol. 1 & 2, Plenum Press, NY (1989); infiltration by, for example, bradykinin or penetrant A-7 (see, for example, U.S. Patent No. 5,112,596, No. 5,268,164, 5, 506, 206 and 5, 686, 416) and transfecting a neuron across the blood-brain barrier with a vector containing the gene encoding the antibody (see, e.g., U.S. Patent Publication No. 2003/0083299).

Lipid-based methods for transporting antibodies or immunoconjugates across the blood-brain barrier include, but are not limited to, encapsulation of antibodies or immunoconjugates with antibody-binding fragments that bind to receptors on the vascular endothelium of the blood-brain barrier In a liposome (see, for example, U.S. Patent Application Publication No. 20040225313) and a low density lipoprotein particle (see, e.g., U.S. Patent Application Publication No. 20040204354) or a lipoprotein E (see, e.g., U.S. Patent Application Publication No. 20040131692 Apply an antibody or immunoconjugate.

A stem cell-based method of transporting antibodies or immunoconjugates across the blood-brain barrier requires genetic engineering of neural progenitor cells (NPCs) to express the antibody or immunoconjugate of interest and then implant the stem cells into the brain of the individual to be treated. See Behrstock et al. (2005) Gene Ther . December 15, 2005 Advanced Online Publication (Reporting that when implanted in the brains of rodent and primate models, NPC is genetically engineered to express Parkinson's disease (Parkinson) Disease) The neurotrophic factor GDNF reduces symptoms).

Receptor- and channel-based methods of transporting antibodies or immunoconjugates across the blood-brain barrier include, but are not limited to, the use of glucocorticoid blockers to increase permeability of the blood-brain barrier (see, for example, US Patent Application Publication No. 2002) /0065259, 2003/0162695 and 2005/0124533); activated potassium channels (see, e.g., U.S. Patent Application Publication No. 2005/0089473); inhibits ABC drug transporters (see, e.g., U.S. Patent Application Publication No. 2003) /0073713); coating an antibody or immunoconjugate with transferrin and modulating the activity of one or more transferrin receptors (see, e.g., U.S. Patent Application Publication No. 2003/0129186) and cations of antibodies or immunoconjugates (see, e.g., U.S. Patent No. 5,004,697).

The antibodies or immunoconjugates of the invention will be formulated, administered and administered in a manner consistent with good pharmaceutical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the schedule of administration, and other factors known to the medical practitioner. The antibody or immunoconjugate is not required (but optionally) to be formulated with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other agents will depend on the amount of antibody or immunoconjugate present in the formulation, the condition or type of treatment, and other factors described above. It is generally administered in the same dosages and routes of administration as described above, or from about 1 to 99% of the dosages described above, or any dosage and any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dose of an antibody or immunoconjugate of the invention (when used alone or in combination with one or more other therapeutic agents such as a chemotherapeutic agent) will depend on the type of disease, antibody or immunoconjugate type to be treated. , severity and progression of the disease, whether to administer antibodies or immunoconjugates for prophylactic or therapeutic purposes, prior therapy, clinical history of the patient, and response to antibodies or immunoconjugates and judgment of the attending physician. The antibody or immunoconjugate is suitable for administration to a patient once or via a series of treatments. Depending on the type and severity of the disease, an antibody or immunoconjugate of about 1 μg/kg to 15 mg/kg (eg, 0.1 mg/kg to 10 mg/kg) can be a candidate starting dose for administration to a patient, regardless of eg Infusion by one or more separate administrations or by ligation. Depending on the above factors, a typical daily dose may range from about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally be continued until the desired inhibition of the symptoms of the disease occurs. An exemplary dosage of an antibody or immunoconjugate is in the range of from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to the patient. Such doses can be administered, for example, intermittently weekly or every three weeks (e.g., such that the patient receives from about two to about twenty, or such as about six doses of antibody or immunoconjugate). A higher initial loading dose can be administered, followed by one or more lower doses. An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of antibody. However, other dosing regimens are applicable. This treatment process is easily monitored by conventional techniques and assays.

3. Assays The anti-TAT226 antibodies and immunoconjugates of the invention can be characterized for their physical/chemical properties and/or biological activity by various assays known in the art.

a) Activity assay In one aspect, a assay is provided to identify a biologically active anti-TAT226 antibody or immunoconjugate thereof. Biological activity can include, for example, the ability to inhibit cell growth or proliferation (e.g., "cell killing" activity) or the ability to induce cell death, including progressive cell death (apoptosis). Antibodies or immunoconjugates having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, the ability of an anti-TAT226 antibody or immunoconjugate thereof to inhibit cell growth or proliferation in vitro is tested. Assays for inhibiting cell growth or proliferation are well known in the art. Cell viability was measured by specific assays for cell proliferation exemplified by the "cell kill" assay described herein. One such assay is a commercially available assay from Promega (Madison, WI) of the CellTiter-Glo ^TM Luminescent Cell Viability. The assay determines the amount of viable cells in the culture based on the quantification of the ATP present, which is indicative of metabolically active cells. See Crouch et al. (1993) J. Immunol . Meth . 160: 81-88, U.S. Patent No. 6,602,677. The assay can be performed in 96 or 384 well format, subject to automatic high throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure includes a single reagent (CellTiter-Glo The reagent) is added directly to the cultured cells. This causes the cells to dissolve and produce a luminescent signal produced by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, and the amount of ATP present is directly proportional to the number of viable cells present in the culture. Data can be recorded by a photometer or a CCD photographic imaging device. The luminescence output is expressed as relative light units (RLU).

