BR122020023278B1 - USE OF AN ANTIBODY POLYPEPTIDE FOR THE MANUFACTURE OF A MEDICINE FOR THE TREATMENT AND/OR DIAGNOSIS OF PROSTATE CANCER - Google Patents

Abstract

The present invention provides antibody polypeptides having binding specificity for human kallikrein-2 (hK2), wherein the antibody polypeptide comprises (a) a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 3 and/or (b) a light chain variable region comprising the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6 and wherein the heavy chain variable region and light chain variable region comprise framework amino acid sequences of one or more human antibodies. The invention further provides use of said antibody polypeptides in the diagnosis and treatment of prostate cancer.

Description

FIELD OF INVENTION

[001] This invention relates generally to the field of therapeutic and diagnostic agents and methods, particularly in the field of prostate cancer.

BACKGROUND

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

[003] The incidence of prostate cancer is highest in northwestern Europe and the United States. Tumor growth is usually a process that takes place over a long period of time. Prostate cancer is usually a mild form of cancer. In fact, most men diagnosed with prostate cancer survive and recover, with only a minority of men experiencing a more aggressive form of prostate cancer that metastasizes at an early stage. This aggressive form of prostate cancer is only curable if it is diagnosed at an early stage, before the cancer has spread to extracapsular tissue.

[004] Today, diagnosis and monitoring of prostate cancer is typically performed by measuring the concentration of a prostate-specific antigen (PSA) in the patient's blood. If the PSA concentration is markedly elevated in several consecutive measurements, taken at different points in time, the assessment is that there is a probability of prostate cancer. At this point, a biopsy may be performed to check for the presence of prostate cancer.

[005] PSA (also known as kallikrein III) is a protein, consisting of a single chain of 237 amino acids, that is produced in the secretory cells of the prostate gland. These secretory cells can be found throughout the prostate gland. PSA is a well-established and thoroughly investigated marker for prostate cancer. Compared to healthy cells, PSA production is lower in malignant cells and higher in hyperplastic cells. It is quite contradictory that, in fact, the concentration of PSA is higher in the blood of men suffering from prostate cancer. However, one explanation may be that malignant cells have a deteriorated cellular structure and are therefore more permeable to PSA.

[006] 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, together with the gene encoding PSA. hK2 is expressed mainly in prostate tissue, as is PSA. In the prostate, PSA is present as an inactive proform and is activated through the action of hK2 peptidase. Immunohistochemical investigation of hK2 has shown that hK2 is expressed in relation to the level of differentiation. This means that hK2 is expressed with greater yield in poorly differentiated tissue, such as tissue subject to prostate cancer, and with less yield in highly differentiated tissue, such as tissue subject to benign prostatic hyperplasia (BPH) which is another common prostate problem.

[007] Current therapies for prostate cancer are surgery (e.g., radical prostatectomy), radiotherapy (comprising brachytherapy and external beam radiotherapy, high-intensity focused ultrasound (HIFU), chemotherapy, oral chemotherapeutic drugs, cryosurgery (freezing the tumor), hormonal therapy (such as antiandrogen therapy), castration, or combinations of the above.

[008] Most of these therapies (surgery and external radiotherapy) are, however, only (or essentially) useful for treating primary tumors and large metastases. Chemotherapy is used for disseminated cancer, but for most of these patients it is a palliative effect and/or prolonged survival. Other treatment modalities, or complementary treatment, are therefore necessary to achieve considerable improvements in disseminated malignancies, particularly in cases of micrometastases.

[009] Therapies, such as immunotherapy or radioimmunotherapy, using targeting molecules such as antibodies and fragments could provide the possibility of therapy for disseminated disease.

[010] Thus, there is a need for new therapeutic agents and methods to treat and diagnose prostate cancer.

SUMMARY OF THE INVENTION

[011] Accordingly, the present invention seeks to mitigate, alleviate or eliminate one or more of the above-identified prior art deficiencies and disadvantages, alone or in any combination, and solves at least the aforementioned problems by providing therapeutic agents and methods in accordance with the appended patent claims.

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

[013] The six amino acid sequences set forth above represent the complementarity determining regions (CDRs) of the antibody polypeptides of the invention, as defined according to Kabat et al., (1991) Sequences of Immunological Interest, 5th edition, NIH, Bethesda, MD (the disclosures of which are incorporated herein by reference).

[014] By “antibody polypeptide” we mean 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, as well as antigen-binding fragments and derivatives thereof.

[015] The term “amino acid” as used herein, comprises the twenty genetically encoded standard amino acids and their corresponding stereoisomers in the ‘D’ form (compared to the naturally occurring ‘L’ form), omega amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derived amino acids (see below).

[016] When an amino acid is specifically enumerated, such as “alanine” or “Ala” or “A”, the term refers to both L-alanine and D-alanine, unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, provided that the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single-letter designation corresponding to the trivial name of the conventional amino acid.

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

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

[019] An exemplary hK2 sequence is described as Transcript: KLK2-201 (ENST00000325321), a product of the gene ENSG00000167751 as given in the joint database, which can be found at the following world-wide-web address: ensembl.org/Homo_sapiens/Transcript/Sequence_Protein?g=ENSG00 000167751;r=19:51376689-51383822;t=ENST00000325321 and has the following sequence: MWDLVLSIAL SVGCTGAVPL IQSRIVGGWE CEKHSQPWQV AVYSHGWAHC GGVLVHPQWV LTAAHCLKKN SQVWLGRHNLFEPEDTGQRV PVSHSFPHPL YNMSLLKHQS LRPDEDSSHDLMLLRLSEPA KITDVVKVLG LPTQEPALGT TCYASGWGSIEPEEFLRPRS LQCVSLHLLS NDMCARAYSE KVTEFMLCAGLWTGGKDTCG GDSGGPLVCN GVLQGITSWG PEPCALPEKP AVYTKVVHYR KWIKDTIAANP [SEQID NO:7](in which the sequence of the mature, active hK2 protein is underlined, which is preceded at its N-terminus by a signal peptide and propeptide sequence)

[020] Most of the hK2 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 show that hK2 can bind to α2-antiplasmin (α2-AP), ACT, AMG, anti-thrombin III (ATIII), C1 inactivator, and plasminogen activator inhibitor-1 (PAI-1).

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

[022] Additional examples of hK2 proteins are identified by the following accession numbers:a) GenBank: AAF08277.1;b) GenBank: AAF08275.1; andc) UniProtKB/Swiss-Prot: P20151.1

[023] Production of recombinant hK2 is described in Lovgren et al., 1999, Eur. J. Biochem. 266:1050-5 (the disclosures of which are incorporated herein by reference).

[024] By “specificity” we mean that the antibody polypeptide is capable of binding to hK2 in vivo, i.e., under the physiological conditions in which hK2 exists in the human body. Preferably, the antibody polypeptide does not bind to any other protein in vivo.

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

[026] Murine antibodies with specificity for hK2 are known in the art. For example, Vaisanen et al., 2004, Clinical Chemistry 50(9):1607-1617 describes the production of monoclonal antibodies in mice with specificity for hK2 (the disclosures of which are incorporated herein by reference). Two of the antibodies, designated “11B6” and “7D7”, are indicated as being selective for hK2.

[027] The amino acid sequences of the component light and heavy chains of the murine antibody 11B6 are disclosed in International Patent Application PCT/GB2012/052675 (WO 2013/061083; the disclosures of which are incorporated herein in their entirety by reference); see, in particular, SEQ ID NO: 4 and 5.

[028] The antibody polypeptides of the present invention are based on a selected humanized version of the 11B6 antibody, which exhibits unexpected favorable properties.

[029] In particular, the humanized antibodies of the invention exhibit an improved therapeutic ratio compared to the parental murine antibody 11B6 (m11B6) from which their CDR sequences were derived (see Example 6).

[030] By “improved therapeutic ratio” we mean that the antibody polypeptide of the invention (a humanized form of the 11B6 antibody), when administered to a patient with a prostate tumor, provides a higher ratio of tumor absorbed dose to (healthy) bone marrow absorbed dose than the parent murine antibody 11B6 (compared to the same radioactivity and route of administration). The ratio of tumor to bone marrow absorbed doses can be calculated using the method described in Example 6.

[031] The unexpectedly better therapeutic profile of the antibodies of the invention allows the use of higher radiation doses (absorbed doses), leading to greater efficacy in the treatment of prostate cancer without increased side effects or “collateral damage” to healthy tissues and organs.

[032] Humanization (also called “reshaping” or CDR transplantation) is a technique to reduce the immunogenicity of monoclonal antibodies from xenogeneic sources (usually rodents such as mice) and to improve their activation by the human immune system (see review by Almagro & Fransson, 2008, Frontiers in Bioscience 13:1619-1633; the disclosures of which are incorporated herein by reference). Several humanized monoclonal antibodies are in clinical trials, and some have been approved for use as drugs. Although the mechanics of monoclonal antibody production using molecular biology techniques are relatively straightforward, simple grafting of rodent complementarity determining regions (CDRs) onto human frameworks does not always reconstitute the binding affinity and specificity of the original monoclonal antibody. To humanize an antibody, the design of the humanized antibody is a critical step in reproducing the function of the original molecule.

[033] The design of a humanized antibody involves several key choices, such as the length of the CDRs to be used and the human frameworks to be used. However, to retain the specificity of the parental antibody, it may also be critical to replace one or more residues of the rodent mAb in the human framework regions (so-called back mutations). Identifying the position of the necessary back mutations requires detailed sequence/structural analysis. Recently, phage libraries have been used to vary the amino acids at chosen positions. Similarly, several approaches have been used to select the most appropriate human frameworks to which to graft the rodent CDRs. Previous experiments have used a limited subset of well-characterized human monoclonal antibodies (often when the framework was available), regardless of sequence similarity to the rodent monoclonal antibody (the so-called fixed-framework approach). Some groups use variable regions with high amino acid sequence similarity to the rodent variable regions (homology matching or “best-fit”); others use consensus or germline sequences while still others select fragments of the framework sequences within each light or heavy chain variable region of several different human monoclonal antibodies. Approaches to humanization have also been developed that replace rodent surface residues with more common residues found in human monoclonal antibodies ("resurfacing" or "veneering") and others that use different definitions of CDR extensions.

[034] However, despite extensive study of antibody humanization, some rodent monoclonal antibodies have proven difficult to humanize.

[035] Development of the antibody polypeptides of the invention requires reverse mutations 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 NO: 1 to 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) relative to the murine parent antibody. These CDR mutations were made to confer optimal specificity and stability in the humanized version of 11B6.

[036] In one embodiment, the antibody polypeptides of the invention bind to hK2 with a KD greater than 0.1x10-9 M.

[037] Methods for measuring the overall affinity (KD) and on- and off-rate (ka) of an interaction (such as an interaction between an antibody and a ligand) are well known in the art. Exemplary in vitro methods are described in Example 3 below. It is also conceivable to use methods based on flow cytometry (Sklar et al., 2002, Annu Rev Biophys Biomol Struct, 31:97-119; the disclosures of which are incorporated herein by reference).

[038] Advantageously, the antibody polypeptide of the invention has an affinity (KD) for hK2 lower than 1.0 x10-10 M, for example a KD of less than 9.0 x10-11 M, 8.0 x10-11 M, 7.0 x10-11 M, 6.0 x10-11 M, 5.0 x10-11 M, 4.0 x10-11 M, 3.0 x10-11 M, 2.0 x10-11 M or less than 1.0 x10-11 M.

[039] It will be recognized by those skilled in the art that the antibody polypeptides of the invention may comprise antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen-binding fragments and derivatives thereof.

[040] In one embodiment, the antibody polypeptide comprised or consists of an intact (i.e., complete) antibody, such as an IgA, IgD, IgE, IgG, or IgM molecule.

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

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

[043] By “antigen-binding fragments and derivatives” of antibodies we mean Fv fragments (e.g., single-chain Fv and Fv with disulfide bonds), Fab-identical fragments (e.g., Fab fragments, Fab’ fragments, and F(ab)2 fragments), and domain antibodies (e.g., single variable VH or variable VL domains).

[044] For example, the antibody polypeptide may comprise or consist of a scFv or Fab fragment.

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

[046] By “framework sequences” we mean the regions of the heavy chain and light chain variable domains other than the CDRs. Typically, each variable domain will comprise four framework regions, designated FR1 to FR4, within which the CDR sequences are located: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4

[047] It will be appreciated that the amino acid sequences of the framework regions may be entirely human or may contain one or more reverse mutations (i.e., the amino acid sequence present in the human framework may be replaced by the amino acid that is found at the corresponding position in the parent rodent variable domain from which the CDRs are derived). Accordingly, the FR1, FR2, FR3 and/or FR4 sequences of the heavy and/or light chain variable domain(s) of the antibody polypeptide of the invention may be non-naturally occurring.

[048] In one embodiment, the framework sequences of the antibody polypeptide share at least 70% sequence identity with framework regions of one or more human antibodies, e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. Thus, the antibody polypeptide may comprise a heavy chain FR1 region that shares at least 70% sequence identity with a FR1 region of a human antibody. It will be appreciated, however, that the heavy and light chains of the antibody polypeptide may share sequence identity with the framework regions of different human antibodies.

