Humanized antibodies against PSMA

Abstract

The present application relates to humanized and / or deimmunized antibodies, antibody fragments or antibody derivatives that bind to prostate-specific membrane antigen (PSMA), and to methods of using the antibodies, antibody fragments or antibody derivatives in the treatment of prostate cancer and other neoplastic and neurological diseases ( FIG. 1 ).

Description

Technical Field

[0001] The present application relates to humanized and / or deimmunized antibodies, antibody fragments or antibody derivatives that bind to prostate specific membrane antigen (PSMA), and to methods of using the antibodies, antibody fragments or antibody derivatives in the treatment of prostate cancer and other neoplastic diseases and neurological diseases. Background Art

[0002] Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) or N-acetyl-aspartylglutamate (NAAG) peptidase, is an enzyme encoded by the human folate hydrolase (FOLH1) gene. PSMA is a membrane-bound cell surface peptidase that exerts diverse physiological effects and is expressed in various tissues such as the prostate, kidney, small intestine, central nervous system, and peripheral nervous system. It is highly expressed by malignant prostate epithelial cells and vascular endothelial cells of many solid tumor malignancies, including glioblastoma, breast cancer, and bladder cancer. The protein has also been implicated in a variety of neurological diseases, including schizophrenia and ALS.

[0003] PSMA is a class II transmembrane glycoprotein with a short N-terminal cytoplasmic tail of 18 amino acids (amino acids (aa) 1-18), a single transmembrane helix (aa19-43), and an extracellular portion consisting of aa 44-750 with a molecular weight of approximately 84 kDa (Barinka et al., 2004). The short N-terminal cytoplasmic tail has been shown to interact with membrane scaffold proteins (such as clathrin, clathrin adaptor protein 2, filamin A (FLNa), and caveolin-1), which control the endocytosis of some PSMA-bound substrates. The extracellular portion is further divided into three domains: the protease (aa 57-116 and aa 352-590), the apical portion (aa117-351), and the C-terminal or dimerization domain (aa 591-750); these portions together perform substrate / ligand recognition ( et al., 2012)( Figure 1 ). With regard to aa sequence and domain organization, the 3-dimensional extracellular structure of PSMA resembles the transferrin receptor (Tfr). PSMA is enzymatically active only in its dimeric form. Dimerization as well as glycosylation are essential for the correct conformation and enzymatic activity of the molecule.

[0004] The expression and localization of PSMA in the normal human prostate is associated with the cytoplasm and apical side of the epithelial cells surrounding the prostatic ducts. Dysplasia and neoplastic transformation of prostatic tissue result in the translocation of PSMA from the apical membrane of the ducts to the luminal surface. Although PSMA was initially thought to be prostate-specific, recent studies have shown that it is also expressed by intestinal epithelial (brush border) cells, proximal renal tubular cells, and salivary glands, as well as in the nervous system.

[0005] In the small intestine, PSMA is located in the brush border of the proximal jejunum, where it acts as a hydrolase of poly-gamma-glutamylated folate and actively transports mono-glutamylated folate into the bloodstream (Navrátil et al., 2014). Therefore, it exhibits both hydrolytic and endocytic functions, at least in humans and pigs. Endocytic internalization occurs via receptor-mediated endocytosis through clathrin invagination pits. In the nervous system, PSMA (also known as NAAG-hydrolase) catalyzes the hydrolysis of NAAG, a neurotransmitter widely distributed in the mammalian brain, into glutamate and N-acetylaspartate (NAA). The different effects of PSMA in different tissues have triggered extensive exploration of multiple therapeutic approaches that target the specific delivery of drugs and small molecules to cells expressing PSMA (Evans et al., 2016).

[0006] PSMA is expressed at higher levels in malignant tissues of different origins than in normal and healthy tissues (Evans et al., 2016; Haffner et al., 2009). This expression pattern directly implies the role of PSMA in cancer progression and invasion, and makes this molecule a desirable target for the diagnosis and treatment of solid tumors.

[0007] PSMA is an ideal target in prostate cancer for several reasons. First, it is significantly overexpressed on almost all prostate cancer cells. The expression level on prostate cancer cells is 100-1,000 times higher than in normal tissue. PSMA is expressed in almost all prostate cancers; only 5%-10% of primary prostate cancers or prostate cancer lesions have negative PSMA results on PET (Eiber et al., 2017).

[0008] Second, its expression is further increased in advanced, highly metastatic disease, hormone-refractory prostate cancer and metastatic castration-resistant prostate cancer (mCRPC).

[0009] Third, in contrast to other prostate-associated antigens (e.g., PSA, prostatic acid phosphatase, and prostatic secretory protein), PSMA, as a type II integral membrane cell surface protein, is not secreted and is therefore an excellent target for, for example, antibody or other ligand-mediated therapy. After binding to the active center of the extracellular domain, the PSMA ligand is internalized. Subsequent endosomal recycling increases deposition, leading to increased tumor uptake, retention, and subsequent high local doses for therapeutic applications.

[0010] In general, prostate cancer metastasis often involves the bone marrow and lymph nodes, which absorb high levels of circulating antibodies and respond well to monoclonal antibody therapy for other tumor types such as lymphoma and breast cancer (Nanus et al., 2003).

[0011] In prostate cancer, the expression of PSMA is negatively regulated by androgens (Israeli et al., 1993). Gene transcription analysis has shown that androgens can inhibit the promoter of the PSMA gene. Therefore, the start of androgen deprivation therapy induces an early but temporary upregulation of PSMA expression, downregulation of PSMA expression under long-term androgen deprivation therapy, and ultimately induces overexpression of PSMA in androgen-resistant tumors. These effects can potentially be measured for improved therapy and diagnosis (Eiber et al., 2017). Although the direct role of PSMA in prostate cancer metastasis is still not fully understood, hormonal androgen ablation therapy on androgen-sensitive cells has been shown to increase PSMA levels, and increased PSMA expression reduces the invasiveness of the cells. On the other hand, PSMA knockout resulted in a five-fold increase in invasive activity.

[0012] Prostate cancer is the most commonly diagnosed cancer in men and the second most common cause of death in Western countries. Due to the significant mortality and morbidity associated with disease progression, new targeted therapies are urgently needed. A variety of approaches are currently being evaluated for the treatment of prostate cancer. Small molecules, aptamers or antibodies are being used as specific ligands to target the delivery of chemotherapeutic drugs using PSMA (see Evans et al., 2016, for review). PSMA-targeted gene therapy approaches based on, for example, RNAi, antisense RNA or aptamers, as well as PSMA-mediated suicide gene therapy are currently being evaluated. In radioimmunotherapy, which is currently used for approximately 25% of patients with localized prostate cancer (Cooperberg et al., 2010), targeting ligands (such as monoclonal antibodies or aptamers) are added to radionuclides to allow targeting of cancer-associated cell surface antigens (such as PSMA), and thus reduce undesirable side effects and dose increases. Immunotherapy based on sensitized dendritic cells (DCs) or T cells (which carry chimeric antigen receptors (CARs) for PSMA to induce CTL activity against PSMA-positive tumor cells) is in the clinical stage.

[0013] Antibodies that bind to PSMA have been described in the prior art. PSMA was originally characterized by the murine monoclonal antibody 7E11, which was derived from mice immunized with a partially purified cell membrane fraction isolated from a human prostate adenocarcinoma (LNCap) cell line (Horoszewicz et al., 1986). This initially published anti-PSMA monoclonal antibody recognized the N-terminus of the intracellular domain of PSMA (MWNLLH) (Troyer et al., 1995).

[0014] Four murine IgG monoclonal antibodies (J591, J415, J533, and E99) have been generated against the extracellular domain of PSMA and, unlike 7E11, recognize two different non-competing epitopes located on the outside of the cell (Liu et al., 1997; Nanus et al., 2003).

[0015] Wolf et al. (2010a) generated three different murine anti-PSMA monoclonal antibodies 3 / A12, 3 / E7 and 3 / F11, which showed strong binding to the PSMA-positive prostate cancer LNCaP subcell line C4-2, with an average half-maximal saturation concentration of 9nM-17nM (EP 1883698 B2). These antibodies stained epithelial cells of all normal and neoplastic prostate tissue samples tested. As revealed in competitive binding studies, the three monoclonal antibodies bind to different extracellular PSMA epitopes. The epitope of 3 / A12 was found to be close to or partially overlapping with the epitope of antibody J591.

[0016] An anti-PSMA single-chain antibody fragment (scFv) from monoclonal antibody 3 / F11 isolated by phage display, called D7, was used to construct an immunotoxin by C-terminally ligating Pseudomonas exotoxin A to the cDNA of D7, which inhibited the growth of PSMA-positive prostate cancer cells both in vitro and in vivo (Wolf et al., 2010b).