Another cell proliferation assay is the "MTT" assay, which measures 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by mitochondrial reductase. Salt oxidation is a colorimetric assay of formazan. As CellTiter-Glo ^TM assay, this assay indicates the number of metabolically active cells present in the culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65: 55-63 and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

In one aspect, the ability of the anti-TAT226 antibody to induce cell death in vitro was tested. A test for inducing cell death is well known in the art. In some embodiments, such assays are for example, loss of membrane integrity as indicated by propidium iodide (PI), trypan blue (see Moore et al. (1995) Cytotechnology , 17: 1-11) or 7AAD absorption. . In an exemplary PI uptake assay, cells were cultured in Dubeco's modified Ig medium (D-MEM) supplemented with 10% heat-inactivated FBS (Hyclone) and 2 mM L-glutamic acid: Ham F-12 (50:50). Therefore, this assay is performed in the absence of complement and immune effector cells. The cells were seeded at a density of 3 x 10 ⁶ per plate in a 100 x 20 mm dish and allowed to attach overnight. The medium is removed and replaced with fresh medium alone or medium containing various concentrations of antibody or immunoconjugate. The cells were incubated for a period of 3 days. After treatment, the monolayer was washed with PBS and separated by trypsinization. The cells were then centrifuged at 1200 rpm for 5 minutes at 4 ° C. The pellet was resuspended in 3 ml of cold Ca ²⁺ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ) and aliquoted The cell mass was removed in a 12 x 75 mm tube (1 ml per tube, 3 tubes per treatment group) covered with a 35 mm filter. The tube was then subjected to PI (10 μg/ml). Analysis of the samples using flow cytometry and FACSCAN ^TM software FACSCONVERT ^TM CellQuest (Becton Dickinson). Thus an antibody or immunoconjugate that induces a statistically significant degree of cell death as determined by PI uptake is identified.

In one aspect, the ability of an anti-TAT226 antibody or immunoconjugate to induce apoptosis (progressive cell death) in vitro is tested. An exemplary assay for an antibody or immunoconjugate that induces apoptosis is an annexin binding assay. In an exemplary annexin binding assay, cells are cultured in plate and inoculated as described in the previous section. The medium was removed and replaced with fresh medium alone or medium containing 0.001 to 10 μg/ml of antibody or immunoconjugate. After a three day incubation period, the monolayer was washed with PBS and separated by trypsinization. The cells were then centrifuged as described in the previous paragraph, resuspended in Ca ²⁺ binding buffer and aliquoted into tubes. The tube is then received with labeled annexin (eg, annexin V-FITC) (1 μg/ml). FACSCAN ^TM analyze samples using flow cytometry and FACSCON VERT ^TM CellQuest software (BD Biosciences). Thus, an antibody or immunoconjugate that induces a statistically significant degree of annexin binding relative to the control group is identified. Another exemplary assay for antibodies or immunoconjugates that induce apoptosis is a histone DNA ELISA colorimetric assay for detecting nucleosome degradation of genomic DNA. This assay can be performed using, for example, a cell death detection ELISA kit (Roche, Palo Alto, CA).

Cells for any of the above in vitro assays include cells or cell lines that naturally express TAT226 or have been engineered to express TAT226. Such cells include tumor cells that overexpress TAT226 relative to normal cells of the same tissue source. Such cells also include cell lines that express TAT226 (including tumor cell lines) and cell lines that have been transfected with a nucleic acid that has been shown to express TAT226. Exemplary cell lines provided herein for use in any of the above in vitro assays include the OVCAR3 human ovarian cancer cell line expressing TAT226 and the HCT116 human colon cancer cell line transfected with a nucleic acid encoding TAT226.

In one aspect, the ability of an anti-TAT226 antibody or its immunoconjugate to inhibit cell growth or proliferation in vivo is tested. In certain embodiments, the ability of an anti-TAT226 antibody or immunoconjugate thereof to inhibit tumor growth in vivo is tested. In vivo model systems, such as xenograft models, can be used for this test. In an exemplary xenograft system, human tumor cells can be introduced into a non-human animal, such as an athymic "naked" mouse, with inadequate immune function. The antibody or immunoconjugate of the invention can be administered to an animal. The ability of an antibody or immunoconjugate to inhibit or reduce tumor growth is measured. In certain embodiments of the above xenograft system, the human tumor cells are tumor cells from a human patient. These xenograft models are available from Oncotest GmbH (Frieberg, Germany). In certain embodiments, as exemplified herein, a human tumor cell is a cell from a human tumor cell line, such as an OVCAR3 cell. In certain embodiments, human tumor cells are introduced into a non-human animal of inadequate immune function by subcutaneous injection or by implantation into a suitable site, such as a mammary fat pad.

b) Binding assays and other assays In one aspect, the anti-TAT226 antibody is tested for antigen binding activity. For example, in certain embodiments, the ability of an anti-TAT226 antibody to bind to TAT226 expressed on the surface of a cell is tested. A FACS assay such as described in Example D can be used for this test.

In one aspect, a competition assay can be used to identify monoclonal antibodies that compete with YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, or YWO.49.H6 for binding to TAT226. . In certain embodiments, the competing antibody binds to the same epitope determinant bound by YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6 (eg linear or conformational epitopes). Exemplary competition assays include, but are not limited to, such routine assays as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). A detailed exemplary method for mapping antigen-binding epitopes of antibodies is provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ). It is stated that if the two antibodies each block 50% or more of the binding of the other, both bind to the same epitope.

In an exemplary competition assay, a first labeled antibody that binds to TAT226 is included (eg, YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, or YWO.49. H6) and fixed TAT226 were incubated in a solution of a second unlabeled antibody that was tested for its ability to compete with the first antibody for binding to TAT226. The second antibody can be present in the supernatant of the fusion tumor. As a control group, fixed TAT226 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing the first antibody to bind to TAT226, excess unbound antibody was removed and the amount of label associated with immobilized TAT226 was measured. If the amount of label associated with the immobilized TAT226 in the test sample is substantially reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to TAT226. In certain embodiments, the immobilized TAT226 is present on the surface of the cell or in a film preparation obtained from cells whose surface exhibits TAT226.

In one aspect, the purified anti-TAT226 antibody can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC). , mass spectrometry, ion exchange chromatography and papain digestion.

In one embodiment, the invention encompasses altered antibodies having some, but not all, of the effector functions, which makes it important in the in vivo half-life of the antibody, while certain effector functions (such as complement and ADCC) are non-essential or harmful. It is an ideal candidate for many applications. In certain embodiments, the Fc activity of the antibody is measured to ensure that only the desired characteristics are maintained. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcyR binding (and thus may lack ADCC activity), but retains FcRn binding ability. The primary cells used to mediate ADCC, ie NK cells express only FcλRIII, while monocytes express FcλRI, FcλRII and FcλRIII. The FcR expression lines on hematopoietic cells are summarized in Table 3 on page 464 of Ravetch and Kinet, Annu . Rev. Immunol 9:457-92 (1991). An example of an in vitro assay for assessing ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural kill (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind to Clq and thus lacks CDC activity. To assess complement activation, a CDC assay such as that described in Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996) can be performed. FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art.

