Humanized Anti-kallikrein-2 antibodies

Abstract

To provide novel therapeutic agents for treating and / or diagnosing non localized prostate cancer, metastatic prostate cancer and / or castration resistant prostate cancer.SOLUTION: A composition for use in the treatment and / or diagnosis of prostate cancer comprises an antibody polypeptide that specifically binds to human kallikrein-2 (hK2), where the antibody polypeptide comprises: (a) a heavy chain variable region comprising compatibility determining region (CDR) CDRH1, CDRH2 and CDRH3 comprising the amino acid sequences shown by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; and (b) a light chain variable region comprising compatibility determining region (CDR) CDRL1, CDRL2 and CDRL3 comprising the amino acid sequences shown by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively.SELECTED DRAWING: Figure 15

Description

[Technical field]

[0001] The present invention relates generally to the field of therapeutic and diagnostic agents and methods, and in particular to the field of prostate cancer. [Background technology]

[0002] Prostate cancer is currently the most common form of cancer in men. The prostate is a walnut-sized gland in men that produces a fluid that is part of semen. The prostate has two or more lobes, or segments, and is surrounded by an outer layer of tissue. The prostate is located just below the bladder in front of the rectum and surrounds the urethra.

[0003] The incidence of prostate cancer is highest in northwestern Europe and the United States. Tumor growth is a process that usually occurs over a long period of time. Prostate cancer is usually a mild form of cancer. In fact, the majority of men diagnosed with prostate cancer survive and recover, and only a small number of men develop a malignant form of prostate cancer that metastasizes at an early stage. This malignant form of prostate cancer is only treatable if it is diagnosed at an early stage, before the cancer spreads to extracapsular tissues.

[0004] Today, prostate cancer is commonly diagnosed and monitored by measuring the concentration of prostate-specific antigen (PSA) in the patient's blood. If the concentration of PSA is significantly elevated in several consecutive measurements taken at different times, prostate cancer is deemed probable. At this point, a biopsy can be performed to confirm prostate cancer.

[0005] PSA (also known as kallikrein III) is a protein consisting of a single chain of 237 amino acids, which is produced in secretory cells of the prostate. These secretory cells can be found throughout the prostate. PSA is a well-established and well-studied marker for prostate cancer. By comparison with healthy cells, the production of PSA is lower in malignant cells and higher in hyperplastic cells. It is rather paradoxical that in fact the concentration of PSA is higher in the blood of men with prostate cancer. However, one explanation is that malignant cells have a deteriorated cellular structure and are therefore more permeable to PSA.

[0006] Another important serine protease suitable as a target for prostate cancer therapy is human glandular kallikrein 2 (hK2). The gene encoding hK2 is located on chromosome 19 along with the gene encoding PSA. hK2 is expressed primarily in prostate tissue, just like PSA. In the prostate, PSA exists as an inactive preform and is activated by the peptidase action of hK2. Immunohistochemical studies on hK2 have shown that hK2 expression correlates with the level of differentiation. This suggests that hK2 is expressed in higher amounts in less differentiated tissues, such as those in prostate cancer, and in lower amounts in well-differentiated tissues, such as those in benign prostatic hyperplasia (BPH), another common prostate problem. This means that it is expressed in terms of production amount.

[0007] Today's therapies for prostate cancer include surgery (e.g., radical prostatectomy), radiation therapy (including brachytherapy and external beam radiation therapy), high intensity focused ultrasound (HIFU), chemotherapy, oral chemotherapy drugs, cryosurgery (freezing the tumor), hormone therapy (such as antiandrogen therapy), castration, or a combination of the above.

[0008] Most of these therapies (surgery and external beam radiation therapy), however, are only (or primarily) useful for treating the primary tumor and large metastases. Chemotherapy is used for disseminated cancer, but provides palliative care and / or prolongs survival for the majority of these patients. Thus, in the case of disseminated malignant disease, especially micrometastases, other or complementary therapeutic modalities are required to achieve substantial improvement.

[0009] Therapies such as immunotherapy or radioimmunotherapy using targeted molecules such as antibodies and fragments offer the possibility of treating disseminated disease.

[0010] Thus, there is a need for new therapeutic agents and methods for treating and diagnosing prostate cancer. Summary of the Invention [Problem to be solved by the invention]

[0011] Accordingly, the present invention seeks to alleviate, alleviate or eliminate one or more of the above defined deficiencies and disadvantages in the art singly or in any combination and solves at least the above mentioned problems by providing therapeutic agents and methods according to the appended claims. [Means for solving the problem]

[0012] The first aspect of the present invention is an antibody having binding specificity for human kallikrein-2 (hK2). A polypeptide comprising: (a) Heavy chain variable region CDRH1 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3: SDYAWN SEQ ID NO:1 CDRH2: YISYSGSTTYNPSLKS SEQ ID NO:2 CDRH3: GYYYGSGF SEQ ID NO:3 and / or (b) Light chain variable region CDRL1 comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6: KASESVEYFGTSLMH SEQ ID NO: 4 CDRL2: AASNRES SEQ ID NO:5 CDRL3: QQTRKVPYT SEQ ID NO:6 wherein the heavy chain variable region and the light chain variable region comprise framework amino acid sequences derived from one or more human antibodies.

[0013] The above six amino acid sequences represent the complementarity determining regions (CDRs) of the antibody polypeptides of the invention, as defined in accordance with Kabat et al. (1991) Sequences of Immunological Interest, 5th ed., NIH, Bethesda, MD, the disclosure of which is incorporated herein by reference.

[0014] "Antibody polypeptides" include substantially intact antibody molecules, single chain antibodies, diabodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen-binding fragments and derivatives thereof.

[0015] As used herein, the term "amino acid" includes the standard 20 genetically encoded amino acids and their corresponding stereoisomers in the "D" form (as compared to the natural "L" form), ω-amino acids, other naturally occurring amino acids, non-canonical amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), as well as chemically derivatized amino acids (see below).

[0016] When an amino acid is specifically recited, such as "alanine" or "Ala" or "A", the term refers to both L-alanine and D-alanine unless expressly stated otherwise. Other non-canonical amino acids can also be suitable components of the polypeptides of the invention, so long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue is represented by a single letter designation, where appropriate, and is in accordance with the conventional Corresponds to the common names of amino acids.

[0017] In one embodiment, the polypeptides defined herein comprise or consist of L-amino acids.

[0018] The antibody polypeptides of the present invention exhibit specificity for hK2.

[0019] An exemplary hK2 sequence can be found at the following World Wide Web address: ensembl.org / Homo_sapiens / Transcript / Sequence_Protein?g=ENSG00000167751;r=19:51376689-51383822;t=ENST00000325321 As seen in the ensemble database, which can be found at: transcript: KLK2-201 (ENST00000325321), gene product: ENSG00000167751, with the following sequence: MWDLVLSIAL SVGCTGAVPL IQSR IVGGWE CEKHSQPWQV AVYSHGWAHC GGVLVHPQWV LTAAHCLKKN SQVWLGRHNL FEPEDTGQRV PVSHSFPHPL YNMSLLKHQS LRPDEDSSHD LMLLRLSEPA KITDVVKVLG LPTQEPALGT TCYASGWGSI EPEEFLRPRS LQCVSLHLLS NDMCARAYSE KVTEFMLCAG LWTGGKDTCG GDSGGPLVCN GVLQGITSWG PEPCALPEKP AVYTKVVHYR KWIKDTIAANP [SEQ ID NO: 7] (the sequence of the mature, active hK2 protein is underlined and is preceded at the N-terminus by the signal peptide and propeptide sequences).

[0020] The majority of hK2 found in seminal plasma is inactive and complexed with protein C inhibitor (PCI). It is also possible that hK2 forms complexes with other extracellular protease inhibitors. In vitro studies indicate that hK2 can bind to α2-antiplasmin (α2-AP), ACT, AMG, antithrombin III (ATIII), C1 inactivating factor, and plasminogen activator inhibitor-1 (PAI-1).

[0021] In one embodiment, the antibody polypeptide has specificity for the free (i.e., uncomplexed) hK2 isoform compared to the complexed hK2 isoform. A binding moiety with specificity for the free hK2 isoform has binding specificity for an epitope that is exposed on the free hK2 isoform but not on the complexed hK2 isoform, which may be a linear or conformational (i.e., non-linear) epitope. For example, an antibody polypeptide may have binding specificity for an epitope that is exposed in free hK2 but not on the complexed hK2 isoform, such as the form present in seminal fluid when hK2 is complexed with PCI. The hK2 protein may have specificity for an epitope that includes one or more amino acid residues that are part of the catalytic groove of hK2 that are not exposed in the hK2 isoform. Epitope mapping of hK2 is described in Vaisanen et al., Clinical Chemistry 50:9, 1607-1617 (2004), the disclosure of which is incorporated herein by reference.

[0022] Further examples of hK2 proteins are identified by the following accession numbers: (a) GenBank: AAF08277.1; (b) GenBank: AAF08275.1; and (c)UniProtKB / Swiss-Prot:P20151.1

[0023] The production of recombinant hK2 is described in Lovgren et al., 1999, Eur. J. Biochem. 266:1050-5, the disclosure of which is incorporated herein by reference.

[0024] By "specificity" is meant that the antibody polypeptide is capable of binding to hK2 in vivo, i.e., under physiological conditions in which hK2 exists in the human body. Preferably, the antibody polypeptide does not bind to any other proteins in vivo.

[0025] Such binding specificity can be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blot and flow cytometry using transfected cells expressing hK2.Advantageously, the antibody polypeptide can selectively bind to hK2, i.e., the antibody binds to hK2 at least 10 times stronger than other proteins (particularly other kallikreins such as prostate specific antigen or PSA).Preferably, the antigen polypeptide does not bind to PSA in vivo.

[0026] Mouse antibodies with specificity for hK2 are known to those skilled in the art. For example, Vaisanen et al., 2004, Clinical Chemistry 50(9):1607-1617, describe the production of monoclonal antibodies with specificity for hK2 in mice (the disclosure of which is incorporated herein by reference). Two antibodies, designated "11B6" and "7D7", are stated to be selective for hK2.

[0027] The amino acid sequences of the heavy and light chain components of murine antibody 11B6 are disclosed in International Application PCT / GB2012 / 052675 (WO2013 / 061083, the disclosure of which is incorporated herein by reference in its entirety); see in particular SEQ ID NOs: 4 and 5 therein.

[0028] The antibody polypeptides of the present invention are based on selected humanized versions of the 11B6 antibody, which exhibit unexpected favorable properties.

[0029] In particular, the humanized antibodies of the present invention exhibit enhanced therapeutic ratios compared to the murine parent 11B6 antibody (m11B6) from which the CDR sequences are derived (see Example 6).

[0030] By "enhanced therapeutic ratio" is meant that the antibody polypeptide of the present invention (humanized form of the 11B6 antibody), when administered to a patient with a prostate tumor, results in a higher ratio of tumor absorbed dose to (healthy) bone marrow absorbed dose than the parent murine 11B6 antibody (compared at the same radioactivity and route of administration). The tumor to bone marrow absorbed dose ratio can be calculated using the method described in Example 6.

[0031] The unexpectedly favorable therapeutic profile of the antibodies of the present invention allows for higher radiation doses (absorbed doses). This allows for the use of ribozymes that can be used in combination with other drugs, resulting in greater efficacy in the treatment of prostate cancer, without increasing side effects or "collateral damage" to healthy tissue and organs.

[0032] Humanization (also called reconstitution or CDR grafting) is a technique for reducing the immunogenicity of monoclonal antibodies from xenogeneic sources (generally from rodents such as mice) and improving their activation by the human immune system (see review by Almagro and Fransson, 2008, Frontiers in Bioscience 13:1619-1633, the disclosure of which is incorporated herein by reference). A number of humanized monoclonal antibodies are in clinical trials, and several have been approved for use as drugs. The techniques for producing engineered monoclonal antibodies using molecular biology techniques are relatively straightforward. However, simple grafting of rodent complementarity determining regions (CDRs) onto a human framework does not necessarily reconstitute the binding affinity and specificity of the original monoclonal antibody. To humanize an antibody, the design of the humanized antibody is an important step in reproducing the function of the original molecule.

[0033] The design of a humanized antibody involves several key choices, including the extent of the CDRs to be used and the human framework to be used. However, to retain the specificity of the parent antibody, it may also be important to substitute one or more residues from the rodent mAb into the human framework regions (so-called backmutations). Identifying the positions of the necessary backmutations requires detailed sequence / structure analysis. In recent years, phage libraries have been used to vary amino acids at selected positions. Similarly, many approaches have been used to select the most suitable human frameworks into which to graft the rodent CDRs. Early experiments used a limited subset of well-characterized human monoclonal antibodies (often for which structures were available) without regard to sequence identity to the rodent monoclonal antibodies (the so-called fixed framework approach). Some groups use variable regions with high amino acid sequence identity (homology matching or best fit) to the rodent variable regions; others use consensus or germline sequences, while still others select fragments of the framework sequences of each light or heavy chain variable region from several different human monoclonal antibodies. Humanization techniques have also been developed that replace rodent surface residues with the most common residues found in human monoclonal antibodies ("resurfacing" or "veneering") and use differently defined CDR regions.

[0034] However, despite extensive research into antibody humanization, some rodent monoclonal antibodies have proven difficult to humanize.

[0035] The development of the antibody polypeptide of the present invention required backmutations not only in the framework regions but also in some of the CDRs (see Example 1 below). Thus, the six CDR sequences represented above in SEQ ID NOs: 1-6 are derived from the murine anti-hK2 antibody 11B6, but contain mutations in CDRH2 (SEQ ID NO: 2) and CDRL1 (SEQ ID NO: 4) compared to the murine parent antibody. These mutations in the CDRs were made to obtain optimal specificity and stability in the humanized version of 11B6.

[0036] In one embodiment, the antibody polypeptide of the invention is administered in a concentration of 0.1×10 -9 K is bigger than M D It binds to hK2.

[0037] The overall affinity (K D Methods for measuring the kd and kk as well as the association rate (ka) and dissociation rate (kd) are well known to those skilled in the art. Exemplary in vitro methods are described in Example 3 below. It is also contemplated that flow cytometry-based methods can be used (Sklar et al., 2002, Annu R ev Biophys Biomol Struct, 31:97-119; the disclosure of which is incorporated herein by reference).