[049] Percent identity may be determined, for example, by the LALIGN program (Huang and Miller, Adv. Appl. Math. (1991) 12:337-357) on the Expasy website (http://www.ch.embnet.org/software/LALIGN_form.html) using as parameters the global alignment option, BLOSUM62 scoring matrix, gap opening penalty -14, gap extension penalty -4. Alternatively, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group, and it will be appreciated that percent identity is calculated with respect to polypeptides whose sequence has been optimally aligned.

[050] The alignment may alternatively be performed using the Clustal W program (as described in Thompson et al., 1994, Nucl. Acid Res. 22:4673-4680, which is incorporated herein by reference). The parameters used may be as follows:o Parameters for fast pairwise alignment: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of upper diagonals; 5. Scoring method: x percentage.o Parameters for multiple alignment: gap opening penalty; 10, gap extension penalty; 0.05.o Matrix and score: BLOSUM

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

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

[053] For example, the framework sequences may be encoded, at least in part, by a VH4-28 germline gene (e.g., FR1, FR2, and FR3 may be encoded by VH4-28 and FR4 may be encoded by JH1).

[054] 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]

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

[056] For example, the framework sequences may be encoded, at least in part, by an IgkV4-B3 germline gene (e.g., FR1, FR2, and FR3 may be encoded by IgkV4-B3 and FR4 may be encoded by JK2).

[057] 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:DIVLTQSPDSLAVSLGERATINCKASESVEYFGTSLMHWYQQKPGQP PKLLIYAASNRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQT RKVPYT FGQGTKLEIK[SEQ ID NO: 9]

[058] By “at least in part” we mean that the framework sequences comprise at least ten contiguous amino acids encoded by the reference gene, for example at least 20 contiguous amino acids. We also understand that one or more, but not all, of the FR regions are encoded by the reference gene (for example, FR1 and FR2 may be encoded by the reference gene, but not FR3).

[059] 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.

[060] Optionally, the antibody polypeptide of the invention further comprises a heavy chain constant region, or part thereof.

[061] In one embodiment, the antibody polypeptide comprises a CH1, CH2, and/or CH3 region 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 regions of an IgG1 heavy chain. For example, the antibody polypeptide can be a Fab fragment comprising CH1 and CL constant regions.

[062] In one embodiment, the antibody polypeptide may comprise an antibody Fc region. It will be recognized by one of skill in the art that the Fc portion may be from an IgG antibody, or from a different class of antibody (such as IgM, IgA, IgD, or IgE). In one embodiment, the Fc region is from an IgG1, IgG2, IgG3, or IgG4 antibody.

[063] The Fc region may be naturally occurring (e.g., part of an endogenously produced antibody) or may be artificial (e.g., comprising one or more point mutations relative to a naturally occurring Fc region and/or modifications to the carbohydrate moieties within the CH2 domain). Fc regions with point mutations that improve their ability to bind to FcR may be advantageous, for example, by altering serum half-life or by modulating (i.e., increasing or reducing) binding to FCY receptors (FCYRs) involved in ADCC and CDC.

[064] Advantageously, the antibody polypeptide may comprise the amino acid sequence of SEQ ID NO: 10, or part thereof: C N V N H K P S N T K V D K K V E P KS C D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L MI S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V L T V L H Q D W L N G EY K C K V Y N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L PP S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K [SEQ ID NO: 10]

[065] Optionally, the antibody polypeptide of the invention further comprises a light chain constant region, or part thereof.

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

[067] For example, the antibody polypeptide may comprise the amino acid sequence of SEQ ID NO: 11, or part thereof: Y A C E V T H Q G L S S P V T K SF N R G E C [SEQ ID NO: 11]

[068] 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.

[069] In a preferred embodiment, the antibody polypeptide of the invention comprises: a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 (wherein the variable region is in bold and the CDR sequences are boxed and italicized) [SEQ ID NO: 12] and/or (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 (where the variable region is in bold and the CDR sequences are boxed and italicized) [SEQ ID NO: 13]

[070] For example, the antibody polypeptide may comprise or consist of two heavy chains of SEQ ID NO: 12 and two light chains of SEQ ID NO: 13, joined by disulfide bridges to form a typical IgG antibody structure.

[071] The antibody polypeptides of the invention may comprise or consist of one or more amino acids that have been modified or derivatized.

[072] Chemical derivatives of one or more amino acids can be obtained by reaction with a functional side group. Such derivative molecules include, for example, those in which free amine groups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. The free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. The free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are peptides containing naturally occurring amino acids derived from the twenty standard amino acids. For example: 4-hydroxyproline can be replaced by proline; 5-hydroxylysine can be replaced by lysine; 3-methyl histidine can be replaced by histidine; homoserine can be replaced by serine and ornithine by lysine. Derivatives also comprise peptides comprising one or more additions or deletions so long as the required activity is retained. Other modifications comprised are amidation, amine-terminal acylation (e.g., acetylation or amidation of thioglycolic acid), terminal carboxylamidation (e.g., with ammonia or methylamine), and similar terminal modifications.

[073] It will also be appreciated by those skilled in the art that peptidomimetic compounds may also be useful. The term “peptidomimetic” refers to a compound that mimics the conformation and desirable characteristics of a particular peptide as a therapeutic agent.

[074] For example, said polypeptide comprises not only molecules in which the amino acid residues are joined by peptide bonds (-CO-NH-), but also molecules in which the peptide bond is reversed. Such retro-inverse peptidomimetics can be carried out using methods known in the art, for example as described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, which is incorporated herein by reference. This approach involves producing pseudo-peptides comprising changes involving the structure rather than the orientation of the side chains. Retro-inverse peptides, which comprise NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, said polypeptide may be a peptidomimetic compound in which one or more of the amino acid residues are joined by a -γ(CH2NH)- bond instead of the conventional amide bond.

[075] In a further alternative, the peptide bond may be dispensed with entirely provided that an appropriate linking moiety is used which retains the spacing between the carbon atoms of the amino acid residues; it may be advantageous for the linking moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond. It will be appreciated that said polypeptide may be conveniently blocked at its N- or C-terminus in order to help reduce susceptibility to exoproteolytic digestion.

[076] Various non-coding or modified amino acids, such as D-amino acids and N-methyl amino acids, have also been used to modify mammalian peptides. Additionally, a putative bioactive conformation can be stabilized by a covalent modification, such as cyclization, or by incorporation of lactam or other types of bridges, e.g., see Veber et al., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem. Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

[077] It will be understood by those skilled in the art that the antibody polypeptides of the invention may be augmented with a functional moiety to facilitate their intended use, for example as an in vivo imaging agent or therapeutic agent.

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

[079] Any suitable therapeutic moiety may be used. A suitable therapeutic moiety is one capable of reducing or inhibiting the growth of, or in particular killing, a prostate cancer cell. For example, the therapeutic agent may be a cytotoxic moiety. A cytotoxic moiety may comprise or consist of one or more radioisotopes. For example, the one or more radioisotopes may each be independently selected from the group comprising beta emitters, Auger emitters, conversion electron emitters, alpha emitters, and low photonic energy emitters. It may be desired for the one or more radioisotopes to each independently have a locally absorbed energy emission pattern that creates a high absorbed dose in the immediate vicinity of the agent. Exemplary radioisotopes may comprise long-range beta emitters such as 90Y, 32P, 186Re/188Re; 166Ho, 76As/77As, 89Sr, 153Sm; medium-range beta emitters such as 131I, 177Lu, 67Cu, 161Tb, 105Rh; low-energy beta emitters such as 45Ca or 35S; Auger or conversion emitters such as 51Cr, 67Ga, 99Tcm, 111In, 114mIn, 123I, 125I, 201Tl; and alpha emitters such as 212Bi, 213Bi, 223Ac, 225Ac, 212Pb, 255Fm, 223Ra, 149Tb, and 221At. Other radionuclides are available that can be used for therapy.

[080] In another embodiment, it may be desired that the therapeutic moiety or cytotoxic moiety is not a moiety as disclosed as a “tracer” in WO 2006/087374 A1, in particular on page 11, lines 7-15.

[081] In a preferred embodiment, the antibody polypeptide is linked to (or labeled with) the radioisotope 177Lu.

[082] Alternatively, the therapeutic moiety may comprise or consist of one or more therapeutic drugs (such as cytotoxic drugs), for example, a cytostatic drug; an anti-androgen drug; cortisone and its derivatives; a phosphonate; a testosterone-5-α-reductase inhibitor; a boron adduct; a cytokine; thapsigargin and its metabolites; a toxin (such as saponin or calichemycin); a chemotherapeutic agent (such as an antimetabolite); or any other therapeutic or cytotoxic drug useful in the treatment of prostatic carcinoma.

[083] Exemplary therapeutic/cytotoxic drugs may comprise, for example:o Cytostatics, particularly those with dose-limiting side effects, comprising, but not limited to, cyclophosphamide, chlorambucil, ifosfamide, busulfan, lomustine, taxanes, estramustine phosphate and other nitrogen mustards, antibiotics (comprising doxorubicin, calichemycins and spiramycin), vinca alkaloids, aziridines, platinum-containing compounds, endostatin, alkylsulfonates, nitrosureas, triazines, folic acid analogues, pyrimidine analogues, purine analogues, enzymes, substituted urea, methylhydrazine derivatives, daunorubicin, amphipathic amines;o Antiandrogens such as flutamide and bicalutamide and their metabolites;o Cortisone and its derivatives;o Phosphonates such as diphosphonate and buphosphonate;o Testosterone-5-α-reductase inhibitors;o Boron additives;o Cytokines;o Thapsigargin and its metabolites;o Other agents used in the treatment of prostatic carcinoma.

[084] Alternatively, the cytotoxic fraction may comprise or consist of one or more fractions suitable for use in activation therapy, such as photonic activation therapy, neutronic activation therapy, neutron-induced Auger electron therapy, synchrotron radiation therapy, or low-energy Rx photon activation therapy.

[085] For example, with the antibody polypeptides of the invention there will be the potential to use synchrotron radiation (or low energy Rx) to advance radiotherapy, focusing primarily on so-called photoactivation radiotherapy (PAT), in which the local deposition of external irradiation Rx energy is increased in cancer tissue by interaction with a pre-administered high impedance agent targeted to the tumor.

[086] The PAT treatment modality uses monochromatic Rx from a synchrotron source, as provided by the “ID17 biomedical beamline” at the European Synchrotron Radiation Facility (ESRF) in Grenoble, and which is anticipated to be available at other facilities in the future, such as the new Swedish synchrotron facility, Max-IV.

[087] As a potential additional treatment modality, research into “Auger-induced electron tumor therapy” is forthcoming at the European Spallation Source (ESS) in Lund, and hopefully an experimental medical station. Thermally and semi-thermally produced neutrons have long been used in boron neutron capture therapy, BNCT, both for preclinical experiments and for treating brain tumors with the induced alpha particles and recoil nucleus (7L) that provide a locally high absorbed energy. A similar approach is the use of neutrons and suitable tumor-targeting molecules labeled with stable nuclei with a high neutron crossover probability. Antibodies or peptides can, for example, be labeled with stable Gadolinium (157Gd) and act as the target molecule for the neutrons that are captured by the Gd nucleus, in so-called Gadolinium Neutron Capture Therapy (GdNCT). The dose distribution in the tumor and surrounding tissues is calculated using Monte Carlo techniques, which results from Y photons, neutrons, nuclear recoils, as well as characteristic Rx, Auger electrons and internal conversion of gadolinium or other potential elements.

[088] As discussed above, the therapeutic moiety (such as a radioisotope, cytotoxic moiety or the like) may be linked directly, or indirectly, to the binding moiety (such as an antibody or fragment thereof). Suitable ligands are known in the art and comprise, for example, prosthetic groups, non-phenolic ligands (derivatives of N-succinimidylbenzoates; dodecaborate), chelating moieties of macrocyclic and acyclic chelators such as 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, deferoxamine (DFO), diethylenetriaminepentaacetic acid (DTPA) derivatives, S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivatives and 1,4,8,11-tetraazacyclodocedane-1,4,8,11-tetraacetic acid (TETA) derivatives, 3,6,9,15-Tetraazabicyclo[9.3.1]-pentadeca- 1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA), 5-S-(4-Aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A) derivatives and other chelating moieties. The use of such linkers may be particularly appropriate in circumstances where the agent comprises or consists of an antibody or fragment thereof as the binding moiety linked, through a linker, to a radioisotope as the therapeutic moiety.

[089] A preferred ligand is DTPA, for example as used in177Lu-DTPA-[antibody polypeptide of the invention].

[090] Another preferred ligand is deferoxamine, DFO, for example as used in 89Zr-DFO-[antibody polypeptide of the invention].