F. Products

In another aspect of the invention, an article of manufacture containing a substance suitable for the treatment, prevention, and/or diagnosis of the above conditions is provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials such as glass or plastic. The container holds a composition that is separate or in combination with another composition effective to treat, prevent, and/or diagnose the condition, and may have a sterile access port (eg, the container may be an intravenous solution bag or have a stopper pierceable by a hypodermic needle) Vial). At least one active agent in the composition is an antibody or immunoconjugate of the invention. The marker or package insert indicates that the composition is used to treat the selected condition. Additionally, the article of manufacture may comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody or immunoconjugate of the invention; and (b) a second container having the composition contained therein, wherein the composition Contains other cytotoxic or therapeutic agents. The article of manufacture in this embodiment of the invention may additionally comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may additionally comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer Ringer's solution and dextrose solution. It may further comprise other materials required from a commercial or user standpoint, including other buffers, diluents, filters, needles and syringes.

IV. Examples

The following are examples of the methods and compositions of the present invention. It will be appreciated that various other embodiments may be practiced in the general description provided above.

Analysis of A.TAT226 gene expression

Use a proprietary database containing gene expression information (GeneExpress) , Gene Logic Inc., Gaithersburg, MD) analyzed human TAT226 gene expression. GeneExpress using the Microarray Distribution Viewer Graphical analysis of the database. Figure 13 is a graphical representation of the TAT226 gene expression in various tissues, which is listed on the left. The scale across the top of the figure indicates the degree of gene expression based on the intensity of the hybridization signal. The points presented above and below the line are adjacent to the respective listed organization. The point presented above the line indicates the gene expression in normal tissues, and the point presented below the line indicates the gene expression in the tumor or diseased tissue. Figure 13 shows the tendency to increase TAT226 gene expression in tumor or diseased tissue relative to its normal counterpart. In particular, TAT226 exhibits substantially overexpression in tumor and diseased ovaries relative to normal ovaries and exhibits substantially overexpression in Wilm's tumors relative to normal kidneys. Other tissues that exhibit excessive performance in tumor or diseased tissue relative to normal tissue include the endometrium, adrenal gland, bone, lung, skin, and soft tissue. In addition, TAT226 is more potent in normal brain tissues such as the olfactory brain, hippocampus, and basal ganglia, and in tumor or diseased brain tissue such as gliomas.

GeneExpress The database is also used to analyze normal ovaries; normal fallopian tubes; ovarian cancers of clear cells, mucinous and serous cystadenocarcinoma subtypes; human TAT226 gene expression in metastatic ovarian cancer and other types of ovarian cancer. The results are reported in Figure 14, where a particular tissue type is indicated below the figure. The scale on the y-axis in the graph indicates the degree of gene expression based on the intensity of the hybridization signal. Serous cystadenocarcinoma and metastatic ovarian cancer show a strong overexpression of TAT226 relative to normal ovaries. Clear cells and mucinous subtypes display comparable performance to normal ovaries. The normal fallopian tube also shows the substantial performance of TAT226. It should be noted that the serous subtype of ovarian cancer is very similar to that of the fallopian tube epithelial tissue, and both the ovary and the fallopian tube are derived from the same embryonic tissue. See Fox et al. (2002) "Pathology of epithelial ovarian cancer," in Chapter 9 of the Ovarian Cancer (edited by Jacobs et al., Oxford University Press, New York).

B. Production of anti-TAT226 antibody

An antibody to TAT226 was generated by screening a phage display library with a recombinant "TAT226-His" fusion protein comprising the amino acid 1-115 of SEQ ID NO: 75 and a C-terminal polyhistamine. The phage display library is a synthetic (Fab') ₂ library generated using the Fab'-zip-phage system. See Lee et al. (2004) J. Immunol. Methods 284: 119-132. The library comprises the huMAb4D5-8 heavy chain variable region (see Figures 5A and 5B, second receptor "B", SEQ ID NO: 50, 51, 57, 35) and the immobilized huMAb4D5- as shown in SEQ ID NO: The heavy chain HVR library in the framework of the 8 light chain variable region. The selected pure lines were presented using phage for TAT226-His screening using phage ELISA (see, eg, Sidhu et al. (2004) J. Mol . Biol. 338: 299-310). Pure lines YWO.32 and YWO.49 were selected for further analysis.

To improve the affinity of YWO.49, a phage display library was generated in the context of YWO.49 with HVR-H3 and HVR-L3 randomized to the targeting software, wherein the selected amino acid residues in the designated HVR were kept constant, Other residues are subject to mutation. The selected pure line was screened by phage ELISA. Affinity matured antibodies designated YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 were selected for further analysis. As shown in Figures 9 and 10, the nucleotides of the VH and VL regions of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 are determined. The encoded polypeptide sequence. The heavy and light chain HVR sequences of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 are shown in Figures 2-4. The consensus HVR-H3 and HVR-L3 sequences derived from YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 are also shown in Figure 4. YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 were "rearranged" to full length by transplanting the Fab' fragment onto the appropriate constant region using recombinant techniques. IgG. The experiments described below were performed using rearranged antibodies.

C. Characterization of Recombinant Antigen Binding Affinity

By using BIACORE Determination of Recombinant Antigen by YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6 by Surface Plasma Resonance Measurement of 3000 System (Biacore, Inc., Piscataway, NJ) The combination of affinity. Briefly, according to the supplier's instructions, N -ethyl- N' -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide ( NHS) Activated carboxymethylated dextran biosensing wafer (CM5, BIAcore Inc). The anti-TAT226 antibody was diluted to 5 μg/ml with 10 mM sodium acetate, pH 4.8, prior to injection at a flow rate of 5 μl per minute to achieve approximately 500 reaction units (RU) of the coupled antibody. Second, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of TAT226-His (0.7 nM to 500 nM) were injected into PBS with 0.05% Tween 20 at 25 °C at a flow rate of 25 μl/min. Using a simple one to one Langmuir binding model to calculate the association rate _{(k on) (one-to} -one Langmuir binding model) (BIA Evaluation Software version 3.2) and dissociation rates (k _off). The equilibrium dissociation constant (Kd) is calculated as the ratio of k _off /k _on . The results of this experiment are shown in Table 2 below.