[0038] Advantageously, the antibody polypeptide of the present invention has an antibody activity of 1.0×10 -10 Affinity (K D ), e.g. 9.0x10 -11 M, 8.0x10 -11 M, 7.0x10 -11 M, 6.0x10 -11 M, 5.0x10 -11 M, 4.0x10 -11 M, 3.0x10 -11 M, 2.0x10 -11 Less than M or 1.0x10 -11 K under M D has.

[0039] The antibody polypeptide of the present invention is an antibody heavy chain, an antibody light chain, an antibody heavy and / or light chain homopolymer. It will be appreciated by those skilled in the art that monodimers and heterodimers, as well as antigen-binding fragments and derivatives thereof, can be constructed.

[0040] In one embodiment, the antibody polypeptide comprises or consists of a complete antibody, such as an IgA, IgD, IgE, IgG or IgM molecule.

[0041] Advantageously, the antibody polypeptide comprises or consists of an intact IgG molecule or an antigen-binding fragment or derivative thereof.

[0042] The IgG molecule may be of any known subtype, for example IgG1, IgG2, IgG3 or IgG4.

[0043] "Antigen-binding fragments and derivatives" of antibodies include Fv fragments (e.g., single chain Fv and disulfide-linked Fv), Fab-like fragments (e.g., Fab fragments, Fab' fragments and F(ab)2 fragments) and domain antibodies (e.g., single V H Variable Domain or V L variable domain).

[0044] For example, an antibody polypeptide may comprise or consist of an scFv or Fab fragment.

[0045] A further characterizing feature of the antibody polypeptides of the present invention is the presence of framework amino acid sequences derived from one or more human antibodies in the heavy and light chain variable regions.

[0046] "Framework sequences" include the regions of the heavy and light chain variable domains other than the CDRs. Generally, each variable domain will contain four framework regions, designated FR1 to FR4, within which the CDR sequences are located: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0047] It goes without saying that the amino acid sequences of the framework regions may be fully human sequences or may contain one or more back mutations (i.e., amino acid sequences present in the human framework may be replaced by amino acids found at the corresponding positions in the rodent variable domain from which the CDRs are derived). Consequently, the sequences of FR1, FR2, FR3 and / or FR4 of the heavy and / or light chain variable domains of the antibody polypeptides of the invention may be non-naturally occurring sequences.

[0048] In one embodiment, the framework sequences of the antibody polypeptides have at least 70%, e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to framework regions derived from one or more human antibodies. Thus, an antibody polypeptide can include a heavy chain FR1 region that shares at least 70% sequence identity with the FR1 region of a human antibody. However, it will be appreciated that the heavy and light chains of an antibody polypeptide can share sequence identity with framework regions of different human antibodies.

[0049] Percent identity can be calculated, for example, using the Expasy institutional site (http: / / www.ch.embnet.org / software / LALIGN_form.html). The LALIGN program [Huang and Miller, Adv. Appl. Math. (1991) 12:337-357] was used with the parameters global alignment option, score matrix BLOSUM62, starting gap penalty -14, An extended gap penalty of -4 may be used to determine the percent sequence identity between two polypeptides. Alternatively, the percent sequence identity between two polypeptides may be determined using a suitable computer program, such as the GAP program of the University of Wisconsin Genetic Computing Group, and it will be appreciated that the percent identity is calculated relative to the polypeptides whose sequences are optimally aligned.

[0050] Alternatively, alignment can be performed using the Clustal W program (described in Thompson et al., 1994, Nucl. Acid Res. 22:4673-4680, incorporated herein by reference). The parameters used were as follows: Fast pairwise alignment parameters: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: Gap opening penalty; 10, Gap extension penalty; 0.05. · Score matrix: BLOSUM.

[0051] Alternatively, the BESTFIT program can be used to determine local sequence alignments.

[0052] In one embodiment, the framework sequences of the heavy chain variable domain of an antibody polypeptide of the invention are encoded by the human immunoglobulin VH4 gene family.

[0053] For example, the framework sequences are encoded, at least in part, by the VH4-28 germline gene (eg, FR1, FR2 and FR3 are encoded by VH4-28, and FR4 is encoded by JH1).

[0054] Thus, in one embodiment, the antibody polypeptide may comprise or consist of a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:8: QVQLQESGPGLVKPSDTLSLTCAVSGNSITSDYAWNWIRQPPGKGLEWIGYISYSGSTTYNPSLKSRVTMSRDTSKNQFSLKLSSVTAVDTAVYYCATGYYYGSGFWGQGTLVTVSS [SEQ ID NO:8]

[0055] In one embodiment, the framework sequences of the light chain variable domain of an antibody polypeptide of the invention are encoded by the human immunoglobulin kappa V4 gene family.

[0056] For example, the framework sequences are encoded, at least in part, by the IgkV4-B3 germline gene (eg, FR1, FR2 and FR3 are encoded by IgkV4-B3, and FR4 is encoded by JK2).

[0057] Thus, in one embodiment, the antibody polypeptide may comprise or consist of a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 9: DIVLTQSPDSLAVSLGERATINCKASESVEYFGTSLMHWYQQKPGQPPKLLIYAASNRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTRKVPYTFGQGTKLEIK [SEQ ID NO: 9]

[0058] "At least a portion" includes that the framework sequence comprises at least 10 contiguous amino acids, e.g., at least 20 contiguous amino acids, encoded by the reference gene. One or more, but not all, of the FR region may be encoded by the reference gene. (e.g., FR1 and FR2 are encoded in the reference gene, but FR3 is not).

[0059] In a preferred embodiment, the antibody polypeptide comprises a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:8, and a light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:9.

[0060] Optionally, the antibody polypeptide of the present invention further comprises a heavy chain constant region or a portion thereof.

[0061] In one embodiment, the antibody polypeptide comprises the CH1, CH2 and / or CH3 regions of an IgG heavy chain (such as an IgG1, IgG2, IgG3 or IgG4 heavy chain). Thus, the antibody polypeptide can comprise part or all of the constant region from an IgG1 heavy chain. For example, the antibody polypeptide can be a Fab fragment comprising the CH1 and CL constant regions.

[0062] In one embodiment, the antibody polypeptide can comprise an antibody Fc region. Those skilled in the art will recognize that the Fc portion is derived from an IgG antibody or from an antibody of a different class (such as IgM, IgA, IgD or IgE). In one embodiment, the Fc region is derived from an IgG1, IgG2, IgG3 or IgG4 antibody.

[0063] The Fc region may be naturally occurring (e.g., part of an endogenously produced antibody) or artificial (e.g., containing one or more point mutations and / or modifications to the carbohydrate moiety in the CH2 domain compared to a naturally occurring Fc region). Fc regions with point mutations that improve their ability to bind to FcRs may be advantageous, for example, by altering serum half-life or by altering (i.e., enhancing or decreasing) binding to Fcγ receptors (FcγRs) that are involved in ADCC and CDC.

[0064] Advantageously, the antibody polypeptide may comprise the amino acid sequence of SEQ ID NO: 10, or a part thereof: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 10]

[0065] Optionally, the antibody polypeptide of the present invention further comprises a light chain constant region or a portion thereof.

[0066] In one embodiment, the antibody polypeptide comprises a CL region of an IgG light chain (such as a kappa or lambda light chain).

[0067] For example, the antibody polypeptide can include the amino acid sequence of SEQ ID NO:11, or a portion thereof: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 11]

[0068] Advantageously, the antibody polypeptide comprises a heavy chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO:10, and a light chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO:11.

[0069] In a preferred embodiment, the antibody polypeptide of the present invention comprises: (a) a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 (variable regions in bold and CDR sequences in boxed italics) [ka] and / or

[0070] (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 (variable regions in bold and CDR sequences in boxed italics) [ka] Includes.

[0071] For example, an antibody polypeptide may comprise or consist of two heavy chains of SEQ ID NO: 12 and two light chains of SEQ ID NO: 13, which are linked together by disulfide bridges to form a typical IgG antibody structure.

[0072] The antibody polypeptides of the invention may comprise or consist of one or more amino acids which are modified or derivatized.

[0073] Chemical derivatives of one or more amino acids can be achieved by reaction with a functional side group. Such derivatized molecules include, for example, molecules in which free amino groups are derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, or formyl groups. Free carboxy groups can be derivatized to form salts, methyl and ethyl esters or other types of esters, and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include peptides that contain naturally occurring amino acid derivatives of the 20 standard amino acids. For example: 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine and ornithine can be substituted for lysine. Derivatives also include peptides containing one or more additions or deletions, so long as the requisite activity is maintained. Other modifications included are terminal modifications such as amidation, amino terminal acylation (eg, acetylation or thioglycolic acid amidation), terminal carboxylamidation (eg, with ammonia or methylamine), and the like.

[0074] Those skilled in the art will further recognize that peptidomimetic compounds are also useful. The term "peptidomimetic" refers to a compound that mimics the conformation and desirable characteristics of a particular peptide as a therapeutic agent.

[0075] For example, the polypeptide includes molecules in which amino acid residues are joined by peptide bonds (-CO-NH-) as well as molecules in which the peptide bonds are reversed. Such retro-inverso peptidomimetics are described, for example, in Meziere et al. (1997) J. Am. Soc. The antibodies can be made using methods known in the art, such as those described in .Immunol. 159, pp. 3230-3237, which is incorporated herein by reference. This method involves creating pseudopeptides that contain backbone changes that do not change the orientation of the side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. The polypeptide may also be a peptidomimetic compound, in which one or more of the amino acid residues are linked by a -y(CH2NH)- bond in place of a conventional amide bond.

[0076] Further alternatively, a peptide bond may be dispensed with entirely if a suitable linker moiety is used which maintains the spacing between the carbon atoms of the amino acid residues; it will be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond.

[0077] It goes without saying that, in order to reduce susceptibility to exoproteolytic digestion, the said polypeptide can advantageously be blocked at its N- or C-terminus.

[0078] Mammalian peptides have also been modified using a variety of uncoded or modified amino acids, such as D-amino acids and N-methyl amino acids. In addition, the putative bioactive conformation can be stabilized by covalent modifications such as cyclization or by the incorporation of lactam or other types of bridges, see, e.g., Veber et al., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem. Biophys. Res. Comm. 111:166, incorporated herein by reference.

[0079] Those skilled in the art will recognize that the antibody polypeptides of the present invention can be extended with functional moieties to facilitate their intended use, for example, as in vivo imaging or therapeutic agents.

[0080] Thus, in one embodiment, the antibody polypeptide is linked, directly or indirectly, to a therapeutic moiety.

[0081] Any suitable therapeutic moiety can be used. Suitable therapeutic moieties are those capable of reducing or inhibiting the growth, and in particular killing, of prostate cancer cells. For example, the therapeutic agent can be a cytotoxic moiety. The cytotoxic moiety can comprise or consist of one or more radioisotopes. For example, the one or more radioisotopes can be independently selected from the group consisting of beta emitters, Auger emitters, conversion electron emitters, alpha emitters, and low photon energy emitters. It is desirable that the one or more radioisotopes independently have a radiation pattern of locally absorbed energy that results in a high absorbed dose in the vicinity of the agent. Exemplary radioisotopes include: 90 Y, 32 P, 186 Re / 188 long-range beta emitters such as Re; 131 I, 177 Lu, 67 Cu, 161 Tb, 105 intermediate-range beta emitters such as Rh; 166 Ho, 76 As / 77 As, 89 Sr, 153 Sm; 45 Ca or 35 low energy beta emitters such as S; 51 Cr, 67 Ga, 99 Tc m , 111 In, 114 mIn, 123 I, 125 I, 201 conversion or Auger emitters such as T; and 212 Bi, 213 Bi, 223 Ac, 225 Ac, 212 Pb, 255 Fm, 223 Ra, 149 Tb and 221 Other radionuclides are available and may be used in therapy.

[0082] In another embodiment, it is desirable that the therapeutic or cytotoxic moiety is not a moiety disclosed as a "tracer" in WO2006 / 087374A1, particularly on page 11, lines 7-15.

[0083] In a preferred embodiment, the antibody polypeptide is radioisotope- 177 Connected to Lu (otherwise it will be marked).

[0084] Alternatively, the therapeutic moiety may comprise or consist of one or more therapeutic agents (such as cytotoxins), e.g., cytostatics; antiandrogens; cortisone and its derivatives; phosphonates; testosterone-5-α-reductase inhibitors; boron addends; cytokines; thapsigargin and its metabolites; toxins (such as saporin or calicheamicin); chemotherapeutic agents (such as antimetabolites); or any other therapeutic or cytotoxic agent useful in the treatment of prostate cancer.

[0085] Exemplary therapeutic / cytotoxic drugs include, for example: Cytostatics, especially those with dose-limiting side effects, including but not limited to cyclophosphamide, chlorambucil, ifosfamide, busulfan, lomustine, taxanes, estramustine phosphate and other nitrogen mustards, antibiotics (including doxorubicin, calicheamicin and esperamicin), vinca alkaloids, aziridines, platinum-containing compounds, endostatin, alkylsulfonates, nitrosoureas, triazenes, folic acid analogues, pyrimidine analogues, purine analogues, enzymes, substituted ureas, methylhydrazine derivatives, daunorubicin, amphipathic amines Antiandrogens such as flutamide and bicalutamide and their metabolites; Cortisone and its derivatives; Phosphonates such as diphosphonates and bisphosphonates; · Testosterone-5-alpha-reductase inhibitors; ·Boron adducts; Cytokines; Thapsigargin and its metabolites; Other drugs used to treat prostate cancer may include.

[0086] Alternatively, the cytotoxic moiety may comprise or consist of one or more moieties suitable for use in activation therapy, such as photon activation therapy, neutron activation therapy, neutron induced Auger electron beam therapy, synchrotron radiation therapy or low energy X-ray photon activation therapy.

[0087] For example, the antibody polypeptides of the present invention offer the possibility of using synchrotron radiation (or low-energy X-rays) to enhance radiation therapy, which is mainly focused on so-called photoactivated radiotherapy (PAT), in which the local energy deposition from external X-ray irradiation is enhanced in cancer tissue by interaction with a pre-administered high-Z tumor-targeting agent.

[0088] PAT therapy utilizes monochromatic X-rays from synchrotron sources, such as those provided by the ID17 biomedical beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, and those expected to become available in the future at other facilities, such as the new Swedish synchrotron facility, Max-IV.