[091] Optionally, the antibody polypeptide of the invention may (or may not) further comprise a detectable moiety. For example, a detectable moiety may comprise or consist of a radioisotope, such as a radioisotope selected from the group comprising 99mTc, 111In, 67Ga, 68Ga, 72As, 89Zr, 123I and 201Ti. Optionally, the agent may comprise a pair of detectable and cytotoxic radionuclides, such as 86Y/90Y or 124I/211At. Alternatively, the agent may comprise a radioisotope that is capable of acting simultaneously, in a multimodal manner, as a detectable moiety and also as a cytotoxic moiety to provide so-called “multimodal theragnostics”. The binding moieties can thus be coupled to nanoparticles that have multi-imaging capabilities (e.g., SPECT, PET, MRI, Optical or Ultrasound) along with therapeutic capabilities using cytotoxic drugs such as radionuclides or chemotherapeutic agents. Also encompassed by the present invention is the possibility of hyperthermia treatment using high frequency alternating magnetic fields accompanied by ultrasound imaging.

[092] Alternatively, the detectable fraction may comprise or consist of a paramagnetic isotope, such as a paramagnetic isotope selected from the group comprising 157Gd, 55Mn, 162Dy, 52Cr and 56Fe.

[093] In the event that the antibody polypeptide comprises a detectable fraction, then the detectable fraction may be detectable by an imaging technique such as SPECT, PET, MRI, or optical or ultrasonic imaging.

[094] Therapeutic and detectable moieties may be conjugated or combined with the antibody polypeptide using methods well known in the art (e.g., the existing immunoconjugate therapy, gemtuzumab ozogamicin [trade name: Mylotarg®], comprises a monoclonal antibody linked to the cytotoxin calichemycin).

[095] In another embodiment, the antibody polypeptide of the invention is used to treat prostate cancer in the form of a formulation comprising a population of antibody polypeptide molecules. In one option, all of the antibody polypeptide molecules (or substantially all, such as greater than 90%, 95%, 99%, 99.9% or more, by weight) in the population comprise the same therapeutic moiety. In another option, the population comprises a mixture of other agents with different therapeutic moieties. This option will provide possibilities for enhancing the effects of targeted radionuclide therapy using multiple agents such as chemotherapeutic agents, hormonal therapy agents or other combination therapies wherein the targeting agent not only delivers therapeutically active radionuclides to tumor-associated antigens but simultaneously also radiosensitizes the target tumor cells by modulating (e.g., triggering or blocking) an intracellular signaling cascade. This option is also useful in the treatment of prostate cancer with a mixture of cytotoxic agents, for example, using a cocktail of alpha emitters and different ranges of beta emitters, or a cocktail of radionuclides with different ranges, LET (linear energy transfer) and RBE (relative biological effect), for combined treatment of large tumors, micrometastases and single tumor cells. In one embodiment, long-range emitters can be used for treatment of large tumors and short-range emitters can be used for treatment of smaller tumors such as micrometastases and single tumor cells.

[096] Optionally, the antibody polypeptide of the present invention may (or may not) further comprise a moiety for increasing the in vivo half-life of the agent. Exemplary moieties for increasing the in vivo half-life of the agent may comprise polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids, and dextran. PEG may be especially contemplated.

[097] It will be understood that the polypeptides of the invention may be lyophilized for storage and reconstituted in a suitable vehicle prior to use, for example, by freeze-drying, spray-drying, spray-cooling, or by supercritical carbon dioxide particle formation (precipitation). Any suitable lyophilization method (e.g., freeze-drying, spray-drying, cake drying) and/or reconstitution techniques may be used. It will be recognized by those skilled in the art that lyophilization and reconstitution may lead to varying degrees of loss of activity and that use levels may have to be adjusted upward to compensate. Preferably, the lyophilized (cold-dried) polypeptide loses no more than about 1% of its activity (prior to lyophilization) when rehydrated, or no more than about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or no more than about 50% of its activity (prior to lyophilization) when rehydrated.

[098] Methods for producing polypeptides of the invention are well known in the art.

[099] Conveniently, the polypeptide is or comprises a recombinant polypeptide. Suitable methods for producing such recombinant polypeptides are well known in the art, such as expression in prokaryotic or eukaryotic host cells (e.g., see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York, the relevant disclosures of which are incorporated herein by reference).

[0100] The antibody polypeptides of the invention may also be produced using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheat germ lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system may be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of producing a suitable mRNA transcript from a coding DNA polynucleotide in the same reaction as translation.

[0101] It will be appreciated by those skilled in the art that the polypeptides of the invention may alternatively be synthesized artificially, for example using well-known liquid phase or solid phase synthesis techniques (such as t-Boc or Fmoc solid phase peptide synthesis).

[0102] A second aspect of the invention provides an isolated nucleic acid molecule encoding an antibody polypeptide of the invention, or a component polypeptide chain thereof. By “nucleic acid molecule” we understand DNA (e.g., genomic DNA or complementary DNA) and mRNA molecules, which may be single- or double-stranded.

[0103] In one embodiment, the nucleic acid molecule is a cDNA molecule.

[0104] It will be appreciated by those skilled in the art that the nucleic acid molecule may be codon-optimized for expression of the antibody polypeptide in a particular host cell, e.g., for expression in human cells (e.g., see Angov, 2011, Biotechnol. J. 6(6):650-659).

[0105] In a preferred embodiment, the nucleic acid molecule of the invention comprises (a) the nucleotide sequence of SEQ ID NO: 14 CAG GTT CAG CTG CAG GAA AGC GGA CCT GGC TTG GTG AAA CCC AGC GAT ACC CTT AGC CTG ACA TGT GCT GTG TCT GGC AAT TCC ATC ACT TCC GAC TAT GCG TGG AAC TGG ATT CGG CAA CCA CCG GGA AAA GGG CTC GAG TGG ATA GGG TAC ATCAGC TAT TCT GGT TCA ACC ACG TAC AAT CCC TCA CTG AAGAGT AGG GTT ACC ATG TCC AGA GAC ACC TCC AAG AAC CAGTTC AGC CTG AAG CTG AGT AGT GTG ACA GCC GTA GAT ACAGCC GTC TAT TAC TGC GCA ACA GGG TAC TAC TAT GGC TCTGGC TTT TGG GGT CAA GGA ACT CTC GTC ACT GTG TCA AGC [SEQ ID NO: 14] and/or b) the nucleotide sequence of SEQ ID NO: 15 GAC ATA GTG CTC ACT CAG AGC CCT GAT AGC TTG GCT GTCAGT CTT GGG GAA AGA GCC ACC ATC AAC TGC AAA GCG TCCGAA AGC GTC GAG TAT TTC GGG ACT AGC CTG ATG CAC TGGTAT CAG CAG AAA CCC GGA CAA CCG CCT AAG CTG CTG ATCTAT GCA GCC TCT AAT CGC GAA AGT GGC GTT CCA GAC AGGTTT TCC GGT TCT GGA TCA GGC ACA GAC TTC ACC CTC ACGATT TCC TCA CTG CAA GCT GAG GAT GTA GCC GTG TAC TACTGT CAG CAG ACA CGG AAA GTG CCC TAC ACC TTT GGT CAGGGC ACA AAG CTG GAG ATT AAG [SEQ ID NO: 15]

[0106] The following are also included within the scope of the 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., 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 making 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 in which said polypeptide is expressed and isolating the polypeptide therefrom.

[0107] A sixth aspect of the invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of an antibody polypeptide of the first aspect of the invention and a pharmaceutically acceptable diluent, carrier or excipient.

[0108] Additional compounds may also be included in the pharmaceutical compositions, comprising chelating agents such as EDTA, citrate, EGTA or glutathione.

[0109] The pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently stable for storage and suitable for administration to humans and animals. For example, the pharmaceutical compositions may be lyophilized, for example by cold dehydration, spray drying, spray cooling, or by using particle formation from supercritical particle formation.

[0110] By "pharmaceutically acceptable" we mean a non-toxic material that does not diminish the effectiveness of the binding activity of the agent of the invention to the kallikrein protein. Such pharmaceutically acceptable buffers, carriers or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R. 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).

[0111] 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, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0112] The term "diluent" is intended to mean an aqueous or non-aqueous solution for the purpose of diluting the agent in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0113] The term "adjuvant" is intended to mean any compound added to the formulation to enhance the biological effect of the agent of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole) and cationic polypeptides such as polyhistidine, polylysine, polyarginine and peptides comprising these amino acids.

[0114] The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol and cyclodextrins, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethyl(hydroxyethyl) cellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonate, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinylacetate of different degrees of hydrolysis and polyvinylpyrrolidone, all with different molecular weights, which are added to the composition, for example, to control viscosity, to achieve bioadhesion, or to protect the lipids from chemical and proteolytic degradation. Examples of lipids include fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids, and glycolipids, all with different acyl chain lengths and saturations, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for similar reasons to polymers. Examples of minerals include talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition to obtain benefits such as reduced fluid accumulation or advantageous pigmentary properties.

[0115] The antibody polypeptides of the invention can be formulated into any type of pharmaceutical composition known in the art as being suitable for their delivery.

[0116] In one embodiment, the pharmaceutical compositions of the invention may be in the form of a liposome, in which the antibody polypeptide is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms such as micelles, insoluble monolayers, and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the above lipids modified by polyethylene glycol at the polar head group to prolong circulation time in the bloodstream. The preparation of such liposomal formulations can be found, for example, in US 4,235,871, the disclosures of which are incorporated herein by reference.

[0117] The pharmaceutical compositions of the invention may also be in the form of biodegradable microspheres. Aliphatic polyesters such as polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA (PLGA) or polycaprolactone (PCL) and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. Preparations of such microspheres can be found in US 5,851,451 and EP 0,213,303, the disclosures of which are incorporated herein by reference.

[0118] In a further embodiment, the pharmaceutical compositions of the invention are provided in the form of polymeric gels, in which polymers such as starch, cellulose ethers, carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, poly(vinylimidazole), polysulfonate, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinylacetate of varying degrees of hydrolysis and polyvinylpyrrolidone are used for thickening the solution comprising the agent. The polymers may also comprise gelatin or collagen.

[0119] Alternatively, the antibody polypeptides may 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), gum tragacanth and/or various buffers.

[0120] It will be appreciated that the pharmaceutical compositions of the invention may comprise ions and a defined pH for potentiation of the action of the active antibody polypeptide. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may comprise conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0121] The pharmaceutical compositions according to the invention may be administered by any suitable route known to the person skilled in the art. Thus, possible routes of administration include parenteral (intravenous, subcutaneous and intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral and rectal. Administration via implants is also possible. Infusion may be a desirable route due to the potentially high cytotoxicity of the administered agent.

[0122] In one embodiment, the pharmaceutical compositions are administered parenterally, e.g., intravenously, intracerebroventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intracisternally, intracranially, intramuscularly, or subcutaneously, or may be administered by infusion techniques. They are conveniently used in the form of a sterile aqueous solution which may comprise other substances, e.g., sufficient salts or glucose to make the solution isotonic with blood. Aqueous solutions should be suitably buffered (e.g., to a pH of 3 to 9), if necessary. Preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0123] Formulations suitable for parenteral administration comprise sterile aqueous and non-aqueous solutions for injection which may comprise antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and sterile aqueous and non-aqueous suspensions which may comprise suspending agents and thickening agents. The formulations may be presented in unit dose or multiple dose containers, for example sealed ampoules and vials, and be stored under cold-dry (lyophilized) conditions requiring only the addition of a sterile liquid vehicle, for example water for injections, immediately before use. Solutions and suspensions for extemporaneous injection may be prepared from sterile powders, granules and tablets of the type previously described.

[0124] Thus, the pharmaceutical compositions of the invention are particularly suitable for parenteral administration, e.g. intravenous, or local administration into a tumor in a patient (e.g. intratumoral or peritumoral).

[0125] The pharmaceutical compositions will be administered to a patient at a pharmaceutically effective dose, i.e., a therapeutically effective absorbed dose of the therapeutic radionuclide.

[0126] In the context of therapeutic use of the antibody polypeptides of the invention, a “pharmaceutically effective amount,” or “effective amount,” or “therapeutically effective,” as used herein, refers to the amount that provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the necessary additive and diluent, i.e., an administration vehicle. Furthermore, it is intended to refer to an amount sufficient to reduce and/or prevent a clinically significant deficit in host activity, function, and response. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is known to those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may comprise a predetermined amount of active composition calculated to produce the desired therapeutic effect in association with the necessary diluent. In the methods and use for manufacturing compositions of the invention, a therapeutically effective amount of the active ingredient is provided. A therapeutically effective amount can be determined by a medical expert based on the characteristics of the patient, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art (see Example 8 below). Administration of the pharmaceutically effective dose can be accomplished by single administration in the form of a single dose unit or several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the dose can be administered as a continuous infusion over an extended period.

[0127] In the context of diagnostic use of the antibody polypeptides of the invention, a “pharmaceutically effective amount,” or “effective amount,” or “diagnostically effective,” as used herein, refers to the amount that provides a detectable signal for in vivo imaging purposes.