D. Characterization of antibodies that bind to the cell surface TAT226

The ability of the anti-TAT226 antibody to bind to TAT226 expressed on the surface of OVCAR3 (human ovarian cancer cell line) was examined. Fluorescence activated cell sorting (FACS) was performed on OVCAR3 cells in the presence and absence of YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 or YWO.49.H6. Briefly, the isolated cells were incubated on ice for 5 hours with 5 μg/ml primary antibody, washed and incubated on ice for 30 minutes with a secondary antibody (conjugated to phycoerythrin anti-human IgG). Use FACScan ^TM flow cytometer (BD Biosciences, San Jose, CA ) were FACS.

The results of FACS analysis for YWO.49, YWO.49.H2 and YWO.H6 are shown in Figure 15. The peaks on the left side of each figure indicate "background" binding, that is, only the binding of secondary antibodies. The peak on the right side of each figure indicates the binding of the designated anti-TAT226 antibody. As seen by FACS, YWO.49.B7 does not significantly bind to OVCAR3, even though it is within the Kd range observed for YWO.49 and other affinity matured antibodies (see BIACORE in Table 2 above). As a result of the analysis, Kd binds to the recombinant antigen. The combination of YWO.49.C9 may correspond to the combination observed for YWO.49.H2 and YWO.H6.

E. Characterization of binding affinity to cell surface antigen

The binding affinities of YWO.49.H2 and YWO.49.H6 for TAT226 expressed on the surface of OVCAR3 cells were tested using competition assays. Briefly, labeled (iodinated) YWO.49.H2 or YWO.49.H6 was conjugated to OVCAR3 cells in the presence of unlabeled antibodies. The binding affinity of the antibodies was determined according to the Scatchard analysis methodology originally described in Munson et al., Anal . Biochem. 107: 220 (1980). The results of this experiment are shown in Table 3 below.

Compared with recombinant TAT226-His, YWO.49.H2 and YWO.49.H6 showed higher Kd of TAT226 on the surface of OVCAR3 cells (compare YWO.49.H2 and YWO.49.H6 in Tables 2 and 3. Kd), this indicates that YWO.49.H2 and YWO.49.H6 bind to recombinant TAT226-His with a slightly higher affinity than TAT226 expressed on the surface of OVCAR3 cells.

F. TAT226 mRNA and protein expression

Use 5' nuclease (TaqMan Quantitative and real-time quantitative PCR analysis of TAT226 mRNA expression in OVCAR3 cells and ovarian cancer sample plates. The ovarian cancer sample named "HF ####" in Figure 16 is a frozen tissue section. RNA was isolated from tissue sections, amplified using Ambion's Message Amp II kit (Ambion, Austin, TX) and reverse transcribed into cDNA. RNA was isolated from OVCAR3 cells and reverse transcribed into cDNA. The TAT226 cDNA was amplified by real-time PCR in the presence of a non-extensible reporter probe specific for the amplification product. The threshold period or "Ct" (the period during which the signal generated by self-reported conductor probe cleavage exceeds the background value) is determined and used to calculate the initial TAT226 mRNA content. As shown in the bar graph of Figure 16, the TAT226 mRNA content in the ovarian cancer sample plate relative to the TAT226 mRNA content in OVCAR3 cells.

Immunohistochemistry (IHC) was used to analyze the expression of TAT226 protein in OVCAR3 cells and the above ovarian cancer sample plates. Tissue sections of ovarian cancer samples (frozen or embedded with papain) were fixed in acetone/ethanol for 5 minutes. Sections were washed in PBS, blocked with avidin and biotin (Vector Laboratories, Inc., Burlingame, CA) for 10 minutes and washed again in PBS. Sections were then blocked with 10% serum for 20 minutes and washed dry to remove excess serum. The primary antibody (YWO.49.H2 or YWO.49.H6) was then added to the sections at a concentration of 10 μg/ml for 1 hour. The sections were then washed with PBS. Biotinylated secondary anti-human antibodies were added to the sections for 30 minutes and then the sections were washed with PBS. The sections were then exposed to the Vector ABC kit (Vector Laboratories, Inc., Burlingame, CA) for 30 minutes and then washed in PBS. The sections were then exposed to diaminobenzidine (Pierce) for 5 minutes and then washed in PBS. The sections were then stained with Mayers hematoxylin, covered with a coverslip and observed. IHC was performed on OVCAR3 cells using the same protocol except that the cells were first granulated, frozen and then sectioned. The sections were then subjected to the above experimental protocol.

The results are qualitatively reported in Figure 16, and the degree of performance is classified as "-", "+/-" or "+". In general, there is an overall correlation between the extent of TAT226 mRNA expression and the performance of TAT226 protein on the surface of OVCAR3 cells. IHC experiments also identified antibodies that recognize TAT226 on the cell surface. The histology of each cell in the ovarian cancer cell plate is also reported in Figure 16, where the abbreviation "adenoca." stands for "adenocarcinoma."

G. Generation of anti-TAT226 ADC

Anti-TAT226 ADCs were generated by ligating YWO.49.H2 and YWO.49.H6 to the following drug-linker moieties: MC-vc-PAB-MMAE, MC-vc-PAB-MMAF and MC-MMAF, Described in Section III C.1.b.2 above. Prior to conjugation, the antibody was partially reduced with TCEP using standard methods according to the methods described in WO 2004/010957 A2. The partially reduced antibody is conjugated to the above drug-linker moiety using standard methods according to the methods described in Doronina et al. (2003) Nat. Biotechnol. 21: 778-784 and US 2005/0238649 A1. Briefly, the partially reduced antibody is combined with a drug linker moiety such that the moieties are joined to a cysteine residue. The ligation reaction was stopped and the ADC was purified. The drug loading rate of each ADC (the average number of drug parts per antibody) was determined by HPLC as follows:

H. Cell killing assay

In the following in vitro and in vivo cell kill assays, the test antibody-drug conjugate (ADC) inhibits the ability of cells expressing TAT226 to proliferate.

1. OVCAR3 in vitro cell kill assay The ability of YWO.49.H2 and YWO.49.H6ADC to inhibit OVCAR3 cell proliferation was tested. OVCAR3 cells were seeded in 96-well plates in RPMI with 20% FBS. As shown in Figure 17, OVCAR3 cells with a density of 3000 cells per well were grown at different concentrations of ADC. An anti-MUC16/CA125 antibody conjugated to MC-Vc-PAB-MMAE was used as a positive control. MUC16/CA125 is a known ovarian cancer antigen. See, for example, Yin et al. (2001) J. Biol. Chem. 276: 27371-27375. An anti-IL-8 antibody conjugated to MC-vc-PAB-MMAE was used as a negative control. Incubated for 5 days, according to the manufacturer's instructions using the CellTiter-Glo ^TM luminescent cell viability assay (Promega, Madison, WI) measuring cell viability. The scale on the y-axis in Figure 17 represents the relative light unit or "RLU" from luciferase luminescence, which is a measure of cell viability.

