bispecific single-chain antibody molecule, its use and production process, as well as pharmaceutical

专利摘要:
BISPECIFIC SINGLE-CHAIN ANTIBODY PSMAXCD3, ITS USE AND PRODUCTION PROCESS, AS WELL AS NUCLEIC ACID SEQUENCE, HOST CELL AND PHARMACEUTICAL COMPOSITION. The present invention relates to a bispecific single chain antibody molecule comprising a first binding domain capable of binding a human epitope and a non-chimpanzee primate of the CD3 epsilon chain, wherein the epitope is part of a amino acid sequence comprised in the group consisting of SEQ ID NOs. 2, 4, 6 and 8, and a second binding domain capable of binding prostate specific membrane antigen (PSMA). The present invention also provides nucleic acids encoding said bispecific single chain antibody molecule, as well as vectors and host cells and a process for their production. The present invention further relates to pharmaceutical compositions comprising said bispecific single chain antibody molecule and medical uses of said bispecific single chain antibody molecule.
公开号:BR112012024964B1
申请号:R112012024964-9
申请日:2011-04-01
公开日:2021-05-25
发明作者:Peter Kufer
申请人:Amgen Research (Munich) Gmbh；
IPC主号:

专利说明:

The present invention relates to a single-chain bispecific antibody molecule comprising a first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 epsilon chain, in which the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NO: 2, 4, 6 and 8, and a second binding domain capable of binding prostate specific membrane antigen (PSMA). The present invention also provides nucleic acids encoding said single-chain bispecific antibody molecule, as well as vectors and host cells and a process for their production. The present invention further relates to pharmaceutical compositions comprising said bispecific single chain antibody molecule and medical uses of said bispecific single chain antibody molecule.
[0002] T cell recognition is mediated through the clonotypically distributed delta, alpha beta and gamma T cell receptors (TCRs), which interact with the peptide-loaded molecules of the MHC peptide (pMHC) (Davis & Bjorkman, Nature 334 (1988), 395 to 402). The antigen-specific TCR chains lack signaling domains, but instead are coupled to the multi-conserved CD3 subunit signaling apparatus (Cell Call, 111 (2002), 967 to 979, Alarcon, Immunol Rev. 191 (2003) , 38 to 46, Malissen Immunol. Rev. 191 (2003), 7 to 27). The mechanism by which TcR binding is directly communicated to the signaling apparatus remains a fundamental question in T cell biology (Alarcon, loc cit, Davis, Cell 110 (2002), 285 to 287). It seems clear that sustained T cell responses involve coreceptor coupling, TcR oligomerization, and a higher order arrangement of TCR-pMHC complexes at the immune synapse (Davis & van der Merwe, Curr. Biol. 11 (2001), R289 to R291, Davis, Nat. Immunol 4. (2003), 217 to 224). However, TcR signaling occurs very early in the absence of these events, and may include a ligand-induced conformational change in CD3 epsilon cells (Alarcon, loc. cit., Davis (2002), loc. cit., Gil, J. Biol Chem 276 (2001), 11174 to 11179, Gil, Cell 109 (2002), 901 to 912). The epsilon, gamma, delta, and zeta subunits of the signaling complex associate with each other to form an epsilon-gamma CD3 heterodimer, an epsilon-delta CD3 heterodimer, and a zeta-zeta CD3 homodimer (Call, loc. cit.).
[0003] Several studies have revealed that CD3 molecules are important for the proper cell surface expression of TcR beta alpha and normal T cell development (Berkhout, J. Biol. Chem. 263 (1988), 8528 to 8536, Wang, J. Exp. Med. Chem. 188 (1998), 1375 to 1380, Kappes, Curr. Opin. Immunol. 7 (1995), 441 to 447). The solution structure of the ectodomain fragments of the mouse CD3 epsilon gamma heterodimer showed that the epsilon gamma subunits are both defined Ig C2 domains, which interact with each other to form an unusual side-by-side dimer configuration (Sun, Cell 105 ( 2001), 913 to 923). Although the cysteine-rich stem appears to play an important role in driving CD3 dimerization (Su, loc. cit., Borroto, J. Biol. Chem. 273 (1998), 12807 to 12816), through the interaction of the extracellular domains of CD3 epsilon and CD3 gamma is sufficient for the assembly of these proteins with TcR beta (Manolios, Eur. J. Immunol. 24 (1994), 84 to 92, Manolios & Li, Immunol. Cell Biol. 73 (1995), 532 to 536 ). Although still controversial, the dominant TcR stoichiometry likely comprises a CD3 epsilon gamma heterodimer, a CD3 epsilon delta heterodimer, and a zeta zeta homodimer (Call, loc. cit.). Given the central role of the human epsilon CD3 gamma heterodimer in the immune response, the crystal structure of the complex bound to the therapeutic OKT3 antibody has recently been elucidated (Kjer-Nielsen, PNAS 101, (2004), 7675 to 7680).
[0004] A number of therapeutic strategies modulate T cell immunity by targeting TcR signaling, especially anti-human CD3 monoclonal antibodies (mAbs) which are widely used clinically in immunosuppressive regimens. The mouse-specific CD3 mAb OKT3 was the first mAb licensed for use in humans (Sgro, Toxicology 105 (1995), 23 to 29) and is widely used clinically as an immunosuppressive agent in transplantation (Chatenoud, Clin. Transplant 7 (1993) ), 422 to 430, Chatenoud, Nat. Transplant Rev. Immunol. 3 (2003), 123 to 132, Kumar,. Proc. 30 (1998), 1351 to 1352), type 1 diabetes (Chatenoud (2003), loc. cit.) and psoriasis (Utset, J. Rheumatol. 29 (2002), 1907 to 1913). Furthermore, anti-CD3 mAbs can induce partial T cell signaling and clonal anergy (Smith, J. Exp. Med. 185 (1997), 1413 to 1422). OKT3 has been described in the literature as a potent T cell mitogen (Van Wauve, J. Immunol. 124 (1980), 2708 to 18) as well as a potent T cell killer (Wong, Transplantation 50 (1990), 683 to 9). OKT3 displays both activities in a time-dependent fashion; the following early activation of T cells leading to the release of cytokines, with further administration of OKT3 after it blocks all known T cell functions. This is because of the subsequent blockade of T cell function that OKT3 has found wide application as an immunosuppressant in therapy regimens for the reduction or even abolition of graft tissue rejection.
[0005] OKT3 reverses tissue allograft rejection, most likely by blocking the function of all T cells, which play an important role in acute rejection. OKT3 reacts with and blocks the function of the CD3 complex on the membrane of human T cells, which is associated with the T cell antigen recognition (TCR) structure and is essential for signal transduction. The subunit to which TCR/CD3 is linked by OKT3 has been the subject of several studies. Although some evidence has pointed to a specificity of OKT3 for the epsilon subunit of the TCR/CD3 complex (Tunnacliffe, Int. Immunol 1 (1989), 546 to 50; Kjer-Nielsen PNAS 101, (2004), 7675 to 7680). Other evidence has shown that OKT3 binding to the TCR/CD3 complex requires other subunits of that complex to be present (Salmeron, J. Immunol. 147 (1991), 3047-52).
[0006] Other well-known antibodies specific to the CD3 molecule are listed in Tunnacliffe, Int. Immunol. 1 (1989), 546 to 50. As indicated above, such specific CD3's are capable of inducing different T cell responses to lymphokine production, such as (Von Wussow, J. Immunol 127 (1981), 1197, Palacious, J Immunol 128 (1982), 337), proliferation (Van Wauve, J. Immunol. 124 (1980), 2708 to 18) and a T cell induction suppressor (Kunicka, in "Lymphocyte Typing II" 1 (1986) , 223). That is, depending on experimental conditions, the CD3-specific monoclonal antibody can inhibit or induce cytotoxicity (Leewenberg, J. Immunol 134 (1985), 3770, Phillips, J. Immunol 136 (1986) 1579; Platsoucas, Proc Natl. Acad Sci USA 78 (1981), 4500; Itoh, Cell Immunol 108 (1987), 283-96, Mentzer, J. Immunol 135 (1985), 34, Landegren, J. Exp. Med. 155. (1982) , 1579; Choi (2001), Eur. J. Immunol 31, 94 to 106; Xu (2000), Cell Immunol 200, 16 to 26; Kimball (1995), Transpl Immunol 3, 212 to 221).
[0007] Although many of the CD3 antibodies described in the art have been reported to recognize the CD3 epsilon subunit of the CD3 complex, most of them actually bind to conformational epitopes and thus only recognize CD3 epsilon in the native context of the TCR. Conformational epitopes are characterized by the presence of two or more discrete amino acid residues that are separated in the primary sequence, but join on the surface of the molecule when the polypeptide nails to the native protein/antigen (Sela, (1969) Science 166, 1365 and Lavide, (1990) Cell 61, 553 to 6). The conformational epitopes linked by CD3 epsilon described in the art can be separated into two groups. In the main group, said epitopes are formed by means of two subunits, for example, CD3 of the epsilon chain CD3 and CD3 gamma or CD3 of the delta chain. For example, the most widely used CD3 epsilon monoclonal antibodies OKT3, WT31, UCHT1, 7D6 and Leu-4 have been found in several studies to not bind to cells transfected individually with the CD3 epsilon chain. However, these antibodies labeled doubly transfected cells with a combination of epsilon CD3 plus either gamma CD3 or delta CD3 (Tunnacliffe, loc cit, Law, Int. Immunol 14 (2002), 389 to 400, Salmeron, J. Immunol. 147 (1991), 3047 to 52; Coulie, Eur. J. Immunol 21 (1991), 1703 to 9). In a second smaller group, the conformational epitope is being formed within the CD3 epsilon subunit by itself. A member of this group is, for example mAb APA 1/1 which was raised against denatured CD3 epsilon (Risueno, Blood 106 (2005), 601 to 8). Taken together, most CD3 epsilon antibodies described in the art recognize conformational epitopes located on two or more CD3 subunits. The discrete amino acid residues that form the three-dimensional structure of these epitopes can thus be located either on the epsilon CD3 subunit or on the epsilon CD3 subunit and other CD3 subunits, such as CD3 gamma or CD3 delta.
[0008] Another problem with respect to the CD3 antibody is that many CD3s have been found to be species specific. Anti-CD3 monoclonal antibodies - as is generally true of all other monoclonal antibodies - through the highly specific recognition function of target molecules. They only recognize a single site, or epitope, on their target CD3 molecule. For example, one of the most widely used and best characterized as monoclonal antibodies specific for the CD3 complex is OKT-3. This antibody reacts with chimpanzee CD3 but not with the CD3 homolog of other primates, such as monkeys or with dog CD3 (Sandusky et al., J. Med. Chem. Primatol. 15 (1986), 441 to 451). Likewise, WO2005/118635 or WO2007/033230 describe human monoclonal antibodies CD3 epsilon, which react with human CD3 epsilon, but not with CD3 epsilon from rat, mouse, rabbit, or non-chimpanzee primates such as rhesus monkeys, cynomolgus monkey, or monkey baboon. The UCHT-1 anti-CD3 monoclonal antibody is also reactive with chimpanzee CD3 but not monkey CD3 (own data). On the other hand, there are also examples of monoclonal antibodies, which recognize monkey antigens but not their human counterparts. An example of this group is the monoclonal antibody FN-18 directed to monkey CD3 (Uda et al., J. Med. Chem. Primatol. 30 (2001), 141 to 147). Interestingly, peripheral lymphocytes from about 12% of cynomolgus monkeys were found to lack reactivity to the anti-rhesus monkey anti-CD3 monoclonal antibody (FN-18), due to a polymorphism of the CD3 antigen in monkeys. Uda et al. described a two amino acid substitution in the CD3 sequence of cynomolgus monkeys, which are not reactive with FN-18 antibodies, compared to animal-derived CD3 cells, which are reactive with FN-18 antibodies (Uda et al., J Med Primatol 32 (2003), 105 to 10; Uda et al, J Med Primatol 33 (2004), 34 to 7).
[0009] The ability to discriminate, that is, the inherent species specificity, not only for CD3 monoclonal antibodies (and fragments thereof), but monoclonal antibodies in general is a significant obstacle to their development as therapeutic agents for the treatment of human diseases. In order to gain market approval, no new drug candidate must pass rigorous testing. This test can be subdivided into pre-clinical and clinical phases: Whereas the latter - subdivided into phases generally known as clinical phases I, II and III - is performed on human patients, the former is performed on animals. The purpose of the preclinical trial is to demonstrate that the candidate drug has the desired activity, and most importantly is safe. Only when the safety in animals and possible efficacy of the candidate drug has been determined in pre-clinical testing of this drug candidate will it be approved for clinical trials in humans through the respective regulatory authority. Drug candidates can be tested for safety in animals in the following three ways: (i) from a given species, that is, from a species in which drug candidates can recognize ortholog antigens, (ii) from an animal transgenic containing the human antigens and (iii) through the use of a substitute for the drug candidate that can bind the orthologous antigens present in the animal. The limitations of transgenic animals are that this technology is typically limited to rodents. Between rodents and humans there are significant differences in physiology and safety results cannot be easily extrapolated to humans. The limitations of a substitute for the drug candidate is the different composition of matter compared to the actual drug candidate and often the animals used are rodents with the limitation as discussed above. Therefore, preclinical data generated in rodents are of limited predictive power regarding the drug candidate. The approach of choice for safety testing is to use a relevant species, preferably a minor primate. The limitation now of monoclonal antibodies suitable for therapeutic intervention in man described in the art is that the corresponding species are higher primates, especially chimpanzees. Chimpanzees are considered to be an endangered species and, due to their human nature, the use of these animals for drug safety testing has been banned in Europe and elsewhere is very restricted. CD3 has also been used successfully as a target for bispecific single-chain antibodies in order to redirect cytotoxic T cells to pathological cells, resulting in the depletion of diseased cells from the organism concerned (WO 99/54440, WO 04/106380). For example, Bargou et al. (Science 321 (2008):974 to 7) recently reported on the clinical activity of a bispecific CD19xCD3 antibody construct called blinatumomab, which has the potential to engage all cytotoxic T cells in human patients for cancer cell lysis. Doses as low as 0.005 mg per square meter per day in non-Hodgkin lymphoma patients led to an elimination of target cells in the blood. Complete and partial tumor regressions were first observed with a dose of 0.015 mg, and all seven patients treated with a dose of 0.06 mg experienced tumor regression. Blinatumomab also led to clearance of tumor cells from bone marrow and liver. Although this study established a clinical proof of concept for the therapeutic potency of the bispecific single-chain antibody format in the treatment of blood cell derived cancer, there is still a need for successful concepts for therapies for other types of cancer.