[0128] The antibody polypeptides of the invention may be formulated at various concentrations depending on the efficacy/toxicity of the compound to be used. The formulation may comprise the polypeptide at a concentration of between 0.1 μM and 1 mM, between 1 μM and 500 μM, between 500 μM and 1 mM, between 300 μM and 700 μM, between 1 μM and 100 μM, between 100 μM and 200 μM, between 200 μM and 300 μM, between 300 μM and 400 μM, between 400 μM and 500 μM, and about 500 μM.

[0129] Typically, the therapeutic dose of the antibody polypeptide (with or without a therapeutic moiety) in a human patient will be in the range of 100 μg to 700 mg per administration (based on a body weight of 70 kg). For example, the maximum therapeutic dose may be in the range of 0.1 to 10 mg/kg per administration, e.g., between 0.1 and 5 mg/kg or between 1 and 5 mg/kg or between 0.1 and 2 mg/kg. It will be appreciated that such a dose may be administered at different intervals as determined by the oncologist/physician; for example, a dose may be administered daily, twice weekly, weekly, biweekly or monthly.

[0130] It will be appreciated by those skilled in the art that the pharmaceutical compositions of the invention can be administered alone or in combination with other therapeutic agents used in the treatment of prostate cancer, or before, after, or concurrently with treatment of the patient with other therapeutic modalities for the treatment of prostate cancer, such as other therapeutic antibodies, surgery (e.g., radical prostatectomy), radionuclide therapy, brachytherapy and external beam radiation therapy, high-intensity focused ultrasound (HIFU), chemotherapy, oral chemotherapeutic drugs, cryosurgery (freezing the tumor), hormone therapy (such as antiandrogen therapy), castration, or combinations of the foregoing.

[0131] A seventh aspect of the invention provides a kit comprising an antibody polypeptide according to the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention, together with instructions for use thereof as described herein.

[0132] An eighth aspect of the invention provides a polypeptide antibody according to the first aspect of the invention for use in medicine.

[0133] A ninth aspect of the invention provides a polypeptide antibody according to the first aspect of the invention for use in the treatment and/or diagnosis of prostate cancer.

[0134] 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.

[0135] By “treatment” we mean both therapeutic and prophylactic treatment of the patient. The term “prophylactic” is used to mean the use of an agent, or formulation thereof, as described herein, that prevents or reduces the likelihood of prostate cancer, or the spread, dissemination, or metastasis of localized prostate cancer in a patient or individual. The term “prophylactic” also means the use of an agent, or formulation thereof, as described herein, to prevent the recurrence of prostate cancer in a patient who has been previously treated for prostate cancer.

[0136] 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.

[0137] By “diagnosis” we mean the detection of prostate cancer cells, either in vivo (i.e., within a patient's body) or ex vivo (i.e., from within a sample of tissue or cells taken from a patient's body).

[0138] The prostate cancer to be treated or diagnosed may be localized to the prostate, or it may be non-localized (i.e., disseminated) prostate cancer. Prostate cancer localized to the prostate may, for example, be clinically classified as T1 or T2 cancers according to the TNM (abbreviated for Tumor/Lymph Nodes/Metastases) system, whereas non-localized/disseminated prostate cancer may, for example, be clinically classified as T3 or T4 cancers.

[0139] The prostate cancer to be treated or diagnosed may be metastatic prostate cancer. Metastasis refers to the spread of a cancer from its original location to other sites in the body. For example, the metastatic prostate cancer to be treated or diagnosed may be a metastasis present in the lymphatic system; in the bone (including spine, vertebrae, pelvis, ribs); metastases within the pelvis, rectum, bladder or urethra. Metastases present in other less common locations may also be treated with the present invention. The metastases may be micrometastases. Micrometastasis is a form of metastasis in which newly formed tumors are generally too small to be detected, or are detected with difficulty. For example, the present invention provides the skilled artisan with means for treating single tumor cells or clusters of cells, even if the presence of such cells or clusters is not possible to diagnose but exists, for example as occult disseminated disease.

[0140] Accordingly, it is anticipated that a particularly important technical advantage of the treatment provided by the present invention compared to previous prostate cancer treatments will be the increased efficacy in the treatment of disseminated and/or metastatic prostate cancer (including micrometastatic prostate cancer).

[0141] Thus, in one embodiment, the invention provides antibody polypeptides and methods for preventing or treating metastases of a primary prostate cancer.

[0142] Prostate cancer tends to develop in men over the age of 50, most commonly in men over the age of 60, 65 or 70, and although it is one of the most prevalent cancers in men, many never have symptoms, do not undergo any therapy and ultimately die from other causes. This is because prostate cancer is, in most cases, slow-growing, asymptomatic and, since men with the condition are elderly, they often die from causes unrelated to prostate cancer, such as heart/circulatory disease, pneumonia, other unrelated cancers, or age. About two-thirds of prostate cancer cases are slow-growing, the other third are more aggressive and fast-growing.

[0143] Accordingly, the development of effective methods for the treatment and diagnosis of prostate cancer is particularly important for the management of more aggressive and rapidly developing forms of the cancer, particularly in younger patients. Suitably, in one embodiment, the invention relates to the treatment or diagnosis of prostate cancer in a patient who is less than 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40 or younger at the time of diagnosis of prostate cancer and/or at the time of treatment.

[0144] It is thought that men who have a first-degree relative (father or brother) with prostate cancer have twice the risk of developing prostate cancer, and that those who have two first-degree relatives affected have a five-fold increased risk compared to men with no family history. Suitably, the invention may relate to the treatment or diagnosis of prostate cancer in a patient who is characterized in that one, two, or more family members, in particular first-degree members (such as a father or a brother) have previously been diagnosed with prostate cancer.

[0145] The 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). CRPC can be characterized by typically becoming refractory to hormonal treatment after one to three years and resuming growth despite hormonal therapy.

[0146] In the medical uses and methods of the invention, the antibody polypeptide is typically injected or infused into the body of a patient. In vivo, the antibody polypeptide then binds to tissues to produce the target antigen, hK2; primarily, prostate cancer cells and metastases thereof. Upon binding, the antibody polypeptide may directly exert a therapeutic effect (e.g., inducing cell death through ADCC, CDC, or by virtue of carrying a radioisotope or other cytotoxic moiety). Alternatively, the bound antibody polypeptide may serve as a diagnostic (imaging) tool, which may guide the choice of therapy or aid in the surgical removal of cancer cells.

[0147] It will be appreciated by those skilled in the art that the antibody polypeptides of the invention may be used in combination with other therapeutic and/or diagnostic agents/treatments, such as external beam radiotherapy, surgery, cytostatic and androgenic treatments.

[0148] The foregoing description focuses on embodiments of the present invention applicable to methods for the treatment and diagnosis of prostate cancer. However, it will be appreciated that the invention is not limited to such applications but may be useful for post-operative examinations and examinations during or after cytostatic, androgen and radiation treatments.

[0149] In another embodiment, Radioguided Surgery (RGS) or Image Guided Surgery (IGS) can be used to identify labeled antibody polypeptides of the invention during and/or prior to surgery. Thus, an antibody polypeptide comprising a detectable moiety, as discussed above, can be administered during and/or prior to surgery. In this embodiment, the antibody polypeptides can be infused first. Then, RGS/IGS can be used to identify hK2-producing tissue with a detection instrument sensitive to the detectable moiety, during or prior to surgery. The detectable moiety can be, for example, a radiation-emitting or magnetically sensitive detectable moiety; it can be, for example, a Cerenkov and/or Bremsstrahlung radiation emitter; it can be a fluorescent marker and/or a magnetic or magnetizable marker. Suitably, RGS/IGS according to the present invention can, for example, be a method based on the detection of optical, Cerenkov, Bremsstrahlung or beta radiation; the detection of a radionuclide marker and/or may involve magnetometry. RGS is well known to the skilled artisan as a surgical technique that allows the surgeon to identify "labeled" tissue by the detectable fraction.

[0150] Views obtained according to the above methods may be combined with other radiological visualization methods, such as SPECT/PET, computed tomography (CT), ultrasound (US), and magnetic resonance imaging (MRI).

[0151] Suitably, in another aspect, the present invention also provides antibody polypeptides for use in medicine by administration to a patient with prostate cancer prior to or during surgery, such as Radioguided Surgery or Image Guided Surgery.

[0152] Yet another aspect of the invention provides an in vitro method for detecting prostate tumor cells in the blood of a subject, the method comprising: a) providing a blood sample 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 is indicative of the presence of prostate tumor cells in the blood of a subject.

[0153] Thus, the method comprises performing an assay to determine whether the blood sample comprises free hK2; the presence of free hK2 being indicative of the presence of prostate tumor cells in the blood of an individual.

[0154] Those skilled in the art will appreciate that there are several ways to perform such an assay. For example, the immunoassay may be homogeneous or, more preferably, heterogeneous. The assay may also be performed in a competitive format or, more preferably, a non-competitive format.

[0155] In the case of the heterogeneous, non-competitive assay, an exemplary protocol could be: a) providing a blood sample from an individual to be tested; b) optionally extracting and/or purifying cells present in the blood sample; c) placing a solid phase immobilized antibody polypeptide according to the first aspect of the invention in contact with cells present in the blood sample; d) washing to remove soluble components (not bound to the solid surface); e) adding the label, i.e., another anti-hK2 specific antibody labeled with a reporter molecule/particle; f) washing to remove unbound label antibody; and g) detecting the signal from the label antibody.

[0156] Between steps b and c or c and d, there would typically be an incubation period to allow the cell to produce soluble hK2, which can then be detected.

[0157] A further aspect of the invention provides an in vitro method for detecting prostate tumor cells in tissue from a subject, the method comprising a) providing 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 is indicative of the presence of prostate tumor cells in the tissue from a subject.

[0158] In one embodiment of the in vitro methods referred to above, step (d) is performed by ELISA. However, any suitable assay for detecting antibody-antigen interactions in vitro may be used.

[0159] In an additional embodiment, the method further comprises quantifying prostate tumor cells in the sample.

[0160] In another embodiment of the in vitro methods referred to above, the method is for diagnosing prostate cancer in an individual.

[0161] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or specification may mean “one” but is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”.

[0162] These and other embodiments of the invention will be best appreciated and understood when considered in conjunction with the above description and the accompanying figures. However, it should be understood that the above description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention encompasses all such substitutions, modifications, additions and/or rearrangements.

[0163] The following figures are part of this specification and are intended to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

[0164] Figure 1: The sequences of the heavy and low variable regions of the exemplary humanized 11B6 Fab fragment of the invention.

[0165] Figure 2: SDS-PAGE gel with native and reduced samples of murine and humanized 11B6 antibodies.

[0166] Figure 3: Association phases after binding of test antibodies11B6 to hK2 on a chip.

[0167] Figure 4: Dissociation phases of 11B6 deteste antibodies.

[0168] Figure 5: Biodistribution of humanized antibodies labeled with 177Lu.

[0169] Figure 6: Exemplary SPECT image showing binding of 177Lu-labeled h11B6 to mouse prostate tumor.

[0170] Figure 7: Percentage uptake of 177Lu-labeled h11B6 and m11B6 in tumor and bone.

[0171] Figure 8: Percent uptake ratio per gram of 177Lu-labeled h11B6 and m11B6 in tumor to bone.

[0172] Figure 9: Kinetics of (a) humanized 11B6 antibody and (b) murine 11B6 antibody.

[0173] Figure 10: Elimination from blood of 177Lu-labeled h11B6 and m11B6.

[0174] Figure 11: Representative photographs of tumor size (top image) and after (bottom image) treatment with 177Lu-11B6.

[0175] Figure 12: Summary of the effect of (a) single ‘D’ radioactivity amount of 177Lu-11B6, (b) double ‘2 x D’ radioactivity amount of 177Lu-11B6 and (c) control treatment on tumor size in LNCaP xenografts.

[0176] Figure 13: (a) Tumor growth data and (b) a SPECT image for a mouse bearing LNCaP xenografts treated with a single dose of 177Lu-11B6.

[0177] Figure 14: Tumor volume as a function of days post-injection for (a) animals receiving 177Lu-labeled h11B6 antibody according to the invention, (b) animals receiving 177Lu-labeled “isotype control” non-specific IgG antibody and (c) animals receiving NaCl alone. Treatment was administered on Day 0. Animals were euthanized in the event of the following occurrences: large tumor volumes (diameter > 14 mm); large weight loss (weight loss > 15% compared to initial weight); negatively affected general condition or a combination of all three of these parameters (the numbers on the right are the ID numbers of each animal).

[0178] Figure 15: Kaplan-Meier curve for the three treatment groups shown in Figure 14. Solid line: 177Lu-h11B6; dashed line: 177Lu-labeled nonspecific IgG “isotype control” antibody; dotted line: NaCl

[0179] The following examples are intended to demonstrate particular embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the examples that follow represent techniques found by the inventor to function well in the practice of the invention, and may thus be considered to constitute specific modes for its practice. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made to the specific embodiments that are disclosed and still obtain an identical or similar result without departing from the spirit and scope of the invention.