Figure 17 shows that, similar to the positive control, YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF have significant cell killing activity, especially at about 0.01 and 0.1. This is especially true at concentrations of μg/ml. YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE also have cell killing activity, but the degree is lower than YWO.49.H2-MC-vc- The extent of PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF. The IC ₅₀ of YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF is about 0.005 nM, and YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB- MMAE the IC ₅₀ of about 0.2 nM. The free MMAE IC ₅₀ of about 0.1 nM. YWO.49.H2-MC-MMAF and YWO.49.H6-MC-MMAF did not exhibit significant cell killing activity in this assay. In this particular assay system, it should be noted that due to the overall high concentration of MMAE and MMAF, cell viability is substantially reduced at high concentrations of ADCs (including negative control ADCs).

2. In vitro cell killing assay using HCT116 transfected cells YWO.49.H2 and YWO.49.H6 ADC inhibits HCT116 cells stably transfected with nucleotides encoding human TAT226 (ie colon cancer cell line) The ability to proliferate. Untransfected HCT116 cells are generally about 5-6 fold less sensitive to free (unjoined) MMAE than to OVCAR3 cells.

Briefly, HCT116 cells were transfected as follows. Nucleotides encoding the epitope-tagged human TAT226 were constructed in the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The epitope tag consists of the amino acid 1-53 of the herpes simplex virus type 1 glycoprotein D ("gD" tag) which replaces the signal sequence from the amino acid 1-22 at the N-terminus of human TAT226. The recombinant vector was transfected into HCT116 cells using Lipofectamine 200 (Invitrogen) according to the manufacturer's protocol. Transfected HCT116 cells were cultured in McCoy 5a medium with 10% FBS and 0.4 mg/ml G418. Cells were stained with anti-gD antibodies and sorted by FACS to select individual lines that express recombinant gD: human TAT226 fusion protein. One of the pure lines designated HCT116#9-4 was selected for further analysis.

For cell killing assay, HCT116#9-4 cells were seeded in 96-well plates. As shown in Fig. 18, HCT116#9-4 cells having a density of 1000 cells per well were cultured with different concentrations of ADC. An anti-gp120 antibody conjugated to MC-vc-PAB-MMAE was used as a negative control. 3 days of cultivation, according to the manufacturer's instructions using the CellTiter-Glo ^TM luminescent cell viability assay (Promega, Madison, WI) measuring cell viability.

Figure 18 shows that YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF have significant cell killing activity, especially at about 0.01 μg/ml and up to the highest concentration tested. This is especially true. The IC ₅₀ of YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF is about 0.05 nM, and the IC ₅₀ of free MMAE is about 0.9 nM. In this particular assay, YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE did not have substantial cell killing activity relative to the negative control. Between the cell killing activity of YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE in this assay compared to the OVCAR3 cell kill assay (above) The difference can be attributed to various factors such as differences in cell density and/or drug sensitivity.

It should be noted that YWO.49.H2 and YWO.49.H6 ADCs do not exhibit significant cell killing activity for some other cell lines tested that typically exhibit TAT226 mRNA or protein. It can be attributed to various factors such as cell type-specific effects, degree of cell surface TAT226 expression, and/or differences in drug sensitivity.

3. In vivo assay using HCT116 #9-4 xenografts The in vivo xenograft model was used to test the ability of unconjugated and conjugated YWO.49.H6 to inhibit the proliferation of tumor cells expressing TAT226 in vivo. Mice were injected subcutaneously into the back side by about 5 × 10 ⁶ HCT116 # 9-4 cells in athymic nude "nu-nu" mice induced tumors. The tumor was allowed to grow until it reached an average tumor volume of 200 mm ³ . Specify this time point as "Day 0". As shown in Figure 19, mice received an intravenous injection of 3 mg/kg of the indicated unconjugated antibody or ADC on days 0, 7, and 16. Unconjugated and conjugated anti-ragweed antibodies (Ab) were used as negative controls. Mean tumor volumes were measured on days 3, 7, 10, 16, and 21. As shown in Figure 19, YWO.49.H6-MC-vc-PAB was compared to the anti-ragweed Ab-MC-vc-PAB-MMAF in this specific xenograft model as measured by the mean tumor volume. - MMAF exhibits significant tumor cell killing activity. In this xenograft model, YWO.49.H6-MC-vc-PAB-MMAE did not exhibit significant tumor cell killing activity relative to the anti-ragweed Ab-MC-vc-PAB-MMAE. However, due to various factors, such as differences in the drug sensitivity or degree of expression of the cell surface TAT226 in the xenograft tumor microenvironment, this xenograft model cannot be reflected in YWO.49.H6-MC- observed in vitro. Cell killing activity of vc-PAB-MMAE.

4. Other xenograft models Other xenograft models can be used to test the ability of unconjugated and conjugated anti-TAT226 antibodies to inhibit the proliferation of tumor cells expressing TAT226 in vivo. For example, xenograft models of ovarian and brain tumors can be provided by published sources such as Oncotest GmbH (Frieberg, Germany) and Southern Research Institute (Birmingham, AL). In particular, the Oncotest model was developed by growing a patient's tumor in immunodeficient nude mice. Xenografts exhibiting TAT226 mRNA and/or protein are suitable for exhibiting the cell killing activity of the anti-TAT226 antibody in vivo.

The ability of the conjugated YWO.49.H6 to inhibit ovarian tumor cell proliferation in vivo was tested in the Oncotest model OVXF1023. The Oncotest model OVXF1023 is derived from poorly differentiated papillary serous adenoma ovarian cancer (stage M1) of cancer metastasis. OVXF1023 mice were treated with the ADC shown in Figure 20. In Fig. 20, YWO.49.H6 was named "H6"; the anti-ragweed (control) antibody was named "RW" and the linker -MC-vc-PAB- was abbreviated as "vc". The ADC was administered at this concentration on the day specified in Figure 20. The results shown in Figure 20 indicate that YWO.49.H6-MC-vc-PAB-MMAF and YWO.49.H6-MC-MMAF significantly reduced tumor volume relative to other ADCs.