[0089] A further potential treatment, "stimulated Auger electron tumour therapy", will be investigated at the upcoming European Spallation Neutron Source (ESS) at RAND and hopefully at a medical experimental station. Thermal and sub-thermal neutrons produced in the reactor will be useful in boron neutron capture therapy, BNCT, both pre-clinical experiments and brain tumour treatment using stimulated alpha particles, as well as the use of recoil nuclei ( 7L) has long been used. A similar approach is to use suitable tumor targeting molecules labeled with neutrons and stable nuclei with high cross sections for neutrons. Antibodies or peptides can be labeled with stable gadolinium ( 157 Gd) This antibody or peptide can be used as a target molecule for neutrons captured by Gd-nuclei. This is the so-called gadolinium neutron capture therapy (GdNCT). By Monte Carlo techniques, the dose distribution in the tumor and surrounding tissues is calculated as it is due to γ-photons, neutrons, nuclear recoils, and characteristic X-rays, internal conversion and Auger electrons from gadolinium or other potential elements.

[0090] As mentioned above, the therapeutic moiety (such as a radioisotope, a cytotoxic moiety, etc.) can be directly or indirectly linked to the binding moiety (such as an antibody or a fragment thereof). Suitable linkers are known to those skilled in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succinimidyl benzoate; dodecaborate), chelating moieties, both macrocyclic and acyclic chelators, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, and the like. conductor (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11 tetraacetic acid (TETA), derivatives of 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9 triacetic acid (PCTA), derivatives of 5-S-(4-aminobenzyl)-1-oxa-4,7,10 triazacyclododecane-4,7,10 tris(acetic acid) (DO3A), as well as other chelating moieties. The use of such linkers may be particularly appropriate in situations where the agent comprises or consists of an antibody or a fragment thereof as the binding moiety linked via the linker to the therapeutic moiety, a radioisotope.

[0091] One preferred linker is, for example 177 Lu-DTPA - the DTPA used in the antibody polypeptide of the present invention.

[0092] Further preferred linkers are e.g. 89 Zr-DFO - Deferoxamine, DFO, used in the antibody polypeptide of the invention.

[0093] Optionally, the antibody polypeptide of the present invention can further comprise (or not comprise) a detectable moiety. For example, the detectable moiety can be 99m Tc, 111 In, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I and 201 Optionally, the agent may comprise or consist of a radioisotope, such as a radioisotope selected from the group consisting of Tl. 86 Y / 90 Y or 124 I / 211 Alternatively, the agent may contain a radioisotope capable of acting simultaneously in a multimodal manner as a detectable moiety and as a cytotoxic moiety to provide so-called "multimodal theragnostics." Thus, the binding moieties can be coupled to nanoparticles that have the capability of multiplex imaging (e.g., SPECT, PET, MRI, optical, or ultrasound) along with the therapeutic capability using cytotoxic drugs such as radionuclides or chemotherapeutic agents. The possibility of treatment by hyperthermia using high frequency alternating magnetic fields and associated ultrasound imaging is also included in the present invention.

[0094] Alternatively, the detectable moiety may comprise or consist of a paramagnetic isotope, such as157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 The metal is selected from the group consisting of Fe.

[0095] In the case where the antibody polypeptide comprises a detectable moiety, then the detectable moiety is detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasound imaging.

[0096] Therapeutic and detectable moieties can be conjugated or otherwise combined with the antibody polypeptides using methods well known in the art {e.g., an existing immunoconjugate therapy, gemtuzumab ozogamicin (trade name: Mylotarg®), comprises a monoclonal antibody linked to the cytotoxin calicheamicin}.

[0097] In a further embodiment, the antibody polypeptide of the present invention is used to treat prostate cancer in the form of a formulation comprising a population of antibody polypeptide molecules. In one option, all (or substantially all, such as more than 90%, 95%, 99%, 99.9% or more by weight) of the antibody polypeptide molecules in the population contain the same therapeutic moiety. In another option, the population contains a mixture of other agents with different therapeutic moieties. This option provides the possibility to enhance the effect of targeted radionuclide therapy using various agents, such as chemotherapeutic agents, hormonal therapy agents or other combinations of therapies, in which the targeted agents not only deliver therapeutically active radionuclides to tumor-associated antigens, but also radiosensitize the targeted tumor cells by simultaneously altering (e.g., triggering or blocking) intracellular signaling cascades. This option is also useful for the treatment of prostate cancer using a mixture of cytotoxic agents, such as a cocktail of alpha emitters and beta emitters with different ranges, or a cocktail of radionuclides with different ranges, LET (linear energy transfer) and RBE (relative biological effectiveness), for the combined treatment of large tumors, micrometastases as well as single tumor cells. In one embodiment, long-range emitters can be used to treat large tumors and short-range emitters can be used to treat smaller tumors, such as micrometastases and single tumor cells.

[0098] Optionally, the antibody polypeptide of the present invention can further comprise (or not comprise) a moiety for increasing the in vivo half-life of the drug.Typical moieties for increasing the in vivo half-life of the drug include polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran.PEG is particularly contemplated.

[0099] It is understood that the polypeptide of the present invention can be lyophilized and stored, for example, by freeze-drying, spray-drying, spray-cooling, or by using particle formation (precipitation) from supercritical carbon dioxide, and then redissolved in a suitable carrier before use. Any suitable freeze-drying method (e.g., freeze-drying, spray-drying, cake-drying) and / or redissolving technique can be utilized. Those skilled in the art will recognize that freeze-drying and redissolving may result in varying degrees of activity loss, and the use level must be adjusted upward to compensate. Preferably, the lyophilized (freeze-dried) polypeptide loses only about 1% of its (pre-lyophilized) activity when rehydrated, or only about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or only about 50% of its (pre-lyophilized) activity when rehydrated.

[0100] The method for producing the polypeptide of the present invention is well known to those skilled in the art. Advantageously, the polypeptide is or comprises a recombinant polypeptide. Suitable methods for producing such recombinant polypeptides, such as expression in prokaryotic or eukaryotic host cells, are well known to those skilled in the art (see, for example, Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor, New York, the relevant disclosures in the document are incorporated herein by reference).

[0101] The antibody polypeptides of the present invention can also be produced using commercially available in vitro translation systems such as rabbit reticulocyte lysate or wheat germ lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system is Coupling the translation system to a transcription system such as the TNT transcription translation system (Promega) This system has the advantage of producing the appropriate mRNA transcript from the encoding DNA polynucleotide in the same reaction as translation.

[0102] Alternatively, those skilled in the art will recognize that the polypeptides of the present invention can be artificially synthesized using, for example, well-known liquid or solid phase synthesis techniques (such as t-Boc or Fmoc solid phase peptide synthesis).

[0103] A second aspect of the invention provides an isolated nucleic acid molecule encoding an antibody polypeptide of the invention, or a polypeptide chain component thereof. "Nucleic acid molecule" includes DNA (e.g., genomic or complementary DNA) and mRNA molecules, which may be single-stranded or double-stranded.

[0104] In one embodiment, the nucleic acid molecule is a cDNA molecule.

[0105] Those skilled in the art will recognize that nucleic acid molecules can be codon optimized for expression of an antibody polypeptide in a particular host cell, e.g., expression in a human cell [see, e.g., Angov, 2011, Biotechnol. J. 6(6):650-659].

[0106] In a preferred embodiment, the nucleic acid molecule of the invention comprises (a) the nucleotide sequence of SEQ ID NO: 14 [ka] and / or

[0107] (b) the nucleotide sequence of SEQ ID NO: 15 [ka] Includes.

[0108] Also within the scope of this invention: (a) A third aspect of the invention provides a vector (such as an expression vector) comprising a nucleic acid molecule according to the second aspect of the invention; (b) a fourth aspect of the invention provides a host cell (such as a mammalian cell, e.g. a human cell) comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention; and (c) A fifth aspect of the invention provides a method of producing an antibody polypeptide according to the first aspect of the invention, comprising culturing a population of host cells according to the fourth aspect of the invention under conditions such that the polypeptide is expressed, and isolating the polypeptide therefrom.

[0109] A sixth aspect of the invention provides a pharmaceutical composition comprising a medicamentously effective amount of an antibody polypeptide of the first aspect of the invention and a pharma- ceutically acceptable diluent, carrier or excipient.

[0110] Additional compounds may also be included in the pharmaceutical composition, including chelating agents such as EDTA, citric acid, EGTA, or glutathione.

[0111] The pharmaceutical compositions can be prepared in a manner known to those skilled in the art that is sufficiently storage stable and suitable for administration to humans and animals. For example, the pharmaceutical compositions can be lyophilized, for example, by freeze drying, spray drying, spray cooling, or by using particle formation from supercritical particle formation.

[0112] "Pharmaceutically acceptable" refers to a non-toxic material that does not reduce the effectiveness of the kallikrein protein binding activity of the agent of the present invention. Such pharma-ceutically acceptable buffers, carriers, or excipients are well known in the art [see Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rd Edition, A. Kibbe, ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference].

[0113] The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, A MPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

[0114] The term "diluent" refers to an aqueous or non-aqueous solution intended for diluting a drug in a pharmaceutical formulation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0115] The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the agent of the present invention.The adjuvant may be one or more of zinc, copper or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartarate, and acetate of different acyl composition.The adjuvant may also be cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic polymers such as cationic dendrimers, cationic synthetic polymers such as poly(vinylimidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0116] The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol and cyclodextrin, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginic acid, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinylpyrrolidone, all of which have different molecular weights, which are added to the composition, for example, to achieve viscosity control, bioadhesion, or to protect lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di- and triglycerides, ceramides, sphingolipids and glycolipids, all of which vary in acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those of the polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of fluid storage or advantageous pigment properties.

[0117] The antibody polypeptides of the present invention can be formulated into any type of pharmaceutical composition known to those of skill in the art for suitable delivery thereof.

[0118] In one embodiment, the pharmaceutical composition of the present invention may be in the form of liposomes in which the antibody polypeptide is combined with an amphiphilic agent such as lipids, in addition to other pharma- ceutically acceptable carriers, and the liposomes exist in aggregated form as micelles, insoluble monolayers, and liquid crystals.Suitable lipids for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like.Suitable lipids include the above lipids whose polar head groups are modified with poly(ethylene glycol) to extend bloodstream circulation time.Preparation of such liposomal formulations can be found, for example, in U.S. Pat. No. 4,235,871, the disclosure of which is incorporated herein by reference.

[0119] The pharmaceutical composition of the present invention can also be in the form of biodegradable microspheres.Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres.The preparation of such microspheres can be found in U.S. Pat. No. 5,851,451 and European Patent No. 0 213 303, the disclosures of which are incorporated herein by reference.

[0120] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, in which polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginic acid, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polyvinylimidazole, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate of different degrees of hydrolysis, and polyvinylpyrrolidone are used to thicken the solution containing the drug. The polymer may also include gelatin or collagen.

[0121] Alternatively, the antibody polypeptides can simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil), tragacanth gum, and / or various buffers.

[0122] It goes without saying that the pharmaceutical compositions of the present invention may contain ions and a defined pH to enhance the action of the active antibody polypeptide. In addition, the compositions may be subjected to conventional pharmaceutical processes such as sterilization and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0123] The pharmaceutical composition according to the present invention can be administered by any suitable route known to those skilled in the art.Thus, possible routes of administration include parenteral (intravenous, subcutaneous, intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral, and rectal.Administration via implants is also possible.Due to the potentially high cytotoxicity of the administered drug, infusion may be a desirable route.

[0124] In one embodiment, the pharmaceutical compositions are administered parenterally, for example, intravenously, intraventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intracisternally, intracranially, intramuscularly or subcutaneously, or they can be administered by infusion techniques. They are conveniently used in the form of a sterile aqueous solution which may contain other substances, for example, sufficient salts or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be suitably buffered (e.g., to a pH of 3 to 9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0125] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes that render the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. Preparations may be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0126] Thus, the pharmaceutical compositions of the present invention are particularly suitable for parenteral, eg, intravenous, or local administration to a tumour (eg, intratumoural or peritumor) in a patient.

[0127] The pharmaceutical compositions will be administered to a patient at a medicamentously effective dose, ie, a therapeutically effective absorbed dose of the therapeutic radionuclide.

[0128] In the context of therapeutic use of the antibody polypeptide of the present invention, a "pharmaceutical effective amount", or an "effective amount", or a "therapeutically effective" as used herein refers to an amount that provides a therapeutic effect for a given condition and administration treatment regimen. This is a predetermined amount of active material calculated to produce a desired therapeutic effect in association with the required additives and diluents, i.e., carriers or administration vehicles. It is further intended to mean an amount sufficient to reduce and / or prevent clinically significant impairments in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to produce an improvement in a clinically significant condition in the host. As recognized by those skilled in the art, the amount of a compound varies depending on its specific activity. A suitable dosage can contain a predetermined amount of active composition calculated to produce a desired therapeutic effect in association with the required diluents. In the method of manufacture and use of the composition of the present invention, a therapeutically effective amount of the active ingredient is provided. A therapeutically effective amount can be determined by an ordinarily skilled medical practitioner based on patient characteristics such as age, weight, sex, symptoms, comorbidities, other diseases, etc., as is well known to those skilled in the art (see Example 8 below). The administration of a medicamentously effective dose can be carried out both by single administration in the form of an individual dosage unit or several other smaller dosage units, and also by multiple administration of doses subdivided at specific intervals. Alternatively, the dose can be given by continuous infusion over a long period of time.

[0129] In the context of diagnostic uses of the antibody polypeptides of the invention, a "therapeutically effective amount," or "effective amount" or "diagnostically effective," as used herein, refers to an amount that provides a detectable signal for in vivo diagnostic imaging purposes.

[0130] The antibody polypeptide of the present invention can be formulated at various concentrations depending on the efficacy / toxicity of the compound used. The formulation can contain the polypeptide at concentrations of 0.1 μM to 1 mM, 1 μM to 500 μM, 500 μM to 1 mM, 300 μM to 700 μM, 1 μM to 100 μM, 100 μM to 200 μM, 200 μM to 300 μM, 300 μM to 400 μM, 400 μM to 500 μM, and about 500 μM.