EXAMPLES Example 1 - Cloning of 11B6 from a hybridoma cell line Reagents

[0180] A monoclonal antibody-producing hybridoma cell line 11B6 was used for mRNA extraction and production of antibodies that were further affinity purified for protein sequencing (Vaisanen et al., 2004).

[0181] Restriction enzymes, FastAP and T4 DNA ligase were from Fermentas, primers from the University of Turku, Department of Biotechnology (WO252) and from Thermo Scientific. DNA purifications were done with gel extraction and PCR purification kits from Qiagen.

mRNA Extraction and cDNA Synthesis

[0182] mRNA was extracted from MAb 11B6-producing hybridoma cells (5E6 cells) with QuickPrep Micro mRNA purification kit (Amersham Biosciences) and cDNA synthesis from mRNA was performed with Applied Biosystems “High-capacity cDNA archive kit” according to the instructions.

Amplification of Antibody Genes from cDNA

[0183] N-terminal sequences of the heavy (H) and light (L) chains of purified mAb 11B6 were determined by Edman degradation at the University of Helsinki protein sequencing facility. The light chain sequence was DIVLTQSPAS [SEQ ID NO: 16] and the heavy chain sequence DVQLQESGPG [SEQ ID NO: 17]. Amino acid comparison with the IMGT database identified the genes: IGKV3 and IGHV3, respectively. Complementary regions for PCR forward (degenerate) primers were designed based on the DNA sequences (found by NCBI BLAST) encoding the N-terminal amino acids. The reverse primer used to clone the heavy chain was designed to bind to CH1. In the case of the light chain, two reverse primers were used; the one used in the first PCR binds to CL and the other, used in the second PCR, to the border of VL and CL. All primers also contain the restriction enzyme recognition sites necessary for cloning (underlined below).

[0184] The light chain forward primer was SfiI_DIVLTQSPAS [SEQID NO: 16]:(5'-TTACTCGCGGCCCAGCCGGCCATGGCGGAYATHGTRYTVACNCA RTCTCC-3'; [SEQ ID NO: 18]) and reverse primers WO252(5'-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3', XbaI;

[0185] The heavy chain forward primer was NotI_DVQLQESGPG[SEQ ID NO: 17](5’-TGCTGCTGGCGGCCGCTCCAGCCATGGCTGAYGTVCARCTKCAG GAGTCDGG-3’; [SEQ ID NO: 21]) and the reverse primer was asCH1_SacI(5’-CGCCACCAGAGCTCTCACAATCCCTGGGCACAATTTTC-3’; [SEQ ID NO: 22]).

[0186] The VL+CL fragment was amplified in a PCR reaction comprising 100 ng of cDNA as template, 0.2 mM dNTPs, 0.5 μM SfiI primers DIVLTQSPAS [SEQ ID NO: 16] and WO252, 1x Phusion HF buffer and 0.6 U Phusion DNA polymerase (Finnzymes). Amplification was performed by a protocol of 98°C 30 sec, 30 cycles of 98°C 7 sec, 50°C 20 sec, 72°C 20 sec and final extension of 72°C 10 min. After sequencing the PCR product and finding the VL-CL boundary sequence, PCR for cloning itself was performed again from cDNA in a reaction as above except with primers SfiI_DIVLTQSPAS [SEQ ID NO: 16] and CpoI_JK2 to clone only the VL part. Amplification was performed by a protocol of 98°C 30 sec, 10 cycles of 98°C 7 sec, 60°C 20 sec, 72°C 20 sec, 25 cycles of 98°C 7 sec, 56°C 20 sec, 72°C 20 sec and a final extension of 72°C 10 min.

[0187] The VH+CH1 fragment was amplified in a reaction like that of VL except with primers NotI_DVQLQESGPG [SEQ ID NO: 17] and asCH1_SacI. The amplification protocol was 98°C 30 sec, 30 cycles of 98°C 7 sec, 64°C 20 sec, 72°C 20 sec and final extension of 72°C 10 min.

Cloning

[0188] The correct size products were purified from preparative agarose gel. VL was digested with SfiI and CpoI, VH+CH1 with SacI and NotI. The recipient vector pAK400 5404 FAb lch (modified from pAK400, Krebber et al., 1997) was digested separately with both enzyme combinations, the fragments dephosphorylated with FastAP and purified from the preparative gel.

[0189] Digested 11B6 VL and the corresponding vector fragment were ligated with T4 DNA ligase and transformed by electroporation into Escherichia coli XL1-Blue cells (Stratagene) to produce pAK400-11B6-VL vector. The ligation product of SacI+NotI digested VH+CH1 and the vector fragment was named pAK400-11B6-VH+CH1. Correct clones were confirmed by DNA sequencing and sequence comparison with the original protein sequences and with antibodies found in the database (BLAST search).

[0190] To construct the full-length 11B6 Fab, both previously made constructs were digested with NotI and SacI. The pAK400-11B6-VL vector was used as the recipient vector into which VH+CH1 from the pAK400-11B6-VH+CH1 vector was inserted. Ligation and transformation were performed as indicated above. The constructed pAK400 11B6 Fab lch vector was confirmed with restriction enzyme analysis. 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, 2nd edition, 1995Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun 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):35-55Lilja H, Christensson A, Dahlén U, Matikainen MT, Nilsson O, Pettersson K, Lovgren T. (1991) Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin Chem. 37(9):1618-25Pajunen 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):192-202Vaisanen 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

Example 2 - Humanization of antibody 11B6

[0191] The variable domain of the murine anti-hK2 antibody 11B6 was humanized using the CDR grafting method. In this approach, the complementarity determining regions (CDRs) of the murine antibody were grafted into the heavy and light variable domain frameworks. Additionally, residues in CDR regions, the residues at critical positions in the framework regions were retained murine-identical rather than becoming human-identical to keep the conformation of grafted CDR loops as similar as possible to their conformation in the murine parental antibodies.

[0192] The Kabat numbering scheme (Kabat et al., 1991) is used throughout this description.

Homology Modeling

[0193] A homology model of the murine 11B6 antibody was generated using an automated antibody modeling web server (VAM; http://antibody.bath.ac.uk/index.html). The model was used to assess the significance of residues differing between the murine parental antibodies and the human immunoglobulin sequences used as frameworks for variable domain humanization, respectively, based on visual inspection.

Design of humanized 11B6 V-domain sequences VL Domain Design

[0194] The amino acid sequence of the murine 11B6 light chain variable domain was compared against the human immunoglobulin germline sequence database at NCBI using the ClustalW sequence alignment program. 11B6 VL was observed to have the highest similarity to the human germline B3 gene (IGKV4-1*01), the only member of the human VK4 family. With respect to the J segment encoding the C-terminal part of the variable domain sequence, human Jk2 was observed to be most similar to the corresponding region of murine 11B6.

[0195] The human B3 gene, together with the IGKJ2 sequence, was used as a framework for grafting the CDR loops (Fig. 1) from the murine parental 11B6 antibody light chain. A residue of murine origin (leucine) was introduced at position 4 of VL in place of the human-identical methionine. This Vernier zone residue (Foote and Winter, 1992) is located directly beneath the CDR1 and CDR3 loops of the light chain. At position 54 in CDR-L2, the human-identical arginine was used in place of the murine-identical valine. According to modeling, the residue at this position is unlikely to form a direct interaction with antigen; however, Arg54 appears to form a salt bridge with the negatively charged aspartate at position 60 in the human framework. The residue at position 24 in CDR-L1 was considered unlikely to be involved in antigen contact. Consequently, human-identical lysine was introduced at this position instead of murine-identical arginine.

VH Domain Design

[0196] The amino acid sequence of the murine 11B6 heavy chain variable domain was compared with the human immunoglobulin germline sequence database at NCBI using the ClustalW sequence alignment program. It was observed that VH 11B6 has the highest similarity to the human VH4 family member VH4-28. With respect to the J segment encoding the C-terminal part of the variable domain sequence, it was observed that human JH1 is most similar to the corresponding region of murine 11B6.

[0197] The human VH4-28 gene, together with the JH1 sequence, were used as a framework for grafting the CDR loops (Fig. 1) of the heavy chain of the murine parental 11B6 antibody.

[0198] Murine-identical asparagine and threonine residues were introduced at positions 27 and 30 of VH, respectively. Although they do not belong to CDR-H1 according to the definition of Kabat (Kabat et al., 1991; Fig. 1), they are classified as CDR residues by other CDR definition procedures, such as those of Chothia (1989). Residues 27 and 30 may affect the structure of the other parts of CDR-H1 and possibly participate in direct contacts 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 here murine-identical arginine was used instead of human-identical valine. At position 94, preceding the important CDR-H3 loop, a threonine residue derived from murine 11b6 was introduced instead of arginine identical to human VH4-28. Additionally, it was considered unlikely that the residue at position 60 in CDR-H2 is involved in antigen contacting. Therefore, human-identical asparagine was introduced at this position instead of murine-identical serine.

[0199] Genes encoding humanized 11B6 as Fab fragment, where the designed VH and VL domains were joined to the human constant domains CHI and CK, respectively, were purchased as a synthetic construct (Genscript, US). The genes were cloned into the pAK400Fab expression vector modified from pAK400 (Krebber et al., 1997) using SfiI restriction enzyme with recognition sites on both sides of the Fab cassette. The vector was transformed into XL-1 blue E. coli cells for expression of the humanized Fab fragment.

[0200] The sequences of the exemplary humanized 11B6 Fab fragment heavy chain and yeast variable regions of the invention are shown in Figure 1.ReferencesChothia, C., Lesk, A. M., Tramontano, A., Levitt, M., SmithGill, S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R., Colman, P. M., Spinelli, S., Alzari, P. M., and Poljak, R. J. (1989) Conformations of immunoglobulin hypervariable regions Nature, 342, 877-883Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S and Foeller, C. (1991) Sequences of Immunoglogical Interest, 5th edit., NIH, Bethesda, MDKrebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun 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):35-55Tramontano, A., Chothia, C. and Lesk, A. M. (1990) Framework Residue 71 is a Major Determinant of the Position and Conformation of the Second Hypervariable Region in the VH Domains of Immunoglobulins. J. Mol. Biol. 215, 175-182

Example 3 - Expression and purification of h11B6

[0201] HEK293 cells were expanded to 2 L of suspension culture in FreeStyle 293 Expression medium (Life Technologies). The cell density was, on the day of transfection, 1 x 106 cells/mL.

[0202] The nucleotide sequences encoding the component heavy or light chains (i.e., SEQ ID NO: 14 and 15, respectively) were codon optimized for expression in mammalian cells, synthesized, and cloned into IgG expression vectors. Plasmid DNA (expression vector) containing the nucleotide sequences for the heavy and light chains was then mixed with the transfection agent and incubated for 10 min at RT. The DNA-transfection agent mixture was slowly added to the cell culture while gently shaking the flask. The transfected cell culture was then incubated at 37°C, 8% CO2 on an orbital shaker platform rotating at approximately 135 rpm, for seven days.

[0203] The culture medium was harvested by centrifugation and filtered through 5 μm, 0.6 μm and 0.22 μm filter systems.

[0204] The antibodies were purified by Protein G chromatography and the buffer was changed to PBS pH 7.4 by dialysis; subsequently, the antibodies were concentrated by ultrafiltration.

[0205] The concentration was measured by absorbance.DNA: Light chain: p11B6VLhV1hk (4300 bp) quantity: 0.35 mgHeavy chain: p11B6VHhV1hIgG1 (4900 bp) quantity: 0.6 mg

[0206] DNA amounts were not optimized.

[0207] Transfection agent: proprietary (however, suitable transfection agents are readily commercially available, such as Xfect™ Transfection Reagent (Clontech), Lipofectamine (Life Technologies), FuGENE® HD Transfection Reagent (Promega), FreeStyle™ Max Reagent (Invitrogen), DEAE-dextran, polyethylenimine, and calcium phosphate).

[0208] Overall yield: 13.1 mg (~6.5 mg/L)

Example 4 - Characterization of h11B6: Affinity Study Objectives

[0209] The aim of the study was to investigate the binding kinetics between four variants of the 11B6 antibody and the hK2 antigen using the Surface Plasmon Resonance (SPR) technique in a Biacore apparatus.

[0210] To investigate the quality of the protein samples (antibodies and antigen), an SDS-PAGE gel was run prior to the SPR experiments.

[0211] In a Pre-Study, different parameters were investigated to find the appropriate conditions for the experiments in the Study.

[0212] In the Study, multiple binding measurements were performed for the four antibodies and the antigen. The association and dissociation rate constants (kon and koff) and the dissociation constants (KD) were calculated from the data obtained and reported here.

Reagent and Apparatus Information

[0213] The following solutions of the four antibodies and one antigen were supplied by Diaprost AB: m11B6 stock: a-ehk211B6 14.12013 PP, 3.41mg/mL: 0.9% NaCl, 100μL h11B6 stock: Innovagen Lot 90476.30 2013-0412, 1mg/mL: PBS pH 7.4, 320μL h11B6-DTPA stock: 0.2M Na-acetate pH 5.5, 0.9mg/mL, 340μL h11B6-DF stock: 5mg/mL gentisic acid in 0.2M ammonium acetate pH 5.5, 1.6mg/mL, 400μL hK2 stock: 26.6 μg/mL frakt 2 fr 7 SL + protein inhibitor 5/2-02 1% BSA

[0214] All samples were aliquoted and kept in a freezer at -20°C prior to analysis.