[00010] In 2008, approximately 186,320 men will be diagnosed with prostate cancer in the United States and approximately 28,660 men will die from the disease. The most recent cancer mortality report shows that, in 2004, the global death rate from prostate cancer among American men was 25 per 100,000. In the late 1980s, the widespread adoption of prostate-specific antigen (PSA) represented a major advance in the treatment of prostate cancer. This test measures the amount of PSA protein in the blood, which is often elevated in patients with prostate cancer. In 1986, the U.S. Food and Drug Administration approved the use of the PSA test to monitor patients with prostate cancer and, in 1994, it additionally approved its use as a screening test for this disease. Due to the widespread implementation of PSA testing in the United States, approximately 90 percent of all prostate cancers are currently diagnosed at an early stage, and as a result, men are surviving longer after diagnosis. However, the results of two ongoing clinical trials, the NCI-Sponsored Prostate, Lung, Colorectal and Ovarian Screening Test (PLCO) and the European Screening Study for Prostate Cancer (ERSPC) will be needed to determine whether APE exam really saves lives. Ongoing clinical trials over the past 25 years have investigated the effectiveness of natural and synthetic compounds in preventing prostate cancer. For example, the Prostate Cancer Prevention Test (PCPT), which enrolled nearly 19,000 healthy men, found finasteride, an approved drug for the treatment of benign prostatic hyperplasia (BPH), which is a benign enlargement of the prostate. , reduces the risk of developing prostate cancer by 25 percent. Another trial, the Selenium and Vitamin E Cancer Prevention Test (SELECT), is studying more than 35,000 men to determine whether daily supplements of selenium and vitamin E can reduce the incidence of prostate cancer in healthy men. Other prostate cancer prevention trials are currently evaluating the protective potential of multivitamins, vitamins C and D, soy, green tea, and lycopene, which is a natural compound found in tomatoes. A study, carried out in 2005, showed that specific genders were fused in 60 to 80 percent of the analyzed prostate tumors. This study represents the first observation of non-random gender rearrangements in prostate cancer. This genetic alteration can eventually be used as a biomarker to aid in the diagnosis and possibly treatment of this disease. Other studies have shown that genetic variations in a specific region of chromosome 8 can increase a man's risk of developing prostate cancer. These genetic variations account for about 25 percent of prostate cancers that occur in white men. They are the first validated genetic variants that increase the risk of developing prostate cancer and may help scientists better understand the genetic causes of the disease. There is also ongoing research that examines how proteins circulating in a patient's blood can be used to improve the diagnosis of prostate cancer and other cancers. In 2005, scientists identified a specific group of proteins that are produced by a patient's immune system in response to prostate tumors. These proteins, a type of autoantibody, were able to detect the presence of prostate cancer cells in blood samples with greater accuracy than 90 percent. When used in combination with PSA, blood proteins and others can eventually be used to reduce the number of false positive results obtained with the PSA test alone and therefore reduce the number of unnecessary prostate biopsies that are performed each year due to false-positive PSA test results.
[00011] In addition to PSA, several other markers for prostate cancer have been identified, including, for example, the six transmembrane epithelial antigen of the prostate (STEAP) (Hubert et al. PNAS 96 (1999), 14523 to 14528) , prostate stem cell antigen (PSCA) (Reiter et al., Proc. Nat. Acad. Sci. 95: 1735 to 1740, 1998) and prostate specific membrane antigen (PSMA; PSM) (Israeli et al., al, Cancer Res 53 (1993.) PSMA was originally defined by the monoclonal antibody (MAb) 7E11 derived from immunization with a partially purified membrane preparation from the prostatic adenocarcinoma lymph node (LNCaP) cell line (Horoszewicz et al. al., Anticancer Res. 7 (1987), 927 to 35) The 2.65-kb cDNA fragment encoding the protein was cloned and PSMA subsequently mapped to chromosome 11p11.2 (Israeli et al, loc cit, O' Keefe et al, Biochem Biophys Acta 1443 (1998), 113 to 127). into the prostatic secretory epithelium cells. Immunohistochemical staining showed that PSMA was absent to moderately expressed in hyperplastic and benign tissues, whereas malignant tissues stained more intensely (Horoszewicz et al., loc. cit.). Subsequent investigations have recapitulated these results and evidenced PSMA expression as a universal feature in virtually all prostate tissues examined to date. These reports further demonstrate that PSMA expression increases abruptly proportional to tumor aggressiveness ( Burger et al, Int. J. Cancer 100 (2002), 228-237, Chang et al, Cancer Res 59 (1999), 3192-98, Chang et al, Urology 57 (2001), 1179 to 1183), and Kawakami Nakayama, Cancer Res. 57 (1997), 2321 to 24;. Liu et al., Cancer Res 57 (1997), 3629 to 34; Lopes et al, Cancer Res. 50 (1990), 6423 to 29, Silver et al, Cancer Res Clin 9 (2003), 6357 to 62; Sweat et al, Urology 52 (1998), 637 to 40, Troyer et al, Int. J. Cancer 62 (1995), 552 to 558; Wright et al, Urology 48 (1996), 326 to 334). Consistent with the correlation between PSMA expression and tumor stage, increased PSMA levels are associated with androgen-independent prostate cancer (CaP). Analysis of tissue samples from patients with prostate cancer demonstrated elevated levels of PSMA after surgical castration or androgen deprivation therapy. Unlike prostate-specific antigen expression, which is dysregulated after androgen ablation, PSMA expression is significantly increased in both primary and metastatic tumor specimens (Kawakami et al., Wright et al., Loc. Cit.). Consistent with elevated expression in androgen-independent tumors, PSMA transcription is also known to be deregulated via steroids, and testosterone administration mediates a dramatic reduction in PSMA protein and mRNA levels (Israeli et al., Cancer Res. 54 (1994), 1807 to 1811, Wright et al, loc cit). PSMA is also highly expressed in secondary prostate tumors and occult metastatic disease. Immunohistochemical analysis revealed relatively intense and uniform expression of PSMA within metastatic lesions localized to lymph nodes, bones, soft tissues, and lungs compared to benign prostatic tissues (Chang et al (2001), loc cit, Murphy et al, Cancer 78 (1996), 809 to 818; Sweat et al, loc cit). Some reports have also indicated limited expression of PSMA in extraprostatic tissues, including a subset of renal proximal tubules, some cells in the brush border membrane of the intestine, and rare cells in intestinal crypts (Chang et al. (1999), Horoszewicz et al. ., Israel et al. (1994), Lopes et al., Troyer et al., loc. cit.). However, PSMA levels in these tissues are generally two to three orders of magnitude less than those seen in the prostate (Sokoloff et al., Prostate 43 (2000), 150 to 157). PSMA is also expressed in tumor-associated neovasculature of most solid tumors examined and is not yet present in normal vascular endothelium (Chang et al. (1999), Liu et al., Silver et al., Loc. Cit.). Although the significance of PSMA expression within the vasculature is unknown, the specificity for tumor-associated endothelium makes PSMA a potential target for the treatment of many forms of malignancy.
[00012] Although much effort has been put into identifying new targets for therapeutic approaches to cancer, cancer is still one of the most diagnosed diseases. In view of this, there is still a need for effective cancer treatments.
[00013] The present invention provides a single chain bispecific antibody molecule comprising a first binding domain capable of binding to an epitope from the human and non-chimpanzee primate CD3ε (epsilon) chain, wherein the epitope is part of a amino acid sequence comprised in the group consisting of SEQ ID NOs: 2, 4, 6 and 8, and a second binding domain capable of binding prostate specific membrane antigen (PSMA).
[00014] Although T cells involve bispecific single-chain antibodies described in the art having great therapeutic potential in the treatment of malignant diseases, most of these bispecific molecules are limited in that they are species-specific and recognize only human antigen, and - due to to genetic resemblance - likely chimpanzee homologue. The advantage of the present invention is the provision of a single-chain bispecific antibody comprising a binding domain that exhibits cross-species specificity for human and non-chimpanzee primates of the epsilon CD3 chain.
[00015] In the present invention, a polypeptide fragment of 1 to 27 amino acid residues from the N-terminus of the extracellular domain of CD3 epsilon was surprisingly identified which - in contrast to all other known epitopes of CD3 epsilon cells described in the art - maintains the its integrity in the three-dimensional structure when taken from its native environment, in the CD3 complex (and optionally fused to a heterologous amino acid sequence such as EpCAM or an immunoglobulin Fc part). The present invention, therefore, provides a single-chain bispecific antibody molecule comprising a first binding domain capable of binding to an epitope of an N-terminal fragment of 1 to 27 amino acid residues of epsilon CD3 extracellular domain polypeptide (CD3 epsilon, which is, for example, taken from its native environment and/or constituted by (displayed on the surface of) a T cell) of the human being and at least one non-chimpanzee primate of CD3 epsilon chain, where the epitope is part of an amino acid of the sequence comprised in the group consisting of SEQ ID NOs: 2, 4, 6 and 8, and a second binding domain capable of binding to prostate specific membrane antigen (PSMA). Preferred non-chimpanzee primates are mentioned elsewhere herein. At least one (or a selection thereof or all) of the primate(s) selected from Callithrix jacchus; Saguinus oedipus, Saimiri sciureus, and Macaca fascicularis (or SEQ ID 631 or 632 or both), is (are) particularly preferred. Mulatta macaque, also known as rhesus monkey is also predicted as another preferred primate. It is, therefore, anticipated that the antibodies of the present invention bind to (are capable of binding to) the context-independent epitope of a 1 to 27 amino acid residue polypeptide fragment of the N-terminal extracellular domain of CD3 epsilon human and from Callithrix jacchus, Saguinus oedipus, Saimiri sciureus, and Macaca fascicularis (or SEQ ID NO: 631 or 632 or both), and optionally also Macaca mulatta. A single chain bispecific antibody molecule comprising a first binding domain as defined in the present invention can be obtained (is obtainable by) or can be manufactured according to the protocol set forth in WO 2008/119567 (in particular in the Example 2 of WO 2008/119567). For this purpose, the (a) immunization of mice with a fragment of 1 to 27 amino acid residues of polypeptide from the extracellular domain of the N-terminus of human and/or Saimiri sciureus CD3 epsilon is foreseen, (b) the generation of a murine immune antibody scFv library, (c) identifying specific CD3 epsilon binders by testing the ability to bind to at least SEQ ID NOs: 2, 4, 6, and 8.
[00016] The CD3 epitope independence context provided in the present invention corresponds to the first 27 amino acids of the N-terminus of CD3 epsilon, or functional fragments of this 27 amino acid stretch. The phrase "context-independent," as used in the present invention in relation to the CD3 epitope means that the binding of the present invention described in the present invention, the binding molecules/antibody molecules does not lead to a change or modification of the conformation, sequence or structure surrounding the antigenic determinant or epitope. In contrast, the CD3 epitope recognized by a conventional CD3 binding molecule (for example, as described in WO 99/54440 or WO 04/106380) is located on the epsilon CD3 chain from the C-terminus to the N-terminus of 1 to 27 context-independent amino acids of the epitope, where it only has the correct conformation if it is integrated into the rest of the epsilon chain and maintained in the steric right position by heterodimerization of the epsilon chain with either the CD3 or delta chain. Anti-CD3 binding domains as part of a bispecific PSMAxCD3 single chain molecule as provided herein have been described in WO 2008/119567. These binding domains are generated (and directed) against a context independent of the CD3 epitope predicting a surprising clinical improvement with respect to T cell redistribution and thus a more favorable safety profile. Without being limited by theory, since the CD3 epitope is context-independent, forming a sufficient autonomous subdomain on its own without influencing the rest of the CD3 complex, the CD3 binding domain of the bispecific single chain molecule PSMAxCD3 provided in the present invention induces less allosteric changes in CD3 conformation than conventional CD3 binding molecules (such as molecules provided in WO 99/54440 or WO 04/106380), which recognize context-dependent CD3 epitopes.
The context of CD3 epitope independence that is recognized by the CD3 binding domain of the single PSMAxCD3 chain bispecific antibody of the present invention is associated with less or no T cell redistribution (T cell redistribution equals one initial episode of drop and subsequent recovery of absolute T cell counts) during the initial phase of treatment with said single-chain bispecific PSMAxCD3 antibody of the present invention. These results in an improved safety profile of the single PSMAxCD3 chain bispecific antibody of the present invention compared to conventional CD3 binding molecules known in the art, which recognize context-dependent CD3 epitopes. Particularly because T cell redistribution during the early phase of treatment with CD3 binding molecules is an important risk factor for adverse events such as CNS adverse events, bispecific single chain PSMAxCD3 antibodies of the present invention by recognizing a context-independent , rather than a context dependent CD3 epitope has a substantial advantage over the safety of CD3 binding molecules known in the art. Patients with such adverse CNS events related to T cell redistribution during the initial phase of treatment with conventional CD3 binding molecules often suffer from confusion and disorientation, in some cases also from urinary incontinence. Confusion is a change in mental state where the patient is not able to think with their normal level of clarity. Generally, the patient finds it difficult to concentrate and think not only blurred and uncertainly, but often significantly slower. Patients with adverse CNS events related to T cell redistribution during the early phase of treatment with conventional CD3 binding molecules may also suffer from memory loss. Confusion often leads to the loss of the ability to recognize people, places, time, or the date. Feelings of disorientation are common in confusion, and decision-making ability is impaired. Adverse CNS events related to T cell redistribution during the initial phase of treatment with conventional CD3 binding molecules can further comprise blurred speech and/or difficulty in finding the words. This disorder can impair both language expression and comprehension, as well as reading and writing. In addition to urinary incontinence, vertigo and dizziness may also accompany CNS adverse events related to T cell redistribution during the initial phase of treatment with conventional CD3-binding molecules in some patients. The maintenance of the three-dimensional structure within said polypeptide fragments of the 27 amino acids of the N-terminus of CD3 epsilon can be used for the generation of, preferably, in humans, the binding domains that are capable of binding to the N-terminal fragment of the polypeptide on CD3 epsilon in vitro and for the native (epsilon CD3 subunit) CD3 complex on T cells in vivo with the same binding affinity. These data strongly indicate that the N-terminal fragment as described in the present invention constitutes a tertiary conformation, which is similar to its structure normally existing in vivo. A very sensitive test for the importance of the structural integrity of said polypeptide fragments of the 27 amino acids of the N-terminus of CD3 epsilon was carried out. The individual amino acids of the N-terminal 27 amino acid polypeptide fragments of CD3 epsilon were changed to alanine (alanine scan) to test the sensitivity of the N-terminal 27 amino acid polypeptide fragments of CD3 epsilon for minor disruptions. CD3-specific binding domains as part of the unique PSMAxCD3 bispecific antibody chain of the present invention were used to test for binding with alanine mutants the N-terminal 27 amino acid polypeptide fragments of CD3 epsilon (see WO 2008/ 119567). Individual exchanges of the first five amino acid residues at the N-terminus of the fragment and two of the amino acids at positions 23 and 25 of the polypeptide fragments from the 27 amino acids of the N-terminus of CD3 epsilon were critical for the binding of antibody molecules. The substitution of amino acid residues in the region of position 1 to 5 residues that make up Q (glutamine at position 1), D (aspartic acid at position 2), G (glycine at position 3), N (asparagine at position 4) , and E (glutamic acid at position 5) to Alanine abrogated the binding of the preferably unique bispecific PSMAxCD3 human chain antibody of the present invention to said fragment. Whereas, for at least some of the, preferably, human chain antibody, the unique bispecific PSMAxCD3 of the present invention, two amino acid residues at the C-terminal end of said fragment of T (threonine at position 23) and I (isoleucine at position) 25) reduced the binding energy for the preferably single bispecific PSMAxCD3 human chain antibody of the present invention.