[0017] Unlike the results obtained with mAbs 3 / A12, 3 / E7, and 3 / F11, which were derived from mice immunized with unpurified LNCaP cell lysates, the same researchers obtained three mAbs, K7, K12, and D20, from mice immunized with purified PSMA that were highly reactive only to the isolated antigen but showed weak or no reactivity with live LNCaP cells ( -Beile et al., 2006).

[0018] Tykvart et al. (2014) characterized thirteen different PSMA-specific murine monoclonal antibodies based on their binding activity to native and denatured antigens.

[0019] WO2001009192 (Medarex) describes the development of human monoclonal antibodies against prostate specific membrane antigen. Human anti-PSMA monoclonal antibodies were generated by immunizing mice with purified PSMA or enriched preparations of PSMA antigen. Such purified antigen is denatured PSMA because it has been purified by immunoadsorption after lysing cells with ionic detergents.

[0020] WO1997035616 (Pacific Northwest Cancer Foundation) describes monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen. Immunization is performed with a C-terminal peptide or a tumor membrane preparation expressing PSMA. The monoclonal antibodies obtained do not bind specifically to cells expressing PSMA and therefore cannot be used for diagnostic or therapeutic purposes.

[0021] Bander et al. (2003) disclosed monoclonal antibodies against prostate-specific membrane antigen. Since immunization was performed with purified antigen, monoclonal antibodies were not suitable for cell binding, and scFv could not be obtained from any of these monoclonal antibodies. In WO1998 / 03873, the same antibody or its binding portion that recognizes the extracellular domain of prostate-specific membrane antigen is described, but it is not shown that the binding portion of the antibody actually binds to the antigen.

[0022] When developing antibody-based drugs, immunogenicity is key (Almagro and Fransson, 2008). All foreign proteins used for therapeutic purposes pose a risk of causing the formation of anti-drug antibodies in the recipient (Schellekens 2002), and it has generally been found that the use of therapeutic proteins and antibodies of non-human origin is associated with the generation of anti-drug antibodies, often leading to the formation of harmful immune complexes and / or neutralizing therapeutic agents. In addition to the origin and nature of the antibody, other factors such as disease type, route of administration and genetic background of the recipient have been shown to affect immunogenicity.

[0023] Therefore, the above-mentioned anti-PSMA antibodies from the prior art are not suitable for human therapy for several reasons, in particular due to their immunogenic potential, lack of target recognition or insufficient binding affinity.

[0024] Brief description of the invention

[0025] In view of the prior art, it is therefore an object of the present invention to provide humanized antibodies, antibody fragments or antibody derivatives that bind with high affinity to an epitope of the prostate specific membrane antigen (PSMA), in particular the extracellular domain of PSMA, as described in the present application.

[0026] The antibodies or antibody fragments or antibody derivatives disclosed herein comprise humanized sequences, particularly sequences of antigen binding regions based on VH and VL, which retain appropriate ligand affinity. The amino acid sequence modifications for obtaining the humanized sequences may occur in CDR regions and / or framework regions of the original antibody and / or antibody constant region sequences.

[0027] It is yet another object of the present invention to provide antibodies that specifically bind to native cell surface PSMA and are therefore valuable in diagnostic and therapeutic applications focused on PSMA as a target antigen for prostate cancer.

[0028] It is a further object of the present invention to provide compounds for use in methods of treating prostate cancer or nervous system disorders.

[0029] It is yet another object of the present invention to provide compounds that destroy prostate cancer cells expressing PSMA.

[0030] Another object of the present invention is to provide an antibody or its antibody fragment or antibody derivative having low or no immunogenicity in humans. Preferably, the antibody is a monoclonal antibody or its antibody fragment or antibody derivative.

[0031] Another object of the present invention is to provide antibody drug conjugates (ADCs) for use in methods of treating prostate cancer and other diseases, preferably comprising amatoxin.

[0032] It is completely surprising that the humanized sequences according to the present invention, especially the CDR sequences of the VH and VL regions involved in binding to the target molecule, show specific and strong binding (as demonstrated in the examples), and retain or even exceed the binding properties of the original mouse antibody 3F11, without any affinity maturation usually required after antibody humanization. The technician would not expect that the binding properties of the humanized variants would be similar to those of the original mouse antibody, let alone even better than its binding properties. Taking into account the sequence changes in the variable domains, especially the CDRs, the beneficial binding properties of the humanized sequences demonstrated herein must be considered as a surprising technical effect.

[0033] In addition, it is completely surprising that the use of humanized sequences according to the present invention when expressed in eukaryotic host cells obtains significantly better expression yields than using the parent antibody sequence. Therefore, the biotechnology production of humanized antibodies according to the present invention or their antibody fragments or antibody derivatives can be carried out at a significantly lower cost than using the parent antibody sequence.

[0034] These and other objects are achieved by methods and means according to the independent claims of the present invention. The dependent claims relate to specific embodiments.

[0035] The present invention and the general advantages of its features are discussed in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 . Schematic representation of the homodimeric PSMA transmembrane protein (from Evans et al., 2016).

[0037] Figure 2 Schematic representation of an antibody. The IgG structure is shown with two identical heavy (H) chains and two identical light (L) chains. The H chain has four domains, three C domains (C H 1 to C H 3) And a V H The L chain has a C L domain and a V L The Fv fragment (indicated by the square) is the part of the molecule that interacts with the antigen. L and V H The Fv fragment is shown in an antigenic view indicating the layout of the hypervariable loops (from Almagro and Fransson, 2008).

[0038] Figure 3 . Sequence alignment of the heavy chain variable (VH) regions of humanized / deimmunized variants of antibody 3F11. Amino acid substitutions relative to murine 3F11-wt are shown in bold.

[0039] Figure 4 . Sequence alignment of the light chain variable (VL) regions of humanized / deimmunized variants of antibody 3F11. Amino acid substitutions relative to murine 3F11-wt are shown in bold.

[0040] Figure 5 SDS-PAGE and Coomassie staining for analysis of humanized 3F11 antibody variants 13-16 under reducing conditions. 1 μg of samples and controls were loaded per well on the gel.

[0041] Figure 6 Binding dose response curves of humanized 3F11 antibody variants. Symbols represent the mean ± SEM of replicate samples of normalized binding data from one experiment (n = 2 for humanized samples and n = 8 for chimeric 3F11-wt antibody). MFI vs. log dose transformed data were normalized using the maximum and minimum MFI values. In all cases, R 2 ≥0.99.

[0042] Figure 7 EC of humanized 3F11 antibody variants 50 Binding. Symbols represent the average EC 50, and the bars show 95% confidence intervals (CI). The black dashed line shows the 95% CI for 3F11-wt antibody (n=8), and the red dashed line shows the 95% CI for 3F11-var7 (n=1) for one experiment. The variant with the highest binding activity (3F11-var7) and the wt antibody are highlighted with solid symbols. DETAILED DESCRIPTION OF THE INVENTION

[0044] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific components of the described device or the process steps of the described method, because such devices and methods can vary. It should be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive. It should be noted that, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" used in this specification and the appended claims include singular and / or plural indicators. It should also be understood that, in the case of giving a parameter range defined by a numerical value, the range is deemed to include these limiting values.

[0045] It is further understood that the embodiments disclosed herein are not meant to be understood as separate embodiments that are unrelated to each other. Features discussed with respect to one embodiment are meant to be disclosed in conjunction with other embodiments shown herein as well. In one case, if a particular feature is not disclosed for one embodiment, but is disclosed for another embodiment, those skilled in the art will understand that this does not necessarily mean that the feature is not meant to be disclosed for the other embodiment. Those skilled in the art will understand that the application intends that the feature is also disclosed for other embodiments, but only for the purpose of clarity and keeping the specification within a manageable amount of text, this is not done.

[0046] In addition, the contents of the prior art documents referred to herein are incorporated by reference. This refers in particular to prior art documents that disclose standards or conventional methods. In this case, the main purpose of incorporating by reference is to provide sufficient enabling disclosure and avoid lengthy repetition.

[0047] As used herein, the term "antibody" shall refer to a protein consisting of one or more polypeptide chains encoded by immunoglobulin genes or fragments of immunoglobulin genes or cDNAs derived from immunoglobulin genes. The immunoglobulin genes include light chain kappa, lambda and heavy chain alpha, delta, epsilon, gamma, mu constant region genes, and any of a number of different variable region genes.