[0131] Generally, the therapeutic dose of an antibody polypeptide (with or without a therapeutic moiety) in a human patient will be between 100 μg and 700 mg per administration (based on a body weight of 70 kg). For example, the maximum therapeutic dose may be between 0.1 and 10 mg / kg per administration, such as between 0.1 and 5 mg / kg or between 1 and 5 mg / kg or between 0.1 and 2 mg / kg. Of course, such doses can be administered at different intervals, as determined by the oncologist / physician; for example, doses can be administered daily, twice weekly, weekly, biweekly or monthly.

[0132] Those skilled in the art will recognize that the pharmaceutical compositions of the present invention can be administered alone or in combination with other therapeutic agents used in the treatment of prostate cancer, or before, after, or simultaneously with treating a patient with other therapeutic modalities for the treatment of prostate cancer, such as other therapeutic antibodies, surgery (e.g., radical prostatectomy), radionuclide therapy, brachytherapy, external beam radiation therapy, high intensity focused ultrasound (HIFU), chemotherapy, oral chemotherapy drugs, cryosurgery (freezing the tumor), hormone therapy (such as antiandrogen therapy), castration, or combinations of the foregoing.

[0133] A seventh aspect of the present invention relates to an antibody polypeptide according to the first aspect of the present invention or a polypeptide of the present invention. A kit is provided comprising the pharmaceutical composition according to the sixth aspect together with instructions for use as described herein.

[0134] An eighth aspect of the invention provides an antibody polypeptide according to the first aspect of the invention for use in medicine.

[0135] A ninth aspect of the invention provides an antibody polypeptide according to the first aspect of the invention for use in the treatment and / or diagnosis of prostate cancer.

[0136] A tenth aspect of the invention provides a method of treating prostate cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody polypeptide according to the first aspect of the invention.

[0137] "Treatment" includes therapeutic and prophylactic treatment of patients. The term "prophylactic" is used to include the use of a drug or its formulation, as described herein, which either prevents or reduces the likelihood of prostate cancer, or the spread, dissemination or metastasis of localized prostate cancer in a patient or subject. The term "prophylactic" also includes the use of a drug or its formulation, as described herein, to prevent the recurrence of prostate cancer in patients who have previously been treated for prostate cancer.

[0138] An eleventh aspect of the invention provides a method of diagnosing prostate cancer in a subject, the method comprising administering to the subject a diagnostically effective amount of an antibody polypeptide according to the first aspect of the invention.

[0139] "Diagnosis" includes the detection of prostate cancer cells either in vivo (ie, within the patient's body) or ex vivo (ie, in a tissue or cell sample removed from the patient's body).

[0140] The prostate cancer to be treated or diagnosed can be localized to the prostate gland, or can be non-localized (i.e., disseminated) prostate cancer. Prostate cancer that is localized to the prostate gland can be classified as, for example, clinical T1 or T2 cancer according to the TNM system (abbreviated as tumor / node / metastasis), while non-localized / disseminated prostate cancer can be classified as, for example, clinical T3 or T4 cancer.

[0141] The prostate cancer to be treated or diagnosed may be metastatic prostate cancer. Metastasis refers to the spread of cancer from its original location to other parts of the body. For example, the metastatic prostate cancer to be treated or diagnosed may be lymphatic system; metastasis present in bone (including spine, vertebrae, pelvis, ribs); pelvic, rectal, bladder, or urethral metastasis. Metastasis present in other less common locations may also be treated by the present invention. The metastasis may be micrometastasis. Micrometastasis is a form of metastasis where newly formed tumors are generally too small to be detected or are difficult to detect. For example, the present invention provides the skilled artisan with the means to treat single cancer cells or cell clusters, even if the presence of such cells or clusters is undiagnosable but present, e.g., very small amount of disseminated disease.

[0142] Thus, a particularly important technical advantage of the treatment provided by the present invention compared to prior art treatments for prostate cancer is expected to be enhanced efficacy in the treatment of disseminated and / or metastatic (including micrometastases) prostate cancer.

[0143] Thus, in one embodiment, the invention provides antibody polypeptides and methods for preventing or treating metastasis of primary prostate tumors.

[0144] Prostate cancer tends to develop in men over 50, more commonly over 60, 65 or 70, and is one of the most common types of cancer in men, but many men never have symptoms, never receive treatment, and eventually die from other causes. This is because cancer of the prostate is most often slow-growing and symptomless, and men who do have symptoms are elderly, so they often die from causes unrelated to prostate cancer, such as heart / circulatory disease, pneumonia, other unrelated cancers, or old age. About two-thirds of prostate cancer cases are slow-growing, while the other third are more aggressive and develop faster.

[0145] Thus, the development of effective methods for the treatment and diagnosis of prostate cancer is particularly important for the management of more aggressive and fast-growing forms of cancer, especially in younger patients.Thus, in one embodiment, the present invention relates to the treatment or diagnosis of prostate cancer in patients under 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40 years of age or younger at the time of diagnosis and / or treatment of prostate cancer.

[0146] Men with one first degree relative (father or brother) with prostate cancer are considered to have twice the risk of developing prostate cancer, and those with two affected first degree relatives are considered to have a five times greater risk compared to men with no family history. Thus, the present invention can relate to the treatment or diagnosis of prostate cancer in patients characterized by one, two or more relatives, particularly first degree relatives (such as father or brother), having been previously diagnosed with prostate cancer.

[0147] The present invention also relates to the treatment or diagnosis of prostate cancer in a patient, wherein the prostate cancer to be treated is castration-resistant prostate cancer (CRPC), which can be characterized by generally becoming intractable to hormone therapy after one to three years and resuming growth despite hormone therapy.

[0148] In the medical use and method of the present invention, antibody polypeptide is generally injected or infused into the patient's body.In vivo, antibody polypeptide then binds to the tissue that produces target antigen, hK2; first, prostate cancer cells and their metastases.Upon binding, antibody polypeptide can directly exert therapeutic effects (e.g., induce cell death by ADCC, CDC, or by carrying radioisotope or other cytotoxic moiety).Alternatively, bound antibody polypeptide can act as a diagnostic (imaging) tool, and can guide therapeutic options or assist in surgical removal of cancer cells.

[0149] Those skilled in the art will appreciate that the antibody polypeptides of the present invention may be used in conjunction with other therapeutic and / or diagnostic agents / treatments, such as external radiation therapy, surgery, cytostatic agents and androgen therapy.

[0150] The foregoing description has focused on embodiments of the invention that are applicable to methods of treating and diagnosing prostate cancer, however, it will be appreciated that the invention is not limited to such applications and is useful for post-operative monitoring, as well as monitoring during and following radiation, cytostatic and androgen therapy.

[0151] In another embodiment, radio-guided surgery (RGS) or image-guided surgery (IGS) can be used to identify the tracer-labeled antibody polypeptide of the present invention during and / or before surgery. Thus, an antibody polypeptide comprising the aforementioned detectable moiety can be administered during and / or before surgery. In this embodiment, the antibody polypeptide can be injected first. RGS / IGS can then be used to detect the antibody polypeptide during and / or before surgery. hK2-producing tissues can be identified by a detection instrument that is sensitive to the detectable moiety or before that. The detectable moiety can be, for example, a radioactive or magnetically sensitive detectable moiety; for example, a Cerenkov radiation emitter and / or a controlled radiation; a fluorescent label and / or a magnetic or magnetizable label. Thus, RGS / IGS according to the present invention can be, for example, a method based on the detection of optical, Cerenkov, controlled radiation or beta radiation; the detection of radionuclide labels, and / or includes magnetometry. RGS is well known to those skilled in the art as a surgical technique that allows surgeons to identify tissues that are "marked" by detectable moieties.

[0152] The visualization obtained by the above methods can be combined with other radiological visualization methods such as SPECT / PET, computed tomography (CT), ultrasound (US) and magnetic resonance imaging (MRI).

[0153] Thus, in a further aspect, the present invention also provides an antibody polypeptide for use in medicine by administration to a patient with prostate cancer prior to or during surgery, such as radiation-guided or image-guided surgery.

[0154] A still further aspect of the invention is an in vitro method for detecting prostate tumor cells in the blood of a subject comprising: (a) obtaining a sample of blood from a subject to be tested; (b) optionally extracting and / or purifying cells present in the blood sample; (c) contacting an antibody polypeptide according to the first aspect of the invention with cells present in the blood sample; (d) determining (directly or indirectly) whether the antibody polypeptide binds to free (i.e., uncomplexed) hK2; wherein binding of the antibody polypeptide to free hK2 indicates the presence of prostate tumor cells in the blood of the subject.

[0155] Thus, the method comprises the steps of performing an assay to determine whether a blood sample contains free hK2; the presence of free hK2 indicating the presence of prostate tumor cells in the subject's blood.

[0156] Those skilled in the art will appreciate that there are many ways to perform such assays. For example, immunoassays can be either homogeneous or, more preferably, heterogeneous. Assays can be performed in either a competitive or, more preferably, non-competitive format.

[0157] For a heterogeneous, non-competitive assay, a typical procedure is: (a) obtaining a sample of blood from a subject to be tested; (b) optionally extracting and / or purifying cells present in the blood sample; (c) contacting a solid phase immobilised antibody polypeptide according to the first aspect of the invention with cells present in the blood sample; (d) washing to remove soluble components (not bound to the solid surface); (e) adding a tracer, i.e., another anti-hK2 specific antibody labeled with a reporter molecule / particle; (f) washing to remove unbound tracer antibody; (g) detecting a signal from the tracer antibody; may be also possible.

[0158] Between steps b and c or c and d there should generally be an incubation period to allow the cells to produce soluble hK2 which can then be detected.

[0159] An additional aspect of the invention is an in vitro method for detecting prostate tumor cells in a tissue of a subject, comprising the steps of: (a) obtaining a tissue sample (such as a histological sample) from a subject to be tested; (b) optionally extracting and / or purifying cells present in the tissue sample; (c) contacting an antibody polypeptide according to the first aspect of the invention with cells present in the tissue sample; (d) determining (directly or indirectly) whether the antibody polypeptide binds to free (i.e., uncomplexed) hK2; wherein binding of the antibody polypeptide to free hK2 indicates the presence of prostate tumor cells in the tissue of the subject.

[0160] In one embodiment of the above in vitro method, step (d) is carried out by ELISA. However, any assay suitable for detecting antibody-antigen interactions in vitro can be used.

[0161] In an additional embodiment, the method further comprises quantifying prostate tumor cells in the sample.

[0162] In a further embodiment of the above in vitro method, the method is for diagnosing prostate cancer in a subject.

[0163] The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "more than one or more."

[0164] These and other embodiments of the invention will be better appreciated and understood when considered in conjunction with the above description and the accompanying drawings. It should be understood, however, that the above description, while indicating numerous various embodiments of the invention and specific details thereof, is given by way of illustration and not limitation. Numerous substitutions, modifications, additions and / or rearrangements can be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and / or rearrangements.

[0165] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0166] [Figure 1] FIG. 1 shows the heavy and light chain variable region sequences of an exemplary humanized 11B6 Fab fragment of the present invention. [Diagram 2] FIG. 1 shows an SDS-PAGE gel with native and reduced samples of mouse and humanized 11B6 antibodies. [Diagram 3] FIG. 13 shows the binding stages of the binding of 11B6 test antibody to hK2 on the chip. [Figure 4] FIG. 1 shows the dissociation stages of the 11B6 test antibody. [Diagram 5]FIG. 1 shows the biodistribution of 177Lu-labeled humanized 11B6 antibody. [Figure 6] FIG. 1 shows representative SPECT images demonstrating the binding of 177Lu-labeled h11B6 to mouse prostate tumors. [Figure 7] FIG. 1 shows the uptake rates of 177Lu-labeled h11B6 and m11B6 in tumors and bones. [Figure 8] FIG. 1 shows tumor to bone ratios of uptake per gram of 177Lu-labeled h11B6 and m11B6. [Figure 9A] FIG. 1 shows the kinetics of humanized 11B6 antibody. [Figure 9B] FIG. 1 shows the kinetics of the murine 11B6 antibody. [Figure 10] FIG. 1 shows the clearance of 177Lu-labeled h11B6 and m11B6 from blood. [Figure 11] Representative photographs of tumor size before (top image) and after (bottom image) treatment with 177Lu-11B6. [Figure 12A] FIG. 13 shows a single radioactive dose "D" of 177Lu-11B6 relative to tumor size in LNCaP xenografts. [Figure 12B] FIG. 13 shows the dual radioactivity dose "2xD" of 177Lu-11B6 relative to tumor size in LNCaP xenografts. [Figure 12C] FIG. 13 shows a summary of the effect of control treatments on tumor size in LNCaP xenografts. [Figure 13] FIG. 1 shows (a) tumor growth data and (b) SPECT images of one LNCaP xenograft mouse treated with a single dose of 177Lu-11B6. [Figure 14A]Figure 1 shows tumor volume as a function of days after injection in animals given 177Lu-labeled h11B6 antibody according to the present invention. Treatment was administered on day 0. Animals were terminated if the following events occurred: large tumor volume (diameter > 14 mm); large weight loss (weight loss > 15% compared to initial weight); general symptoms were adversely affected; or a combination of all three of these indicators occurred. (The numbers on the right are the ID numbers of each animal.) [Figure 14B] Tumor volume as a function of days after injection in animals that received 177Lu-labeled non-specific IgG "isotype control" antibody. Treatment was administered on day 0. Animals were terminated if the following events occurred: large tumor volume (diameter >14 mm); large weight loss (weight loss >15% compared to initial weight); adverse general symptoms; or a combination of all three of these indicators. (Numbers to the right are ID numbers for each animal) [Figure 14C] Tumor volume as a function of days after injection in animals receiving NaCl only. Treatment was administered on day 0. Animals were terminated if the following events occurred: large tumor volume (diameter >14 mm); large weight loss (weight loss >15% compared to initial weight); adverse general symptoms; or a combination of all three of these indicators. (Numbers to the right are ID numbers for each animal) [Figure 15] Figure 15 shows Kaplan-Meier curves for the three treatment groups shown in Figure 14. Solid line: 177Lu-h11B6; dashed line: 177Lu-labeled non-specific IgG "isotype control" antibody; dotted line: NaCl. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0167] The following examples are included to demonstrate specific embodiments of the invention. Those of skill in the art should recognize that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute specific modes for its practice. However, those of skill in the art should recognize in light of this disclosure that many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLES

[0168] Cloning of 11B6 from a hybridoma cell line reagent A monoclonal antibody 11B6 producing hybridoma cell line was used for mRNA extraction and antibody production, which was further affinity purified for protein sequencing (Vaisanen et al., 2004).