[0215] All binding experiments were performed on a CM4 chip in a Biacore 3000 apparatus. The chip and all reagents required for activation, immobilization, deactivation, binding and regeneration were purchased from GE Healthcare and used according to the manufacturer's directions.

SDS-PAGE (a) Description of the Experiment

[0216] Reagents supplied by Diaprost AB were run on a Novex TRIS-Tricine 10-20% acrylamide gel according to the manufacturer's instructions.

[0217] Two series of protein samples, native and reduced, were run simultaneously on the same gel together with a standard sample.

[0218] Each sample in the native series comprised: 1-1.3μg of protein, TRIS buffer pH 8.8, SDS and loading buffer.

[0219] Each sample in the reduced series comprised: 1-1.3μg of the protein, TRIS buffer pH 8.8, SDS and loading buffer and 0.04% v/v beta2-mercaptoethanol (the reducing agent).

[0220] Gel staining was performed in Coommasie brilliant blue solution of acetic acid, ethanol and water with corresponding proportions of 0.7, 3.0, 6.3.

[0221] Gel decolorization was performed in acetic acid, ethanol, and water solution with corresponding proportions of 0.7, 3.0, 6.3.(b) Results and Conclusions

[0222] The results are represented in Figure 2.

[0223] It is evident from these results that the antibody and antigen samples are of high quality and purity.

Affinity Study (a) Antigen immobilization on a CM4 chip

[0224] Activation of the CM4-2 chip was performed according to the manufacturer's guidelines for amine coupling using a mixture of EDC and NHS.

[0225] A solution comprising 2.96μg/mL of hK2 antigen (hK2 stock solution diluted in 10 mM NaAc buffer pH 3.8) was dropped onto the fc2-4 channels on the CM4-2 chip to immobilize the antigen on the chip. Flow rate: 5 μL/min, volume: 200 μL. RU Target < MW/1 0 Mw(hk2) = 25900 Da RU Target (hk2) < 2590

[0226] The fc1 channel was used blank.

[0227] The following immobilization has been reached: fc2 = 1104 RU fc3 = 731 RU fc4 = 688 RU

[0228] All channels (fc1-4) were blocked by ethanolamine after activation and immobilization.

[0229] These data demonstrate that adequate immobilization was achieved using 2.96μg/mL of antigen.(d) Investigation of the Association Phase

[0230] The association phase of the four antibodies to the CM4-2 chip was followed for 4-5 minutes when solutions of 5 different concentrations of each antibody (stock solutions diluted in HSP buffer) were flowed over the fc2-4 channels on the CM4-2 chip at a rate of 30μL/min.

[0231] The concentrations investigated for each antibody were: 100, 50, 25, 12.5 and 6.25 nM.

[0232] Additionally, association data were obtained from experiments in which the dissociation process was followed for 480 minutes.

[0233] In total, 18 individual association experiments were performed for each antibody.

[0234] The blank channel signal, fc1, has been subtracted for all data.

[0235] In figure 3, the association phases in the fc2 channel on the CM-2 chip are represented for each of the antibodies at the 5 different concentrations.

[0236] We observed that after 4-5 minutes, we were able to fit the data to the association processes.(e) Investigation of the Dissociation Phase

[0237] The dissociation phase was followed for 480 minutes for each of the antibodies after flowing a 50nM solution of the antibody for 5 minutes over the fc2-4 channels on the CM4-2 chip at a rate of 30μL/min (Figure 4).

[0238] The blank channel signal, fc1, is subtracted from all data used in dissociation rate constant calculations.

[0239] The data indicate that the dissociation processes are very slow. For all four antibodies, the signal in the fc4 channel was drifting and the dissociation process could not be followed in that channel.(f) Estimation of the Dissociation Rate Constant (koff)

[0240] The dissociation phase data were fitted and the dissociation rate constants (koff) were estimated (see Table 2).Table 2

[0241] Based on the two measurements made for each antibody, there does not appear to be a significant difference between the dissociation rate constants (koff) of the antibodies tested.(g) Estimation of the Association Rate Constant (kon)

[0242] To estimate the association rate constants, the dissociation rate constants (Table 2) were used in the fitted equations.

[0243] All adjusted data were used to calculate an average value of the association rate constant and standard deviation for each antibody (see Table 3).Table 3

[0244] Based on the 15-18 measurements made for each antibody, there does not appear to be a significant difference between the association rate constants (kon) of the antibodies tested.(h) Estimation of Dissociation Constant (kD)

[0245] The dissociation constants (KD) for each of the antibodies tested are presented in Table 4.Table 4

[0246] The dissociation constants (KD) are in the order of 10-12M for all four antibodies.

[0247] Although not statistically significant, the dissociation constant for the humanized antibody appears to be higher than that of the murine parental antibody.

[0248] Conjugation of the humanized antibody does not appear to noticeably affect affinity as the K D is not significantly different for h11B6-DTPA or h11B6-DFO. Summary The association processes are very fast for all four antibodies and the association rate constants (kon) are all on the order of 105 M-1 s-1 based on 15-18 experiments for each antibody. The dissociation processes are very slow and almost in the range of Biacore's technical limitations. The dissociation constants (koff) are all on the order of 10-5 s-1 based on two experiments for each antibody. The dissociation constants (K D ) are on the order of 10-12 M for all four antibodies.

Example 5 - Characterization of h11B6: Aggregation Executive Summary

[0249] Dynamic Light Scattering (DLS) studies were performed on 4 IgG variants to study their propensity to aggregate. DLS results show that all constructs are of reasonable size (200 kDa or slightly above 200 kDa assuming a spherical protein) and exhibit little or no aggregation.

Objective

[0250] To characterize four IgG constructs with respect to oligomeric state using Dynamic Light Scattering. Insulin was used as a control.

Results Dynamic Light Scattering

[0251] Phosphate buffered saline (PBS pH 7.4) was filtered through a 0.22 micron filter. The protein obtained was diluted to 0.1 mg/mL in PBS pH 7.4. Dynamic light scattering was measured at 20°C on duplicate samples using the Malvern APS instrument. Each sample was measured three times. The dilution buffer was used as a control to ensure that the buffer was reasonably free of dust and aggregates, figure 1c. All samples could be measured reliably using the number distribution function. The mean radius of the most abundant species was calculated together with the polydispersity of the species. The mean mass distribution of this species was also calculated, see table 5. Table 5: Dynamic Light Scattering Data Derived from Size Distribution Polydispersity = Standard deviation of radius / Mean radius x 100%

[0252] The insulin control (4 mg/mL 20 mM Na2HPO4, 10 mM Na3EDTA) has an average radius of 2.8 nm which is about 37 kDa. Insulin is known to form hexamers of about 35 kDa in solution. 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 reasonably close to the molecular weight of 150 kDa for IgG molecules, meaning that most samples consist essentially 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 and this makes it difficult to separate monomers and dimers, but easy to separate large aggregates of monomers or monomers from hexamers (as in the case of insulin).

Conclusions

[0253] Dynamic Light Scattering shows that all constructs are of reasonable size and have little or no aggregation. The size distributions for all four constructs are in overlap (data not shown).

Example 6 - Characterization of h11B6: In vivo biodistribution

[0254] This study compares the in vivo biodistribution of murine 11B6 and human 11B6 when labeled with 177Lu.

Material and Methods Materials

[0255] 177Lu was purchased from Mallinkrodt Medical BV, Petten, Netherlands.

[0256] All chemicals were obtained from Sigma Aldrich and buffers were prepared in-house using analytical grade water (unless otherwise specified).

[0257] The parental murine antibody m11B6, with specificity for human parakallikrein 2, was obtained from the University of Turku, Finland.

[0258] m11B6 heavy chain [SEQ ID NO: 23]:DVQLQESGPGLVKPSQSLSLTCTVTGNSITSDYAWNWIRQFPGNRLE WMGYISYSGSTTYSPSLKSRFSITRDTSKNQFFLQLNSVTPEDTATYF CATGYYYGSGFWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVT LGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVP SSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFI FPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQ PREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNG QPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEG LHNHHTEKSLSHSPGK

[0259] m11B6 light chain [SEQ ID NO: 24]:DIVLTQSPASLAVSLGQRATISCRASESVEYFGTSLMHWYRQKPGQP PKLLIYAASNVESGVPARFSGSGSGTDFSLNIQPVEEDFSMYFCQQT RKVPYTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDE YERHNSYTCEATHKTSTSPIVKSFNRNEC

[0260] A humanized homologous antibody, h11B6, was produced as described in Examples 2 and 3 above (see Figure 1).

[0261] For in vivo studies, the prostate carcinoma cell lines LNCaP expressing hK2 (ATCC, Manassas, VA, USA) and DU145 (ATCC, Manassas, VA, USA) were used. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and PEST (penicillin 100 IU/mL and 100 μg/mL streptomycin). Cells were maintained at 37°C in a humidified incubator with 5% CO2 and were detached with trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Thermo Scientific). Matrigel matrix from BD-biosciences (San-Jose, CA, USA) was used to perform LNCaP cell xenografts. NMRI-Nu (Charles River) and Balb/c-Nu (in-house bred) mice were inoculated with both cell lines.

Conjugation and Radiolabeling

[0262] Conjugation of CHX-A’’-DTPA with 11B6: Solutions of murine and humanized 11B6 demAbs in PBS were adjusted to pH 9.2 using 0.07 M sodium borate buffer, before being concentrated in an Amicon Ultra-2 centrifugal filter (2 mL, 100 K). The resulting protein solution was then conjugated with CHX-A’’-DTPA chelator (Macrocyclics, USA) at a 3:1 chelator to antibody molar ratio at 40°C. The reaction was terminated after 4 h and CHX-A’’-DTPA-11B6 (DTPA-11B6) was separated from 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 antibodies were eluted with 1 mL of ammonium acetate buffer.

[0263] Radiolabeling of DTPA-11B6: Humanized murine DTPA-11B6, in ammonium acetate buffer pH 5.5, was mixed with a predetermined amount of 177LuCl3. A final activity of 0.5-0.6 MBq per individual was used for biodistribution or a final activity of 18-20 MBq per individual for SPECT studies. After incubation at room temperature for 2h, the labeling was terminated and purified on a NAP-5 column, equilibrated with PBS.

Animal Studies

[0264] All animal experiments were carried out in accordance with national legislation on the protection of laboratory animals.

[0265] Immunodeficient male nude mice, NMRI-Nu, (6-8 weeks old) and Balb/c-Nu, were used for this study. All mice were xenografted with LNCaP or DU145 cells in their left or right flank, 8-10 million cells, in 100 μL of growth medium and 100 μL of Matrigel.

Biodistribution Studies

[0266] Biodistribution studies were performed on h11B6 in11B6.

[0267] Six groups (n=4) of mice were injected intravenously with 20 μg of 177Lu-labeled h11B6 or 20 μg of m11B6. Animals were sacrificed 24 h after injection, 48 h after injection, and 72 h after injection, and organs of interest were analyzed with an automated NaI(Tl) well counter with a 3-inch NaI(Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland).

[0268] The tissue uptake value, expressed as a percentage of injected dose per gram of tissue (%AI/g), was calculated as: %AI/g = (tissue radioactivity/injected radioactivity)/organ weight x 100where, for intravenous injections:Injected radioactivity = Average radioactivity in control syringes - radioactivity in used syringe - radioactivity in tail

[0269] The organs were also weighed after dissection.

Kinetic Data

[0270] The time-%DI curve is represented as a straight line DI(t)=k*t+m up to 48. Based on the data from the second and third time points (48 and 72h respectively), a monoexponential curve (DI(t)=DI(0)e-Àt) is applied for the time interval [48,~[. If, however, lambda becomes a negative value, i.e. that DI increases between 48 and 72h, the time-DI curve in this time interval is modeled as a straight line, and it is assumed that the drug is retained in the organ from 72 hours onwards. To obtain the time-activity curves, the physical half-life is applied.

[0271] Note - in some figures, DI is designated ”IA”; these expressions are used interchangeably here.

Results Biodistribution of humanized 177Lu-11B6

[0272] The biodistribution of 177Lu-h11B6 is shown in Figure 5.

[0273] The antibody accumulates rapidly in the LNCaP tumor within 24 hours and radioactivity remains elevated at 72 hours.

[0274] Initially, elevated levels of h11B6 are also evident in the blood and kidneys, which decrease over the next 48 hours as the antibody is eliminated from the body. Such biokinetics are an inevitable and expected consequence of intravenous injection of any radiolabeled antibody.

[0275] All other organs, such as bone and muscle, have low levels of radioactivity.

[0276] These data demonstrate that 177Lu-h11B6 can be effectively targeted to prostate cancer cells in vivo.