[0048] The basic immunoglobulin (antibody) structural unit is usually a tetramer composed of two identical pairs of polypeptide chains: a light chain (L, molecular weight about 25 kDa) and a heavy chain (H, molecular weight about 50 kDa-70 kDa). Each heavy chain includes a heavy chain variable region (abbreviated as VH or VH ) and the heavy chain constant region (abbreviated as CH or C H The heavy chain constant region consists of three domains, namely CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated as VL or VL) and a light chain constant region (abbreviated as CL or C L ). The VH and VL regions can be further subdivided into hypervariable regions, also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region includes three CDRs and four FRs, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains form a binding domain that interacts with an antigen.

[0049] CDR is the most important for the binding of antibodies or their antigen-binding portions. FR can be replaced by other sequences, provided that the three-dimensional structure required for antigen binding is retained. Structural changes in the construct usually result in the loss of sufficient binding to the antigen.

[0050] The term "antigen-binding portion" of a (monoclonal) antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to prostate-specific membrane antigen in its native form. Examples of antigen-binding portions of antibodies include: Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains), F(ab')2 fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), Fd fragments (consisting of VH and CH1 domains), Fv fragments (consisting of the VL and VH domains of a single arm of an antibody), and dAb fragments (consisting of the VH domain) and isolated complementarity determining regions (CDRs).

[0051] As used herein, the term "monoclonal antibody (mAb)" shall refer to an antibody composition having a homogeneous antibody population (i.e., a homogeneous population consisting of whole immunoglobulins or fragments or derivatives thereof). Particularly preferably, such antibodies are selected from IgG, IgD, IgE, IgA and / or IgM or fragments or derivatives thereof.

[0052] The term "fragment" as used herein shall refer to fragments of such antibodies that retain target binding ability, for example, CDR (complementarity determining region), hypervariable region, variable domain (Fv), IgG heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions), IgG light chain (consisting of VL and CL regions) and / or Fab and / or F(ab)2.

[0053] As used herein, the term "derivative" should refer to a protein construct that is structurally different from the common antibody concept (e.g., scFv, Fab and / or F(ab)2 and bispecific, trispecific or higher specific antibody constructs) but still has some structural relationship thereto. All of these items are explained below.

[0054] Other antibody derivatives known to the skilled person are diabodies; camel antibodies; domain antibodies; bivalent homodimers with two chains consisting of scFv, IgA (two IgG structures joined by J chain and secretory component); shark antibodies; antibodies consisting of a primate framework plus non-primate CDRs; dimerization constructs comprising CH3+VL+VH; other scaffold protein formats comprising CDRs; and antibody conjugates (e.g., antibodies or fragments or derivatives thereof linked to drugs, toxins, cytokines, aptamers, nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), therapeutic polypeptides, radioisotopes or labels). The scaffold protein formats may include, for example, anchor proteins and affilin proteins, etc.

[0055] The term "Fab" as used herein relates to an IgG fragment containing the antigen binding region, which fragment is composed of one constant domain and one variable domain from each of the heavy and light chains of the antibody.

[0056] The term "F(ab)2" as used herein relates to an IgG fragment consisting of two Fab fragments linked to each other by a disulfide bond.

[0057] The term "scFv" as used herein refers to a single-chain variable fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked together by a short linker that generally contains serine (S) and / or glycine (G) residues. Despite the removal of the constant region and the introduction of a linker peptide, the chimeric molecule retains the specificity of the original immunoglobulin.

[0058] Modified antibody formats are, for example, bispecific or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, and the like.

[0059] IgG, scFv, Fab and / or F(ab)2 are antibody formats well known to the skilled person. The relevant enabling technologies can be obtained from the corresponding textbooks.

[0060] Monoclonal antibodies (mAbs) derived from mice may cause undesirable immunological side effects because they contain proteins from another species, which may induce antibodies. To overcome this problem, antibody humanization and maturation methods have been designed to generate antibody molecules with minimal immunogenicity when applied to people, while ideally still retaining the specificity and affinity of non-human parent antibodies (reviewed in Almagro and Fransson 2008). Using these methods, for example, the framework regions of mouse mAbs are replaced with corresponding human framework regions (so-called CDR transplants). WO200907861 discloses that the CDR regions of non-human antibodies are connected to human constant regions to generate humanized forms of mouse antibodies by recombinant DNA technology. US6548640 of the Medical Research Council describes CDR transplantation technology, and US5859205 of Celltech describes the production of humanized antibodies.

[0061] However, humanization by CDR grafting often results in a significant reduction or even loss of binding affinity because a set of supporting framework residues in the so-called Vernier zone are important for maintaining the conformation of the CDRs (Foote and Winters 1992). These residues are responsible for stabilizing the hypervariable loop structure and modifying its positioning; they fine-tune the antibody affinity. Reintroducing mouse residues into the human framework (Queen et al., 1989) can solve this problem. Such substitutions are often referred to as "back mutations".

[0062] Immunogenicity is the ability to induce a T helper (Th) cell response, which is triggered when a unique T cell receptor recognizes a peptide bound to an HLA (human leukocyte antigen) class II molecule displayed on an antigen presenting cell (APC). The peptide generates a protein that is internalized by the free antigen presenting cell and then processed through the endosomal cleavage pathway. Only peptides with sufficient affinity for HLA class II molecules are presented on the cell surface of APCs and may trigger a Th response.

[0063] It is possible to further reduce the immunogenic potential by removing Th epitopes using a process known as deimmunization (Chamberlain 2002; Baker and Jones 2007). This is achieved by predicting which peptides in the corresponding therapeutic protein can bind to HLA class II molecules and then introducing substitutions that eliminate or reduce the peptide binding affinity for HLA class II molecules.

[0064] There are several HLA class II genes, and HLA class II molecules consist of an alpha chain and a beta chain, each expressed by a different gene, increasing variation (DRA / DRB, DQA / DQB and DPA / DPB) and almost all highly polymorphic; only DRA is non-polymorphic. The focus during deimmunization is on the DR allotype, which is known to be expressed at higher levels than DQ and DP. The assessment of the relevance of each epitope is based on the criteria of promiscuity, i.e. the number of HLA allotypes bound by a particular epitope, as well as the importance (frequency) of the allotype in the population and a qualitative assessment of the strength of binding of the HLA-peptide complex. Since the individual's T cell population has been selected not to recognize "self-peptides" of endogenous proteins (such as antibodies), it is possible to screen the protein to be deimmunized for peptide screening corresponding to (known) self-peptides, which should not normally induce a Th response.

[0065] The term "humanized antibody" as used herein relates to an antibody, fragment or derivative thereof, in which at least a portion of the constant region and / or framework region, and optionally a portion of the CDR region, of the antibody is derived from or adapted to human immunoglobulin sequences.

[0066] Sequence variants of the nucleic acid sequences or amino acid sequences claimed herein are also included in the scope of the present invention, which sequence variants are defined, for example, by the claimed % sequence identity (homology), and retain the described properties of the antibodies, fragments of antibodies or derivatives according to the present invention. The term "sequence identity" relates to the percentage of identical nucleotides or amino acids when performing a sequence comparison.

[0067] The term "amino acid substitution" as used herein relates to a modification of the amino acid sequence of a protein in which one or more amino acids are replaced with the same number of different amino acids, thereby producing a protein containing an amino acid sequence different from that of the original protein. Conservative amino acid substitutions are understood to involve substitutions that do not significantly affect the structure and function of the protein due to similar size, charge, polarity and / or conformation. In this sense, the group of conservative amino acids represents, for example, the non-polar amino acids Gly, Ala, Val, Ile and Leu; the aromatic amino acids Phe, Trp and Tyr; the positively charged amino acids Lys, Arg and His; and the negatively charged amino acids Asp and Glu.

[0068] The present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof that binds to the extracellular domain of prostate specific membrane antigen (PSMA).

[0069] Preferred CDR sequences are discussed below. It is important to understand that the CDRs are contained within an appropriate protein framework to enable binding to the extracellular domain of prostate specific membrane antigen (PSMA).

[0070] The present invention also provides a humanized antibody or an antibody fragment or antibody derivative thereof that binds to the extracellular domain of PSMA and comprises a VH domain, wherein the VH domain comprises the following CDR sequence:

[0071] -GX1TX2TX3X4 (CDR H1), where X1, X2, X3 and X4 are Y, F, W respectively,

[0072] -GISPGDX5NX6NYX7X8X9FX 10 G (CDR H2), wherein X5 is G, S; X6 is T, V; X7 is N, A; X8 is E, Q; X9 is N, K; X 10 is K, Q; and

[0073] -DGNX 11 PX 12 X 13 AMDS (CDR H3), where X 11 , X 12 , X 13 They are Y, F, and W respectively.

[0074] The present invention also provides the humanized antibody or its antibody fragment or antibody derivative, wherein the VH domain comprises the CDR H1 sequence GYTFTYF (SEQ ID No. 1).