[0169] Restriction enzymes, FastAP and T4 DNA ligase were from Fermentas, primers were from the University of Turku Department of Biotechnology (WO252) and Thermo Scientific. DNA purification was performed with Qiagen gel extraction and PCR purification kits.

[0170] mRNA extraction and cDNA synthesis mRNA was extracted from 11B6 MAb-producing hybridoma cells (5E6 cells) using the QuickPrep Micro mRNA purification kit (Amersham Biosciences), and cDNA synthesis from the mRNA was performed using the High-capacity cDNA archive kit from Applied Biosystems according to the manufacturer's instructions.

[0171] Amplification of antibody genes from cDNA The N-terminal sequences of the heavy (H) and light (L) chains of the purified 11B6 MAb were determined by Edman degradation at the Protein Sequencing Service of the University of Helsinki. The light chain sequence was DIVLTQSPAS [SEQ ID NO: 16] and the heavy chain sequence was DVQLQESGPG [SEQ ID NO: 17]. The genes: IGKV3 and IGHV3, respectively, were identified by amino acid IMGT database comparison. The complementary regions of the forward PCR primers (degenerate) were designed based on the DNA sequence (found by NCBI BLAST) encoding the N-terminal amino acids. The reverse primer used to clone the heavy chain was C H1 For the light chain, two reverse primers were used; the one used in the first PCR was C L The other was used in the second PCR, V L and C. L All primers also contained the restriction enzyme recognition sites necessary for cloning (underlined below).

[0172] The light chain forward primer was SfiI_DIVLTQSPAS [SEQ ID NO: 16]: (5'-TTACTCGC GGCC CAGCC GGCC ATGGCGGAYATHGTRYTVACNCARTCTCC-3'; [SEQ ID NO: 18] and the reverse primer is (5'-GCGCCG TCTAG ATTAACACTCATTCCTGTTGAA-3', XbaI; [SEQ ID NO: 19]) and CpoI_JK2 (5'-GATACAGTTGGTGCAGCAT CGGTCCG TTTTATTTCCAGCTTGGTCCCCCCT-3'; [SEQ ID NO: 20] It was.

[0173] The heavy chain forward primer was NotI_DVQLQESGPG [SEQ ID NO: 17] (5'-TGCTGCTG GCGGCCGCTCCAGCCATGGCTGAYGTVCARCTKCAGGAGTCDGG-3'; [SEQ ID NO: 21] and the reverse primer was asCH1_SacI (5'-CGCCACCA GAGTC TCACAATCCCTGGGCACAATTTTC-3'; [sequence number 22]). It was.

[0174] V L +C L The fragment was amplified in a PCR reaction containing 100 ng of template cDNA, 0.2 mM dNTPs, 0.5 μM primers SfiI DIVLTQSPAS [SEQ ID NO: 16] and WO252, 1× Phusion HF buffer, and 0.6 U Phusion DNA polymerase (Finnzymes). Amplification was performed at 98° C. for 30 seconds, 30 cycles of 98° C. for 7 seconds, 50° C. for 20 seconds, 72° C. for 20 seconds, and a final extension at 72° C. for 10 minutes. PCR products were sequenced and V L -C L After elucidating the sequence of the borders, PCR for the actual cloning was performed. L The cDNA was again amplified in the same reaction as above, except for the primers SfiI_DIVLTQSPAS [SEQ ID NO: 16] and CpoI_JK2 for cloning only the portion. Amplification was performed using the following procedure: 98°C for 30 seconds, 10 cycles of 98°C for 7 seconds, 60°C for 20 seconds, 72°C for 20 seconds, 25 cycles of 98°C for 7 seconds, 56°C for 20 seconds, 72°C for 20 seconds, and a final extension at 72°C for 10 minutes.

[0175] V H +C H1 The fragment was amplified using primers NotI_DVQLQESGPG [SEQ ID NO: 17] and V L The amplification procedure was 98°C for 30 seconds, 30 cycles of 98°C for 7 seconds, 64°C for 20 seconds, and 72°C for 20 seconds, with a final extension at 72°C for 10 minutes.

[0176] Cloning The correct size product was purified from a preparative agarose gel. L , with SfiI and CpoI, H +C H1 was digested with SacI and NotI. The recipient vector pAK400 5404FAb lch (modified from pAK400, Krebber et al., 1997) was digested with both enzyme combinations separately and the fragments were dephosphorylated with FastAP and purified from a preparative gel.

[0177] Digested 11B6 V L and the corresponding vector fragment were ligated with T4 DNA ligase and transformed into E. coli XL1-Blue cells (Stratagene) by electroporation to generate vector pAK400-11B6-VL. H +C H1 The ligation product of the and vector fragment was named pAK400-11B6-VH+CH1. Correct clones were confirmed by DNA sequencing and the sequences were compared with the original protein sequences and antibodies found in databases (BLAST search).

[0178] To construct the complete 11B6 Fab, both of the previous constructs were digested with NotI and SacI. The vector pAK400-11B6-VL was fused with the VH+CH1 derived vector pAK400-11B6-VH+CH1. H +C H1 was used as the recipient vector for insertion. Ligation and transformation were performed as described above. The constructed pAK400 11B6 FAb lch vector was confirmed by restriction enzyme analysis.

[0179] References Barbas CF 3rd, Kang AS, Lerner RA, Benkovic SJ. (1991) Assembly of combinatorial antibody libraries on phage surfaces: The gene III site. Proc. Nat. Acad. Sci., Vol. 88, pp. 7978-7982 Biomagnetic Techniques in Molecular Biology: Technical handbook. Dynal A.S, 2 nd edition, 1995 Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Pluckthun A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 201(1):35-55 Lilja H, Christensson A, Dahlen U, Matikainen MT, Nilsson O, Pettersson K, Lovgr en T. (1991) Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin Chem. 37(9):1618-25 Pajunen M, Saviranta P, Jauria P, Karp M, Pettersson K, Mantsala P, Lovgren T. (1997) Cloning, sequencing, expression and characterization of three anti-estradiol-17beta Fab fragments. Biochim Biophys Acta. 1351(1-2):192-202 Vaisanen V, Eriksson S, Ivaska KK, Lilja H, Nurmi M, Pettersson K. (2004) Development of sensitive immunoassays for free and total human glandular kallikrein 2. Clin Chem. 50(9):1607-17 EXAMPLES

[0180] Humanization of 11B6 antibody The variable domains of the murine anti-hK2 antibody 11B6 were humanized using the CDR grafting method. In this approach, the complementarity determining regions (CDRs) of a murine antibody were grafted onto the variable heavy and light domain frameworks. In addition to the residues in the CDR regions, residues in certain key positions in the framework regions were kept murine-like rather than changed to human-like, to keep the conformation of the grafted CDR loops as similar as possible to their conformation in the murine parent antibody.

[0181] The Kabat numbering scheme (Kabat et al., 1991) has been used throughout this description.

[0182] Homology modelling A homology model of the murine 11B6 antibody was generated using the Automated Web Antibody Modeling Server (VAM; http: / / antibody.bath.ac.uk / index.html). The model was used to visually assess the importance of residues that differ between the parent murine antibody and the human immunoglobulin sequence used as a framework for humanization of the variable domains, respectively.

[0183] Design of 11B6 humanized V-domain sequences V L Domain Design The amino acid sequence of the mouse 11B6 light chain variable domain was compared to a database of human immunoglobulin germline sequences using the ClustalW sequence alignment program from NCBI. L is a human V κ4 It was found to share the highest similarity with the only other member of the family, the human germline gene B3 (IGKV4-1*01). With regard to the J-segment, which encodes the C-terminal part of the variable domain sequence, the human J κ2 was found to be most similar to the corresponding region of mouse 11B6.

[0184] Using the human B3 gene together with the sequence of IGKJ2 as a framework, we grafted the CDR loops from the light chain of the mouse parent antibody 11B6 (Figure 1). The residue of mouse origin (leucine) was replaced by V instead of the human-like methionine. L A human-like arginine was introduced at position 4 of CDR-L2. This Vernier zone (Foote and Winter, 1992) residue is located just below the CDR1 and CDR3 loops of the light chain. At position 54 of CDR-L2, a human-like arginine was used instead of the mouse-like valine. Modeling suggested that residues at this position would not form direct interactions with the antigen, but Arg54 was likely to form a salt bridge with the negatively charged aspartic acid at position 60 of the human framework. The residue at position 24 of CDR-L1 was not thought to be involved in antigen contacts. Consequently, a human-like lysine was introduced at this position instead of the mouse-like arginine.

[0185] V H Domain Design The amino acid sequence of the mouse 11B6 heavy chain variable domain was determined by the ClustalW sequence of NCBI. An alignment program was used to compare the sequence with a database of human immunoglobulin germline sequences. H is a human V H4 It was found to have the highest similarity to family member VH4-28. Regarding the J-segment, which encodes the C-terminal part of the variable domain sequence, human J H1was found to be most similar to the corresponding region of mouse 11B6.

[0186] J H1 The human VH4-28 gene was used as a framework together with the sequence of the CDR loops from the heavy chain of the murine parent antibody 11B6 (Figure 1).

[0187] The mouse-like residues asparagine and threonine are H The mouse 11B6-derived residues threonine and human VH4-28-like arginine were introduced at positions 27 and 30, respectively. Although they do not belong to CDR-H1 according to the Kabat definition (Kabat et al., 1991; Figure 1), they were classified as CDR residues by some other CDR definition procedures, such as the definition by Chothia (1989). Residues 27 and 30 could affect the structure of other parts of CDR-H1 and could be involved in direct contact with the antigen. The residue at position 71 is known to play an important role in maintaining the conformation of CDR-H2 (Tramontano et al., 1990), and a mouse-like arginine was used here instead of a human-like valine. At position 94, before the critical CDR-H3 loop, a mouse 11B6-derived residue threonine was introduced instead of a human VH4-28-like arginine. In addition, the residue at position 60 of CDR-H2 was not thought to be involved in antigen contact. Therefore, a human-like asparagine was introduced at this position instead of a mouse-like serine.

[0188] Designed V H and V L The domain is human C H1 and human C κ The genes encoding the humanized 11B6, Fab fragments each tethered to a constant domain, were purchased as synthetic constructs (Genscript, US). The genes were cloned into the expression vector pAK400Fab, modified from pAK400 (Krebber et al., 1997), using the SfiI restriction enzyme, which has recognition sites on either side of the Fab cassette. The vector was transformed into E. coli XL-1 blue cells to express the humanized Fab fragments.

[0189] The sequences of the heavy and light chain variable regions of an exemplary humanized 11B6 Fab fragment of the present invention are shown in FIG.

[0190] References Chothia, C., Lesk, AM, Tramontano, A., Levitt, M., Smith-Gill, SJ, Air, G., Sheriff, S., Padlan, EA, Davies, D., Tulip, WR, Colman, PM, Spinelli, S., Alzari, PM, and Poljak, RJ (1989) Conformations of immunoglobulin hypervariable regions Nature, 342, 877-883 Kabat, EA, Wu, TT, Perry, HM, Gottesman, K. S and Foeller, C. (1991) Sequences of Immunological Interest, 5 th editor., NIH, Bethesda, MD Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Pluckthun A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 201(1):35-55 Tramontano, A., Chothia, C. and Lesk, AM (1990) Framework Residue 71 is a Major Determinant of the Position and Conformation of the Second Hypervariable Region in the V H Domains of Immunoglobulins. J. Mol. Biol. 215, 175-182 EXAMPLES

[0191] Expression and purification of h11B6 HEK293 cells were cultured in FreeStyle 293 expression medium (Life Technologies, The cells were expanded into 2 L suspension cultures in 1000 sucrose transfection tubes (logies). The cell density was 1 × 10 6 The cell density was 100 cells / mL.

[0192] Nucleotide sequences encoding the heavy or light chain components (i.e., SEQ ID NO: 14 and 15, respectively) were codon-optimized for expression in mammalian cells, synthesized, and cloned into an IgG expression vector. Plasmid DNA (expression vector) containing the heavy and light chain nucleotide sequences was then mixed with transfection agent and incubated at RT for 10 minutes. The DNA-transfection agent mixture was slowly added to the cell culture while gently swirling the flask. The transfected cell culture was then incubated at 37°C, 8% CO2 for 7 days on an orbital shaker platform rotating at approximately 135 rpm.

[0193] The medium was harvested by centrifugation and filtered through 5 μm, 0.6 μm and 0.22 μm filter systems.

[0194] The antibody was purified by protein G chromatography and the buffer exchanged against PBS pH 7.4 by dialysis; the antibody was then concentrated by ultrafiltration.

[0195] The concentration was determined by absorbance. DNA: Light chain: p11B6VLhV1hk (4300bp) Quantity: 0.35mg Heavy chain: p11B6VHhV1hIgG1 (4900bp) Amount: 0.6mg

[0196] The amount of DNA was not optimized.

[0197] Transfection agents: Proprietary [however, suitable commercial transfection agents are readily available, such as Xfect™ Transfection Reagent (Clontech), Lipofectamine (Life Technologies), FuGENE® HD Transfection Reagent (Promega), FreeStyle™ Max Reagent (Invitrogen), DEAE dextran, polyethyleneimine and calcium phosphate].

[0198] Total yield: 13.1mg (about 6.5mg / L) EXAMPLES

[0199] Characterization of h11B6: affinity Research Objective The aim of this study was to probe the binding kinetics of four variants of antibody 11B6 with the antigen hK2 by using surface plasmon resonance (SPR) technology on a Biacore instrument.

[0200] To examine the quality of the protein samples (antibody and antigen), they were run on SDS-PAGE gels prior to the SPR experiments.

[0201] To find suitable conditions for the experiments in this study, different indicators were examined in a preliminary study.

[0202] In this study, multiplex binding measurements were performed for four antibodies and antigens. From the collected data, the association and dissociation rate constants (k on and k off ) and dissociation constant (KD ) were calculated and reported here.