[0277] Figure 6 shows an exemplary SPECT image, in which the binding of 177Lu-h11B6 to LNCaP tumor cells in a xenograft mouse is clearly evident. 177Lu-h11B6 exhibits an unexpectedly better therapeutic ratio

[0278] Comparison of biodistribution data of humanized 177Lu-11B6 with that of the murine parental antibody (177Lu-m11B6) revealed an unexpected and advantageous difference.

[0279] As shown in Figure 7, the uptake of 177Lu-h11B6 by the LNCaP tumor is elevated by approximately 20% at 72 h post injection compared to 177Lu-m11B6. Concomitantly, the uptake of 177Lu-h11B6 by healthy bone is reduced by approximately 40% at 72 h post injection compared to 177Lu-m11B6.

[0280] Figure 8 depicts data expressed as a ratio of antibody uptake in tumor versus healthy bone at 24, 48, and 72 hours post injection. At 72 hours, this ratio is markedly increased for the humanized 11B6 antibody.

Dosimetry Calculations

[0281] Calculation of absorbed doses provides a more sophisticated measure of differences in the kinetics of the humanized antibodies of the invention relative to that of the murine parental 11B6 antibody.

[0282] The absorbed dose was calculated according to the MIRD scheme D(r^rs)=Ã(rs) S(rp^rs), where à is the total number of disintegrations in a source organ, and S is the absorbed dose per unit disintegrations (see Bolch et al., 2009, J. Nucl. Med. 50:477-484, the disclosures of which are incorporated herein by reference). à was calculated as the time integral of the time-activity curve. The S factor was based on mouse-specific Monte Carlo simulations using the Moby-phantom (see Larsson et al., 2011, Acta Oncol. 50:973-980 and Keenan et al., 2010, J. Nucl. Med. 50:471-476). To obtain the total absorbed dose, all organs were considered to be source as well as target sources.

[0283] The calculated absorbed dose values for different tissues are presented in Table 6.Table 6

[0284] As can be seen from Table 6, the tumor absorbed dose increases from 1.21 Gy/MBq for m11B6 to 2.2 Gy/MBq for h11B6, i.e. an increase of 80%. The tumor absorbed dose ratios to bone marrow increase from 3.9 for m11B6 to 5.6 for h11B6, approximately 40%. Here the increased therapeutic efficacy for h11B6 compared to m11B6 is shown and indicates that higher tumor absorbed doses can be given with less normal organ toxicity.

Elimination of Antibody from the Blood

[0285] Analysis of blood levels for humanized and murine 11B6 antibodies is shown in Figure 10.

[0286] The results suggest that h11B6 may be cleared from the blood slightly faster than the murine 11B6 antibody. If so, such an increased clearance rate for the humanized antibody may also be of therapeutic benefit from a safety perspective, potentially allowing for the administration of higher activities.

[0287] An increased elimination rate may also be beneficial for external imaging.

Conclusions

[0288] The results of this study demonstrate the following: the humanized 11B6 antibody, 177Lu-h11B6, effectively targets prostate tumors in vivo; the humanized 11B6 antibody exhibits an unexpectedly better therapeutic ratio than its parent murine antibody (as determined by the ratio of uptake in tumors to uptake in healthy bone); and the humanized 11B6 antibody can be cleared from the blood slightly faster than the murine 11B6 antibody.

[0289] Taken together, these findings provide compelling evidence for the enhanced therapeutic efficacy of humanized 11B6 antibodies in the treatment (and diagnosis) of prostate cancer.

[0290] Since the humanized and murine antibodies are directed to the same antigen (namely, human kallikrein 2), the calculated difference in the ratio of uptake in tumor to healthy bone marrow cannot be readily predicted or explained (especially since the humanized antibody appears to exhibit a lower affinity for the target hK2 antigen compared to the murine parent antibody; see Example 4).

[0291] The difference in relative uptake in tumor compared to healthy bone between the humanized and murine 11B6 antibodies is of considerable importance since this comparison provides a measure of the therapeutic ratio. A higher value for this ratio (as is evident for the humanized 11B6 antibody) is indicative of a better therapeutic antibody. In particular, a higher therapeutic ratio means that larger absorbed doses of therapeutically radiolabeled h11B6 can be administered to achieve a better therapeutic effect (since the binding of the humanized antibody to healthy tissue and organs is much lower than for the murine antibody). The higher ratio also indicates that h11B6 will be better than the murine antibody for diagnostic purposes (since it equates to a lower signal to noise ratio, allowing visualization of smaller tumors, including metastases).

[0292] In conclusion, the data demonstrate that humanization of the 11B6 antibody offers an improved possibility for early diagnosis and an unexpectedly higher therapeutic efficacy in the treatment of prostate cancer.

Example 7 - Demonstration of Diagnostic and Therapeutic Efficacy

[0293] The objective of this study was to confirm the utility of 11B6, an mAb that specifically targets an epitope within the catalytic cleft of hK2, as a vehicle to deliver highly toxic radionuclides specifically to prostate cancer growth sites. In this proof-of-concept study, we labeled the murine parental 11B6 antibody with 177Lu, a low-energy beta particle that also employs gamma emission, enabling SPECT imaging.

Materials and methods Materials

[0294] 177Lu was purchased from Mallinkrodt Medical BV, Petten, The Netherlands. The CycloneTM Storage Phosphor System and OptiQuantTM image analysis software (Perkin Elmer, Wellesley, MA, USA) were used to measure radioactivity on ITLC (instant thin layer chromatography) strips (Biodex, US) to determine labeling kinetics and radiochemical purity. All chemicals were obtained from Sigma Aldrich and buffers were prepared in-house using analytical grade water unless otherwise stated. mAb 11B6 is an antibody specific for human kallikrein 2 with an affinity for this antigen of about 1.2 nM; see Figure 1 (obtained from the University of Turku, Finland). For in vivo studies, the hK2-expressing LNCaP prostate carcinoma cell lines (ATCC, Manassas, VA, USA) were used. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and PEST (penicillin 100 IU/mL and streptomycin 100 μg/mL). Cells were maintained at 37°C in a humidified incubator with 5% CO2 and were detached with trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Thermo Scientific).

Conjugation and Radiolabeling

[0295] Conjugation of CHX-A’’-DTPA with 11B6: A solution of demAb 11B6 in PBS was adjusted to pH 9.2 using 0.07 M sodium borate buffer. The sample was concentrated on an Amicon Ultra-2 centrifugal filter (2 mL, 100 K). The protein solution was conjugated with the chelator CHX-A’’-DTPA (Macrocyclics, USA) at a 3:1 chelator to antibody molar ratio at 40°C. The reaction was terminated after 4 h and CHX-A’’-DTPA-11B6, designated DTPA-11B6 from here on, was separated from the free chelate by size exclusion chromatography on a NAP-5 column (GE Healthcare) equilibrated with 20 mL 0.2 M ammonium acetate buffer, pH 5.5.

[0296] Radiolabeling of DTPA-11B6: DTPA-11B6 in ammonium acetate buffer pH 5.5 was mixed with a predetermined amount of 177LuCl3. After incubation at room temperature for 2 h, the labeling was terminated and purified on a NAP-5 column equilibrated with PBS. The labeling efficiency and labeling kinetics were monitored with ITLC strips eluted with 0.2 M citric acid. In this system, the radiolabeled conjugate remains on the parent line, while Lu-177 migrates with the solvent front. The radioactivity distribution was determined with a PhosphorImager system (Perkin Elmer, Wellesley, MA, USA) using Optiquant as quantification software (Perkin Elmer, Wellesley, MA, USA).

Animal Studies

[0297] All animal experiments were performed in accordance with national legislation on the protection of laboratory animals. The animal study was approved by the local Ethics Committee for Animal Experimentation. Male immunodeficient nude mice, NMRI, (6-8 weeks old) purchased from Taconic Europe (Bomholt, Denmark) were used for this study.

[0298] Xenografts of LNCaP prostate carcinoma cells expressing hK2 were subcutaneously implanted into the right flank and/or left flank at approximately 10*106 cells per injection.

[0299] Animals that developed LNCaP tumors were divided into groups and injected with the therapeutic agent 177Lu-DTP-11B6 or a control, see Table 7 below:Table 7

[0300] All animals included were continuously measured and weighed at an interval of 3-4 days.

[0301] Initially some animals showed lower activity (8MBq) of 177Lu-DTPA-11B6 for investigation of the localization of the therapeutic agent using SPECT. One mouse from group 8 was also studied with SPECT. Organs were removed from these animals and an automated NaI(Tl) well counter with a 3-inch NaI(Tl) detector (1480 WIZARD, Wallac Oy, Turku, Finland) was used to quantify radioactivity in these tissue samples.

[0302] To study the effect on the bone marrow, blood samples (10 μL) were collected regularly. Blood samples were collected twice weekly for 8 weeks after injection and leukocyte, erythrocyte and platelet counts were analyzed on a Medonic Cell Analyzer-Vet CA530 Vet (Boule Medical, Stockholm, Sweden). At the time of blood collection, the weight and physical condition of the animals were monitored. Toxicity was assessed by monitoring the animals for body weight loss, decline in general condition and hematologic toxicity.

[0303] The tumor volume was measured with a caliper. The length l, w and thickness t were measured and the volume was calculated.

Therapy Planning

[0304] Based on the relationship between absorbed dose and biological effect in bone marrow in rats subjected to radioimmunotherapy with 90Y and 177Lu (see Larsson et al., 2012, Med. Phys. 39(7):4434-43), it can be estimated that the LD50 for bone marrow would be in the order of 12 Gy. In the literature, LD50 for acute irradiation of rats and mice are the same, about 9 Gy (for example, see Radiobiology for the radiologist, Hall & Giacca (Eds), 2006, 6th edition).

[0305] The therapies were then designed based on the assumption of a tolerable absorbed dose of 12 Gy for the bone marrow. Then, from the dosimetry calculations, the activity corresponding to this absorbed dose was calculated.

[0306] Corresponding doses/activities were used for the controls.

Results Tumor Reduction in Animals

[0307] Figure 11 shows how the tumor in one of the mice (visible on the animal's flank, under the skin) decreases in volume after treatment.

Radioimmunotherapy Results

[0308] Figure 12 presents the results for the study groups with activities administered (a) D, (b) 2xD and (c) a control group (where D = 26.7 MBq).

[0309] There is a clear trend towards a decrease in tumor volume in both treatment groups. The onset of tumor reduction is seen within a few days after injection of 177Lu-m11B6. In the control group there is an increase in tumor volume after injection of NaI solution.

[0310] Figure 13(a) presents the results for one of the mice in the group injected with activity A. Here, the tumor grows steadily from day one to day six when 177Lu-m11B6 activity A is administered. After treatment, a rapid decrease in tumor volume is observed.

[0311] In the SPECT study (8 days after injection) the tumor volume is shown with activity still present; see Figure 13(b).

Conclusion

[0312] The present study with exemplary 177Lu-m11B6 antibody clearly demonstrates the therapeutic efficacy of hK2-directed antibodies against prostate cancer tumors in vivo.

Example 8 - Therapeutic Efficacy of a 177Lu-Labeled Humanized 11B6 Antibody Exemplary of the Invention in Prostate Cancer Xenografts Materials and methods Antibodies, Conjugation and Radiolabeling

[0313] Antibodies: Exemplary humanized monoclonal antibody 11B6 (IgG1/kappa, transiently expressed in HEK 293 cells), comprising a heavy chain according to SEQ ID NO: 12 and a light chain according to SEQ ID NO: 13, was provided by Innovagen AB, Lund (1 mg/mL in PBS pH 7.4, Lot No. 90476.30). A nonspecific antibody was used as isotype control (mouse serum IgG antibody, Sigma I-8765).

[0314] Conjugation: The exemplary nonspecific control IgG antibody for h11B6 was conjugated to the chelator CHX-A’’-DTPA (Macrocyclics, USA) as follows: a solution of the antibody was concentrated in an Amicon Ultra-2 centrifugal filter (2 mL, 100 K) and was later adjusted to pH 9.2 using 0.07 M sodium borate buffer (Sigma Aldrich).

[0315] Coupling of the chelating compound CHX-A’’-DTPA to the protein solution at a molar ratio of approximately 3:1 (chelator to antibody) was performed similarly to the method previously described (see Almqvist et al). The coupling efficiency, i.e. the number of chelators obtained per antibody, can be determined by a spectrophotometric method (Pippin et al) but was not analyzed in this study. However, coupling should preferably not exceed 3 chelators/antibody to avoid protein damage. The chelator was added to the protein and the solution was incubated with gentle shaking at 40°C.

[0316] The reaction was terminated after 4 h and CHX-A”-DTPA-h11B6, designated as DTPA-h11B6, was separated from free chelate by size exclusion chromatography on a NAP-5 column (GE Healthcare) equilibrated with 20 mL 0.2 M ammonium acetate buffer (Sigma Aldrich), pH 5.5. The h11B6 conjugate was eluted with 1 mL ammonium acetate buffer and aliquoted samples were stored at -20°C.

[0317] Conjugation of the control IgG antibody was controlled in a similar manner as above.