[0075] The present invention further provides the humanized antibody or its antibody fragment or antibody derivative that binds to the extracellular domain of PSMA, wherein the VH domain comprises the CDR H2 sequence of SEQ ID No.2 or SEQ ID No.7 or SEQ ID No.9 or SEQ ID No.11.

[0076] The present invention further relates to the humanized antibody or antibody fragment or antibody derivative thereof that binds to the extracellular domain of PSMA, wherein the VH domain comprises the CDR H3 sequence DGNFPYYAMDS (SEQ ID No. 3).

[0077] In a preferred embodiment, the antibody or antibody fragment or antibody derivative that binds to the extracellular domain of PSMA comprises a VH domain comprising

[0078] SEQ ID No. 1 and

[0079] SEQ ID No.2 or SEQ ID No.7 or SEQ ID No.9 or SEQ ID No.11

[0080] · and SEQ ID No.3.

[0081] In a further preferred embodiment, the antibody or antibody fragment or antibody derivative thereof that binds to the extracellular domain of PSMA comprises a VH domain comprising any one of the sequences of SEQ ID No. 13, 15, 17 or 19.

[0082] The present invention also relates to a humanized antibody or an antibody fragment or an antibody derivative thereof that binds to the extracellular domain of PSMA, wherein the humanized antibody or the antibody fragment or the antibody derivative thereof comprises a VL domain, wherein the VL domain comprises the following CDR sequence:

[0083] -RSSQSLVHSX1GX2TX3LH (CDR L1), where X1 is N, S; X2 is N, Q; X3 is Y, F, W,

[0084] -TVSNRX4S (CDR L2), where X4 is A, Y, F, W, and

[0085] -SQSTHVPT(CDR L3).

[0086] In a preferred embodiment, the antibody or its antibody fragment or antibody derivative comprises a VL domain, and the VL domain comprises the CDR L1 sequence SEQ ID No.4 or SEQ ID No.8 or SEQ ID No.10.

[0087] In a further preferred embodiment, the antibody or its antibody fragment or antibody derivative comprises a VL domain, and the VL domain comprises the CDR L2 sequence SEQ ID No.5 or SEQ ID No.12.

[0088] In a further preferred embodiment, the antibody or its antibody fragment or antibody derivative comprises a VL domain, and the VL domain comprises a combination of:

[0089] SEQ ID No. 4 or SEQ ID No. 8 or SEQ ID No. 10, and

[0090] SEQ ID No. 5 or SEQ ID No. 12, and

[0091] ·SEQ ID No.6.

[0092] The present invention particularly relates to an antibody or an antibody fragment or an antibody derivative thereof comprising a VL domain, wherein the VL domain comprises any one of the sequences SEQ ID No. 14, 16, 18 or 20.

[0093] The present invention also relates to an antibody or an antibody fragment or antibody derivative thereof comprising a combination of at least one of the above-mentioned VH CDR sequences and at least one of the above-mentioned VL CDR sequences.

[0094] The present invention also relates to the antibody or its antibody fragment or antibody derivative comprising a combination of at least one of the VH domain sequences and at least one of the VL domain sequences, wherein the VH domain sequence is selected from SEQ ID No. 13, 15, 17 and 19, and the VL domain sequence is selected from SEQ ID No. 14, 16, 18 and 20.

[0095] The present invention also relates to any such antibody or antibody fragment or antibody derivative described above, wherein the antibody or antibody fragment or antibody derivative is expressed in the form of EC 50 Affinity <0.4μg / ml, preferably EC 50 Affinity <0.3μg / ml, most preferably EC 50 Binds to the extracellular domain of PSMA with an affinity of <0.4 μg / ml.

[0096] The humanized anti-PSMA antibody or antibody fragment or antibody derivative thereof may be glycosylated. The glycan may be an N-linked oligosaccharide chain at asparagine 297 of the heavy chain.

[0097] The prostate specific membrane antigen may be a mammalian, non-primate, primate prostate specific membrane antigen, and in particular a human prostate specific membrane antigen.

[0098] The antibody or antibody fragment or antibody derivative thereof according to the present invention may be a monoclonal antibody.The antibody may be of IgA, IgD, IgE, IgG or IgM isotype.

[0099] The present invention relates to antibody fragments selected from the group consisting of variable domain (Fv), Fab fragments and F(ab)2 fragments. The present invention also relates to antibody derivatives which are preferably single chain Fv (scFv).

[0100] In a preferred embodiment, the humanized antibodies or fragments or derivatives thereof (such as scFv) described in this application specifically bind to native cell surface PSMA, and therefore will be valuable in diagnostic and therapeutic applications focused on PSMA as a target antigen for, for example, prostate cancer. Because PSMA is expressed in specific tertiary and quaternary structures on prostate cancer cells, antibodies specifically directed against this cell conformation can recognize and firmly bind to live prostate cancer cells and PSMA-expressing tissues. Therefore, one objective of this study is to generate such antibodies or fragments or derivatives thereof that can be used for targeted therapy and diagnosis of prostate cancer.

[0101] In a preferred embodiment, the present invention also relates to an antibody drug conjugate (ADC, also known as immunoconjugate) comprising a humanized antibody or a fragment or derivative thereof as described above conjugated to a therapeutic agent, optionally via a linker moiety.

[0102] The therapeutic agent can be, for example, a drug, a chemotherapeutic agent, a cytotoxic agent (such as, for example, paclitaxel, cytochalasin B, gramicidin D), a toxin, a growth inhibitory agent, an aptamer, a nucleic acid (such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), a therapeutic polypeptide, or a radioactive substance.

[0103] The ADC may comprise a linker region between the therapeutic agent and the anti-PSMA antibody or antibody fragment or antibody derivative thereof. The linker is cleavable by, for example, but not limited to, a cleavage agent or acidic pH conditions.

[0104] In another aspect of the present invention, there is provided a humanized anti-PSMA antibody or a fragment or derivative thereof, said antibody being formulated as a pharmaceutical formulation.

[0105] In aqueous form, the pharmaceutical formulation may be ready for administration, while in lyophilized form, the formulation may be converted to liquid form prior to administration, for example, by the addition of water for injection which may or may not contain preservatives such as, but not limited to, benzyl alcohol, antioxidants (such as vitamin A, vitamin E, vitamin C, retinyl palmitate and selenium), amino acids (cysteine ​​and methionine), citric acid and sodium citrate, synthetic preservatives (such as parabens: methylparaben and propylparaben).

[0106] The pharmaceutical formulation may further comprise one or more stabilizers, which may be, for example, amino acids, sugar polyols, disaccharides and / or polysaccharides. The pharmaceutical formulation may further comprise one or more surfactants, one or more isotonic agents, and / or one or more metal ion chelators, and / or one or more preservatives.

[0107] The pharmaceutical formulations as described herein may be suitable for at least intravenous, intramuscular or subcutaneous administration. Alternatively, the antibodies according to the invention may be provided in the form of a depot formulation, which allows for sustained release of the biologically active agent over a certain period of time.

[0108] In yet another aspect of the present invention, a primary package, such as a prefilled syringe or pen, a vial or an infusion bag, is provided, comprising the formulation and / or the humanized anti-PSMA antibody or a fragment or derivative thereof according to the aforementioned aspects of the present invention.

[0109] Prefilled syringes or pens may contain the formulation in lyophilized form (which must then be dissolved with, for example, water for injection before administration) or in aqueous form. The syringe or pen is typically a disposable for single use and may have a volume of 0.1 ml to 20 ml. However, the syringe or pen may also be a multiple use or multidose syringe or pen.

[0110] The vial may also contain a formulation in a lyophilized form or an aqueous form and may be used as a single-use or multiple-use device. As a multiple-use device, the vial may have a larger volume. The infusion bag typically contains a formulation in an aqueous form and may have a volume of 20 ml-5000 ml.

[0111] The pharmaceutical formulation is intended for use in treating a medical condition associated with PSMA overexpression and comprises a humanized antibody or antibody fragment or antibody derivative thereof as described herein together with at least one pharmaceutically acceptable carrier.

[0112] The medical condition associated with PSMA overexpression can be, for example, prostate cancer or a neurological disorder. Humanized anti-PSMA antibodies or fragments or derivatives thereof can mediate anti-tumor effects by activating humoral or cellular immune functions, or by targeting tumors with conjugated cytotoxins or radioactivity as described above.

[0113] The present invention also relates to an antibody or antibody fragment or antibody derivative thereof as described herein for use in treating prostate cancer.

[0114] The present invention also relates to a method of treating a patient suffering from prostate cancer, the method comprising administering an effective amount of an antibody or antibody fragment or antibody derivative thereof as described herein.