[0203] Reagent and Instrument Information The following four antibody and one antigen solutions were obtained from Diaprost AB: m11B6 Stock: a-ehk211B6 14.12013 PP, 3.41mg / mL: 0.9% NaCl, 100μL h11B6 Stock: Innovagen Lot 90476.30 2013-04-12, 1mg / mL: PBS pH 7.4, 320μL h11B6-DTPA stock: 0.2M sodium acetate pH 5.5, 0.9mg / mL, 340μL h11B6-DFO stock: 5 mg / mL gentisic acid in 0.2 M ammonium acetate pH 5.5, 1.6 mg / mL, 400 μL hK2 stock: 26.6μg / mL frakt 2 fr 7 SL+ protein inh 5 / 2-02 1% BSA

[0204] All samples were aliquoted and kept in a −20°C freezer prior to analysis.

[0205] All binding experiments were performed on a CM4 chip on a Biacore3000 instrument. The chips and all reagents required for activation, immobilization, deactivation, binding and regeneration were supplied by GE. It was purchased from Healthcare and used according to the manufacturer's guidelines.

[0206] SDS-PAGE (a) Experimental description Reagents provided by Diaprost AB were run on a TRIS-Tricine 10-20% acrylamide gel from Novex according to the manufacturer's guidelines.

[0207] Two series of protein samples, native and reduced, were run simultaneously on the same gel along with standards.

[0208] Each sample in the native series contained the following: 1–1.3 μg protein, Tris buffer pH 8.8, SDS, and loading buffer.

[0209] Each sample in the reduction series contained the following: 1–1.3 μg protein, Tris buffer pH 8.8, SDS, loading buffer, and 0.04% vol / vol β2-mercaptoethanol (reducing agent).

[0210] Staining of the gel was performed with Coomassie brilliant blue solution of acetic acid, ethanol and water in proportions corresponding to 0.7, 3.0 and 6.3.

[0211] Destaining of the gel was carried out with a solution of acetic acid, ethanol and water in proportions corresponding to 0.7, 3.0 and 6.3.

[0212] (b) Results and conclusions The results are shown in Figure 2.

[0213] It was evident from these results that the antibody and antigen samples were of high quality and purity.

[0214] Affinity research (a) Immobilization of antigen onto a CM4 chip Activation of the CM4-2 chip was performed using an amine coupling assay using a mixture of EDC and NHS. The assay was performed according to the manufacturer's guidelines.

[0215] A solution containing 2.96 μg / mL antigen hK2 (stock solution of hK2 diluted in 10 mM NaAc buffer pH 3.8) was flowed over channels fc2-4 of the CM4-2 chip to immobilize the antigen on the chip. Flow rate: 5 μL / min, volume: 200 μL. Target Ru≦M W / 10M W(hk2)=25900Da Target Ru(hk2)≦2590

[0216] Channel fc1 was used as a blank.

[0217] The following fixations were achieved: fc2=1104RU fc3=731RU fc4=688RU

[0218] After activation and fixation, all channels (fc1-4) were blocked with ethanolamine.

[0219] These data demonstrated that adequate immobilization was achieved using 2.96 μg / mL of antigen.

[0220] (b) Examination of the binding step The binding phase of the four antibodies to the CM4-2 chip was tracked for 4-5 min when five different concentrations of each antibody solution (stock solution diluted in HSP-buffer) were flowed over channels fc2-4 of the CM4-2 chip at a rate of 30 µL / min.

[0221] The concentrations explored for each antibody were: 100, 50, 25, 12.5 and 6.25 nM.

[0222] In addition, binding data were obtained from experiments where the dissociation process was followed for 480 min.

[0223] In total, 18 separate binding experiments were performed for each antibody.

[0224] The signal from the blank, fc1, was subtracted for all data.

[0225] The binding phase in channel fc2 of the CM-2 chip for each of the five different concentrations of antibody is shown in FIG.

[0226] After 4–5 min, it was found that the binding process could be fitted to the data.

[0227] (c) Examination of the dissociation stage After flowing a 50 nM antibody solution over channels fc2-4 of the CM4-2 chip at a rate of 30 µL / min for 5 min, the dissociation phase was followed for 480 min for each of the antibodies (Figure 4).

[0228] In all data used to calculate the dissociation rate constants, the signal from the blank, fc1, was subtracted.

[0229] The data showed that the dissociation process was very slow: for all four antibodies, the signal in channel fc4 drifted and the dissociation process could not be tracked in that channel.

[0230] (d) Dissociation rate constant (k off ) Estimation The dissociation step data was fitted to estimate the dissociation rate constant (koff) (see Table 2).

[0231] [Table 1]

[0232] Based on two measurements taken for each antibody, the dissociation rate constants (k off ) did not appear to be significantly different.

[0233] (e) Binding rate constant (k on ) Estimation To estimate the association rate constants, the dissociation rate constants (Table 2) were used in the fitted equations.

[0234] All fitted data were used to calculate the mean binding rate constants and standard deviation for each antibody (see Table 3).

[0235] [Table 2]

[0236] Based on 15–18 measurements obtained for each antibody, the binding rate constants (k on ) did not appear to be significantly different.

[0237] (f) Dissociation constant (k D ) Estimation The dissociation constants (K D ) are shown in Table 4.

[0238] [Table 3]

[0239] Dissociation constant (K D ) was 10 for all four antibodies. -12 It was in the range of M.

[0240] Although not statistically significant, the dissociation constant for the humanized antibody appeared to be higher than that of the murine parent antibody.

[0241] K D was not significantly changed for h11B6-DTPA or h11B6-DFO, so conjugation of the humanized antibody did not appear to noticeably affect affinity.

[0242] overview The binding process is very fast for all four antibodies, with binding rate constants (k on ) are all 10 5 M -1 s -1 was in the range. The dissociation process was very slow, almost within the technical limits of the Biacore. The dissociation rate constant (k off ) are all based on two experiments for each antibody. -5 s -1 The range was. Dissociation constant (K D ) was 10 for all four antibodies. -12 It was in the range of M. EXAMPLES

[0243] Characterization of h11B6: Aggregation overview Dynamic light scattering (DLS) studies were performed on the four variants of IgG to study their tendency to aggregate. DLS results showed that all constructs had reasonable sizes (200 kDa or slightly above 200 kDa assuming globular proteins) and showed little or no aggregation.

[0244] the purpose To characterize the oligomeric state of the four IgG constructs using dynamic light scattering, insulin was used as a control.

[0245] result Dynamic Light Scattering Phosphate buffered saline (PBS pH 7.4) was filtered through a 0.22 micron filter. The resulting protein was diluted to 0.1 mg / mL in PBS pH 7.4. Dynamic light scattering was measured at 20° C. on duplicate samples using a Malvern APS instrument. Each sample was measured in triplicate. The dilution buffer was used as a control to ensure that the buffer was reasonably free of dust and aggregates (FIG. 1c). All samples could be reliably measured using number distribution functions. The average radius of the most abundant molecular species was calculated along with the polydispersity of the molecular species. The average mass distribution of this molecular species was also calculated, see Table 5.

[0246] [Table 4]

[0247] The insulin control (4 mg / mL 20 mM Na2HPO4, 10 mM Na3EDTA) had an average radius of 2.8 nm, which is about 37 kDa. In solution, insulin is known to form hexamers of about 35 kDa. A radius of 5.7 nm corresponds to a molecular weight of about 200 kDa for a protein with a perfectly spherical shape. A radius of 6.1 nm corresponds to a molecular weight of about 230 kDa for a protein with a perfectly spherical shape. This is fairly close to the molecular weight of 150 kDa of an IgG molecule, which means that most samples consist mainly of monomeric and / or dimeric IgG molecules. The reason for not excluding dimers is that light scattering gives a rough size estimate based on molecular shape, so it is difficult to separate monomers and dimers, but it is easy to separate monomers and large aggregates or monomers and hexamers (as in the case of insulin).

[0248] conclusion Dynamic light scattering showed that all constructs were of reasonable size with little or no aggregation. The size distributions of all four constructs overlapped (data not shown). EXAMPLES

[0249] Characterization of h11B6: in vivo biodistribution This study 177 The in vivo biodistribution of mouse 11B6 and human 11B6 when labeled with Lu was compared.

[0250] material and method material 177 Lu was purchased from Mallinkrodt Medical BV, Petten, Holland.

[0251] All chemicals were obtained from Sigma Aldrich and buffers were prepared in-house using analytical quality water (unless otherwise stated).

[0252] The mouse parent antibody m11B6 specific for human kallikrein 2 was obtained from the University of Turku, Finland.

[0253] m11B6 heavy chain [SEQ ID NO:23]: DVQLQESGPGLVKPSQSLSLTCTVTGNSITSDYAWNWIRQFPGNRLEWMGYISYSSGSTTYSPSLKSRFSITRDTSKNQFFLQLNSVTPEDTATYFCATGYYYGSGFWGQG TLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0254] m11B6 light chain [SEQ ID NO:24]: DIVLTQSPASLAVSLGQRATISCRASESVEYFGTSLMHWYRQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIQPVEEDDFSMYFCQQTRKVPYTFGGGTKLE IKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0255] The humanized counterpart antibody, h11B6, was produced as described above in Examples 2 and 3 (see FIG. 1).

[0256] For in vivo studies, the prostate cancer cell line LNCaP expressing hK2 (ATCC, Manassas, VA, USA) and DU145 (ATCC, Manassas, VA, USA) was used. Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and PEST (penicillin 100 IU / mL and streptomycin 100 μg / mL). Cells were maintained in a humidified incubator at 37°C with 5% CO2 and detached with trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Thermo Scientific). For xenografting of LNCaP cells, Matrigel matrix from BD-biosciences (San Jose, California, USA) was used. NMRI-Nu, (Charles River) and Balb / c-Nu (in-house bred) mice were implanted with the two cell lines.

[0257] Conjugation and radiolabeling Conjugation of CHX-A″-DTPA with 11B6: A solution of mouse and humanized 11B6 mAb in PBS was adjusted to pH 9.2 using 0.07 M sodium borate buffer before being concentrated with an Amicon Ultra-2 centrifugal filter (2 mL, 100K). The resulting protein solution was then conjugated with the chelator CHX-A″-DTPA (Macrocylics, USA) at 40° C. in a 3:1 molar ratio of chelator to antibody. The reaction was stopped after 4 h and CHX-A″-DTPA-11B6 (DTPA-11B6) was separated from the free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare), equilibrated with 20 mL of 0.2 M ammonium acetate buffer, pH 5.5. The conjugated 11B6 antibody was eluted with 1 mL of ammonium acetate buffer.

[0258] Radiolabeling of DTPA-11B6: Mouse and Humanized DTPA-11B6 was added to a given amount of 177 Mixed with LuCl3. 0.5 per subject A final activity of ~0.6MBq was used for biodistribution or 18-20MBq per subject for SPECT studies. After 2 hours of incubation at room temperature, labeling was stopped and purified on a NAP-5 column equilibrated with PBS.

[0259] Animal testing All animal experiments were performed in accordance with national legislation on the protection of laboratory animals.

[0260] Male immunodeficient nude mice, NMRI-Nu, (6-8 weeks old) and Balb / c-Nu, were used in this study. All mice were xenografted with LNCaP cells or DU145, 8 million to 10 million cells in 100 μL of growth medium and 100 μL of Matrigel, in the left or right flank.

[0261] Biodistribution study Biodistribution studies were performed with both h11B6 and m11B6.

[0262] Groups of six mice (n=4) were intravenously injected with either 20 μg h11B6 or 20 μg m11B6 labeled with 177Lu. Animals were sacrificed 24, 48, and 72 hours after injection and organs of interest were analyzed with an automated NaI(Tl) well-counter (1480 WIZARD, Wallac Oy, Turku, Finland) equipped with a 3-inch NaI(Tl) detector.

[0263] Tissue uptake values ​​expressed as percent of injected dose per gram of tissue (%IA / g) are: %IA / g = (tissue radioactivity / injected radioactivity) / organ weight x 100 where, for IV injection: Injected radioactivity = average radioactivity in control syringe - radioactivity in used syringe - radioactivity in tail It was decided.

[0264] Organs were also weighed after dissection.

[0265] Kinetic Data Time-%ID curves were expressed as a straight line, ID(t) = k*t + m, up to 48 h. Based on the data from the second and third time points (48 and 72 h, respectively), a monoexponential curve [ID(t) = ID(0)e -λt ] was applied to the time interval [48,∞]. However, when lambda becomes negative, i.e., the ID increases between 48 and 72 hours, the time-ID curve for this time interval was instead modeled as a straight line, and the drug was assumed to be retained in the organs beyond 72 hours. To obtain the time activity curves, the physical half-life was applied.

[0266] NOTE - In some figures, the ID is referred to as "IA"; these terms are used interchangeably herein.

[0267] result Humanization 177 Biodistribution of Lu-11B6 177 The biodistribution of Lu-h11B6 is shown in FIG.

[0268] The antibody rapidly accumulated in LNCaP tumors by 24 hours, and radioactivity remained high at 72 hours.

[0269] Initially, high levels of h11B6 were also evident in the blood and kidneys, which declined after 48 hours as the antibody was cleared from the body. Such biokinetics are an inevitable and expected outcome of intravenous administration of any radiolabeled antibody.

[0270] All other organs, including bone and muscle, showed low levels of radioactivity.

[0271] These data are 177 We demonstrated that Lu-h11B6 could effectively target prostate cancer cells in vivo.

[0272] Figure 6 shows a typical SPECT image of LNCaP tumor cells in xenografted mice. 177The binding of Lu-h11B6 was quite clear.

[0273] 177 Lu-h11B6 exhibits an unexpectedly favorable therapeutic ratio Humanization 177 Biodistribution data of Lu-11B6 and the parent mouse antibody ( 177 Comparison with that of Lu-m11B6) revealed unexpected favorable differences.

[0274] As shown in FIG. 7, 72 hours after injection, 177 The uptake of Lu-h11B6 was 177 The increase was approximately 20% compared to Lu-m11B6. 177 The uptake of Lu-h11B6 was 177 This was reduced to approximately 40% compared to Lu-m11B6.

[0275] Figure 8 shows the data expressed as the ratio of antibody uptake in tumor to healthy bone at 24, 48 and 72 hours after injection. By 72 hours, this ratio was significantly increased for the humanized 11B6 antibody.