[00018] Unexpectedly, it was found that in this way isolated, preferably human, PSMAxCD3 single chain bispecific antibody of the present invention not only recognizes the human N-terminal fragment of CD3 epsilon, but also the corresponding homologous CD3 epsilon fragments of primates, including a number of New -WORLD Monkeys (Mico, Callithrix jacchus, Saguinus oedipus; Saimiri sciureus) and Old - World Monkeys (Macaca fascicularis, also known as Cynomolgus monkeys, or Macaca mulatta, also known as rhesus monkey). Thus, the multi-primate specificity of the single PSMAxCD3 chain bispecific antibody of the present invention was detected. The following sequence analyzes confirmed that humans and primates share a highly homologous sequence in stretch at the N-terminal end of the extracellular domain of CD3 epsilon.
[00019] The amino acid sequence of said above-mentioned N-terminal fragments of CD3 epsilon is represented in SEQ ID NO: 2 (human), SEQ ID NO: 4 (Callithrix jacchus), SEQ ID NO: 6 (Saguinus oedipus), SEQ ID NO: 8 (Saimiri sciureus), SEQ ID NO: 631 QDGNEEMGSI TQTPYQVSISGTTILTC or SEQ ID NO: 632 QDGNEEMGSITQTPYQVSISG TTVILT (Macaca fascicularis, also known as a cynomolgus monkey), and SEQ ID NO: 633 QDSISQVSISGTTILTC like rhesus monkey).
[00020] The second binding domain of the single PSMAxCD3 chain bispecific antibody of the present invention binds to the prostate specific membrane antigen (PSMA). Preferably, the second binding domain of the bispecific single chain antibody PSMAxCD3 binds to human PSMA or a non-chimpanzee primate PSMA, more preferred binds to human PSMA and a non-chimpanzee primate PSMA and therefore is transspecies specific ; even more preferred for human PSMA and PSMA macaque (and therefore is cross-species specific as well). Particularly preferred, the PSMA macaque is the Cynomolgus monkey PSMA and/or the PSMA rhesus monkey. However, it is not excluded from the scope of the present invention that the second binding domain may also bind to PSMA homologs from other species, such as to the PSMA homolog in rodents.
[00021] Prostate cancer is the second largest case of cancer in men. For 2008, an estimated 186,320 men will be diagnosed with prostate cancer in the United States and approximately 28,660 men will die from the disease. The risk of prostate cancer is strongly related to age: very few cases are reported in men under 50 and three quarters of cases occur in men over 65 years. The greatest number of cases is diagnosed in people aged 70 to 74 years old. Currently, the growth rate of the elderly population is significantly higher than that of the total population. By 2025 to 2030, projections indicate that the population over 60 years old will grow 3.5 times faster than the total population. The proportion of elderly people is projected to more than double worldwide over the next half century, meaning that an increase in the incidence of diagnosed prostate cancer is expected in the future. The highly restricted expression of PSMA and its upregulation in later stages of disease and metastatic prostate cancer, as well as its role as a neoantigen in the tumor vasculature of several different types of other solid tumors qualifies PSMA as an attractive target antigen for Antibody-based cancer therapy. As shown in the examples below, the bispecific single-chain PSMAxCD3 antibody of the present invention provides an advantageous tool in order to kill cells expressing human PSMA-cancer, as exemplified by the human prostate cancer cell line LNCaP. Furthermore, the cytotoxic activity of the single PSMAxCD3 chain bispecific antibody of the present invention is higher than the cytotoxic activity of the antibodies described in the prior art. Since, preferably, both the CD3 and PSMA binding domain of the PSMAxCD3 single chain bispecific antibody of the present invention are cross-species reactive, i.e., specific with human and non-chimpanzee primate antigens, it can be used to the preclinical assessment of activity, safety and/or pharmacokinetic profile of these binding domains in primates and - in identical form - as a drug in humans.
Advantageously, the present invention also provides bispecific single-chain PSMAxCD3 antibodies comprising a second binding domain that binds both to human PSMA and to the macaque PSMA homolog, i.e., the homolog of a non-chimpanzee primate. In a preferred embodiment, the single-chain bispecific antibody thus comprises a second binding domain that exhibits cross-species specificity for human and a non-chimpanzee PSMA primate. In this case, the single-chain identical bispecific antibody molecule can be used both for preclinical assessment of activity, safety and/or pharmacokinetic profile of these binding domains in primates and as a drug in humans. To put it in other words, the same molecule can be used in preclinical animal studies as well as in human clinical studies. This leads to very comparable results and a much greater predictive potency of animal studies compared to species-specific surrogate molecules. Since both the CD3 and the PSMA binding domain of the single PSMAxCD3 chain bispecific antibody of the present invention are cross-species reactive, that is, with the specific antigens of human and non-chimpanzee primates, it can be used for both evaluation preclinical safety activity, and/or the pharmacokinetic profile of these binding domains in primates and - in identical form - as a drug in humans. It should be understood that, in a preferred embodiment, the cross-species specificity of the first domain and the second binding of the antibodies of the present invention is identical.
[00023] It has been found in the present invention that it is possible to generate a, preferably human, bispecific single-chain PSMAxCD3 antibody in which the identical molecule can be used in preclinical animal testing, in this way as clinical trials and also in therapy in humans. This is due to the unexpected identification of the preferably human chain antibody, single bispecific PSMAxCD3, which, in addition to binding to human CD3 epsilon and PSMA, respectively, (and due to the likely genetic similarity to the chimpanzee counterpart), binds also refer to the counterparts of the aforementioned non-primate antigens, including chimpanzees New World Monkeys and Old World Monkeys. As shown in the Examples that follow, preferably so-called human, the single PSMAxCD3 chain bispecific antibody of the present invention can be used as a therapeutic agent against various diseases, including, but not limited to, cancer. The single-chain bispecific antibody PSMAxCD3 is particularly advantageous for the treatment of cancer, solid tumors, preferably, most preferably, carcinomas and prostate cancer. In view of the above, the need to construct a bispecific single-chain surrogate PSMAxCD3 antibody to test in a distant phylogenetic species (from humans) disappears. As a result, the identical molecule can be used in preclinical animal testing as it is intended to be administered to humans in clinical trials, as well as after market approval and therapeutic drug administration. The ability to use the same molecule from preclinical animal tests as in administration to humans thereafter virtually eliminates, or at least reduces, the danger that data obtained from preclinical animal tests having limited applicability to the human case. In short, obtaining preclinical safety data in animals using the same molecule that will actually be administered to humans does much to ensure the applicability of the data to a relevant human setting. In contrast, in conventional methods using substitution molecules, said substitution molecules have to be molecularly adapted to the animal test system used for preclinical safety assessment. In this way, the molecule to be used in human therapy, in fact, differs in sequence and also probably in structure from the substitute molecule used in pre-clinical tests in pharmacokinetic parameters and/or biological activity, with the consequence that the data obtained in preclinical animal trials have limited applicability/transfer to the human case. The use of replacement molecules requires the construction, production, purification and characterization of a completely new construct. This leads to additional development costs and time needed to obtain this molecule. In short, substitutes have to be developed separately, in addition to the actual drug to be used in human therapy, so two lines of development of two molecules have to be carried out. Therefore, a major advantage of the preferably human bispecific single chain PSMAxCD3 antibodies of the present invention exhibit cross-species specificity described in the present invention is that the identical molecule can be used for therapeutic agents in humans and in pre-test animals. clinical trials.
[00024] It is preferable that at least one of said first and second binding domains of the bispecific single chain antibody of the present invention is CDR-grafted, humanized or human, as defined in more detail below. Preferably, both the first and second binding domains of the bispecific single chain antibody of the present invention are CDR-grafted, humanized or human. For the preferably human bispecific PSMAxCD3 chain antibody of the present invention, the generation of an immune response against said binding molecule is precluded, as far as possible, from administering the molecule to human patients.
[00025] Another major advantage of the unique bispecific PSMAxCD3 preferably human chain antibody of the present invention is its applicability for preclinical testing in different primates. The behavior of a drug candidate in animals should ideally be indicative of the expected behavior of this drug candidate after administration to humans. As a result, data obtained from such preclinical tests should therefore, in general, possess a highly predictive power for the human case. However, as has already been learned from the tragic outcome of the recent phase I clinical trials on TGN1412 (a CD28 monoclonal antibody), a drug candidate may act differently in a primate species than in humans: Whereas, in preclinical testing of said antibody no or only limited adverse effects were observed in animal studies performed with cynomolgus monkeys, six human patients developed multiple organ failure after administration of said antibody (Lancet 368 (2006), 2206-7) . The results of these dramatic, unwanted negative events suggest that it may not be sufficient to limit preclinical testing to just one (non-primate chimpanzee) species. The fact that the bispecific single chain antibody PSMAxCD3 of the present invention binds to a number of New World and Old World monkeys may help to overcome the problems faced in the case mentioned above. Therefore, the present invention provides means and methods to minimize species differences in effects when drugs for human therapy are being developed and tested.
[00026] With the, preferably human, cross-species specific bispecific single chain PSMAxCD3 antibody of the present invention it is also no longer necessary to adapt the test animal to the drug candidate intended for administration to humans, for example, the creation of transgenic animals. The PSMAxCD3, preferably human, bispecific single chain antibody of the present invention exhibits cross-species specificity according to the uses and the methods of the present invention can be used directly for preclinical testing in non-chimpanzee primates, without any genetic manipulation of the animals. As is well known to those of skill in the art, approaches in which the test animal is adapted to the drug candidate always risk that results obtained in preclinical safety trials are less representative and predictive for humans, because to the modification of the animal. For example, in transgenic animals, the proteins encoded by the transgenes are often highly overexpressed. In this way, data obtained for the biological activity of an antibody against this protein antigen may be limited in its predictive value for humans where the protein is expressed at much lower, more physiological levels.
[00027] Another advantage of the uses of the human PSMAxCD3 antibody, preferably bispecific single chain of the present invention exhibit specific cross-species is the fact that chimpanzees as an endangered species are avoided for animal testing. Chimpanzees are the closest relatives to humans and have recently been grouped into the hominid family based on genome sequencing data (Wildman et al., PNAS 100 (2003), 7181). Therefore, data obtained with chimpanzees is generally considered to be highly predictive for humans. However, due to their status as an endangered species, the number of chimpanzees that can be used for medical experiments is very limited. As indicated above, keeping chimpanzees for animal testing is therefore costly and ethically problematic. The uses of the preferably single bispecific human chain PSMAxCD3 antibody of the present invention avoid both ethical objections and financial burden during preclinical testing, without impairing applicability quality, i.e., animal test data obtained. In view of this, the uses of the preferably unique bispecific human chain PSMAxCD3 antibody of the present invention provide a reasonable alternative for studies in chimpanzees.
[00028] Yet another advantage of the preferably human, single-chain bispecific PSMAxCD3 antibody of the present invention is the ability to extract multiple blood samples when using it as part of preclinical animal testing, for example in the course of pharmacokinetic studies with animals. Multiple blood draws can be much more easily accomplished with a non-chimpanzee primate than with lower animals, eg, a mouse. The extraction of multiple blood samples allows continuous testing of blood parameters for the determination of biological effects induced by the unique, preferably bispecific human chain PSMAxCD3 antibody of the present invention. Furthermore, the extraction of multiple blood samples allows the researcher to assess the pharmacokinetic profile of the preferably unique bispecific human chain PSMAxCD3 antibody of the present invention as defined in the present invention. Furthermore, the potential side effects, which can be induced by said, preferably human, single-chain bispecific PSMAxCD3 antibody of the present invention reflects the parameters in the blood can be measured in different blood samples extracted during the course of administration of said antibody. This allows determination of the toxicity profile of the preferably unique bispecific human chain PSMAxCD3 antibody of the present invention as defined in the present invention.
[00029] The advantages of the bispecific single chain PSMAxCD3 antibodies of the present invention as defined in the present invention exhibits the cross-species specificity which can be briefly summarized as follows:
[00030] First, the bispecific single-chain PSMAxCD3 antibody of the present invention, preferably human, as defined in the present invention, in preclinical tests used is the same as that used in human therapy. Thus, it is no longer necessary to develop two molecules, which may differ in their pharmacokinetic properties and biological activity. This is highly advantageous as, for example, pharmacokinetic results are more directly transferable and applicable to human fixation than for example in conventional surrogate approaches.
[00031] Second, the uses of the PSMAxCD3 antibody, preferably human, bispecific single chain of the present invention as defined in the present invention for the preparation of therapeutic agents in humans is less costly and labor intensive than replacement approaches.
[00032] Thirdly, the preferably human, bispecific single-chain PSMAxCD3 antibody of the present invention as defined in the present invention can be used for preclinical testing, not only in a primate species, but in a series of different primate species, thereby limiting the risk of potential differences between primate and human species.
[00033] Fourth, the chimpanzee as an endangered species for animal testing can be avoided if desired.
[00034] Fifth, multiple blood samples can be extracted by extensive pharmacokinetic studies.
[00035] Sixthly, due to the human origin of the binding molecules, preferably human, according to a preferred embodiment of the present invention, the generation of an immune reaction against said binding molecules is minimized when administered to human patients. Induction of an immune response with antibodies specific to a drug candidate derived from a non-human species, such as a mouse, which leads to the development of human anti-mouse antibodies (HAMA) against therapeutic molecules of murine origin is excluded.
[00036] Last but not least, the therapeutic use of the single-chain bispecific PSMAxCD3 antibody of the present invention provides a new and inventive therapeutic approach for cancer, solid tumors, preferably, more preferably, carcinomas and prostate cancer. As shown in the examples below, the bispecific single-chain PSMAxCD3 antibody of the present invention provides an advantageous tool for killing human prostate cancer cells expressing PSMA. Furthermore, the cytotoxic activity of the bispecific single-chain PSMAxCD3 antibody of the present invention is higher than the activity of the antibodies described in the prior art.