[0115] Another aspect of the invention relates to a diagnostic kit for detecting tumor cells, the diagnostic kit comprising an isolated monoclonal antibody or antigen binding fragment or derivative thereof, thereby allowing detection thereof after binding to suitable detection reagents and devices. The invention also provides a method for in vitro identification of tumor cells, by which the tumor cells to be identified are contacted with an isolated monoclonal antibody or antigen binding fragment or derivative thereof carrying a marker detectable by a suitable analytical device. The marker allows diagnostic identification of tumor cells in human tissue sections obtained, for example, after surgery or biopsy.

[0116] The present invention also relates to a host cell capable of producing the humanized anti-PSMA antibody or a fragment or derivative thereof, and to a cell line transfected with a recombinant plasmid containing the coding sequence of the antibody of the present invention.

[0117] According to another preferred embodiment of the present invention, the host cell is a mammalian cell. According to another preferred embodiment of the present invention, the host cell is selected from at least one of, for example, baby hamster kidney cells (e.g., BHK21), Chinese hamster ovary cells (CHO), mouse myeloma cells (e.g., SP2 / 0 or NS0), human embryonic kidney cells (e.g., HEK-293), human retina-derived cells (e.g., PER-C6), and amniotic fluid cells (e.g., CAP).

[0118] In one embodiment, the mammalian cell is a CHO cell line (eg, CHO-K1, CHO-DG44, CHO-DXB, or CHO-dhfr-).

[0119] The present invention also relates to a nucleic acid encoding a humanized antibody or an antibody fragment or antibody derivative thereof that binds to PSMA as described in the present application.

[0120] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof that binds to the extracellular domain of prostate-specific membrane antigen (PSMA), comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the heavy chain variable (VH) domain comprises:

[0121] According to the CDR H1 sequence of SEQ ID No.1,

[0122] A CDR H2 sequence selected from any one of SEQ ID No. 2, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11,

[0123] According to the CDR H3 sequence of SEQ ID No. 3,

[0124] and the light chain variable (VL) domain comprises:

[0125] a CDR L1 sequence selected from any one of SEQ ID No. 4, SEQ ID No. 8 and SEQ ID No. 10, a CDR L2 sequence selected from any one of SEQ ID No. 5 and SEQ ID No. 12, and

[0126] According to the CDR L3 sequence of SEQ ID No.6,

[0127] However, the humanized antibody or its antibody fragment or antibody derivative does not comprise a combination of a CDR H1 sequence according to SEQ ID No.1, a CDR H2 sequence according to SEQ ID No.2, a CDR H3 sequence according to SEQ ID No.3, a CDR L1 sequence according to SEQ ID No.4, a CDR L2 sequence according to SEQ ID No.5 and a CDR L3 sequence according to SEQ ID No.6.

[0128] For the avoidance of doubt, all possible 24 combinations of said CDRs (ie combinations of one CDR H1, one CDR H2, one CDR H3, one CDR L1, one CDR L2 and one CDR L3) are herein deemed to be disclosed individually in this specification.

[0129] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof comprising a VH domain, wherein the VH domain has at least 90%, preferably 95% sequence homology with a sequence selected from any one of SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No. 19.

[0130] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof comprising a VH domain sequence, wherein the VH domain sequence is selected from any one of SEQ ID No.13, SEQ ID No.15, SEQ ID No.17 and SEQ ID No.19.

[0131] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof comprising a VL domain, wherein the VL domain has at least 90%, preferably 95% sequence homology with a sequence selected from any one of SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 and SEQ ID No. 20.

[0132] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof comprising a VL domain sequence, wherein the VL domain sequence is selected from any one of SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20.

[0133] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof, comprising: a VH domain sequence according to SEQ ID No.13 and a VL domain sequence selected from any one of SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20; or a VH domain sequence according to SEQ ID No.15 and a VL domain sequence selected from any one of SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20; or a VH domain sequence according to SEQ ID No.17 and a VL domain sequence selected from any one of SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20; or a VH domain sequence according to SEQ ID No.19 and a VL domain sequence selected from any one of SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20.

[0134] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof, wherein the heavy chain comprises any one of the sequences SEQ ID No.21, SEQ ID No.23, SEQ ID No.25 or SEQ ID No.27.

[0135] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof, wherein the light chain comprises any one of the sequences SEQ ID No.22, SEQ ID No.24, SEQ ID No.26 or SEQ ID No.28.

[0136] In one embodiment, the present invention relates to a humanized antibody or its antibody fragment or antibody derivative comprising at least one heavy chain and at least one light chain, wherein the at least one heavy chain comprises any one of the sequences SEQ ID No.21, 23, 25 or 27, and the at least one light chain comprises any one of the sequences SEQ ID No.22, 24, 26 or 28.

[0137] In a preferred embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof comprising at least one heavy chain and at least one light chain, wherein the at least one heavy chain comprises SEQ ID No.27, and the at least one light chain comprises SEQ ID No.28.

[0138] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or an antibody derivative thereof. According to the present invention, the humanized antibody or an antibody fragment or an antibody derivative thereof is expressed as 50 Binds to the extracellular domain of PSMA with an affinity of <0.4 μg / ml.

[0139] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof, according to the present invention, wherein the antibody is glycosylated. The glycosylation may involve glycans of N-linked oligosaccharide chains at asparagine 297 of the heavy chain.

[0140] In one embodiment, the present invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof, according to the present invention, wherein the prostate-specific membrane antigen is human prostate-specific membrane antigen.

[0141] The humanized antibody or antibody fragment or antibody derivative according to the present invention may be a monoclonal antibody or an antibody fragment or antibody derivative thereof. The monoclonal antibody may preferably be of IgG isotype.

[0142] The humanized antibody fragment according to the present invention may be selected from the group consisting of a variable domain (Fv), a Fab fragment and a F(ab)2 fragment.

[0143] The humanized antibody derivative according to the present invention may be a single chain Fv (scFv).

[0144] In one embodiment, the invention relates to an antibody drug conjugate (ADC) comprising a humanized antibody or fragment or derivative thereof according to the invention coupled to a therapeutic agent, optionally via a linker moiety.

[0145] In one embodiment, the present invention relates to a pharmaceutical formulation for treating a medical condition associated with overexpression of PSMA, comprising a humanized antibody or antibody fragment or antibody derivative thereof according to the present invention together with at least one pharmaceutically acceptable carrier. The medical condition associated with overexpression of PSMA may be prostate cancer. The pharmaceutical formulation according to the present invention may be a formulation suitable for at least intravenous, intramuscular or subcutaneous administration.

[0146] In one embodiment, the invention relates to a humanized antibody or an antibody fragment or antibody derivative thereof, which is used according to the invention for the treatment of prostate cancer.

[0147] In one embodiment, the present invention relates to a method of treating a patient suffering from prostate cancer, comprising administering to the patient an effective amount of a humanized antibody according to the present invention, or an antibody fragment or antibody derivative thereof.

[0148] In one embodiment, the invention relates to a nucleic acid encoding a humanized antibody or an antibody fragment or antibody derivative thereof according to the invention.

[0149] In one embodiment, the present invention relates to a host cell capable of producing a humanized antibody or an antibody fragment or antibody derivative thereof according to the present invention. The host cell according to the present invention may be a mammalian cell. The mammalian cell according to the present invention is preferably a CHO cell line.

[0150] Antibodies or fragments or derivatives of the present invention can be produced by transfecting host cells with an expression vector comprising the coding sequence of the antibody according to the present invention. Expression vectors or recombinant plasmids are produced by placing the antibody coding sequence under the control of suitable gene regulatory elements (including promoter and enhancer sequences, such as, for example, CMV promoters). Heavy chain and light chain sequences can be expressed from a single expression vector of cotransfection or from a dual expression vector. The transfection can be a transient transfection or a stable transfection. Subsequently, transfected cells are cultivated to produce transfected antibody constructs. When stable transfection is performed, then a stable clone of an antibody with appropriately associated heavy and light chains is selected for secretion by screening with appropriate assays (such as, for example, ELISA), which is subcloned and bred for future production. Example

[0151] Although the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description should be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments may be understood and effected by a person skilled in the art in practicing the claimed invention by studying the drawings, the disclosure and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a / an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0152] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus. All nucleic acid sequences disclosed herein are shown 5'->3'.

[0153] Example 1: Humanization and deimmunization of murine antibody 3F11

[0154] The murine anti-PSMA antibody 3 / F11 (Wolf et al., 2010a; EP 1883698 B2) was humanized by grafting the complementarity determining regions (CDRs) into an acceptor framework selected from the human germline repertoire.