The above experiment was repeated with OVXF1023 under similar conditions, but with varying doses and control ADC. In repeated experiments, higher dose (5 mg/kg) of YWO.49.H6-MC-vc-PAB-MMAE and lower dose (5 mg/kg) of YWO.49.H6-MC-vc- Mice were treated with PAB-MMAF and YWO.49.H6-MC-MMAF. The results indicate that the H6 ADC (and especially YWO.49.H6-MC-vc-PAB-MMAE) showed relative to their respective control ADCs (6.4 mg/kg anti-gp120-MC-vc-PAB _- MMAE; 7.2 mg/ Kg anti-gp120-MC-vc-PAB-MMAF; and 5.4 mg/kg anti-gp120-MC-MMAF) reduced tumor volume, although the difference in efficacy between H6 ADC and its respective control ADC was for some data points Not statistically significant. (The information is not shown). The difference between these results and the results obtained in the first OVXF1023 experiment can be attributed to the dose difference of the H6 ADC and the observed anti-gp120 ADC exhibiting unexpected activity in reducing tumor volume.

In addition, H6 ADC was also tested in another Oncotest model, OVXF899, which was derived from moderately differentiated papillary serous ovarian cancer (primary tumor). The H6 ADC does not reduce tumor volume in this model. (The information is not shown). However, this particular Oncotest model demonstrates the lower performance of TAT226, which illustrates the observed results.

I.ThioMAb

A cysteine engineered antibody or "thioMAb" is produced in which the selected YWO.49.H6 residue is substituted with cysteine to provide additional sites for conjugating the linker-drug moiety. Specifically, the A118C substitution (EU numbering) is carried out in the heavy chain of YWO.49.H6, or the V205C substitution (Kabat numbering) is carried out in the light chain of YWO.49.H6. The resulting A118C thioMAb was then ligated to MC-MMAF and the resulting V205C thioMAb was then ligated to MC-MMAF or MC-vc-PAB-MMAE. It can be seen from FACS analysis that all thioMAbs can bind to OVCAR3 cells. (The information is not shown).

The present invention has been described in detail by way of illustration and example, and the description The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety.

Figure 1 shows the alignment of TAT226 from human, cynomolgus ("cyno"), mouse and rat. The shaded residues are identical in each species. The unshaded residue is based on at least two of the four species. The percent amino acid identity between the TAT226 sequences from humans, cynomolgus monkeys, mice and rats is shown in the table below. The percentage of consistency is calculated using the ClustalW program.

Figure 2 shows the H1, H2 and H3 heavy chain hypervariable region (HVR) sequences of the anti-TAT226 monoclonal antibodies designated YWO.32 and YWO.49 as described in Example B. The amino acid positions are numbered according to the Kabat numbering system as described below.

Figure 3 shows the L1, L2 and L3 light chain HVR sequences of the anti-TAT226 monoclonal antibodies designated YWO.32 and YWO.49 as described in Example B. The amino acid positions are numbered according to the Kabat numbering system as described below.

Figure 4 shows YWO.49 and YWO.49.B7, YWO.49.C9, YWO produced by affinity maturation of YWO.49 using the HVR-H3 and HVR-L3 software randomization libraries as described in Example B. .49. H2 and HWO-H3 and HVR-L3 sequences of YWO.49.H6. The consistent HVR-H3 and HVR-L3 sequences are also shown.

Figures 5A and 5B show exemplary receptor human variable heavy (VH) consensus framework sequences having the following sequence identifiers for practicing the invention: - human VH subgroup I consensus framework "A" minus Kabat CDR (SEQ ID NO: 32, 33, 34, 35). - Human VH subgroup I consistent framework "B", "C" and "D" minus extended hypervariable regions (SEQ ID NO: 36, 37, 34, 35; SEQ ID NO: 36, 37, 38, 35 ; and SEQ ID NO: 36, 37, 39, 35). - Human VH subgroup II consensus framework "A" minus Kabat CDRs (SEQ ID NO: 40, 41, 42, 35). - Human VH subgroup II consensus frameworks "B", "C" and "D" minus extended hypervariable regions (SEQ ID NO: 43, 44, 42, 35; SEQ ID NO: 43, 44, 45, 35 ; and SEQ ID NOS: 43, 44, 46 and 35). - Human VH subgroup III consensus framework "A" minus Kabat CDRs (SEQ ID NO: 47, 48, 49, 35). - Human VH subgroup III consensus framework "B", "C" and "D" minus extended hypervariable regions (SEQ ID NO: 50, 51, 49, 35; SEQ ID NO: 50, 51, 52, 35 And SEQ ID NO: 50, 51, 53, 35). - Human VH receptor framework "A" minus Kabat CDRs (SEQ ID NO: 54, 48, 55, 35). - Human VH receptor frameworks "B" and "C" minus the extended hypervariable regions (SEQ ID NO: 50, 51, 55, 35; and SEQ ID NO: 50, 51, 56, 35). - Human VH Receptor 2 framework "A" minus Kabat CDRs (SEQ ID NO: 54, 48, 57, 35). - Human VH receptor 2 framework "B", "C" and "D" minus extended hypervariable regions (SEQ ID NO: 50, 51, 57, 35; SEQ ID NO: 50, 51, 58, 35: And SEQ ID NO: 50, 51, 59, 35).

Figures 6A and 6B show exemplary receptor human variable light (VL) consensus framework sequences having the following sequence identifiers for practicing the invention: - human VL Subgroup I consistent framework V1): SEQ ID NO: 60, 61, 62, 63. -human VL Subgroup II Consistent Framework V2): SEQ ID NO: 64, 65, 66, 63. -human VL Subgroup III Consistent Framework V3): SEQ ID NO: 67, 68, 69, 63. -human VL Subgroup IV Consistent Framework V4): SEQ ID NO: 70, 71, 72, 63.

Figure 7 shows the framework sequences of the huMAb4D5-8 light and heavy chains. The superscript/bold numbers indicate the position of the amino acid according to Kabat.

Figure 8 shows the huMAb4D5-8 light and heavy chain framework sequences with the indicated modifications. The superscript/bold numbers indicate the position of the amino acid according to Kabat.

Figure 9 shows the heavy chain variable region (VH) sequences of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The HVR is underlined.