[00037] As noted above in the present invention, the present invention provides polypeptides, that is, bispecific single chain antibodies, comprising a first binding domain capable of binding to an epitope of the human and non-chimpanzee primate CD3ε chain and a second binding domain capable of binding to PSMA. The second binding domain preferably binds to human PSMA and a non-chimpanzee PSMA primate. The advantage of bispecific single chain antibody molecules as drug candidates meeting the requirements of the preferred bispecific single chain antibody of the present invention is the use of such molecules in preclinical animal assays, as well as in clinical studies and for therapy in humans. In a preferred embodiment, the transspecies-specific bispecific single-chain antibodies of the present invention, the second binding domain binds to human PSMA. In a specific cross-species bispecific molecule according to the present invention, the human and non-chimpanzee epitope binding domain of the primate epsilon chain CD3 is in the order of VH-VL or VL-VH at the N-terminus or at the C-terminus of the bispecific molecule. Examples of bispecific cross-species specific molecules according to the present invention in different VH-VL arrangements and the chain in the first and second binding domains are described in the appended examples.
[00038] As used in the present invention, a "single-chain bispecific antibody" denotes a single polypeptide chain comprising two binding domains. Each binding domain comprises a variable region of an antibody heavy chain ("VH region"), wherein the VH region of the first binding domain specifically binds to the CD3 ε molecule, and the VH region of the second binding domain binds specifically to PSMA. The two binding domains are optionally linked together by a short polypeptide spacer. A non-limiting example of a polypeptide spacer is Gly-Gly-Gly-Gly-Ser (GGGGS) and its repeat. Each binding domain may further comprise an antibody light chain variable region ("VL region"), the VH region and the VL region within each of the first and second binding domains are linked together through a polypeptide linker, for example, of the type described and claimed in EP 623679 B1 but in any case long enough to allow the VH region and VL region of the first binding domain and the VH region and VL region of the second binding domain paired with each other such that together they are able to specifically bind to respective first and second binding domains.
[00039] The term "protein" is well known in the art and describes biological compounds. Proteins comprise one or more chains of amino acids (polypeptides), in which the amino acids are linked together via a peptide bond. The term "polypeptide" as used in the present invention describes a group of molecules, which consists of more than 30 amino acids. According to the present invention, the group of polypeptides comprises "proteins", since the proteins consist of a single polypeptide chain. Also in accordance with the definition of the term "polypeptide", it describes protein fragments, provided that such fragments consist of more than 30 amino acids. Polypeptides can further form multimers, such as dimers, trimers and larger oligomers, that is, consisting of more than one polypeptide molecule. The polypeptide molecules that form such dimers, trimers, etc. can be identical or non-identical. The corresponding higher order structures of such multimers are therefore called homo- or heterodimers, homo- or heterotrimers etc. An example for a hereteromultimer is an antibody molecule, which, in its naturally occurring form, is made up of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides/proteins, wherein the modification is effected for example by means of post-translational modifications such as glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
[00040] The term "binding domain" characterizes binding with the present invention, a domain of a polypeptide that specifically binds to/interacts with a given structure/target/antigen/epitope. In this way, the binding domain is an "antigen-local interaction". The term "antigen-site interaction" defines, according to the present invention, a motif of a polypeptide which is capable of interacting specifically with a specific antigen or a specific group of antigens, e.g., the identical antigen in different species. Said link/interaction is also understood to define a "specific recognition". The term "specifically recognizes" means, according to the present invention, that the antibody molecule is capable of specifically interacting with and/or binding to at least two, preferably at least three, more preferably at least four amino acids of an antigen , for example, the human CD3 antigen as defined herein. Such linkages can be exemplified through the specificity of a "principle lock and key". In this way, the specific motifs in the amino acid sequence of the binding domain and the antigen bind each other as a result of their primary, secondary or tertiary structure, such as the result of secondary modifications of said structure. The specific interaction of the antigen interaction site with its specific antigen may also result in a simple binding of the site to said antigen. In addition, the specific interaction of the antigen binding domain/interacting site with its specific antigen may alternatively trigger the development of a signal, for example, due to the induction of an antigen conformation change, an oligomerization of the antigen , etc. A preferred example of a binding domain in accordance with the present invention is an antibody. The binding domain can be a monoclonal or polyclonal antibody or derived from a monoclonal or polyclonal antibody.
[00041] The term "antibody" comprises functional derivatives or fragments thereof that still retain binding specificity. Techniques for producing antibodies are well known in the art and described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988, Harlow and Lane "Antibodies: Using Anticorpies: A Laboratory Manual ", Cold Spring Harbor Laboratory Press, 1999, and Little "Recombinant Antibodies for Immunotheraphy" Cambridge University Press 2009. The term "antibody" also includes immunoglobulins (Igs) of different classes (eg, IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc.).
[00042] The definition of the term "antibody" also includes modalities such as chimeric single chain and humanized antibodies, as well as antibody fragments such as, inter alia, Fab fragments. Antibody fragments or derivatives thereof further comprise F(ab') 2 fragments, Fv, scFv or single domain antibodies, single variable domain immunoglobulin antibodies or single domain variable domain which comprises only one variable, which may be from VH or VL, which specifically binds to a antigen or epitope independent of other regions or V domains, see, for example, Harlow and Lane (1988) and (1999) and Little (2009), loc. cit. Such a single immunoglobulin variable domain encompasses not only an isolated single polypeptide variable domain antibody, but also larger polypeptides that comprise one or more monomers of a single polypeptide antibody variable domain sequence.
[00043] Several procedures are known in the art and can be used for the production of such antibodies and/or fragments. In this way, derivatives (of the antibody) can also be produced by means of peptidomimetics. Furthermore, the techniques described for the production of single chain antibodies (see, inter alia, U.S. Patent 4,946,778) can be adapted to produce single chain antibodies specific for the chosen polypeptide(s). Furthermore, transgenic animals can be used to express humanized or human antibodies specific to the polypeptides and fusion proteins of the present invention. For the preparation of monoclonal antibodies, any technique providing the antibodies produced by means of continuous cell line cultures can be used. Examples of such techniques include the hybridoma technique (Kohler and Milstein Nature 256 (1975), 495 to 497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the hybridoma-EBV technique for producing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77 to 96). Surface plasma resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to an epitope of a target polypeptide, such as CD3 epsilon or PSMA (Schier, Human Antibodies Hybridomas 7 (1996) , 97 to 105; Malmborg, J. Immunol. Methods 183 (1995), 7 to 13). It is also envisaged within the scope of the present invention that the term "antibody" comprises antibody constructs, which can be expressed in a host, as described in the present invention below, for example, antibody constructs which can be transfected and /or transduced by, inter alia, plasmid vectors or viruses.
[00044] The term "specific binding" as used in accordance with the present invention means that the binding domain does not or significantly does not cross-react with polypeptides that have similar structure, such as those that have gone to the domain. binding, and which can be expressed by the same cell as the polypeptide of interest. The cross-reactivity of a panel of binding domains under investigation can be tested, for example, by evaluating the binding of said panel of binding domains under conventional conditions (see, for example, Harlow and Lane (1988) and (1999) ) and Little (2009), loc. cit. Examples for the specific interaction of a binding domain with a specific antigen comprise the specificity of a ligand for its receptor. Said definition particularly comprises the interaction of ligands, which induce a signal in binding to its specific receptor. The so-called examples of interaction, which is also specially constituted by the aforementioned definition, is the interaction of an antigenic determinant (epitope) with the binding domain (antigen binding site) of an antibody.
The term "cross-species specificity" or "interspecies specificity", as used in the present invention, means the binding of a binding domain described in the present invention to the target molecule even in humans and non-chimpanzee primates. Thus, "cross-species specificity" or "inter-species specificity" is to be understood as the interspecies of reactivity for the same "X" molecule, expressed in different species, but not for a different "X" molecule . The cross-species specificity of a monoclonal antibody recognizing human CD3 epsilon, for a non-chimpanzee CD3 epsilon primate, e.g., macaque epsilon CD3 can be determined, e.g., by FACS analysis. The FACS analysis is performed in such a way that the respective monoclonal antibody is tested for binding to human and non-chimpanzee primate cells from, for example, monkey cells, expressing said human and non-chimpanzee primate epsilon CD3 antigens, respectively. A suitable test is shown in the following examples. The above mentioned object is applicable to mutatis mutandis for the PSMA antigen: The cross-species specificity of a monoclonal antibody that recognizes, for example, human PSMA, with a non-chimpanzee primate PSMA, for example, monkey PSMA, can be determined by example, through FACS analysis. The FACS analysis is performed in such a way that the respective monoclonal antibody is tested for binding to human and non-chimpanzee primate cells, for example monkey cells, expressing said human PSMA and non-chimpanzee primate antigens, respectively.
[00046] As used in the present invention, CD3 epsilon denotes a molecule expressed as part of the T cell receptor and has the meaning as commonly ascribed to it in the prior art. In humans, it encompasses individually or independently combined all known CD3 subunits, for example, CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta. Non-chimpanzee, non-human primate antigens as referred to herein are, for example, Macaca fascicularis CD3 and Macaca mulatta CD3. In Macaca fascicularis, it encompasses CD3 epsilon FN-18 negative and CD3 epsilon FN-18 positive, CD3 gamma and CD3 delta. In Macaca mulatta, it includes CD3 epsilon, CD3 gamma and CD3 delta. Preferably, said CD3 as used in the present invention is CD3 epsilon.
[00047] Human epsilon CD3 is indicated in GenBank accession number No.NM_000733 and comprises SEQ ID NO: 1. Human gamma CD3 is indicated in GenBank accession number NO. NM_000073. Human CD3 delta is indicated in GenBank accession number NM_000732.
The "FN-18 negative" CD3 epsilon from Macaca fascicularis (i.e. CD3 epsilon not recognized by the FN-18 monoclonal antibody due to a polymorphism as described above) is indicated in GenBank accession number AB073994.
The CD3 epsilon "FN-18 positive" from Macaca fascicularis (ie CD3 epsilon recognized by the FN-18 monoclonal antibody) is indicated in GenBank accession number AB073993. The gamma CD3 of Macaca fascicularis is listed under GenBank accession number AB073992. The CD3 delta of Macaca fascicularis is listed under GenBank accession number AB073991.
The nucleic acid sequences and amino acid sequences of the respective CD3 epsilon gamma and delta homologues of Macaca mulatta can be identified and isolated by means of recombinant techniques described in the art (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd edition, 2001). This applies, mutatis mutandis, for the epsilon, gamma and delta CD3 homologs of other non-chimpanzee primates as defined in the present invention. The identification of the amino acid sequence of Callithrix jacchus, Saimiri sciureus and Saguinus oedipus is described in the attached examples. The amino acid sequence of the extracellular domain of the CD3 epsilon of Callithrix jacchus is represented in SEQ ID NO: 3, one of Saguinus oedipus is represented in SEQ ID NO: 5 and one of Saimiri sciureus is represented in SEQ ID NO: 7.
[00051] Human PSMA is indicated under GenBank accession number 'AY101595'. Cloning of the monkey PSMA homolog is demonstrated in the examples below, the corresponding cDNA and amino acid sequences are shown in SEQ ID NOs: 223 and 224, respectively.
[00052] In accordance with the above, the term "epitope" defines an antigenic determinant, which is specifically bound/identified by a binding domain as defined in the present invention. The binding domain can specifically bind to/interact with conformational or continuous epitopes that are unique to the target structure, for example, the epsilon CD3 chain from human and non-chimpanzee primate or human and non-chimpanzee primate PSMA. A conformational or discontinuous epitope is characterized by polypeptide antigens by the presence of two or more discrete amino acid residues that are separated in the primary sequence, but come together on the surface of the polypeptide molecule when folds in the native protein/antigen (Sela, (1969) ) Science 166, 1365 and Lavide, (1990) Cell 61, 553 to 6). The two or more discrete amino acid residues that contribute to the epitope are present in separate sections of one or more polypeptide chain(s). These residues assemble on the surface of the molecule that the polypeptide chain(s) fold into a three-dimensional structure in order to constitute the epitope. In contrast, a continuous or linear epitope composed of two or more discrete amino acid residues, which are present in a single linear segment of a polypeptide chain. Within the present invention, a "context-dependent" CD3 epitope refers to the conformation of said epitope. Such a context-dependent epitope, located on the epsilon CD3 strand, can only develop its correct conformation if it is integrated into the rest of the epsilon strand and maintained in the correct position by heterodimerization of the epsilon strand with any CD3 gamma or delta strand. In contrast, a context-independent CD3 epitope as provided in the present invention refers to a 1 to 27 amino acid residue of the polypeptide at the N-terminus or a functional fragment thereof of CD3 epsilon. This 1 to 27 amino acid residue of the polypeptide at the N-terminus, or a functional fragment thereof, maintains its three-dimensional structural integrity and correct conformation when removed from its native environment, in the CD3 complex. The context of independence of the 1 to 27 amino acid residue of the polypeptide at the N-terminus or a functional fragment thereof, which is part of the extracellular domain of CD3 epsilon, therefore represents an epitope that is completely different from the described epsilon CD3 epitopes in connection with a method for the preparation of human binding molecules in WO 2004/106380. Said method used exclusively expresses recombinant CD3 epsilon. The conformation of the present uniquely recombinant CD3 epsilon expressed differed from that adopted in its natural form, i.e. the form in which the CD3 epsilon subunit of the TCR/CD3 complex exists as part of a non-covalent complex with the delta CD3 or gamma CD3 subunit of the RCT/CD3 complex. When such uniquely express recombinant protein CD3 epsilon is used as an antigen for selecting antibodies from an antibody library, antibodies specific for that antigen are identified from the library although such library does not contain antibodies with specificity for self antigens /autoantigens. This is due to the fact that only the recombinant protein expressing CD3 epsilon does not exist in vivo, but it is not an autoantigen. Therefore, B cell subpopulations expressing antibodies specific to this protein have not yet been depleted in vivo; an antibody library constructed from such B cells that contain the genetic material for antibodies specific for the uniquely expressed recombinant protein CD3 epsilon.