[0155] Epibase of parental 3 / F11 against 85 HLA class II allotypes TM Immunoassays, clusters of epitopes or adjacent epitopes were analyzed for possible substitutions that remove or reduce binding to HLA allotypes (focusing on HLA-DRB1 allotypes). Key positions that constitute the VH / VL interchain interface or are responsible for the discontinuous arrangement of the CDR classical structure were identified. Based on sequence alignments of the parental antibody sequences with human germline sequences, the closest match (particularly taking into account the combination of heavy chain germline and light chain germline present in the expressed antibody, sequence identity of the framework, identical or compatible interchain interface residues, and supporting loops with the parent CDR classical conformation) identified the best human germline sequence as the receptor. 3 / F11 was found to be most compatible with human light chain germline VK2-A17 of the VK2 light chain germline family VK2 and human heavy chain germline VH1-1-69 of the human heavy chain germline family VH1, respectively. For CDR grafting, amino acids in the parental framework that differ from the selected receptor are replaced with the corresponding human amino acids.

[0156] The sequences were analyzed for potential post-translational modification sites (asparagine deamidation, aspartate isomerization, C-terminal lysine cleavage, free Cys thiol groups, N and O glycosylation, N-terminal cyclization, oxidation and pyroglutamate formation) that could cause problems during the development of therapeutic products. An in silico modeling platform was used to generate structural models of the FV region of 3 / F11 and variants, and candidate structural fragments of the framework and CDRs as well as the complete FV were scored and ranked.

[0157] In order to evaluate the impact of all substitutions considered in an efficient manner, substitutions were grouped where appropriate and a total of four humanized / deimmunized light chains and four humanized / deimmunized heavy chains were selected. Table 2 lists the names of the engineered amino acid chains along with a description of the modifications. Table 1 gives the amino acid sequences of the engineered chains. Figure 3 and Figure 4 Sequence alignments of the humanized / deimmunized light and heavy chains are shown in .

[0158] The heavy chain variable domain variant 3F11-VH-1 contains all three CDRs of the parent murine VH sequence. In variant 3F11-VH-2, the two CDRs are identical to the parent murine VH sequence, however, CDR H2 differs from the parent murine sequence at four positions (amino acids 61-63: NEN>AQK, amino acids 65: K>Q). In variant 3F11-VH-3, the two CDRs are identical to the parent murine VH sequence, however, CDR H2 differs from the parent murine sequence at five positions (amino acids 58: T>V, amino acids 61-63: NEN>AQK, amino acids 65: K>Q). In variant 3F11-VH-4, the two CDRs are identical to the parent murine VH sequence, however, CDR H2 differs from the parent murine sequence at six positions (amino acids 56: G>S, amino acids 58: T>V, amino acids 61-63: NEN>AQK, amino acids 65: K>Q).

[0159] The light chain variable domain variant 3F11-VL-1 comprises all three CDRs of the parent murine VL sequence. In variant 3F11-VL-2, both CDRs are identical to the parent murine VL sequence, however CDR L1 comprises a substitution at amino acid 35 (N>Q). In variant 3F11-VL-3, both CDRs are identical to the parent murine VL sequence, however CDR L1 differs from the parent murine sequence at two positions (amino acid 33: N>S, amino acid 35: N>Q). In variant 3F11-VL-4, CDR L1 differs from the parent murine sequence at two positions (amino acid 33: N>S, amino acid 35: N>Q) and CDR L2 differs from the parent murine sequence at one position (amino acid 60: F>A), and CDR L3 is identical to the parent murine sequence.

[0160] Table 2: CDR composition of 3F11 wt and humanized / deimmunized 3F11 variants with corresponding substitutions (amino acid numbering according to Kabat et al.)

[0161]

[0162]

[0163] 3F11-VL-2 optionally has the following mutations: amino acid 41: F>Y. 3F11-VL-3 optionally has the following mutations: amino acid 41: F>Y. 3F11-VL-4 optionally has the following mutations: amino acid 41: F>Y; amino acid 42: L>Q and / or amino acid 90: V>T.

[0164] To evaluate the effects of substitution combinations, an experimental design of variant combinations was established, as summarized in Table 3. Through the combination of four humanized / deimmunized heavy chains and four humanized / deimmunized light chains, 16 different humanized / deimmunized full-length antibodies were obtained, and their names are shown in Table 3.

[0165] Table 3: Combinations of 3F11 humanized / deimmunized variant VH and VL chains forming 16 different variants of humanized variant domains and full length antibodies, respectively. 3F11 wt comprises murine VH and VL chains.

[0166] Variant Name Light chain name Heavy chain name 3F11 wt 3F11-VL 3F11-VH 3F11-var 1 3F11-VL-1 3F11-VH-1 3F11-var 2 3F11-VL-1 3F11-VH-2 3F11-var 3 3F11-VL-1 3F11-VH-3 3F11-var 4 3F11-VL-1 3F11-VH-4 3F11-var 5 3F11-VL-2 3F11-VH-1 3F11-var 6 3F11-VL-2 3F11-VH-2 3F11-var 7 3F11-VL-2 3F11-VH-3 3F11-var 8 3F11-VL-2 3F11-VH-4 3F11-var 9 3F11-VL-3 3F11-VH-1 3F11-var 10 3F11-VL-3 3F11-VH-2 3F11-var 11 3F11-VL-3 3F11-VH-3 3F11-var 12 3F11-VL-3 3F11-VH-4 3F11-var 13 3F11-VL-4 3F11-VH-1 3F11-var 14 3F11-VL-4 3F11-VH-2 3F11-var 15 3F11-VL-4 3F11-VH-3 3F11-var 16 3F11-VL-4 3F11-VH-4

[0167] The predicted critical epitopes for HLA DRB1, DRB3 / 4 / 5, DQ and DP for the parental and humanized / deimmunized sequences are presented in Table 4. The differences between the parental and variant antibodies in Table 4 explain the corresponding removal of potential epitopes.

[0168] Table 4: Predicted critical epitopes for HLA DRB1, DRB3 / 4 / 5, DQ and DP for parental and humanized / deimmunized sequences are presented.

[0169]

[0170]

[0171] Example 2: Expression and purification of humanized anti-PSMA antibody variants

[0172] The heavy and light chain variable domains of the antibody were synthesized and subcloned into the GS Xceed™ vector (Lonza). The light chain variable domain coding region was transferred into the pXC-κ vector and the heavy chain variable domain coding region was transferred into the pXC-IgG1zaδK vector, respectively.

[0173] Single gene vector (SGV) was transiently co-transfected with reference mouse antibody into Chinese hamster ovary GS knockout cells (CHOK1SV GS-KO) at a 200 ml scale. Transient transfection was performed by electroporation (Gene Pulse XCell, BioRad) using CHOK1SV GS-KO cells that had been cultured for a minimum of two weeks. Cells were subcultured for 24 hours before transfection, and cell viability was >99% at the time of transfection. Six days after transfection, cell viability and viable cell concentration were measured at harvest. The range of cells found was 4.4×10 6 -7.5×10 6 Living cells / ml, viability 89.0%-96.1%.

[0174] The clear supernatant was purified by protein A chromatography using a pre-packed 5ml HiTrap MabSelect SuRE column on an AKTA purifier according to standard procedures. After elution with 10mM sodium formate (pH 3.5), the pH of the protein-containing fraction was adjusted immediately by adding 2× phosphate buffered saline (PBS) to produce a final buffer condition of 1×PBS, 5mM sodium formate, pH 7.4. Table 5 below shows the titer and product yield of 3F11 parent mouse antibody and a variety of humanized antibody variants.

[0175] A surprising effect is that the inventors observed significantly higher expression titers for some humanized anti-PSMA antibody variants compared to the parental murine 3F11 antibody (see Table AA). Expression titers of 9.5 mg / L to 13 mg / L were obtained with the humanized variants 3F11-var2, 3F11-var6, 3F11-var9, 3F11-var11, 3F11-var13, 3F11-var15, and 3F11-var16, compared to 3.88 mg / L expression titers for the parental murine 3F11 antibody. Due to the 3-4 fold increase in expression titers, it is expected that the production costs of therapeutic products based on the humanized / deimmunized variants will be significantly reduced.

[0176] Table 5: Characterization results of humanized 3F11 antibody variants

[0177]

[0178]

[0179] Example 3: Biochemical Characterization of Humanized Anti-PSMA Antibody Variants

[0180] Purified samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion (SE)-HPLC using a Zorbax GF-250 (9.4 mm ID x 25 cm) column (Agilent) on an Agilent 1200 series system.