Figure 10 shows the light chain variable region (VL) sequences of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The VL sequences of humanized monoclonal antibody 4D5-8 ("huMAb4D5-8") and "modified" huMAb4D5-8 are also shown in SEQ ID NO: 31 and SEQ ID NO: 26, respectively. YWO.32 and YWO.49 have the same VL sequence as the "modified" huMAb4D5-8 VL (SEQ ID NO: 26), which contains the following substitutions associated with SEQ ID NO: 31: N30S, R66G and H91S. The HVR is underlined.

Figure 11 shows the alignment of the heavy chain variable region sequences of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The HVR system is enclosed in the box. The HVR-H3 residues of YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, which are different from the corresponding residues of HVR-H3 of YWO.49, are shaded.

Figure 12 shows the alignment of the light chain variable region sequences of YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6. The HVR system is enclosed in the box. The HVR-L3 residues of YWO.49.B7, YWO.49.C9, YWO.49.H2 and YWO.49.H6, which are different from the corresponding residues of HVR-L3 of YWO.49, are shaded.

Figure 13 shows a graphical representation of the extent of human TAT226 gene expression in various tissues as described in Example A.

Figure 14 shows the degree of expression of the human TAT226 gene in normal ovaries; normal fallopian tubes; ovarian cancers of clear cells, mucinous and serous cystadenocarcinoma subtypes; metastatic ovarian cancer and other types of ovarian cancer, as described in Example A. Icon.

Figure 15 shows the results of fluorescent activated cell sorting (FACS) of OVCAR3 cells in the presence or absence of the indicated anti-TAT226 antibody as described in Example D.

Figure 16 shows the 5' nuclease (TaqMan) by OVCAR3 cells and ovarian cancer sample plates as described in Example F. Identification and expression of TAT226 mRNA and protein as determined by immunohistochemistry (IHC).

Figure 17 shows the in vitro activity of various YWO.49.H2 and YWO.49.H6 antibody-drug conjugates (ADCs) in the OVCAR3 cell kill assay as described in Example H.

Figure 18 shows the in vitro activity of various YWO.49.H2 and YWO.49.H6 ADCs in a cell kill assay using HCT116 #9-4 stable transfectants as described in Example H.

Figure 19 shows the in vivo activity of a YWO.49.H6 ADC using mouse xenografts as described in Example H.

Figure 20 shows the in vivo activity of YWO.49.H6 ADC using mouse xenografts derived from human patient tumors as described in Example H.

<110> American firm Nandek <120> Anti-TAT226 antibody and immunoconjugate <130> P2324R1 <140> 096109168 <141> 2007-03-17 <150> US 60/783,746 <151> 2006-03- 17 <160> 78 <210> 1 <211> 10 <212> PRT <213> Manual sequence <220><223> HVR-H1 <400> 1 <210> 2 <211> 18 <212> PRT <213> Artificial sequence <220><223> HVR-H2 <400> 2 <210> 3 <211> 13 <212> PRT <213> Manual sequence <220><223> HVR-H3 <400> 3 <210> 4 <211> 10 <212> PRT <213> Manual sequence <220><223> HVR-H1 <400> 4 <210> 5 <211> 18 <212> PRT <213> Manual sequence <220><223> HVR-H2 <400> 5 <210> 6 <211> 11 <212> PRT <213> Manual sequence <220><223> HVR-H3 <400> 6 <210> 7 <211> 11 <212> PRT <213> Manual sequence <220><223> HVR-H3 <400> 7 <210> 8 <211> 11 <212> PRT <213> Manual sequence <220><223> HVR-H3 <400> 8 <210> 9 <211> 11 <212> PRT <213> Manual sequence <220><223> HVR-H3 <400> 9 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <220><223> HVR-H3 <400> 10 <210> 11 <211> 11 <212> PRT <213> Artificial sequence <220><223> HVR-H3 consistent sequence <220><221> Other <222> 4 <223> Xaa=Val or Ile <220><221> Other <222> 5 <223> Xaa=Ser or Thr <220><221> Other <222> 6 <223> Xaa=Arg, Leu or Ile <220><221> Other <222> 8 <223 > Xaa=Gly, Ala, Ser or Pro <400> 11 <210> 12 <211> 11 <212> PRT <213> Manual sequence <220><223> HVR-L1 <400> 12 <210> 13 <211> 7 <212> PRT <213> Manual sequence <220><223> HVR-L2 <400> 13 <210> 14 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 14 <210> 15 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 15 <210> 16 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 16 <210> 17 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 17 <210> 18 <211> 9 <212> PRT <213> Manual sequence <220><223> HVR-L3 <400> 18 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220><223> HVR-L3 consistent sequence <220><221> Other <222> 2 <223> Xaa=Arg, Lys, His, Asn or Gln <220><221> Other <222> 4 <223> Xaa=Tyr or Val <220><221> Other <222> 5 <223> Xaa=Thr, Phe, Asn, Gly or Ala <220><221> Other <222> 8 <223> Xaa=Pro or Phe <220><221> Other <222> 9 <223> Xaa=Thr, Ile or Ala <400> 19 <210> 20 <211> 122 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of YWO.32 <400> 20 <210> 21 <211> 120 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of YWO.49 <400> 21 <210> 22 <211> 120 <212> PRT <213> Artificial sequence <220><223> Heavy chain variable region of YWO.49.B7 <400> 22 <210> 23 <211> 120 <212> PRT <213> Artificial sequence <220><223> YWO.49.C9 heavy chain variable region <400> 23 <210> 24 <211> 120 <212> PRT <213> Artificial sequence <220><223> YWO.49.H2 heavy chain variable region <400> 24 <210> 25 <211> 120 <212> PRT <213> Artificial sequence <220><223> YWO.49.H6 heavy chain variable region <400> 25 <210> 26 <211> 108 <212> PRT <213> Artificial sequence <220><223> YWO.32, YWO.49 and modified light chain variable region of huMAb4D5-8 <400> 26 <210> 27 <211> 108 <212> PRT <213> Artificial 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<210> 47 <211> 30 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 47 <210> 48 <211> 14 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 48 <210> 49 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 49 <210> 50 <211> 25 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 50 <210> 51 <211> 13 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 51 <210> 52 <211> 31 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 52 <210> 53 <211> 30 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 53 <210> 54 <211> 30 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 54 <210> 55 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 55 <210> 56 <211> 31 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 56 <210> 57 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 57 <210> 58 <211> 31 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 58 <210> 59 <211> 30 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 59 <210> 60 <211> 23 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 60 <210> 61 <211> 15 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 61 <210> 62 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 62 <210> 63 <211> 10 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 63 <210> 64 <211> 23 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 64 <210> 65 <211> 15 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 65 <210> 66 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 66 <210> 67 <211> 23 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 67 <210> 68 <211> 15 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 68 <210> 69 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 69 <210> 70 <211> 23 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 70 <210> 71 <211> 15 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 71 <210> 72 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 72 <210> 73 <211> 32 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 73 <210> 74 <211> 10 <212> PRT <213> Artificial sequence <220><223> Sequence is synthesized <400> 74 <210> 75 <211> 141 <212> PRT <213> Human <400> 75 <210> 76 <211> 141 <212> PRT <213> Cynomolgus <400> 76 <210> 77 <211> 141 <212> PRT <213> Mus musculus <400> 77 <210> 78 <211> 141 <212> PRT <213> Rattus norvegicus <400> 78