[00053] However, since the context independent of the 1 to 27 amino acid residue of the polypeptide at the N-terminus or a functional fragment thereof, it is an epitope, which folds, in its native form, the binding domains of according to the present invention cannot be identified by methods based on the approach described in WO 2004/106380. Therefore, it was found in tests that binding molecules as described in WO 2004/106380 are not able to bind the 1 to 27 amino acid residues of the polypeptide at the N-terminus of the CD3 epsilon chain. In this way, conventional anti-CD3 binding molecules or anti-CD3 antibody molecules (for example, as described in WO 99/54440) bind to the epsilon CD3 chain, at a position that is more at the C-terminus than the context-independent position of the 1 to 27 amino acid residue of the polypeptide at the N-terminus or a functional fragment thereof provided in the present invention. The prior art antibody molecules OKT3 and UCHT-1 also have a specificity for the epsilon subunit of the TCR/CD3 complex between amino acid residues 35 to 85 and therefore the epitope of these antibodies is also more located at the C-terminus. In addition, UCHT-1 binds to the epsilon CD3 chain, in a region between amino acid residues 43 to 77 (Tunnacliffe, Int. Immunol 1 (1989), 546-50, Kjer-Nielsen, PNAS 101, (2004 ), 7675 to 7680; Salmeron, J. Immunol 147 (1991), 3047 to 52). Therefore, from the prior art, anti-CD3 molecules do not bind to and are not directed against that defined in the present invention context-independent epitope of residues from 1 to 27 amino acids at the N-terminus (or a functional fragment thereof). In particular, the prior art does not disclose anti-CD3 molecules, which specifically bind to the context-independent N-terminal amino acid residues 1 to 27 epitope and which are cross-linking species, i.e., specific for humans and non-chimpanzee primates CD3 epsilon.
[00054] For the generation of a preferably human binding domain composed of a bispecific single chain antibody molecule of the present invention, for example, monoclonal antibodies that bind to both human epsilon CD3 and non-chimpanzee primates ( eg monkey CD3 epsilon) or monoclonal antibodies that bind to both human PSMA and non-chimpanzee primates can be used.
[00055] As used in the present invention, "human" and "man" refer to the species Homo sapiens. As far as medical uses of the constructs described in this document are concerned, human patients are being treated with the same molecule.
[00056] It is preferable that at least one of said first and second binding domains of the bispecific single chain antibody of the present invention is CDR-grafted, humanized or human. Preferably, both the first and second binding domains of the bispecific single chain antibody of the present invention are CDR-grafted, humanized or human.
[00057] The term "human" antibody as used in the present invention is to be understood in the sense that the bispecific single chain antibody as defined herein comprises (a) the sequence(s) of amino acid(s) contained in the human germline antibody repertoire. For the purposes of the present definition, said bispecific single-chain antibody may therefore be considered human if it is constituted by such a germline human acid sequence(s) of amino acid, i.e. if the amino acid sequence (s) of the bispecific single chain antibody in question is (are) identical to (a) human germline of the expressed amino acid sequence(s). A bispecific single chain antibody as defined in the present invention may also be considered to be human if it consists of (a) sequence(s) that deviate from its nearest germline sequence. human(s) by no more than would be expected due to the somatic mutation marker. Furthermore, antibodies from many non-human mammals, e.g., rodents such as mice and rats, comprise the VH CDR3 amino acid sequences that can be expected to exist in the repertoire of human expressed antibodies. Any sequence of such human or non-human origin(s) that can be expected to exist in the expressed human repertoire would also be considered "human" for the purposes of the present invention.
[00058] As used in the present invention, the term "humanized", "humanization", "human-like" variants or grammatically related thereto are used interchangeably to refer to a bispecific single chain antibody comprising at least one of its binding domains from at least one complementarity determining region ("CDR") of a non-human antibody or a fragment thereof. Humanization approaches are described, for example, in WO 91/09968 and U.S. 6,407,213. As non-limiting examples, the term encompasses the case where a variable region of at least one binding domain comprises a unique CDR region, e.g. the CDR3 region of the VH (CDRH3), from another non-human animal, for example. example a rodent, as well as the case where one or both of the variable regions comprise in each of their respective first, second and third CDRs of said non-human animal CDRs. In the case where all the CDRs of a single-chain bispecific antibody binding domain have been replaced by their corresponding counterparts from, for example, a rodent, one typically talks about "grafting CDR", and this term should be understood to be encompassed by variants of the term "humanized" or grammatically related thereto as used in the present invention. Variants of the term "humanized" or grammatically related thereof also encompass cases where, in addition to the substitution of one or more CDR regions in a VH and/or VL of the first and/or second additional binding domain of the mutation/s (e.g., substitutions) of at least a single amino acid in the residue/s regions in the framework ("FR") between the CDRs was/were made in such a way that the amino acids at which/those positions correspond/s to the amino acid/s where the position/s, where the animal from which the CDR regions used for substitution is/are derived. As is known in the art, such individual mutations are often made in the in-frame regions of the graft CDR sequence in order to restore the original binding affinity of the non-human antibody used as a CDR donor for its target molecule. The term "humanization" may further encompass (a) replacement of amino acid(s) in the CDR regions from a non-human animal with the amino acid(s) of a corresponding CDR region of a human antibody, in addition to the amino acid substitutions in the framework regions as described above.
[00059] As used herein, the term "homology" or the term "homology" is to be understood as follows: Homology between proteins and DNA is often celebrated on the basis of sequence similarity, especially in bioinformatics. For example, in general, if two or more genera have highly similar DNA sequences, they are likely to be homologous. But sequence similarity can arise from different ancestors: short sequences can be similar at random, and sequences can be similar because both were selected to bind to a specific protein, such as a transcription factor. Such sequences are similar but not homologous. Regions of sequences that are homologous are also called conserved. This is not to be confused with conservation in amino acid sequences where the amino acid at a specific position has been altered, but the physicochemical properties of the amino acid remain unchanged. Homologous sequences are of two types: orthologous and paralogous. Homologous sequences are orthologous if they were separated by a speciation event: when a species diverges into two separate species, the different copies of a single genus in the resulting species are considered orthologous. Orthologs, or orthologous genera, are genera in different species that are similar to one another because they originated from a common ancestor. The strongest evidence that the two genera are orthologous is the result of a phylogenetic analysis of the lineage of genera. Genres that are found within a clade are orthologous, descended from a common ancestor. Orthologs often, but not always, have the same function. Orthologous sequences provide useful information in taxonomic studies of classification of organisms. The pattern of genetic divergence can be used to trace relatedness between organisms. Two organisms that are closely related are likely to show very similar DNA sequences between two orthologs. On the other hand, an organism that is evolutionarily more distant from another organism is likely to present a greater divergence in the sequence of orthologs under study. Homologous sequences are paralogous if they were separated by a genus duplication event: if a genus in an organism is repeated to occupy two different positions in the same genome, then the two copies are paralogous. A set of sequences that are paralogs is called paralogs of the other. Paralogs typically have the same or similar function, but sometimes not: due to the absence of selective pressure on the original of a duplicated genus copy, this copy is free to mutate and acquire new functions. An example can be found in rodents such as rats and mice. Rodents have a pair of paralogous insulin genera, although it is unclear whether any divergence in function has occurred. Paralogous genera often belong to the same species, but this is not necessary: for example, the human hemoglobin genus and the chimpanzee myoglobin genus are paralogous. This is a common problem in bioinformatics: when genomes of different species have been sequenced and homologous genera have been found, it cannot be immediately concluded that these genera have the same or similar function, since they could be paralogs whose function diverged.
[00060] As used in the present invention, a "non-chimpanzee primate" or its grammatical variants refers to any primate animal (eg, non-human), but different from the chimpanzee, that is, with the exception of an animal belonging to the genus Pan, and including the species Pan paniscus and Pan troglodytes, also known as Anthropopithecus troglodytes or Satyrus Simia. It should be understood, however, that it is possible that the antibodies of the present invention may also bind the first and/or second binding domains to the respective epitopes of the fragments/etc. of said chimpanzees. The intention is simply to avoid animal tests, which are carried out with chimpanzees if desired. Thus, it is also envisioned that, in another embodiment, the antibodies of the present invention also bind with their first and/or second binding domains to the respective chimpanzee epitopes. The "primate", "primate species", "primates" or grammatical variants thereof denote an order of eutherian mammals divided into two suborders of prosimians and anthropoids and monkeys comprising, apes and lemurs. Specifically, "primates" as used in the present invention comprise the suborder Strepsirrhini (non-tarsier prosimians), including the above order Lemuriformes (the same including the superfamilies Cheirogaleoidea and Lemuroidea), the above order Chiromyiformes (the same including the family Daubentoniidae) and the above order Lorisiformes (the same including the families Lorisidae and Galagidae). "Primates" as used in the present invention also comprise the suborder Haplorrhini, including the above order Tarsiiformes (the same including the family Tarsiidae), the above order Simiiformes (the same including Platyrrhini, or New World monkeys, and Catarrhini, including Cercopithecidea, or old world monkeys).
[00061] The non-chimpanzee primate species can be understood within the meaning of the present invention as being a lemur, a tarsier, a gibbon, a tamarin (belonging to New World monkeys of the Cebidae family) or an old world monkey (belonging to superfamily Cercopithecoidea).
[00062] As used in the present invention, the term "old world monkey" comprises any monkey that fits into the superfamily Cercopithecoidea, the same subdivided into the families: Cercopithecinae, which are mainly African, but include the various genera of monkeys which they are Asians and North Africans; and Colobinae, which include most of the Asian genera but also the African colobus monkeys.
[00063] Specifically, within the subfamily Cercopithecinae, an advantageous non-chimpanzee primate may be from the tribe Cercopithecini, within the genus Allenopithecus (Allen's Swamp Monkey, Allenopithecus nigroviridis); within the genus Miopithecus (Angolan Talapoin, Miopithecus talapoin; Gabon Talapoin, Miopithecus ogouensis); within the genus Erythrocebus (Patas Monkey, Erythrocebus patas); within the genus Chlorocebus (Green Monkey, Chlorocebus sabaceus; Grivet, Chlorocebus aethiops; Bale Mountains Vervet, Chlorocebus djamdjamensis; Monkey Tantalus, Chlorocebus tantalus; Monkey Vervet, Chlorocebus pygerythrus; Malbrouck, Chlorocebus); or within the genus Cercopithecus (Monkey Dryas or Monkey Salongo, Cercopithecus dryas; Monkey Diana, Cercopithecus diana; Monkey Roloway, Cercopithecus roloway; Monkey Greater Spot-nosed, Cercopithecus nictitans; Monkey Blue, Cercopithecus mitis; Monkey Silvide, Cercopithecus; , Cercopithecus kandti; Sykes's monkey, Cercopithecus albogularis; Mona monkey, Cercopithecus mona; Campbell's Mona monkey, Cercopithecus campbelli; Lowe's Mona monkey, Cercopithecus lowei; Crested Mona, Cercopithecus mona, Cercopithecus de Monaco's Mona, Cerco wolf's monkey; ; Lesser Spot-nosed monkey, Cercopithecus petaurista; White-throated Guenon, Cercopithecus erythrogaster; Sclater's Guenon, Cercopithecus sclateri; Red-eared Guenon, Cercopithecus erythrotis; Moustached Guenon, Cercopithecus cephus L'scanius; , Cercopithecus lhoesti; Preuss's Monkey, Cercopithecus preussi; Sun-tailed Monkey, Cercopit hecus solatus; Hamlyn's Monkey or Owl-faced Monkey, Cercopithecus hamlyni; Brazza's monkey, Cercopithecus neglectus).
[00064] Alternatively, an advantageous non-chimpanzee primate, also within the subfamily Cercopithecinae, but within the tribe Papionini, may be from within the genus Macaca (Barbary Macaque, Macaca sylvanus; Lion-tailed Macaque, Macaca silenus; Southern Pig- tailed Macaque or Beruk, Nemestrina Macaca; Northern Pig-tailed Macaque, Leonine Macaque; Pagai Island Macaque or Bokkoi, Macaca pagensis; Siberut Macaque, Siberu Macaca; Moor Macaque, Maura Macaque; Booted Macaque, Ochreata Macaque; Tonkean Macaca, Macaca; Heck's Macaque, Macaca hecki; Gorontalo Macaque, Macaca nigriscens; Celebes Crested Macaque or Black "Ape", Macaca nigra; Cynomolgo monkey or Crab-eating Macaque or Long-tailed Macaque or Kera, Macaca fascicularis; Stump-tailed Macaque or Bear Macaque Arctoides Macaque; Rhesus Macaque, Mulatta Macaque; Formosan Rock Macaque, Cyclopis Macaque; Japanese Macaque, Fuscata Macaque; Toque Macaque, Sinica Macaque; Bonnet Macaque, Radiata Macaque; Barbary Macaque, Sylv Macaca anmus; Assam Macaque, Macaca assamensis; Tibetan Macaque or Milne-Edwards' Macaque, Thibetana Macaca; Arunachal Macaque or Munzala, Macaca munzala); within the genus Lophocebus (Gray-cheeked Mangabey, Lophocebus albigena; Lophocebus albigena albigena; Lophocebus albigena osmani; Lophocebus albigena johnstoni; Black Crested Mangabey, Lophocebus aterrimus; Opdenbosch's Mangabey, Lophocebus albigena osmani); within the genus Papio (Hamadryas Baboon, Papio hamadryas; Guinea Baboon, Papio papio; Olive Baboon, Papio anubis; Yellow Baboon, Papio cynocephalus; Chacma Baboon, Papio ursinus); within the genus Theropithecus (Gelada, Theropithecus gelada); within the genus Cercocebus (Sooty Mangabey, Cercocebus atys; Cercocebus atys atys; Cercocebus atys lunulatus; Collared Mangabey, Cercocebus torquatus; Agile Mangabey, Cercocebus agilis; Golden-bellied Mangabey, Cercocebus, Cercocebe; sanji); or within the genus Mandrillus (Mandrill, Mandrillus sphinx; Drill, Mandrillus leucophaeus).
[00065] Most preferred are Macaca fascicularis (also known as cynomolgus monkey and therefore in Examples named "Cinomolgus") and Macaca mulatta (rhesus monkey, named "rhesus").
[00066] Within the subfamily Colobinae, an advantageous non-chimpanzee primate may be from the African group, within the genus Colobus (Black Colobus, Colobus satanas; Angola Colobus, Colobus angolensis; King Colobus, Colobus polykomos; Ursine Colobus, Colobus vellerosus; Mantled Guereza, Colobus Guereza); within the genus Piliocolobus (Western Red Colobus, Piliocolobus badius; Piliocolobus badius badius; Piliocolobus badius temminckii; Piliocolobus badius waldronae; Pennant's Colobus, Piliocolobus pennantii; Piliocolobus pennantii pennantii; Piliocolobus pennanticolous' pennantii; Piliocolobus pennanticoloi epien; Red Colobus, Piliocolobus tholloni; Central African Red Colobus, Piliocolobus foai; Piliocolobus foai foai; Piliocolobus foai ellioti; Piliocolobus foai oustaleti; Piliocolobus foai semlikiensis; Piliocolobus foai parmentierorum; Ugandan Red Colorum, Piliocolobus foai ellioti; Colobus, Piliocolobus kirkii; Tana River Red Colobus, Piliocolobus rufomitratus); or within the genus Procolobus (Olive Colobus, Procolobus verus).