[0276] Dose measurement calculation Absorbed dose calculations provided a greater measure of the difference in the kinetics of the humanized antibodies of the invention compared to that of the murine 11B6 parent antibody.

[0277] The absorbed dose is calculated according to the MIRD-schema,

number

number

[0278]

number

[0279] The calculated absorbed dose values ​​for different tissues are given in Table 6.

[0280] [Table 5]

[0281] As can be seen from Table 6, the tumor absorbed dose increased from 1.21 Gy / MBq for m11B6 to 2.2 Gy / MBq for h11B6, an increase of 80%. The tumor to bone marrow absorbed dose ratio increased by approximately 40%, from 3.9 for m11B6 to 5.6 for h11B6. The enhanced therapeutic efficacy of h11B6 compared to m11B6 is shown herein, indicating that higher absorbed doses to the tumor can be obtained with less normal organ toxicity.

[0282] Antibody clearance from the blood An analysis of the blood levels of humanized and murine 11B6 antibodies is shown in FIG.

[0283] The results suggested that h11B6 was cleared from the blood slightly faster than the murine 11B6 antibody, and if so, such an enhanced clearance rate of the humanized antibody could be therapeutically useful from a safety standpoint, potentially allowing the administration of higher potency.

[0284] Enhanced clearance rate may also be beneficial for external imaging

[0285] conclusion The results of this study demonstrated that: humanized 11B6 antibody, 177 Lu-h11B6 effectively targets prostate tumors in vivo; The humanized 11B6 antibody exhibits an unexpectedly better therapeutic ratio (as determined by the ratio of uptake in tumors versus healthy bone) than its murine parent antibody; and The humanized 11B6 antibody is cleared from the blood slightly faster than the murine 11B6 antibody.

[0286] Taken together, these findings support the potential of humanized 11B6 in the treatment (and diagnosis) of prostate cancer. This provided strong evidence of enhanced therapeutic efficacy of the antibody.

[0287] The humanized and mouse antibodies are targeted to the same antigen (i.e., human kallikrein 2) Therefore, the calculated differences in the latest tumor to healthy bone marrow ratios cannot be easily predicted or explained (especially when considering that the humanized antibodies appear to exhibit lower affinity for the target hK2 antigen compared to the mouse parent antibody; see Example 4).

[0288] The difference in relative uptake in tumors compared to healthy bones between humanized and murine 11B6 antibodies was of considerable importance, as this comparison provided a measure of the therapeutic ratio. A higher value of this ratio (as evident for the humanized 11B6 antibody) was indicative of a better therapeutic antibody. In particular, a higher therapeutic ratio meant that a higher absorbed dose of therapeutically radiolabeled h11B6 could be administered to achieve a better therapeutic effect (as the binding of the humanized antibody to healthy tissues and organs is much lower than that of the murine antibody). A higher ratio also indicated that h11B6 would be better than the murine antibody for diagnostic purposes (which is synonymous with a lower signal-to-noise ratio, allowing imaging of smaller tumors, including metastases).

[0289] In conclusion, the data demonstrate that humanization of the 11B6 antibody increases the likelihood of earlier diagnosis and unexpectedly greater therapeutic efficacy in the treatment of prostate cancer. EXAMPLES

[0290] Demonstration of diagnostic and therapeutic efficacy The purpose of this study was to confirm the utility of 11B6, a mAb that specifically targets an epitope within the catalytic groove of hK2, as a vehicle for the specific delivery of highly toxic radionuclides to sites of prostate cancer growth. In this proof of concept study, the murine 11B6 parent antibody was labeled with 177Lu, a low-energy beta particle that also utilizes gamma emission, making SPECT-imaging feasible.

[0291] material and method material 177Lu was purchased from Mallinkrodt Medical BV, Petten, Holland. Radioactivity was measured in ITLC (real-time thin-layer chromatography) strips (Biodex, US) using a Cyclone™ Storage Phosphor system and OptiQuant™ image analysis software (Perkin Elmer, Wellesley, MA, USA) to determine labeling kinetics and radiochemical purity. All chemicals were obtained from Sigma Aldrich, and buffers were prepared in-house using analytical quality water unless otherwise stated. mAb 11B6 is an antibody specific for human kallikrein 2 with an affinity of about 1.2 nM for the antigen; see FIG. 1 (obtained from University of Turku, Finland). For in vivo studies, the prostate cancer cell line LNCaP expressing hK2 (ATCC, Manassas, VA, USA) was used. Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and PEST (penicillin 100 IU / mL and streptomycin 100 μg / mL). Cells were maintained in a humidified incubator at 37°C with 5% CO2 and detached with trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Thermo Scientific).

[0292] Conjugation and radiolabeling Conjugation of CHX-A″-DTPA with 11B6: A solution of mAb 11B6 in PBS was adjusted to pH 9.2 using 0.07 M sodium borate buffer. The pool was concentrated with an Amicon Ultra-2 centrifugal filter (2 mL, 100K). The protein solution was conjugated with the chelator CHX-A″-DTPA (Macrocylics, USA) at a molar ratio of chelator to antibody of 3:1 at 40° C. After 4 h The reaction was stopped at 0.5 °C and CHX-A″-DTPA-11B6, hereafter referred to as DTPA-11B6, was separated from the free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare) equilibrated with 20 mL of 0.2 M ammonium acetate buffer, pH 5.5. Conjugated 11B6 and 5A10 were eluted with 1 mL of ammonium acetate buffer.

[0293] Radiolabeling of DTPA-11B6: DTPA-11B6 in ammonium acetate buffer, pH 5.5, was added to a given volume of 177 LuCl3. After 2 h incubation at room temperature, the labeling was stopped and purified on a NAP-5 column and equilibrated with PBS. Labeling efficiency and labeling kinetics were monitored on ITLC strips and eluted with 0.2 M citric acid. In this system, free Lu-177 migrated with the solvent front line, while the radiolabeled conjugate remained in the original line. Radioactivity distribution was determined with a PhosphorImager system (Perkin Elmer, Wellesley, MA, USA) using Optiquant (Perkin Elmer, Wellesley, MA, USA) as quantification software.

[0294] animal research All animal experiments were performed in accordance with national legislation on the protection of laboratory animals. Animal studies were approved by the local animal research ethics committee. Male immunodeficient nude mice, NMRI, (6–8 weeks old), purchased from Taconic Europe (Bomholt, Denmark) were used in this study.

[0295] Xenografts of LNCaP prostate cancer cells expressing hK2 were administered at approximately 10x10 per injection. 6 Individual cells were subcutaneously transplanted into the right and / or left flank.

[0296] Animals bearing LNCaP tumors were divided into groups and injected with either the therapeutic agent 177Lu-DTP-11B6 or a control, see Table 7 below:

[0297] [Table 6]

[0298] All included animals were measured and weighed consecutively at intervals of 3–4 days.

[0299] To investigate the localization of therapeutic agents using SPECT, we first administered a lower activity (8MBq) 177 Lu-DTPA-11B6. One mouse from group 8 was also studied by SPECT. These animals were organised and the radioactivity in these tissue samples was quantified using an automated NaI(Tl) well-counter with a 3-inch NaI(Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland).

[0300] To study the effects on bone marrow, blood samples (10 μL) were taken periodically. Blood samples were collected twice a week for 8 weeks after injection and white blood cell, red blood cell and platelet counts were analyzed with a Medonic Cell Analyzer-Vet CA530 Vet (Boule Medical, Stockholm, Sweden). At the time of blood collection, the weight and health status of the animals were monitored. Toxicity was evaluated by monitoring the animals for weight loss, general symptom decline and hematological toxicity.

[0301] Tumor volumes were measured with calipers: length l, and w and thickness t were measured and the volume was calculated.

[0302] Treatment plan Based on the relationship between absorbed dose and biological effect on the bone marrow of rats receiving radioimmunotherapy with 90Y and 177Lu [see Larsson et al., 2012, Med. Phys. 39(7):4434-43], it was possible to estimate that the LD50 for bone marrow is approximately 12 Gy. In the literature, the LD50 for acute irradiation in rats and mice is approximately 9 Gy [see, for example, Radiobiology for the rad iologist, Hall and Giacca (eds.), 2006, 6th ed.]

[0303] The regimen was then designed by assuming an acceptable absorbed dose to the bone marrow of 12 Gy, and the activity corresponding to this absorbed dose was then calculated from dosimetric calculations.

[0304] The corresponding dose / activity was used as a control.

[0305] result Tumor reduction in animals FIG. 11 shows how the tumor in one of the mice (visible subcutaneously, in the abdomen of the animal) was reduced in volume after treatment.

[0306] Radioimmunotherapy results FIG. 12 shows the results of the study group with administered activity (a) D, (b) 2xD and (c) control group (D=26.7MBq).

[0307] In both treatment groups, there was a clear trend towards a decrease in tumor volume. The onset of tumor shrinkage was already seen a few days after injection of 177Lu-m11B6. In the control group, there was an increase in tumor volume after injection of NaI solution.

[0308] Figure 13(a) shows the results of one of the mice in the group injected with active A. Here, when active A of 177Lu-m11B6 was administered, the tumor grew continuously from day 1 to day 6. After the treatment, a rapid decrease in tumor volume was observed.

[0309] In a SPECT study (8 days after injection), tumor volumes indicated that activity was still present; see FIG. 13(b).

[0310] conclusion This study using the exemplary antibody 177Lu-m11B6 clearly demonstrated the in vivo therapeutic efficacy of hK2-targeted antibody against prostate cancer tumors. EXAMPLES

[0311] Representative of the invention in prostate cancer xenografts 177 Therapeutic efficacy of Lu-labeled humanized 11B6 antibody material and method Antibodies, conjugation and radiolabeling Antibodies: Exemplary humanized monoclonal antibody 11B6 (IgG1 / κ, transiently expressed in HEK293 cells) comprising a heavy chain according to SEQ ID NO: 12 and a light chain according to SEQ ID NO: 13 was obtained from Innovagen AB, Lund (1 mg / mL in PBS pH 7.4, Lot No. 90476.30). A non-specific IgG antibody was used as an isotype control (IgG antibody from mouse serum, Sigma I-8765).

[0312] Conjugation: A typical h11B6 non-specific IgG control antibody was conjugated with the chelator CHX-A″-DTPA (Macrocylics, USA) as follows: The antibody solution was concentrated with an Amicon Ultra-2 centrifugal filter (2 mL, 100K) and then adjusted to pH 9.2 using 0.07 M sodium borate buffer (Sigma Aldrich).

[0313] Chelation to protein solution at a molar ratio of approximately 3:1 (chelator to antibody) Coupling of the chelator compound CHX-A″-DTPA was carried out similarly to the method described previously (see Almqvist et al.). The coupling efficiency, i.e. the number of resulting chelators per antibody, can be determined by spectrophotometry (Pippin et al.) but was not analyzed in this study. However, to avoid damage to the protein, coupling should preferably not exceed 3 chelators / antibody. The chelator was added to the protein and the solution was incubated with gentle shaking at 40°C.

[0314] The reaction was stopped after 4 hours and CHX-A"-DTPA-h11B6, designated DTPA-h11B6, was separated from the free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare) equilibrated with 20 mL of 0.2 M ammonium acetate buffer (Sigma Aldrich), pH 5.5. Conjugated h11B6 was eluted with 1 mL of ammonium acetate buffer and aliquots were stored at -20°C.

[0315] Conjugation of an IgG control antibody was controlled in a similar manner as above.

[0316] Radiolabeling: Conjugated h11B6 or IgG control antibody (typically 200-300 μL of approximately 1 μg / μL in 0.2 M sodium acetate buffer pH 5.5) is added to a given volume (approximately 200-300 MBq) of 177 LuCl3 (IDB Holland) and incubated for 1.5-2 hours at room temperature. After incubation, labeling was stopped and purified on a NAP-5 column (GE Healthcare) and equilibrated with PBS (Thermo Scientific). Labeling efficiency was monitored by real-time thin-layer chromatography (Biodex, USA) and eluted with 0.2 M citric acid (Sigma Aldrich). In this system, free 177 Lu migrated with the solvent front line, while the radiolabeled conjugate remained in the original line. Radioactivity distribution was determined with a Cyclone Storage Phosphor system (both from Perkin Elmer) using Optiquant as the quantification software.

[0317] Radiolabeling of an IgG control antibody was carried out in a similar manner as above.

[0318] Therapeutic Research Cell line: LNCaP (hK2+) was purchased from American Type Culture Collection (ATCC). Cells were cultured in RPMI1640 medium (Thermo Scientific) supplemented with 10% fetal bovine serum (Thermo Scientific), 100 IU / mL penicillin and 100 μg / mL streptomycin (Thermo Scientific). Cells were maintained in a humidified incubator at 37°C with 5% CO2 and detached with trypsin-EDTA solution (Thermo Scientific).

[0319] All animal experiments were performed in accordance with national legislation on the protection of laboratory animals and with approval from the Animal Research Ethics Committee (Lund University, Sweden). Laboratory-bred male immunodeficient Balb / c nude mice (6-8 weeks old) were used. Mice were xenografted in the right flank by sc injection (8-10 million cells) with LNCaP cells in 100 μL growth medium and 100 μL matrigel (BD Matrigel™ Basement Membrane Matrix Growth Factor Reduced, phenol red-free, catalog number 356231). Mice with established tumors with a diameter of at least about 3 mm were included in the study and divided into three groups as described in Table 8 below. Animals were injected iv into the tail vein. A dose with an activity level of 20 MBq was chosen because it was used in studies with m11B6 and showed good therapeutic effects (see Example 7).

[0320] [Table 7]

[0321] Therapeutic efficacy was determined by repeated measurements of tumor size using calipers. Tumor volume was calculated by measuring the length (L) and width (W) of the tumor and then calculating the volume V as 0.5×L×W×W.

[0322] In addition, hematological (white blood cell count, red blood cell count, platelet count, and hemoglobin count) and weight measurements were performed repeatedly on all animals to identify any potential hematological toxicity and to monitor the animals' general condition. Monitoring hematological toxicity was particularly important when evaluating radioimmunotherapy, since radioactivity will distribute in the blood and eventually reach the bone marrow where blood stem cells reside.

[0323] Mice that developed tumor length / width greater than 14 mm or weight loss greater than 15% compared to initial weight, or otherwise exhibited adverse general symptoms, or had a combination of all three of these indicators, were terminated in accordance with ethical guidelines.