(no component symbol description)

Claims (34)

A monoclonal antibody that binds to TAT226, wherein the antibody comprises: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (2) HVR- comprising the amino acid sequence of SEQ ID NO: H2; (3) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 7-10; (4) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) comprising SEQ ID NO: the amino acid sequence of HVR-L2; and (6) HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 15-18; wherein the antibody that binds to TAT226 has a dissociation constant of ≦10 nM (K _D ). The monoclonal antibody of claim 1 which binds to TAT226, wherein the antibody comprises (a), (b), (c) or (d): (a) (1) comprises the amino acid sequence of SEQ ID NO: HVR-H1; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7; (4) comprising SEQ ID NO HVR-L1 of the amino acid sequence of 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (6) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15. (b) (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (3) comprising SEQ ID NO: HVR-H3 of the amino acid sequence of 8; (4) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; (6) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; (c) (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (3) comprising SEQ ID NO: 9 HVR-H3 of the amino acid sequence; (4) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; 6) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 17; (d) (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (2) comprising SEQ ID NO: HVR-H2 of the amino acid sequence; (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 10; (4) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (5) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (6) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18. The antibody of claim 2, wherein the antibody comprises (c). The antibody of claim 2, wherein the antibody comprises (d). The antibody of claim 2, which additionally comprises at least one framework selected from the group consisting of a VH subgroup III consensus framework and a VL subgroup I consensus framework. A monoclonal antibody that binds to TAT226, wherein the antibody comprises a heavy chain variable domain having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-25, and is selected from the group consisting of SEQ ID NO The amino acid sequence of 27-30 has a light chain variable domain with at least 95% sequence identity. The antibody of claim 6, wherein the antibody comprises a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 24, and the amino acid sequence of SEQ ID NO: 29 A light chain variable domain of at least 95% sequence identity. The antibody of claim 7, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:29. The antibody of claim 6, wherein the antibody comprises a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the amino acid sequence of SEQ ID NO: 30 A light chain variable domain of at least 95% sequence identity. The antibody of claim 9, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:30. An antibody of 2 or 6, wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv or (Fab') ₂ fragments. An antibody of 2 or 6, wherein the antibody is humanized. An antibody of 2 or 6, wherein the antibody is human. An immunoconjugate comprising an antibody that binds to a cytotoxic agent, which binds to TAT226 as defined in any one of claims 1 to 13. The immunoconjugate of claim 14, wherein the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope, and a nucleolytic enzyme. An immunoconjugate having the formula Ab-(LD)p, wherein: (a) Ab is a monoclonal antibody as defined in any one of claims 1 to 13; (b) L is a linker; (c) D is a drug of formula D _E or D _F And wherein R ² and R ⁶ are each methyl, R ³ and R ⁴ are each isopropyl, R ⁵ is -H or methyl, R ⁷ is a second butyl group, and each R ⁸ is independently selected from CH ₃ , O-CH ₃ , OH and H; R ⁹ is H; R ¹⁰ is aryl; Z is -O- or -NH-; R ¹¹ is H, C ₁ -C ₈ alkyl or -(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -O-CH ₃ ; and R ¹⁸ is -C(R ⁸ ) ₂ -C(R ⁸ ) ₂ -aryl; and (d)p is It is in the range of about 1 to 8. The immunoconjugate of claim 16, wherein the linker is linked to the antibody via a thiol group on the antibody. The immunoconjugate of claim 16, wherein the drug is selected from the group consisting of MMAE and MMAF. The immunoconjugate of claim 18, wherein the drug is MMAE. The immunoconjugate of claim 18, wherein the drug is MMAF. The immunoconjugate of claim 18, wherein the linker is cleaved by a protease. The immunoconjugate of claim 21, wherein the linker comprises a val-cit dipeptide. The immunoconjugate of claim 21, wherein the linker comprises a p-aminobenzyl unit. The immunoconjugate of claim 21, wherein the linker comprises 6-maleimidocaproyl. The immunoconjugate of claim 19, wherein the immunoconjugate has the formula Wherein S is a sulfur atom and p is in the range of 2 to 5. The immunoconjugate of claim 20, wherein the immunoconjugate has the formula Wherein S is a sulfur atom and p is in the range of 2 to 5. A pharmaceutical composition comprising an immunoconjugate as defined in any one of claims 14 to 26 for use in the treatment of a cell proliferative disorder. The pharmaceutical composition according to claim 27, wherein the cell proliferative disorder is selected from the group consisting of ovarian cancer, uterine cancer, brain tumor, and Wilms' tumor. Use of an immunoconjugate as defined in any one of claims 14 to 26 for the manufacture of a medicament for the treatment of a cell proliferative disorder. The use of claim 29, wherein the cell proliferative disorder is selected from the group consisting of ovarian cancer, uterine cancer, brain tumor, and Wilm's tumor. The immunoconjugate of claim 25, wherein Ab is a monoclonal antibody as defined in claim 2 (c) or claim 8. An immunoconjugate according to claim 25, wherein Ab is as in claim 2 (d) or Request a monoclonal antibody as defined in item 10. An immunoconjugate according to claim 26, wherein Ab is a monoclonal antibody as defined in claim 2 (c) or claim 8. An immunoconjugate according to claim 26, wherein Ab is a monoclonal antibody as defined in claim 2 (d) or claim 10.