[00067] Within the subfamily Colobinae, an advantageous non-chimpanzee primate may alternatively be from the Langur group (leaf monkey), within the genus Semnopithecus (Nepal Gray Langur, Semnopithecus schistaceus; Kashmir Gray Langur, Semnopithecus ajax; Tarai Gray Langur, Semnopithecus hector; Northern Plains Gray Langur, Semnopithecus entellus; Black-footed Gray Langur, Semnopithecus hypoleucos; Southern Plains Gray Langur, Semnopithecus dussumieri; Tufted Gray Langur, Semnopithecus priam); within the T. vetulus group or the genus Trachypithecus (Purplefaced Langur, Trachypithecus vetulus; Nilgiri Langur, Trachypithecus johnii); within the T. cristatus group of of the genus Trachypithecus (Javan Lutung, Trachypithecus auratus; Silvery Leaf Monkey or Silvery Lutung, Trachypithecus cristatus; Indochinese Lutung, Trachypithecus germaini; Tenasserim Lutung, Trachypithecus barbei); within the T. obscurus group of the genus Trachypithecus (Dusky Leaf Monkey or Spectacled Leaf Monkey, Trachypithecus obscurus; Phayre's Leaf Monkey, Trachypithecus phayrei); within the T. pileatus group of the genus Trachypithecus (Capped Langur, Trachypithecus pileatus; Shortridge's Langur, Trachypithecus shortridgei; Gee's Golden Langur, Trachypithecus geei); within the T. francoisi group of the genus Trachypithecus (Francois' Langur, Trachypithecus francoisi; Hatinh Langur, Trachypithecus hatinhensis; White-headed Langur, Trachypithecus poliocephalus; Laotian Langur, Trachypithecus laotum; Delacour's Langur, Trachypithecus Blackchillus and delaco) ; or within the genus Presbytis (Sumatran Surili, Presbytis melalophos; Banded Surili, Presbytis femoralis; Sarawak Surili, Presbytis chrysomelas; White-thighed Surili, Presbytis siamensis; White-fronted Surili, Presbytis frontata; Javan Surili, Presbytis comata; Thomas's Langur, Thomas's Langur thomasi; Hose's Langur, Presbytis hosei; Maroon Leaf Monkey, Presbytis rubicunda; Mentawai Langur or Joja, Presbytis potenziani; Natuna Island Surili, Presbytis natunae).
[00068] Within the Colobinae subfamily, an advantageous non-chimpanzee primate may alternatively be from an Odd-Nosed group, within the Pygathrix genus (Red-shanked Douc, Pygathrix nemaeus; Black-shanked Douc, Pygathrix nigripes; Gray-shanked Douc; , Pygathrix cinerea); within the genus Rhinopithecus (Golden Snub-nosed Monkey, Rhinopithecus roxellana; Black Snub-nosed Monkey, Rhinopithecus bieti; Gray Snub-nosed Monkey, Rhinopithecus brelichi; Tonkin Snubnosed Langur, Rhinopithecus avunculus); within the genus Nasalis (Proboscis Macaco, Nasalis larvatus); or within the genus Simias (Pigtailed Langur, Simias concolor).
[00069] As used in the present invention, the term "mica" refers to any of the New World Apes of the genus Callithrix, eg belonging to Atlantic marmosets of the subgenus Callithrix (sic!) (Common Marmoset, Callithrix (Callithrix) ) jacchus; Black-tufted Marmoset, Callithrix (Callithrix) penicillata; Wied's Marmoset, Callithrix (Callithrix) kuhlii; White-headed Marmoset, Callithrix (Callithrix) geoffroyi; Buffy-headed Marmoset, Callithrix (Callithrix) Callithrix (Callithrix) aurite); belonging to the Amazonian marmosets of the subgenus Mico (Rio Acari Marmoset, Callithrix (Mico) acariensis; Manicore Marmoset, Callithrix (Mico) manicorensis; Silvery Marmoset, Callithrix (Mico) argentata; White Marmoset, Callithrix (Mico) leuithrix, Callithrix, Emilia's Mico) emiliae; Black-headed Marmoset, Callithrix (Mico) nigriceps; Marca's Marmoset, Callithrix (Mico)marcai; Black-tailed Marmoset, Callithrix (Mico) melanura; Santarem Marmoset, Callithrix (Mico) humeralifera; Maués Marmoset, Callithrix (Mico) ) mauesi; Gold-e-white Marmoset, Callithrix (Mico) chrysoleuca; Hershkovitz's Marmoset, Callithrix (Mico) intermedia; Satéré Marmoset, Callithrix (Mico) saterei); Roosmalens' Dwarf Marmoset belonging to the subgenus Callibella (Callithrix (Callibella) humilis); or Pygmy Marmoset belonging to the subgenus Cebuella (Callithrix (Cebuella) pygmaea).
[00070] Another genus of New World Monkeys comprise marmosets of the genus Saguinus (comprising the S. oedipus group, the S. midas group, the S. nigricollis group, the S. mystax group, the S. bicolor group and the S group . inustus) and squirrel monkeys of the genus Samiri (eg Saimiri sciureus, Saimiri oerstedii, Saimiri ustus, Saimiri boliviensis, Saimiri vanzolini)
[00071] In a preferred embodiment of the bispecific single chain antibody molecule of the present invention, a non-chimpanzee primate is a new world monkey. In a more preferred modality, the New World monkey is a monkey of the Papio genus Macaque genus. More preferably, the monkey of the genus Macaque is Assamese macaque (Macaca assamensis), Barbary macaque (Macaca sylvanus), Bonnet macaque (Macaca radiata), Booted or Sulawesi-Booted macaque (Macaca ochreata), Sulawesi-crested macaque (Macaca nigra), Formosan rock macaque (Macaca cyclopsis), Japanese snow macaque or Japanese macaque (Macaca fuscata), Cynomologus monkey or crab-eating macaque or long-tailed macaque or Java macaque (Macaca fascicularis), Lion-tailed macaque (Macaca silenus) , Pigtailed macaque (Macaca nemestrina), Rhesus macaque (Macaca mulatta), Tibetan macaque (Macaca thibetana), Tonkean macaque (Macaca tonkeana), Toque macaque (Macaca sinica), Stump-tailed macaque or Red-faced macaque or Bear macaque (Macaca arctoides), or Moor macaque (Macaca maurus). More preferably, the monkey of the Papio genus is Hamadryas Baboon, Papio hamadryas; Guinea Baboon, Papio papio; Olive Baboon, Papio anubis; Yellow Baboon, Papio cynocephalus; Chacma Baboon, Papio ursinus.
[00072] In an alternative preferred embodiment, the bispecific single chain antibody molecule of the present invention, a non-chimpanzee primate is a new world monkey. In a more preferred embodiment of polypeptide, the New World monkey is a monkey of the genus Callithrix (marmoset), of the genus or species Saguinus Samiri. More preferably, the monkey of the genus Callithrix is Callithrix jacchus, the monkey of the genus Saguinus is Saguinus oedipus, and the monkey of the genus Samiri is Saimiri sciureus.
[00073] The term "cell surface antigen" as used in the present invention denotes a molecule, which is displayed on the surface of a cell. In most cases, this molecule will be located within or on the cell's plasma membrane in such a way that at least a portion of this molecule remains accessible from outside the cell in tertiary form. A non-limiting example of a cell surface molecule which is located on the plasma membrane is a transmembrane protein which comprises, in its tertiary conformation, regions of hydrophilicity and hydrophobicity. Here, at least one hydrophobic region allows the cell surface molecule to be incorporated or inserted into the hydrophobic cell plasma membrane, while the hydrophilic regions extend on both sides of the plasma membrane into the cytoplasm and extracellular space, respectively. Non-limiting examples of cell surface molecules that are located on the plasma membrane are proteins that have been modified at a cysteine residue to support a palmitoyl group, proteins modified at a C-terminal cysteine residue to support a farnesyl group, or proteins that have been modified at the C-terminus to give a glycosyl phosphatidyl inositol ("GPI") anchor. These groups allow the covalent attachment of proteins to the outer surface of the plasma membrane, where they will remain accessible for recognition by extracellular molecules such as antibodies. Examples of cell surface antigens are CD3 epsilon and PSMA. As described in the present invention above, PSMA is a cell surface antigen that is a target for cancer therapy, including but not limited to solid tumors, preferably carcinomas and prostate cancer.
[00074] In light of this, PSMA can also be characterized as a tumor antigen. The term "tumor antigen" as used in the present invention can be understood as those antigens that are presented on tumor cells. These antigens can be presented on the cell surface, with an extracellular part, which is often combined with a transmembrane and cytoplasmic part of the molecule. These antigens can be presented, sometimes only by tumor cells, and never to normal ones. Tumor antigens may be exclusively expressed on tumor cells or may represent a tumor-specific mutation compared to normal cells. In this case they are called tumor specific antigens. Most common are antigens that are presented through tumor cells and normal cells, and are called tumor associated antigens. These tumor associated antigens may be over-expressed relative to normal cells or are accessible for antibody binding on tumor cells due to the less compact structure of the tumor tissue compared to normal tissue. An example for a tumor antigen in accordance with the present invention is PSMA.
[00075] As described in the present invention above the bispecific single chain antibody molecule of the present invention binds with the first binding domain to an epitope of the human and non-chimpanzee primate CD3ε (epsilon) chain, wherein the epitope is part of an amino acid sequence in the group consisting of 27 amino acid residues as illustrated in SEQ ID NO: 2, 4, 6, or 8, or a functional fragment thereof.
[00076] In line with the present invention, it is preferred for the bispecific single chain antibody molecule of the present invention, which says epitope is part of an amino acid sequence comprising 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids.
[00077] More preferably, wherein said epitope comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (Q-D-G-N-E).
[00078] Within the present invention, a functional fragment of amino acid residues 1 to 27 N-terminal means that said functional fragment is still a context-independent epitope maintaining its three-dimensional structural integrity when removed from its native environment, in the CD3 complex (and fused to a heterologous amino acid sequence such as EpCAM or an immunoglobulin Fc part, for example, as shown in Example 3.1 of WO 2008/119567). Maintenance of the three-dimensional structure within the N-terminal 27 amino acid polypeptide fragment or functional fragment thereof of CD3 epsilon can be used to generate binding domains that bind to the N-terminal epsilon CD3 polypeptide fragment in vitro and for the native CD3 complex (epsilon subunit CD3) on T cells in vivo with the same binding affinity. Within the present invention, a functional fragment of 1 to 27 amino acid residues from the N-terminus means that the CD3 binding domains provided in the present invention can still bind these functional fragments in a context-independent manner. One of skill in the art is aware of epitope mapping methods to determine which amino acid residues of an epitope are recognized by the anti-CD3 such binding domains (eg, alanine scan, see examples of WO 2008/119567) .
[00079] In one embodiment of the present invention, the bispecific single chain antibody molecule of the present invention comprises a (first) binding domain capable of binding to a human and non-chimpanzee primate CD3ε chain epitope and a second domain of binding capable of binding to the cell surface antigen PSMA.
[00080] Within the present invention, it is even preferred that the second binding domain that binds to the cell surface antigen human PSMA and/or a non-chimpanzee primate PSMA. Particularly preferred, the second binding domain binds to human PSMA and a non-chimpanzee primate PSMA, preferably a monkey PSMA. It is to be understood that the second binding domain binds to at least one non-chimpanzee primate PSMA, however, it may also bind to two, three, or more homologous non-chimpanzee primate PSMA. For example, the second binding domain can bind to cynomolgus monkey PSMA and rhesus monkey PSMA.
[00081] The present invention, including all methods, uses, kits, etc. described in the present invention, also relates to secondary binding domains as such (i.e., not in the context of a single-chain bispecific antibody). "As such" further includes bispecific antibody formats other than the single chain antibodies as described in the present invention, e.g. antibody fragments (comprising the secondary domain), humanized antibodies, fusion proteins comprising the secondary domains, etc. Antibody formats other than the bispecific single chain antibodies of the present invention are also described above in the present invention.
[00082] For the generation of the second binding domain of the bispecific single-chain antibody molecule of the present invention, for example, bispecific single-chain antibodies as defined in the present invention, the monoclonal antibodies that bind to both the antigen of respective PSMA cell surface, such as human and/or non-chimpanzee primate can be used. Suitable binding domains for the bispecific polypeptide as defined in the present invention, for example, can be derived from cross-species of specific monoclonal antibodies by means of the recombinant methods described in the art. A monoclonal antibody binding to a human cell surface antigen and to the homolog of said cell surface antigen in non-chimpanzee primates can be tested by FACS assays as described above. It is evident to those skilled in the art that interspecies specific antibodies can also be generated by hybridoma techniques described in the literature (Milstein and Kohler, Nature 256 (1975), 495 to 7). For example, mice can alternatively be immunized with human and non-chimpanzee primate cell surface antigens, such as PSMA. From these mice, specific transspecies of antibody-producing hybridoma cells are isolated by hybridoma technology and analyzed by FACS, as defined above. The generation and analysis of bispecific polypeptides, such as single-chain bispecific antibodies that exhibit cross-species specificity, as described in the present invention is shown in the following examples. The advantages of bispecific single chain antibodies that exhibit cross-species specificity include the points enumerated in the present invention.
It is particularly preferred for the bispecific single chain antibody molecule of the present invention, that the first binding domain capable of binding a human and non-chimpanzee primate CD3 ε chain epitope comprises a VL region comprising CDR-L1 , CDR-L2 and CDR-L3 selected from: (a) CDR-L1 as set out in SEQ ID NO: 27, CDR-L2 as set out in SEQ ID NO: 28 and CDR-L3 as set out in SEQ ID NO: 29; (b) CDR-L1 as set out in SEQ ID NO: 117, CDR-L2 as set out in SEQ ID NO: 118 and CDR-L3 as set out in SEQ ID NO: 119; and (c) CDR-L1 as set out in SEQ ID NO: 153, CDR-L2 as set out in SEQ ID NO: 154 and CDR-L3 as set out in SEQ ID NO: 155.
Variable regions, i.e. the variable light chain ("L" or "VL") and the variable heavy chain ("H" or "VH") are known in the art to provide the binding domain of an antibody . These variable regions house the complementary determining regions.
[00085] The term "complementarity determining region" (CDR) is well known in the art to dictate the antigen specificity of an antibody. The term "CDR-L" or "L CDR" or "LCDR" refers to CDRs in VL, while the term "CDR-H" or "H CDR" or HCDR "refers to CDRs in VH.