[0324] result Assessment of treatment efficacy As shown in FIG. 14(a), a representative humanized 11B6 antibody of the present invention ( 177 Administration of Lu-h11B6) prevented tumor growth in mice (resulting in a significant reduction in tumor volume in all but one of the animals tested), whereas tumors continued to grow rapidly in mice treated with an IgG control antibody (see FIG. 14b) or NaCl (see FIG. 14c).

[0325] Data from individual animals shown in FIG. 14 are summarized in the form of Kaplan-Meier curves in FIG. 177 Administration of Lu-h11B6 resulted in a significant increase in mouse survival over the course of the experiment, with over 80% of animals still alive at the end of the experiment 60 days after injection (compared to 0% survival in the two control groups).

[0326] Assessment of hematological toxicity The white blood cell count, red blood cell count, platelet count, hemoglobin count and weight were determined. 177 Lu-h11B6 administration did not reveal any toxic effects (data not shown).

[0327] Consideration The results of this study showed that177 A significant therapeutic effect of Lu-h11B6 treatment was evident.

[0328] The activity administered to mice (20 MBq) corresponds to an absorbed dose to the bone marrow of approximately 10 Gy and was well tolerated by these animals. Even with this low activity of 20 MBq, a significant therapeutic effect was observed. As previously estimated, a tumor absorbed dose of at least 60 Gy could be expected.

[0329] No signs of hematological toxicity were observed.

[0330] References Almqvist Y., et al. In vitro and in vivo characterization of 177Lu-huA33: a radio-immunoconjugate against colorectal cancer. Nucl Med Biol. 2006;33:991-998. Pippin CG et al. Spectrophotometric method for the determination of a bifunctional DTPA ligand in DTPA-monoclonal antibody conjugates. Bioconjug Chem. 1992;3:342-5. EXAMPLES

[0331] Dosimetry planning for radionuclide therapy and treatment of prostate cancer in patients In radionuclide therapy (RNT), the radioactive source is distributed throughout the body, and the radioactivity is usually administered systemically as a radiopharmaceutical. The distribution of radioactivity is determined by the amount of radiopharmaceutical that accumulates over time in different tissues, which varies between patients (1).

[0332] RNT treatment should be based on the prescribed absorbed dose (2). Then, first, a pretreatment study should be performed using a tracer dose of the radiopharmaceutical to determine the tumor and organ absorbed dose. This information is usually given in units of mGy / MBq; D, which describes the organ absorbed dose per unit of administered activity. P T (organ).

[0333] This factor can then be used to determine the activity required to administer and thereby deliver the prescribed absorbed dose to a given organ, tissue, or tumor when therapeutic administration is under similar conditions (4, 6).

[0334] For the treatment of prostate cancer with radiolabeled h11B6 antibody, pretreatment studies were performed. 111 By In-h11B6 111I n should be based on imaging. 177 If Lu is to be used as a therapeutic radionuclide, 111I n is most suitable for quantitative (2D / SPECT) imaging diagnosis. D P T When determining (organ), the therapy is a therapeutic activity A that gives the prescribed therapeutic effect. T During therapy, the activity distribution as well as the corresponding dose rate should be calculated based on imaging to obtain the actual therapeutic absorbed dose delivered to the tumor and normal organs, which is necessary for the evaluation of the treatment.

[0335] In the event that therapy results in bone marrow toxic levels, then bone marrow support must be planned and the time of stem cell reinfusion must be determined based on dosimetric calculations for the bone marrow cavity.

[0336] Therefore, in summary, the following treatment scheme should be planned:

[0337] Pretreatment dosimetry study 1.111In-labeled h11B6 (200~300MBq) injection 2. Blood sampling - activity concentrations in blood and plasma determined during week 1. 3. Imaging diagnosis (SPECT / 2D) for one week (7 times) 4. Organ dosimetry based on the LundaDose scheme (3) 5. Therapeutic activity determined is in bone marrow (2-3 Gy), kidney (20-30 Gy) and liver ( The radiation dose was limited by the specific absorbed dose to radiosensitive organs (12–36 Gy).

[0338] Therapy including internal therapy dosimetry 1. 177Lu-labeled h11B6 is administered (based on pre-therapy dosimetry) 2. Blood sampling - Activity concentration in blood and plasma 3. Imaging diagnosis over one week (6 times) 4. Organ dosimetry => Verification of prescribed therapeutic absorbed dose.

[0339] Special comments on dosimetry Cumulative activity is the number of disintegrations that occur in a given area over a period of time. The units are Bq seconds or Bq hours. When ionizing radiation travels through matter, it interacts and deposits energy. The energy deposited is the sum of all the deposited energies in a given volume. The absorbed dose is the ratio of the average deposited energy and the amount of volume. The unit of absorbed dose is the gray (Gy), where 1 Gy is the same as 1 J / kg.

[0340] From the values ​​of activity in tissues at different times, the accumulated activity could be determined by integration and the average absorbed dose determined. Activity measurements were performed by using two-dimensional imaging for whole-organ dosimetry. Quantitative SPECT / CT allowed dosimetry in smaller volumes using voxel-based methods.

[0341] From the 3D distribution of activity concentration values, the absorbed dose rate distribution can be calculated using the so-called point dose kernel or voxel S-values, which describe the energy deposition pattern around a point source located in water (or bone). This method assumes that anatomical regions, such as the soft tissues in the torso, are homogeneous in terms of density. For body regions with heterogeneous density, as in the case of the lungs, a direct Monte Carlo calculation is preferred. In this case, the activity distribution obtained from SPECT or PET was used as input to a Monte Carlo dose calculation code.

[0342] References 1. Strand SE, Zanzonico P, Johnson TK. Pharmacokinetic modeling. Med Phys 1993;20(2):515-27 2. ICRU report nr 67 - Dose Specifications in Nuclear Medicine. Adelstein SJ, DeLuca P, Feinendegen LE, Green L, Howell RW, Humm JL, Strand SE ICRU; 2002 3. The LundADose Method for Planar Image Activity Quantification and Absorbed-Dose Assessment in Radionuclide Therapy. Sjogreen,K., Ljungberg,M., Wingardh,K., Minarik,D., and Strand,SE (2005): Cancer Biother.Radiopharm., 20:92-97 4. Quantitative imaging for clinical dosimetry. Bardies M, Flux G, Lassman M, Monsieurs N, Savolainen S, Strand SE Nucl Instr and Methods 2006:569:467-471. 5. 177Lu-[DOTA0,Tyr3] octreotate therapy in patients with disseminated neuroendocrine tumors: Analysis of dosimetry with impact on future therapeutic strategy. Garkavij Michael, Nickel Mattias, Sjogreen-Gleisner Katarina, Ljungberg Michael, Ohlsson Tomas, Wingardh Karin, Strand Sven-Erik, Tennvall Jan. Cancer 2010:116(4 Suppl):1084-92. 6. Dosimetry in patients with B-cell lymphoma treated with [(90)Y]ibritumomab tiuxetan or [(131)I]tositumomab Sjogreen-Gleisner K., Dewaraja YK., Chisea C., Tennvall J., Linden O., Strand SE, Ljungberg M.. Q J Nucl Med Mol Imaging, 2011 April;55(2):126-54.

Claims

1. 1. A composition for use in the treatment and / or diagnosis of prostate cancer, said composition comprising an antibody polypeptide having binding specificity for human kallikrein-2 (hK2), said antibody polypeptide comprising: (a) a heavy chain variable region, including complementarity determining regions (CDRs) CDRH1, CDRH2, and CDRH3, of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, CDRH1: SDYAWN SEQ ID NO: 1 CDRH2: YISSYSGSTTYNPSLKS SEQ ID NO:2 CDRH3: GYYYGSGF SEQ ID NO:3 And (b) a light chain variable region, including CDRL1, CDRL2, and CDRL3, of the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, CDRL1: KASESVEYFGTSLMH SEQ ID NO:4 CDRL2: AASNRES SEQ ID NO:5 CDRL3: QQTRKVPYT SEQ ID NO:6 Including, the heavy chain variable region and the light chain variable region of the antibody polypeptide comprise framework amino acid sequences derived from one or more human antibodies; the antibody polypeptide exhibits an enhanced therapeutic ratio compared to an antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 23 and a light chain amino acid sequence of SEQ ID NO: 24; The composition for use, wherein the prostate cancer to be treated is non-localized prostate cancer, metastatic prostate cancer, and / or castration-resistant prostate cancer (CRPC).

2. The composition for use according to claim 1 , wherein the prostate cancer is micrometastatic prostate cancer.

3. 3. The composition for use according to claim 1 or 2, wherein the prostate cancer is metastatic prostate cancer, including lymphatic metastasis, bone metastasis, or intrapelvic, intrarectal, intravesical, or intraurethral metastasis.

4. The composition for use according to any one of claims 1 to 3, wherein the antibody polypeptide comprises or consists of a complete antibody or an antigen-binding fragment selected from the group consisting of an Fv fragment and a Fab-like fragment.

5. 5. A composition for use according to any one of claims 1 to 4, comprising a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:8, and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

9.

6. The composition for use according to any one of claims 1 to 5, further comprising a heavy chain constant region and / or a light chain constant region.

7. 7. The composition for use of claim 6, wherein the heavy chain constant region is of an immunoglobulin subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

8. The composition for use according to claim 6 , wherein the heavy chain constant region is of the immunoglobulin subtype IgG1.

9. 9. The composition for use according to any one of claims 1 to 8, comprising a heavy chain constant region, wherein the CH1, CH2 and / or CH3 regions of the heavy chain have 100% identity with the CH1, CH2 and / or CH3 regions of an IgG heavy chain constant region having the amino acid sequence of SEQ ID NO: 10, respectively, and / or a light chain constant region, wherein the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

11.

10. 10. The composition for use according to any one of claims 1 to 9, comprising a heavy chain constant region, wherein the CH1, CH2 or CH3 region of the heavy chain has 100% identity with the CH1, CH2 or CH3 region of an IgG heavy chain constant region having the amino acid sequence of SEQ ID NO: 10, respectively, and / or a light chain constant region, wherein the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

11.

11. 11. The composition for use according to any one of claims 1 to 10, comprising a heavy chain, the CH1, CH2 and / or CH3 regions of which have 100% identity with the CH1, CH2 and / or CH3 regions of an IgG heavy chain, respectively, having the amino acid sequence of SEQ ID NO: 12, and / or a light chain, the light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

13.

12. 12. The composition for use according to any one of claims 1 to 11, comprising a heavy chain, the CH1, CH2 or CH3 region of which has 100% identity with the CH1, CH2 or CH3 region of an IgG heavy chain, respectively, having the amino acid sequence of SEQ ID NO: 12, and / or a light chain, the light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

13.

13. A composition for use according to any one of claims 1 to 12, comprising a heavy chain comprising or consisting of one or more point mutations relative to the amino acid sequence of SEQ ID NO: 12, and / or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

13.

14. 7. The composition for use according to claim 6, comprising a heavy chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and / or a light chain constant region comprising or consisting of the amino acid sequence of SEQ ID NO:

11.

15. 15. A composition for use according to claim 14, comprising a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and / or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:

13.

16. The composition for use according to any one of claims 1 to 15, wherein the antibody polypeptide is directly or indirectly linked to a therapeutic moiety.

17. The composition for use according to any one of claims 1 to 16, wherein the antibody polypeptide further comprises a detectable moiety.

18. 18. The composition for use according to claim 16 or 17, wherein the therapeutic moiety and / or the detectable moiety are indirectly linked to the antibody polypeptide via a linking moiety.

19. 19. The composition for use of claim 18, wherein the linking moiety is a chelating agent selected from the group consisting of derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7 triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11 tetraacetic acid (TETA).

20. A composition for use according to any one of claims 1 to 19, comprising or consisting of an intact antibody directly or indirectly linked to a radioisotope.

21. 21. The composition for use of claim 20, wherein the radioisotope is selected from the group consisting of beta emitters, Auger emitters, conversion electron emitters, alpha emitters, and low photon energy emitters.

22. The radioisotope is ２１２ Bi, ２１３ Bi, ２２３ A c, ２２５ Ac and ２２１ 22. The composition for use according to claim 21 , wherein the alpha emitter is selected from:

23. The radioisotope ２２５ 23. The composition for use according to claim 22, wherein said compound is Ac.

24. A composition for use according to any one of claims 1 to 23, wherein the therapeutic ratio is defined as the ratio of uptake in tumour to healthy bone in mice containing implanted human prostate cancer cells.

25. The composition for use described in claim 24, wherein the ratio of uptake in tumor to healthy bone in mice containing transplanted human prostate cancer cells is determined 72 hours after injection of the antibody polypeptide or the antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 23 and the light chain amino acid sequence of SEQ ID NO:

24.

26. 26. The composition for use according to claim 24 or 25, wherein the transplanted human prostate cancer cells are LNCaP cells.

27. A composition for use according to any one of claims 1 to 26, wherein the enhanced therapeutic ratio is defined as an increase in the therapeutic ratio of at least 40%.

28. 28. The composition for use according to any one of claims 1 to 27, wherein the therapeutic ratio is defined as the ratio of uptake in tumor to healthy bone in mice containing implanted human prostate cancer cells, and wherein the therapeutic ratio of the antibody polypeptide at 72 hours after administration is at least 40% higher than the therapeutic ratio of an antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 23 and a light chain amino acid sequence of SEQ ID NO: 24 at 72 hours after administration.

29. The composition for use according to any one of claims 1 to 28, wherein the enhanced therapeutic ratio is defined as an antibody polypeptide having a higher ratio of tumor absorbed dose to bone marrow absorbed dose compared to an antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 23 and a light chain amino acid sequence of SEQ ID NO: 24, when administered to a patient with a prostate tumor and compared at the same radioactivity and route of administration.

30. 30. An isolated nucleic acid molecule for use in the treatment and / or diagnosis of prostate cancer, said nucleic acid molecule encoding an antibody polypeptide or a polypeptide chain component thereof according to any one of claims 1 to 29.

31. 31. A nucleic acid molecule for use according to claim 30, comprising the nucleotide sequence of SEQ ID NO: 14 and / or SEQ ID NO:

15.

32. A pharmaceutical composition comprising an antibody polypeptide according to any one of claims 1 to 29 and a pharma- ceutically acceptable excipient, diluent or carrier.

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