[0318] Radiolabeling: h11B6 conjugate or control IgG antibody (typically 200-300 μL of ~1 μg/μL in 0.2 M ammonium acetate buffer pH 5.5) was mixed with a predetermined amount (~200-300 MBq) of 177LuCl3 (IDB Holland) and incubated at room temperature for 1.5-2h. After incubation, the labeling was terminated and purified on a NAP-5 column (GE Healthcare), equilibrated with PBS (Thermo Scientific). The labeling efficiency was monitored with flash thin layer chromatography (Biodex, USA), eluted with 0.2 M citric acid (Sigma Aldrich). In this system, the radiolabeled conjugate remains in the source line, while free 177Lu migrates with the solvent front. Radioactive distribution was determined with a Cyclone Storage Phosphor System using Optiquant quantification software (both from Perkin Elmer).

[0319] Radiolabeling of the control IgG antibody was performed in a similar manner to that described above.

Study of Therapy

[0320] Cell Lines: LNCaP (hK2+) were purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 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 at 37°C in a humidified incubator at 5% CO2 and were detached with trypsin-EDTA solution (Thermo Scientific).

[0321] All animal experiments were performed in accordance with national legislation on the protection of laboratory animals and with the approval of the Ethics Committee for Animal Research (Lund University, Sweden). In-house bred male immunodeficient Balb/c nude mice (6-8 weeks old) were used. Mice were xenografted with LNCaP cells into their right flank by s.c. injection (8-10 million cells) in 100 μL growth medium and 100 μL Matrigel (BD Matrigel TM Basement Membrane Matrix Growth Factor Reduced, Phenol Red Free, Cat No 356231). Mice with established tumors with a diameter of at least ~3 mm were included in the study and divided into the three groups described below in Table 8. Animals received i.v. injections into the tail vein. The activity level of 20 MBq was chosen because doses in this quantity were used in a study with m11B6, showing good therapeutic effect (see Example 7).

[0322] Table 8: Three groups of animals were comprised: one group injected with 177Lu-h11B6, one with nonspecific 177Lu-mAb (to show the specificity of h11B6) and one with NaCl (as a control group).

[0323] Therapeutic efficacy was assessed by repeated measurement of tumor size using a caliper. Tumor volume was calculated by measuring the length (L) and width (W) of the tumor and then calculating the volume V as 0.5 x L x W x W.

[0324] Additionally, hematologic measurements (white blood cell, red blood cell, platelet, and hemoglobin counts) and weight measurements were performed repeatedly for all animals to identify any potential hematologic toxicity and to monitor the general condition of the animals. It is especially important to monitor hematologic toxicity when evaluating radioimmunotherapy since the radioactivity will be distributed in the blood and eventually reach the bone marrow, where the blood stem cells are located.

[0325] Mice that developed a tumor with a length/width exceeding 14 mm, or a weight loss exceeding 15% compared to initial weight, or had an otherwise negatively affected general condition, or had a combination of all three of these parameters, were euthanized in accordance with ethical guidelines.

Results Therapeutic Efficacy Assessment

[0326] As shown in Figure 14(a), administration of the exemplary humanized 11B6 antibody of the invention (177Lu-h11B6) prevented tumor growth in the mice (and resulted in a pronounced reduction in tumor volume in all but one animal tested). In contrast, tumors continued to grow rapidly in mice treated with the control IgG antibody (see Figure 14b) or NaCl (see Figure 14c).

[0327] The individual animal data presented in Figure 14 are summarized as Kaplan-Meier curves in Figure 15. Administration of 177Lu-h11B6 produced a marked increase in the survival rate of mice after the end of the experiment, with over 80% of the animals still alive after the end of the experiment 60 days after injection (compared to 0% survival in the two control groups).

Hematological Toxicity Assessment

[0328] Evaluation of leukocyte, erythrocyte, platelet, hemoglobin and weight counts did not reveal any toxicity effects of 177Lu-h11B6 administration (data not shown).

Discussion

[0329] The results of this study reveal a significant therapeutic effect of 177Lu-h11B6 treatment in the prostate cancer xenograft model.

[0330] The activity administered to the mice (20 MBq) corresponds to a bone marrow absorbed dose of approximately 10 Gy, which was well tolerated by these animals. Even at this low activity of 20 MBq, a large therapeutic effect was observed. As previously estimated, a tumor absorbed dose of at least 60 Gy can be expected.

[0331] No indications of hematologic toxicity were observed.ReferencesAlmqvist 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.Evaluation of a maleimido derivative of CHX-A” DTPA for site-specific labeling of Affibody moleculesPippin CG et al. Spectrophotometric method for the determination of a bifunctional DTPA ligand in DTPA-monoclonal antibody conjugates. Bioconjug Chem. 1992; 3:342-5.

Example 9 - Dosimetry Planning and Treatment of Prostate Cancer in a Patient with Radionuclide Therapy

[0332] For radionuclide therapy (RNT), the radiation source is distributed throughout the body and the radioactivity is typically administered systemically as a radiopharmaceutical. The distribution of radioactivity depends on the amount of radiopharmaceutical that accumulates over time in different tissues, which varies between patients (1).

[0333] RNT treatment should be based on a prescribed absorbed dose (2). Then, a pre-therapy study should first be performed using a tracer amount of the radiopharmaceutical and the absorbed doses per tumor and organ should be determined. Typically, this information is expressed as a factor describing the absorbed dose per organ per unit of administered activity, in units of mGy/MBq; DPT(organ).

[0334] If therapeutic administration is then carried out under identical conditions, this factor can be used to determine the activity that is necessary to administer to deliver a prescribed absorbed dose to a given organ, tissue or tumor (4,6).

[0335] In the case of treatment of prostate cancer with radiolabeled h11B6 antibodies, a pre-therapy study should be based on 111In imaging with 111In-h11B6. 111In is most suitable for quantitative imaging (planar/SPECT) when 177Lu is the therapeutic radionuclide. When DPT(organ) is determined, therapy can be administered with an AT therapy activity giving a prescribed therapy effect. During therapy, the activity distribution and corresponding dose rate should be calculated based on imaging to obtain the actual absorbed dose of therapy given to tumor and normal organs, necessary for treatment evaluation.

[0336] In the case of therapy where the level of toxicity in the bone marrow is reached as a result of treatment planning, then bone marrow support will be necessary and the time for stem cell reinfusion has to be determined based on dosimetry calculations for the bone marrow cavity.

[0337] In summary, the following treatment regimen should be appropriately planned: Pre-Therapy Dosimetry Study 1. Injection of 111In-labeled h11B6 (200-300 MBq) 2. Blood sampling - activity concentration in blood and plasma determined in the first week. 3. Imaging (SPECT/Planar) for 1 week (7 times) 4. Organ dosimetry based on LundaDose scheme (3) 5. Activity of therapy determined limited by specified absorbed dose for radiosensitive organs such as bone marrow (2-3 Gy), kidneys (20-30 Gy) and liver (12-36 Gy). Therapy including Intra-therapy Dosimetry 1. Administration of 177Lu-labeled h11B6 (based on pre-therapy dosimetry) 2. Blood sampling - activity concentration in blood and plasma 3. Imaging for 1 week (6 times) 4. Organ dosimetry => Verification of absorbed dose of prescribed therapy.

Specific Comments on Dosimetry

[0338] Cumulative activity is the number of decays that occur in a given region over a period of time. The unit is Bq s or Bq h. When ionizing radiation travels through matter, it interacts and deposits energy. The energy transmitted is the sum of all the energy deposits in a given volume. The absorbed dose is the ratio of the average energy transmitted to the mass of the volume. The unit of absorbed dose is Gray (Gy), 1 Gy is equal to 1 J/kg.

[0339] From the activity values in a tissue at different times, the cumulative activity is determined by integration, and the average absorbed dose can be determined. Activity measurements are made using planar imaging for whole-organ dosimetry. Quantitative SPECT/CT allows dosimetry to be performed on smaller volumes using voxel-based methods.

[0340] From the 3D distribution of activity concentration values, the distribution of the absorbed dose rate can be calculated using so-called “point dose kernels” or voxel S values, describing the pattern of energy deposition around a point source located in water (or bone). This method assumes that the anatomical region is homogeneous in terms of density, such as soft tissues within the trunk. For regions of the body where the density is heterogeneous, such as the lungs, a direct Monte Carlo calculation is preferable. Here, the SPECT or PET activity distribution is used as input to a Monte Carlo dose calculation code.References1. Strand S-E, Zanzonico P, Johnson TK. Pharmacokineticmodeling. Med Phys 1993; 20(2):515-272. ICRU report nr 67 - Dose Specifications in Nuclear Medicine.Adelstein SJ, DeLuca P, Feinendegen LE, Green L, Howell RW, Humm JL, Strand SE ICRU; 20023. The LundADose Method for Planar Image ActivityQuantification and Absorbed-Dose Assessment in Radionuclide Therapy. Sjogreen,K., Ljungberg,M., Wingardh,K., Minarik,D., and Strand,S.E. (2005): Cancer Biother. Radiopharm., 20:92-974. Quantitative imaging for clinical dosimetry.Bardies M, Flux G, Lassman M, Monsieurs N, Savolainen S, Strand S-E 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 strategies.Garkavij Michael, Nickel Mattias, Sjogreen-Gleisner Katarina, Ljungberg Michael, Ohlsson Tomas, Wingârdh 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., Lindén O., Strand SE, Ljungberg M.. Q J Nucl Med Mol Imaging, 2011 April;55(2):126-54.

Claims (16)

1. Use of an antibody polypeptide, characterized by the fact that it is for the manufacture of a medicament for the treatment and/or diagnosis of prostate cancer, wherein the antibody polypeptide has binding specificity for human kallikrein-2 (hK2) and comprises: a) a heavy chain variable region comprising a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and b) a light chain variable region comprising a light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3, of the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6, respectively; wherein the heavy chain variable region and light chain variable region comprise amino acid framework sequences of one or more human antibodies, optionally, wherein the antibody polypeptide comprises a therapeutic moiety optionally comprising a radioisotope. 2. Use according to claim 1, characterized in that the one or more radioisotopes is or are independently selected from the group consisting of beta emitters, auger emitters, conversion electron emitters, alpha emitters and low photonic energy emitters. 3. Use according to claim 2, characterized in that one of the one or more radioisotopes are independently selected from the group consisting of long-range beta emitters such as 90Y, 32P, 186Re/186Re; 166Ho, 76As/77As, 153Sm; medium-range beta emitters such as 131I, 177Lu, 67Cu, 161Tb; low-energy beta emitters such as 45Ca, 35S or 14C; auger or conversion emitters such as 51Cr, 67Ga, 99Tcm, 111In, 123I, 125I, 201Tl and alpha emitters such as 212Bi, 213Bi, 225Ac and 221At. 4. Use according to claim 3, characterized in that the one or more radioisotopes are selected from the group consisting of 177Lu and 225Lu. 5. Use according to any one of claims 1 to 4, characterized in that it comprises a detectable fraction optionally comprising a radioisotope. 6. Use according to claim 3, characterized in that the radioisotope is selected from the group consisting of 99mTc, 111In, 67Ga, 68Ga, 72As, 89Zr, 123I and 201Tl. 7. Use according to any one of claims 1 to 6, characterized in that the therapeutic moiety and/or the detectable moiety is linked to the antibody polypeptide indirectly, via a binding moiety. 8. Use according to claim 6, characterized in that the binding moiety is a chelator selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, deferoxamine (DFO), diethylenetriaminepentaacetic acid (DPTA) derivatives, S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivatives and 1,4,8,11-tetraazacyclodocedane-1,4,8,11-tetraacetic acid (TETA) derivatives. 9. Use according to any one of claims 1 to 8, characterized in that the prostate cancer to be treated is metastatic prostate cancer, optionally metastatic prostate cancer, wherein the metastatic prostate cancer to be treated is metastases of the lymphatic system; metastases of the bone (including spine, vertebrae, pelvis, ribs); metastases inside the pelvis, rectum, bladder, urethra. 10. Use according to claim 9, characterized in that the prostate cancer to be treated is castration-resistant prostate cancer (CRPC). 11. Use according to claim 9, characterized in that the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 8 and the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 9. 12. Use according to claim 9, characterized in that the antibody polypeptide comprises or consists of an intact antibody or an antigen-binding fragment selected from the group consisting of Fv fragments and Fab-like fragments. 13. Use according to claim 9, characterized in that the antibody polypeptide comprises a heavy chain constant region, or part thereof, wherein the heavy chain constant region is from an immunoglobulin subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. 14. Use according to claim 9, characterized in that the antibody polypeptide comprises a constant region of the light chain, or part thereof, wherein the constant region of the light chain is of a kappa or lambda light chain. 15. Use according to claim 9, characterized in that 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. 16. Use according to claim 9, characterized in that the antibody polypeptide comprises a heavy chain, which comprises or consists of the amino acid sequence of SEQ ID NO: 12, and a light chain, which comprises or consists of the amino acid sequence of SEQ ID NO: 13.