[00086] In an alternative preferred embodiment, the bispecific single chain antibody molecule of the present invention, the first binding domain capable of binding a human and non-chimpanzee primate CD3 ε chain epitope comprises a VH region comprising CDR- H1, CDR-H2 and CDR H3 selected from: (a) CDR-H1 as described in SEQ ID NO: 12, CDR-H2 as described in SEQ ID NO: 13 and CDR-H3 as described in SEQ ID NO: 14; (b) CDR-H1 as set out in SEQ ID NO: 30, CDR-H2 as set out in SEQ ID NO: 31 and CDR-H3 as set out in SEQ ID NO: 32; (c) CDR-H1 as set out in SEQ ID NO: 48, CDR-H2 as set out in SEQ ID NO: 49 and CDR-H3 as set out in SEQ ID NO: 50; (d) CDR-H1 as set out in SEQ ID NO: 66, CDR-H2 as set out in SEQ ID NO: 67 and CDR-H3 as set out in SEQ ID NO: 68; (e) CDR-H1 as set out in SEQ ID NO: 84, CDR-H2 as set out in SEQ ID NO: 85 and CDR-H3 as set out in SEQ ID NO: 86; (f) CDR-H1 as set out in SEQ ID NO: 102, CDR-H2 as set out in SEQ ID NO: 103 and CDR-H3 as set out in SEQ ID NO: 104; (g) CDR-H1 as set out in SEQ ID NO: 120, CDR-H2 as set out in SEQ ID NO: 121 and CDR-H3 as set out in SEQ ID NO: 122; (h) CDR-H1 as set out in SEQ ID NO: 138, CDR-H2 as set out in SEQ ID NO: 139 and CDR-H3 as set out in SEQ ID NO: 140; (i) CDR-H1 as set out in SEQ ID NO: 156, CDR-H2 as set out in SEQ ID NO: 157 and CDR-H3 as set out in SEQ ID NO: 158; and (j) CDR-H1 as set out in SEQ ID NO: 174, CDR-H2 as set out in SEQ ID NO: 175 and CDR-H3 as set out in SEQ ID NO: 176.
[00087] It is further preferred that the binding domain capable of binding a human and non-chimpanzee primate CD3 ε chain epitope comprises a VL region selected from the group consisting of a VL region as illustrated in SEQ ID NO : 35, 39, 125, 129, 161 or 165.
[00088] It is preferable that, alternatively, the first binding domain capable of binding a human and non-chimpanzee primate CD3 ε chain epitope comprises a VH region selected from the group consisting of a VH region, as depicted in SEQ ID NO : 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181.
[00089] More preferably, the bispecific single chain antibody molecule of the present invention is characterized by the first binding domain capable of binding a human and non-chimpanzee primate CD3 ε chain epitope, which comprises a VL region and a VH region selected from the group consisting of: (a) a VL region as set forth in SEQ ID NO: 17 or 21 and a VH region as set forth in SEQ ID NO: 15 or 19; (b) a VL region as set out in SEQ ID NO: 35 or 39 and a VH region as set out in SEQ ID NO: 33 or 37; (c) a VL region as set out in SEQ ID NO: 53 or 57 and a VH region as set out in SEQ ID NO: 51 or 55; (d) a VL region as set out in SEQ ID NO: 71 or 75 and a VH region as set out in SEQ ID NO: 69 or 73; (e) a VL region as set out in SEQ ID NO: 89 or 93 and a VH region as set out in SEQ ID NO: 87 or 91; (f) a VL region as set out in SEQ ID NO: 107 or 111 and a VH region as set out in SEQ ID NO: 105 or 109; (g) a VL region as set out in SEQ ID NO: 125 or 129 and a VH region as set out in SEQ ID NO: 123 or 127; (h) a VL region as set forth in SEQ ID NO: 143 or 147 and a VH region as set forth in SEQ ID NO: 141 or 145; (i) a VL region as set out in SEQ ID NO: 161 or 165 and a VH region as set out in SEQ ID NO: 159 or 163; and (j) a VL region as set forth in SEQ ID NO: 179 or 183 and a VH region as set forth in SEQ ID NO: 177 or 181.
[00090] According to a preferred embodiment of the bispecific single chain antibody molecule of the present invention, the pairs of VH regions and VL regions of the first epsilon CD3 binding binding domain are in the format of a single chain antibody (scFv ). The VH and VL regions are arranged in the order of VH-VL or VL-VH. It is preferable that the VH region is positioned at the N-terminus to a binding sequence. The VL region is positioned C-terminally from the linker sequence. Put in other words, the domain arrangement in the CD3 binding domain of the bispecific single chain antibody molecule of the present invention is preferably VH-VL, with said CD3 binding domain located at the C-terminus for the second (antigen cell surface, such as PSMA) binding domain. Preferably, the VH-VL comprises or is SEQ ID NO: 185.
[00091] A preferred embodiment of the above-described bispecific single chain antibody molecule of the present invention is characterized by the first binding domain capable of binding to a human and non-chimpanzee primate CD3ε chain epitope comprising a selected amino acid sequence from the group consisting of SEQ ID NO: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187 .
[00092] The present invention further relates to a bispecific single chain antibody described above, in which the second binding domain binds to the cell surface antigen PSMA.
[00093] According to a preferred embodiment of the present invention, a bispecific single chain antibody molecule characterized above comprises a group of one of the following sequences as CDR H1, H2 CDR, CDR H3, L1 CDR, CDR L2 and L3 CDR in the second binding domain selected from the group consisting of: a) H1-3 CDR of SEQ ID NO: 226-228 and L1-3 CDR of SEQ ID NO: 231-233; b) H1-3 CDR of SEQ ID NO: 240-242 and L1-3 CDR of SEQ ID NO: 245-247; c) H1-3 CDR of SEQ ID NO: 254-256 and L1-3 CDR of SEQ ID NO: 259-261; d) H1-3 CDR of SEQ ID NO: 268-270 and L1-3 CDR of SEQ ID NO: 273-275; e) H1-3 CDR of SEQ ID NO: 618-620 and L1-3 CDR of SEQ ID NO: 623-625; f) H1-3 CDR of SEQ ID NO: 282-284 and L1-3 CDR of SEQ ID NO: 287-289; g) H1-3 CDR of SEQ ID NO: 296-298 and L1-3 CDR of SEQ ID NO: 301-303; h) H1-3 CDR of SEQ ID NO: 310-312 and L1-3 CDR of SEQ ID NO: 315-317; i) H1-3 CDR of SEQ ID NO: 324-326 and L1-3 CDR of SEQ ID NO: 329-331; j) H1-3 CDR of SEQ ID NO: 338-340 and L1-3 CDR of SEQ ID NO: 343-345; k) H1-3 CDR of SEQ ID NO: 352-354 and L1-3 CDR of SEQ ID NO: 357-359; l) H1-3 CDR of SEQ ID NO: 366-368 and L1-3 CDR of SEQ ID NO: 371-373; m) H1-3 CDR of SEQ ID NO: 380-382 and L1-3 CDR of SEQ ID NO: 385-387; n) H1-3 CDR of SEQ ID NO: 394-396 and L1-3 CDR of SEQ ID NO: 399-401; o) H1-3 CDR of SEQ ID NO: 408-410 and L1-3 CDR of SEQ ID NO: 413-415; p) H1-3 CDR of SEQ ID NO: 422-424 and L1-3 CDR of SEQ ID NO: 427-429; q) H1-3 CDR of SEQ ID NO: 436-438 and L1-3 CDR of SEQ ID NO: 441-443; r) H1-3 CDR of SEQ ID NO: 450-452 and L1-3 CDR of SEQ ID NO: 455-457; s) H1-3 CDR of SEQ ID NO: 464-466 and L1-3 CDR of SEQ ID NO: 469-471; t) H1-3 CDR of SEQ ID NO: 478-480 and L1-3 CDR of SEQ ID NO: 483-485; u) H1-3 CDR of SEQ ID NO: 492-494 and L1-3 CDR of SEQ ID NO: 497-499; v) H1-3 CDR of SEQ ID NO: 506-508 and L1-3 CDR of SEQ ID NO: 511-513; w) H1-3 CDR of SEQ ID NO: 520-522 and L1-3 CDR of SEQ ID NO: 525-527; x) H1-3 CDR of SEQ ID NO: 534-536 and L1-3 CDR of SEQ ID NO: 539-541; y) H1-3 CDR of SEQ ID NO: 548-550 and L1-3 CDR of SEQ ID NO: 553-555; z) H1-3 CDR of SEQ ID NO: 562-564 and L1-3 CDR of SEQ ID NO: 567-569; aa) H1-3 CDR of SEQ ID NO: 576-578 and L1-3 CDR of SEQ ID NO: 581-583; ab) H1-3 CDR of SEQ ID NO: 590-592 and L1-3 CDR of SEQ ID NO: 595-597; and ac) H1-3 CDR of SEQ ID NO: 604-606 and L1-3 CDR of SEQ ID NO: 609-611.
[00094] A preferred group of bispecific single chain antibody molecule comprises a group of one of the following sequences as CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3 in the second binding domain selected from the group consisting of: a) H1-3 CDR of SEQ ID NO: 226-228 and L1-3 CDR of SEQ ID NO: 231-233; b) H1-3 CDR of SEQ ID NO: 240-242 and L1-3 CDR of SEQ ID NO: 245-247; c) H1-3 CDR of SEQ ID NO: 254-256 and L1-3 CDR of SEQ ID NO: 259-261; d) H1-3 CDR of SEQ ID NO: 268-270 and L1-3 CDR of SEQ ID NO: 273-275; and e) H1-3 CDR of SEQ ID NO: 618-620 and L1-3 CDR of SEQ ID NO: 623-625.
[00095] These molecules comprise a second binding domain that binds to the PSMA cell surface antigen consisting of a VH chain derived from a parent specific binding PSMA molecule and a VL chain derived from a binding molecule that has a specificity for a different antigen, for example, to the epithelial cell adhesion molecule (EpCAM), also known as CD326. It was surprisingly found that binding molecules with such a specific combination of a VH chain derived from a specific binding PSMA parent molecule and a VL chain derived from a specific EpCAM parent specific binding molecule exclusively bind to PSMA, but not to EpCAM. The binding specificities of this group of PSMA specific binding domains comprised in the bispecific single chain antibody molecule of the present invention are described in Example 3 attached.
[00096] Another preferred group of bispecific single chain antibody molecule comprises a group of one of the following sequences as CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3 in the second binding domain selected from the group consisting of: a) H1-3 CDR of SEQ ID NO: 282-284 and L1-3 CDR of SEQ ID NO: 287-289; b) H1-3 CDR of SEQ ID NO: 296-298 and L1-3 CDR of SEQ ID NO: 301-303; c) H1-3 CDR of SEQ ID NO: 310-312 and L1-3 CDR of SEQ ID NO: 315-317; d) H1-3 CDR of SEQ ID NO: 324-326 and L1-3 CDR of SEQ ID NO: 329-331; e) H1-3 CDR of SEQ ID NO: 338-341 and L1-3 CDR of SEQ ID NO: 343-345; f) H1-3 CDR of SEQ ID NO: 352-354 and L1-3 CDR of SEQ ID NO: 357-359; and g) H1-3 CDR of SEQ ID NO: 366-368 and L1-3 CDR of SEQ ID NO: 371-373.
[00097] Another preferred group of bispecific single chain antibody molecule also comprises a group of the following sequences as CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3 in the second binding domain selected from the group consisting of in: a) H1-3 CDR of SEQ ID NO: 380-382 and L1-3 CDR of SEQ ID NO: 385-387; b) H1-3 CDR of SEQ ID NO: 394-396 and L1-3 CDR of SEQ ID NO: 399-401; c) H1-3 CDR of SEQ ID NO: 408-410 and L1-3 CDR of SEQ ID NO: 413-415; d) H1-3 CDR of SEQ ID NO: 422-424 and L1-3 CDR of SEQ ID NO: 427-429; e) H1-3 CDR of SEQ ID NO: 436-438 and L1-3 CDR of SEQ ID NO: 441-443; f) H1-3 CDR of SEQ ID NO: 450-452 and L1-3 CDR of SEQ ID NO: 455-457; g) H1-3 CDR of SEQ ID NO: 464-466 and L1-3 CDR of SEQ ID NO: 469-471; h) H1-3 CDR of SEQ ID NO: 478-480 and L1-3 CDR of SEQ ID NO: 483-485; i) H1-3 CDR of SEQ ID NO: 492-494 and L1-3 CDR of SEQ ID NO: 497-499; j) H1-3 CDR of SEQ ID NO: 506-508 and L1-3 CDR of SEQ ID NO: 511-513; k) H1-3 CDR of SEQ ID NO: 520-522 and L1-3 CDR of SEQ ID NO: 525-527; l) H1-3 CDR of SEQ ID NO: 534-536 and L1-3 CDR of SEQ ID NO: 539-541; m) H1-3 CDR of SEQ ID NO: 548-550 and L1-3 CDR of SEQ ID NO: 553-555; n) H1-3 CDR of SEQ ID NO: 562-564 and L1-3 CDR of SEQ ID NO: 567-569; o) H1-3 CDR of SEQ ID NO: 576-578 and L1-3 CDR of SEQ ID NO: 581-583; p) H1-3 CDR of SEQ ID NO: 590-592 and L1-3 CDR of SEQ ID NO: 595-597; and q) H1-3 CDR of SEQ ID NO: 604-606 and L1-3 CDR of SEQ ID NO: 609-611.
[00098] It is preferred that the bispecific single chain antibody molecule of the present invention that the second binding domain to which PSMA cell surface antigen binds comprises a group of the following sequences as the VH- and VL chains in the second domain of binding selected from the group consisting of: a) of SEQ ID NO: b) of SEQ ID NO: c) of SEQ ID NO: d) of SEQ ID NO: e) of SEQ ID NO: f) of SEQ ID NO : g) of SEQ ID NO: VH of SEQ ID VH of SEQ ID VH of SEQ ID VH of SEQ ID VH of SEQ ID VH of SEQ ID VH of SEQ ID one chain VL one chain VL one chain VL chain one chain VL a VL chain a VL chain h) a VH chain of SEQ ID NO: 314; i) a VH chain of SEQ ID NO: 328; j) a VH chain of SEQ ID NO: 342; k) a VH chain of SEQ ID NO: 356; l) a VH chain of SEQ of SEQ ID NO: 370; m) a VH chain of SEQ ID NO: 384; n) a VH chain of SEQ ID NO: 398; o) a VH chain of SEQ ID NO: 412; p) a VH chain of SEQ ID NO: 426; q) a VH chain of SEQ ID NO: 440; r) a VH chain of SEQ ID NO: 454; s) a VH chain of SEQ ID NO: 468; t) a VH chain of SEQ ID NO: 482; u) a VH chain of SEQ ID NO: 496; v) a VH chain of SEQ ID NO: 510; w) a VH chain of SEQ ID NO: 519 and a VL chain of SEQ ID NO: 524; x) a VH chain of SEQ ID NO: 533 and a VL chain of SEQ ID NO: 538; y) a VH chain of SEQ ID NO: 547 and a VL chain of SEQ ID NO: 552; z) a VH chain of SEQ ID NO: 561 and a VL chain of SEQ ID NO: 566; aa) a VH chain of SEQ ID NO: 575 and a VL chain of SEQ ID NO: 580; ab) a VH chain of SEQ ID NO: 589 and a VL chain of SEQ ID NO: 594; and ac) a VH chain of SEQ ID NO: 603 and a VL chain of SEQ ID NO: 608.