[0181] In SDS-PAA gel, under non-reducing conditions, all variants have visible signals at >98 kDa, which is comparable to the control antibody (IgG1) and the parent 3F11, which is consistent with the full-length antibody. Under reducing conditions, all variants have visible signals at about 28 kDa and a signal at about 49 kDa, which is comparable to the control antibody and the parent 3F11, which is consistent with the size of the antibody light chain and heavy chain, respectively. Figure 5 Representative results are shown in .

[0182] The retention time of humanized 3F11 variant monomer antibody in SE-HPLC (7.96-8.00 minutes) is comparable to that of 3F11 mouse parent antibody (7.968 minutes). SE-HPLC chromatograms of all samples showed additional secondary peaks with an area of ​​up to 5.4% at a lower retention time (about 7.2 minutes), which corresponded to higher molecular weight impurities such as soluble aggregates.

[0183] Example 4: Cell Binding Studies of Humanized Anti-PSMA Antibody Variants

[0184] The quality and binding activity of all humanized 3F11 antibody variants were evaluated in a cell binding assay using a 96-well plate-based assay format and a human prostate cancer cell line LNCAP (German Collection of Microorganisms and Cell Cultures, DSMZ No.: ACC 256) in a cell-based FACS analysis. Pre-fused LNCAP cells reaching 80%-90% confluence were washed and detached from the plate with trypsin before incubation in a flask at 37°C, 5% CO2 for 1 hour to restore cell surface proteins. The cells were washed and adjusted to 1×10 in staining medium (RPMI-1640, 25 mM HEPES, 3% hiFBS, and 0.02% sodium azide). 6 Cells / ml. Monoclonal antibody samples (mouse wt and 16 humanized variants) with concentrations ranging from 50 μg / ml to 0.0032 μg / ml were prepared in staining medium and incubated on ice for 30 minutes with cells in precooled 96-well plates to allow antibodies to bind to the LNCAP cell surface. Subsequently, cells were washed with PBS at 4 °C to remove free antibodies, and the bound antibodies were labeled on ice for 30 minutes with FITC goat anti-human IgG H&L pre-adsorbed (Abcam) secondary antibodies. Finally, cells were washed, fixed and measured by flow cytometry (GUAVA 8HT blood cell counter) for FITC fluorescence associated with antibodies bound to cells. Incyte GuavaSoft 2.7 software (Merck-Millipore) was used to analyze the data. The dose-response data obtained (plotted as logarithmic transformed dose vs. response) generally produce an S-shaped curve with a variable slope, which is fitted to a 4-parameter logarithmic dose-response equation using GraphPad Prism software. EC 50 The dose was defined as the antibody concentration that provided half-maximal target cell binding and was used to compare the biological activities of different antibody variants. The data points represent the mean of replicate wells with standard error of the mean (SEM). Figure 6 Binding curves for the parental 3F11 wt antibody and sixteen humanized variants are shown in .

[0185] Quite surprisingly, the cell-based binding assay showed that all sixteen 3F11 humanized variants bound to PSMA-expressing cells with higher affinity than that obtained with the parental murine antibody 3F11. 50 ) ranged from 0.1784 μg / ml to 0.3732 μg / ml and in most cases (based on 95% confidence interval, CI) were significantly higher than the binding activity (EC 50 =0.4189 μg / ml). 3F11-var7 showed the highest binding activity (EC 50 =0.1784 μg / ml), followed by 3F11-var8 and 3F11-var6 (EC 50 =0.2040 μg / ml and EC 50 =0.2187 μg / ml, see Table 6). Figure 7 Results of a single experiment are shown in .

[0186] Table 6: EC of humanized 3F11 antibody variants 50 Combined activity and 95% CI.

[0187]

[0188]

[0189] Because most humanized variants (all variants not comprising heavy chain variant 3F11_VH_1) did not require any back mutations in the framework regions spanning between CDRs, and especially because none of the variants underwent affinity maturation (which is known to require affinity maturation after humanization of antibodies due to a significant reduction or loss of affinity), these findings were particularly unexpected. Surprisingly, the procedure for generating humanized variants and variants according to the invention were found to not require such additional effort.

[0190] Example 5: Cell Binding Studies of Anti-PSMA scFv Constructs

[0191] Single chain Fv (scFv) constructs were also generated using a combination of 3F11-VH-1 and 3F11-VL-1 chains (3F11-var1) and a combination of 3F11-VH-4 and 3F11-VL-4 chains (3F11-var16). For both constructs, the respective variable domain sequences were cloned into the expression vector pHOG21 in a VL-VH combination and expressed in bacterial cells. After affinity chromatography purification, the two scFv constructs were tested for binding to PSMA-expressing cells (LNCAP subcell line C4-2) and to PSMA-negative DU145 prostate cancer cells. Both scFv constructs showed strong binding to C4-2 cells, but not to negative control DU145 cells.

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[0208] Nanus DM,Milowsky MI,Kostakoglu L,Smith-Jones PM,Vallabahajosula S,Goldsmith SJ,and Bander NH.(2003).Clinical use of monoclonal antibody HuJ591therapy:targeting prostate specific membrane antigen.Journal of UrologyVol.170:S84-S89.

[0209] Navrátil M, J, P,Starková J,Lubkowski J, C et al.(2014).Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.FEBS J281:3228-3242.

[0210] Queen C.et al.(1989).A humanized antibody that binds to theinterleukin 2 receptor.Proc.Natl.Acad.Sci.86:10029-10033.

[0211] Schellekens,H(2002).Immunogenicity of therapeutic proteins:clinicalimplications and future prospects.Clin.Ther.Vol.4:1720-40.

[0212] Troyer J,Feng Q,Beckett M,Wright GJ.(1995).Biochemicalcharacterization and mapping of the 7 E11-C5.3 epitope of the prostatespecific membrane antigen.Urol Oncol 1(1):29-37.

[0213] Tykvart J,Navrátil V,Sedlák F,Corey E,Colombatti M,Fracasso G,Koukolík F,Bárinka C,Sácha P,and Konvalinka J.(2014).Comparative analysis ofmonoclonal antibodies against prostate-specific membrane antigen(PSMA).TheProstate 1-14.

[0214] Wolf P, Freudenberg N, Bühler P, Alt K, Schultze-Seemann W, Wetterauer U, and -Beile U.(2010a).Three conformational antibodies specific fordifferent PSMA epitopes are promising diagnostic and therapeutic tools forprostate cancer.The Prostate 70:562-569.

[0215] Wolf P, Alt K, Wetterauer D, Bühler P, Gierschner D, Katzenwadel A, Wetterauer U, and -Beile U.(2010b).Preclinical evaluation of arecombinant anti-prostate specific membrane antigen single-chain immunotoxinagainst prostate cancer.J Immunother.33:262-271.

[0216] sequence

[0217] The following sequences form part of the disclosure of this application. This application also provides an electronic sequence listing compatible with WIPO ST 25. For the avoidance of doubt, if there is a difference between the sequence in the table below and the sequence in the electronic sequence listing, the sequence in the table below should be regarded as the correct sequence.

[0218] h / d, humanized / deimmunized; CDR, complementarity determining region; FR, framework region; CR, constant region

[0219]

[0220]

[0221]

[0222] Sequence Listing <110> Heidelberg Pharma Res. GmbH <120> Humanized antibodies against PSMA <130> HD40496 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 1 Gly Tyr Thr Phe Thr Tyr Phe 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 2 Gly Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Asn Glu Asn Phe Lys 1 5 10 15 Gly <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 3 Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser 1 5 10 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 4 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 5 Thr Val Ser Asn Arg Phe Ser 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 6 Ser Gln Ser Thr His Val Pro Thr 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 7 Gly Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 8 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 8 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Gln Thr Tyr Leu His 1 5 10 15 <210> 9 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 9 Gly Ile Ser Pro Gly Asp Gly Asn Val Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 10 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 10 Arg Ser Ser Gln Ser Leu Val His Ser Ser Gly Gln Thr Tyr Leu His 1 5 10 15 <210> 11 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 11 Gly Ile Ser Pro Gly Asp Ser Asn Val Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 12 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 12 Thr Val Ser Asn Arg Ala Ser 1 5 <210> 13 <211> 120 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ile Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 112 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 14 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 15 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ile Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 16 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Gln Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 17 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Gly Asn Val Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ile Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 112 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 18 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Ser Gly Gln Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Ser Asn Val Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ile Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Ser Gly Gln Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 21 <211> 450 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ile Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 22 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 22 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 23 <211> 450 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ile Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 24 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 24 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Gln Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 25 <211> 450 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Gly Asn Val Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ile Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Light 450 <210> 26 <211> 218 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 26 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Ser Gly Gln Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 27 <211> 450 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Phe 20 25 30 Asp Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ser Pro Gly Asp Ser Asn Val Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ile Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Light 450 <210> 28 <211> 218 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 28 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Ser Gly Gln Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Thr Val Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Thr Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 29 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 30 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 30 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 31 <211> 259 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 31 Met Ala Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Glu Pro 1 5 10 15 Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Tyr Phe Asp Ile Asn Trp Leu Arg Gln Arg Pro Glu Gln Gly Leu Glu 35 40 45 Trp Ile Gly Gly Ile Ser Pro Gly Asp Gly Asn Thr Asn Tyr Asn Glu 50 55 60 Asn Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Thr Thr 65 70 75 80 Ala Tyr Ile Gln Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Phe Cys Ala Arg Asp Gly Asn Phe Pro Tyr Tyr Ala Met Asp Ser Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Lys 115 120 125 Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg Val Asp Ile Glu Leu Thr 130 135 140 Gln Ser Pro Leu Ser Leu Pro Val Ile Leu Gly Asp Gln Ala Ser Ile 145 150 155 160 Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 165 170 175 Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 180 185 190 Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly 195 200 205 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 210 215 220 Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Thr 225 230 235 240 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Ala 245 250 255 Ala Gly Ser <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> Xaa <222> (2)..(2) <223> Xaa = Y, F or W <220> <221> Xaa <222> (4)..(4) <223> Xaa = Y, F or W <220> <221> Xaa <222> (6)..(6) <223> Xaa = Y, F or W <220> <221> Xaa <222> (7)..(7) <223> Xaa = Y, F or W <400> 32 Gly Xaa Thr Xaa Thr Xaa Xaa 1 5 <210> 33 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> Xaa <222> (7)..(7) <223> Xaa = G or S <220> <221> Xaa <222> (9)..(9) <223> Xaa = T or V <220> <221> Xaa <222> (12)..(12) <223> Xaa = N or A <220> <221> Xaa <222> (13)..(13) <223> Xaa = E or Q <220> <221> Xaa <222> (14)..(14) <223> Xaa = N or K <220> <221> Xaa <222> (16)..(16) <223> Xaa = K or Q <400> 33 Gly Ile Ser Pro Gly Asp Xaa Asn Xaa Asn Tyr Xaa Xaa Xaa Phe Xaa 1 5 10 15 Gly <210> 34 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> Xaa <222> (4)..(4) <223> Xaa = Y, F or W <220> <221> Xaa <222> (6)..(6) <223> Xaa = Y, F or W <220> <221> Xaa <222> (7)..(7) <223> Xaa = Y, F or W <400> 34 Asp Gly Asn Xaa Pro Xaa Xaa Ala Met Asp Ser 1 5 10 <210> 35 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Xaa <222> (10)..(10) <223> Xaa = N or S <220> <221> Xaa <222> (12)..(12) <223> Xaa = N or Q <220> <221> Xaa <222> (14)..(14) <223> Xaa = Y, F or W <400> 35 Arg Ser Ser Gln Ser Leu Val His Ser Xaa Gly Xaa Thr Xaa Leu His 1 5 10 15 <210> 36 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Xaa <222> (6)..(6) <223> Xaa = A, Y, F or W <400> 36 Thr Val Ser Asn Arg Xaa Ser 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 37 Ser Gln Ser Thr His Val Pro Thr 1 5

Claims

1. A humanized antibody or antigen-binding fragment or antigen-binding portion thereof that binds to the extracellular domain of prostate-specific membrane antigen (PSMA), comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR H1, CDR H2 and CDR H3, wherein the amino acid sequences of CDR H1, CDR H2 and CDR H3 are: As shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 respectively; As shown in SEQ ID No. 1, SEQ ID No. 7 and SEQ ID No. 3 respectively; As shown in SEQ ID No. 1, SEQ ID No. 9 and SEQ ID No. 3 respectively; or As shown in SEQ ID No. 1, SEQ ID No. 11 and SEQ ID No. 3 respectively; And the VL domain comprises CDR L1, CDR L2 and CDR L3, wherein the amino acid sequences of CDR L1, CDR L2 and CDR L3 are: As shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 respectively; As shown in SEQ ID No. 8, SEQ ID No. 5 and SEQ ID No. 6 respectively; As shown in SEQ ID No. 10, SEQ ID No. 5 and SEQ ID No. 6 respectively; or As shown in SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 6, respectively, However, the humanized antibody or its antigen-binding fragment or antigen-binding portion does not comprise a combination of the CDRH1 sequence shown in SEQ ID No. 1, the CDR H2 sequence shown in SEQ ID No. 2, the CDR H3 sequence shown in SEQ ID No. 3, the CDR L1 sequence shown in SEQ ID No. 4, the CDR L2 sequence shown in SEQ ID No. 5 and the CDRL3 sequence shown in SEQ ID No.

6.

2. A humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 1, comprising a VH domain having at least 90% sequence homology with a sequence as shown in any one of SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No.

19.

3. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 1, comprising a VH domain having at least 95% sequence homology with a sequence as shown in any one of SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No.

19.

4. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 1, comprising a VH domain sequence, wherein the VH domain sequence is shown in any one of the sequences selected from SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No.

19.

5. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 1, comprising a VL domain having at least 90% sequence homology with a sequence as shown in any one of SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 and SEQ ID No.

20.

6. A humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 1, comprising a VL domain having at least 95% sequence homology with a sequence as shown in any one of SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 and SEQ ID No.

20.

7. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 1, comprising a VL domain sequence, wherein the VL domain sequence is shown in any one of the sequences selected from SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 and SEQ ID No.

20.

8. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 1, comprising: The VH domain sequence shown in SEQ ID No.13 and the VL domain sequence shown in any one of SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20, or The VH domain sequence shown in SEQ ID No.15 and the VL domain sequence shown in any one of SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20, or The VH domain sequence shown in SEQ ID No.17 and the VL domain sequence shown in any one of SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.20, or The VH domain sequence is shown as SEQ ID No.19 and the VL domain sequence is shown as any one sequence selected from SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 and SEQ ID No.

20.

9. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 4, wherein the amino acid sequence of the heavy chain is any one of SEQ ID No.21, SEQ ID No.23, SEQ ID No.25 or SEQ ID No.

27.

10. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to claim 7, wherein the amino acid sequence of the light chain is any one of SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26 or SEQ ID No.

28.

11. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to any one of claims 1 to 10, wherein the humanized antibody or antigen-binding fragment or antigen-binding portion thereof is expressed in terms of EC 50 Binds to the extracellular domain of PSMA with an affinity of <0.4 µg / ml.

12. The humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to any one of claims 1 to 10, wherein the prostate-specific membrane antigen is human prostate-specific membrane antigen.

13. The humanized antibody or antigen-binding fragment or portion thereof according to any one of claims 1 to 10, wherein the antibody is of the IgG isotype.

14. A humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is selected from the group consisting of a variable domain (Fv), a Fab fragment and a F(ab)2 fragment, or wherein the antigen-binding portion is a single-chain Fv (scFv).

15. A humanized antibody or antigen-binding fragment or antigen-binding portion thereof comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is any one of SEQ ID No. 21, 23, 25 or 27, and the amino acid sequence of the light chain is any one of SEQ ID No. 22, 24, 26 or 28, However, the humanized antibody or antigen-binding fragment or antigen-binding portion thereof does not comprise a combination of a heavy chain amino acid sequence of SEQ ID No. 21 and a light chain amino acid sequence of SEQ ID No.

22.

16. A humanized antibody or antigen-binding fragment or antigen-binding portion thereof comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is SEQ ID No. 27, and the amino acid sequence of the light chain is SEQ ID No.

28.

17. An antibody drug conjugate (ADC) comprising the humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to any one of claims 1 to 16 conjugated to a therapeutic agent.

18. The antibody drug conjugate (ADC) according to claim 17, wherein the humanized antibody or antigen-binding fragment or antigen-binding portion thereof is conjugated to the therapeutic agent via a linker moiety.

19. A pharmaceutical formulation comprising a humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to any one of claims 1 to 16 or an antibody drug conjugate (ADC) according to claim 17 or claim 18, and at least one pharmaceutically acceptable carrier, wherein the formulation is suitable for at least intravenous, intramuscular or subcutaneous administration.

20. Use of a humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to any one of claims 1 to 16, an antibody drug conjugate (ADC) according to claim 17 or claim 18, or a pharmaceutical formulation according to claim 19 in the preparation of a medicament for treating prostate cancer.

21. A nucleic acid encoding a humanized antibody or antigen-binding fragment or portion thereof according to any one of claims 1 to 16.

22. A host cell capable of producing a humanized antibody or antigen-binding fragment or antigen-binding portion thereof according to any one of claims 1 to 16, wherein the host cell is a mammalian cell.

23. The host cell according to claim 22, wherein the mammalian cell is a CHO cell line.

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