[00099] The above VH- and VL- chains are also shown in SEQ ID NOs: 235, 249, 263, 277, 627, 291, 305, 319, 333, 347, 361, 375, 389, 403, 417, 431 , 445, 459, 473, 487, 501, 515, 529, 543, 557, 571, 585, 599 and 613, respectively.
[000100] The sequences (amino acid sequence and nucleotide sequence) of the corresponding VL- and VH regions of the second binding domain of the bispecific single-chain antibody molecule of the present invention, as well as of the respective scFvs are shown in the sequence listing.
[000101] In the bispecific single chain antibody molecule of the present invention, the binding domains are arranged in the order VL-VH-VH-VL, VL-VH-VL-VH, VH-VL-VH-VL or VH-VL -VH-VL, as exemplified in the attached examples. Preferably, the binding domains are arranged in the order VH PSMA-VL PSMA-VH CD3-VL CD3 or VL PSMA-VH PSMA-VH CD3-VL CD3.
[000102] A particularly preferred embodiment of the present invention concerns a polypeptide characterized above, wherein the single-chain bispecific antibody molecule comprises a sequence selected from: (a) an amino acid sequence as described in any one of the SEQ ID NOs: 237, 251, 265, 279, 629, 293, 307, 321, 335, 349, 363, 377, 391, 405, 419, 433, 447, 461, 475, 489, 503, 517, 531, 545 , 559, 573, 587, 601 or 615; (b) an amino acid sequence encoded by a nucleic acid sequence as described in any one of SEQ ID NOs: 238, 252, 266, 280, 630, 294, 308, 322, 336, 350, 364, 378, 392, 406, 420, 434, 448, 462, 476, 490, 504, 518, 532, 546, 560, 574, 588, 602 or 616.
[000103] The present invention relates to a bispecific single chain antibody molecule comprising an amino acid sequence as depicted in any one of SEQ ID NOs: 237, 251, 265, 279, 629, 293, 307, 321, 335, 349, 363, 377, 391, 405, 419, 433, 447, 461, 475, 489, 503, 517, 531, 545, 559, 573, 587, 601 or 615, as well as the hair amino acid sequences less 96% identical, preferably 97%, more preferred at least 98% identical, more preferred at least 99% identical to the amino acid sequence of SEQ ID NOs: 237, 251, 265, 279, 629, 293, 307, 321, 335 , 349, 363, 377, 391, 405, 419, 433, 447, 461, 475, 489, 503, 517, 531, 545, 559, 573, 587, 601 or 615. The present invention also relates to the corresponding nucleic acid sequences as described in any one of SEQ ID NOs: 238, 252, 266, 280, 630, 294, 308, 322, 336, 350, 364, 378, 392, 406, 420, 434, 448, 462 , 476, 490, 504, 518, 532, 546, 560, 574, 588, 602 or 616, as well as the sequences Nucleic acid copies of at least 96% identical, preferably 97%, more preferred at least 98% identical, most preferred at least 99% identical to the nucleic acid sequences shown in SEQ ID NOs: 238, 252, 266, 280, 630, 294, 308, 322, 336, 350, 364, 378, 392, 406, 420, 434, 448, 462, 476, 490, 504, 518, 532, 546, 560, 574, 588, 602 or 616. It is to be understood that sequence identity is determined over the entire nucleotide or amino acid sequence. For sequence alignments, for example, the Gap or BestFit programs can be used (Needleman and Wunsch J. Mol. Biol 48 (1970), 443-453, Smith and Waterman, Adv. Appl Math 2 (1981), 482 to 489 ), which is contained in the GCG software package (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991). It is a routine method for those skilled in the art to determine and identify a nucleotide sequence or amino acids having, for example, 96% (97%, 98% or 99%) sequence identity with the nucleotide or amino acid sequences of the bispecific single-chain antibody of the present invention, using an example of the aforementioned programs. according to the Crick's Wobble hypothesis, the 5' base in the anti-codon is not as spatially constrained as the other two bases, and may therefore have non-standard base pairing. To put it in other words: the third position in the triplet a codon may vary by mod o that two triplets that differ at this third position can encode the same amino acid residue. Said hypothesis is well known to one skilled in the art (see, for example, http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19 (1966): 548 to 55).
[000104] Preferred domain arrangements in the bispecific single-chain PSMAxCD3 antibody constructs of the present invention are shown in the following examples.
[000105] In a preferred embodiment of the present invention, the bispecific single chain antibodies are cross-species specific for CD3 epsilon and for the human and non-chimpanzee primate PSMA cell surface antigen, recognized by their second binding domain.
[000106] In an alternative embodiment of the present invention provides a nucleic acid sequence encoding an above-described bispecific single chain antibody molecule of the present invention.
[000107] The present invention also relates to a vector comprising the nucleic acid molecule of the present invention.
[000108] Many suitable vectors are known to experts in molecular biology, the choice of which will depend on the desired function and include plasmids, cosmids, viruses, bacteriophages and other vectors conventionally used in genetic engineering. Methods that are well known to one of skill in the art can be used to construct various plasmids and vectors, see, for example, the techniques described in Sambrook et al. (Loc cit.) And Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989), (1994). Alternatively, the polynucleotides and vectors of the present invention can be reconstituted into liposomes for delivery to target cells. As discussed in more detail below, a cloning vector was used to isolate the individual DNA sequences. The relevant sequences can be transferred to expression vectors where expression of a specific polypeptide is required. Typical pBluescript SK cloning vectors include, pGEM, pUC9, pBR322 and pGBT9. Typical expression vectors include PTRE, PCAL-n-EK, PESP-1, pOP13CAT.
Preferably, said vector comprises a nucleic acid sequence which is a regulatory sequence operably linked to said nucleic acid sequence defined herein.
[000110] The term "regulatory sequence" refers to DNA sequences, which are necessary to effect the expression of coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eukaryotes, control sequences generally include promoters, terminators, and, in some cases, enhancers, or transactivating transcription factors. The "control sequence" is intended to include, at a minimum, all components whose presence is required for expression, and may also include additional advantageous components.
[000111] The term "operatively linked" refers to a juxtaposition in which the components thus described are in a relationship that allows them to function in their intended form. A control sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. In case the control sequence is a promoter, it is obvious to one of skill in the art that double-stranded nucleic acid is preferably used.
[000112] In this way, the vector is preferably recited an expression vector. An "expression vector" is a construct that can be used to transform a selected host for expression and provides a coding sequence in the selected host. Expression vectors can be, for example, cloning vectors, binary vectors or integration vectors. Expression comprises the transcription of the nucleic acid molecule, preferably into a translatable mRNA. Regulatory elements that ensure expression in prokaryotes and/or eukaryotic cells are well known to one of skill in the art. In the case of eukaryotic cells, they normally include promoters that ensure the initiation of transcription and optionally poly-A signals that ensure the termination of transcription and stabilization of the transcript. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, PL, lac, trp or tac in E. coli, the promoter, and examples of regulatory elements that allow expression in eukaryotic host cells are the promoter AOX1 or GAL1 in yeast or the CMV-, SV40, RSV (Rous sarcoma virus) promoter, CMV- enhancer, SV40 enhancer or a globin intron in mammalian and other animal cells.
[000113] Beside elements, which are responsible for the initiation of transcription, such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Furthermore, depending on the expression system used, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium can be added to the coding sequence of the recited nucleic acid sequence and are well known in the art, see for example , WO 2008/119567. The leader sequence(s) is (are) assembled in appropriate phase with the initiation of translation, and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein , or a portion thereof, to the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, for example, stabilization or simplified purification of expressed recombinant product; see above. In this context, suitable expression vectors are known in the art, such as Okayama-Berg expression vector cDNA pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pEF-DHFR, pEF-ADA or pEF-neo (Mack et al. PNAS (1995) 92, 7021-7025 and Raum et al. Cancer Immunol Immunother (2001) 50 (3), 141 to 150) or pSPORT1 (GIBCO BRL).
[000114] Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming the transfection of eukaryotic host cells, but control sequences for prokaryotic hosts can also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, collection and purification of the bispecific single chain antibody molecule of the present invention can below, see, for example, the examples attached.
[000115] An alternative expression system that can be used to express a cell cycle interacting protein in an insect system. In such a system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express exogenous genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The coding sequence of a recited nucleic acid molecule can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of said coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking the coat protein. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the protein of the present invention is expressed (Smith, J. Virol 46 (1983), 584, Engelhard, Proc Nat Acad Sci USA 91. (1994), 3224 to 3227).
[000116] Additional regulatory elements can include transcriptional as well as translational enhancers. Advantageously, the above-described vectors of the present invention comprise a selectable and/or scorable marker.
[000117] Selectable marker genes useful for selection of transformed cells and, for example, plant tissues, and plants are well known to those skilled in the art and include, for example, antimetabolite resistance as a basis for selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol (Life Sci. Adv.) 13 (1994), 143 to 149), npt, which confers resistance to the aminoglycosides neomycin, kanamycin, and paromycin (Herrera-Estrella, EMBO J. 2 (1983) , 987 to 995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481 to 485). Additional selectable genera have been described, namely trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to use histinol in place of histidine (Hartman, Proc Natl Acad Sci USA 85 (1988), 8047); Mannose-6-phosphate isomerase, which allows cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed) or Aspergillus terreus deaminase, which confers resistance to blasticidin S (Tamura, Biosci Biotechnol Biochem 59 (1995.), 2336 to 2338).
[000118] Useful scorable markers are also known to one of skill in the art and are commercially available. Advantageously, said marker is a gene encoding luciferase (Giacomin, Pl Sci 116 (1996), 59 to 72, Scikantha, J. Bact 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett 389 ( 1996), 44 to 47) or beta-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901 to 3907). This modality is particularly useful for simple and rapid screening of cells, tissues and organisms that contain a recited vector.
[000119] As described above, the recited nucleic acid molecule can be used alone or as part of a vector to express the bispecific single chain antibody molecule of the present invention in cells, for, for example, purification, but also for gene therapy purposes. Nucleic acid molecules or vectors that contain the DNA sequence(s) coding for any of the above bispecific single-chain antibody molecules described in the present invention is introduced into the cells, which in turn, produce the polypeptide of interest. Gene therapy, which is based on the introduction of therapeutic genes into cells by ex vivo or in vivo techniques, is one of the most important applications of gene transfer. Suitable vectors, methods or gene delivery systems for in vitro or in vivo gene therapy are described in the literature and known to the person skilled in the art, see, for example, Giordano, Nature Medicine 2 (1996) , 534 to 539; Schaper, Circ. Res. 79 (1996), 911 to 919; Anderson, Science 256 (1992), 808 to 813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370 to 374; Muhlhauser, Circ. Res. 77 (1995), 10771086; Onodera, Blood 91 (1998), 30 to 36; Verma, Genus Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci. 811 (1997), 289 to 292; Verzeletti, Hum.. Genus Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714 to 716; WO 94/29469, WO 97/00957, US 5,580,859; US 5,589,466, or Schaper, Current Opinion in Biotechnology 7 (1996), 635 to 640; dos Santos Coura and Nardi Virol J. (2007), 4:99. The recited nucleic acid molecules and vectors can be designed for direct introduction or for introduction through liposomes or viral vectors (eg, adenoviral, retroviral) into the cell. Preferably, said cell is a germinal cell line, embryonic cell, or egg cell or derivatives thereof, most of the cells, preferably said is a stem cell. An example of an embryonic stem cell may be, inter alia, a stem cell as described in Nagy, Proc. Natl. Academic Sci. US 90 (1993), 8424 to 8428.
[000120] The present invention also provides a host transformed or transfected with a vector of the present invention. Said host can be produced by introducing the above-described vector of the present invention, or the above-described nucleic acid molecule of the present invention into the host. The presence of at least one vector, or at least one nucleic acid molecule in the host can mediate the expression of a gene encoding the antibodies to the single chain constructs described above.
[000121] The described nucleic acid molecule or vector of the present invention, which is introduced into the host or can integrate into the host genome or can be maintained extrachromosomally.
[000122] The host can be any prokaryotic or eukaryotic cell.
[000123] The term "prokaryote" is intended to include all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of a protein of the present invention. Prokaryotic hosts can include gram negative in this way as gram positive bacteria, such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryote" is intended to include yeast, higher plants, insect cells and preferably mammals. Depending on the host used in a recombinant production process, the protein encoded by the polynucleotide of the present invention can be glycosylated or can be non-glycosylated. Especially preferred is the use of a plasmid or virus which contains the coding sequence of the bispecific single-chain antibody molecule of the present invention and genetically fused to an N-terminal and/or His-C-terminal FLAG-tag. Preferably, the length of said FLAG tag is about 4 to 8 amino acids, more preferably 8 amino acids. A polynucleotide described above can be used to transform or transfect the host using any of the techniques commonly known to those skilled in the art. Furthermore, methods for preparing fused genes, operably linked and expressing themselves in, for example, mammalian cells and bacteria, are well known in the art (Sambrook, loc cit.).
[000124] Preferably, said host is a bacterium or an insect, plant, fungal or animal cell.
[000125] It is especially predicted that the recited host may be a mammalian cell. Particularly preferred host cells comprise CHO cells, COS cells, myeloma cell lines such as SP2/0 or NS/0. As illustrated in the examples of WO 2008/119567 for other molecules of the same class, particularly preferred are CH cells as hosts.
Said host cell is most preferably a human cell or a human cell line, e.g. PER.C6 (Kroos, Biotechnol. Prog., 2003, 19: 163 to 168).
[000127] In another embodiment, the present invention thus relates to a process for the production of a bispecific single chain antibody molecule of the present invention, said process comprising culturing a host of the present invention under conditions that allow the expression of the bispecific single chain antibody molecule of the present invention and the recovery of the polypeptide produced from the culture.
[000128] Transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The bispecific single chain antibody molecule of the present invention can then be isolated from growth media, cell lysates or cell membrane fractions. Isolation and purification of bispecific single-chain antibody molecules expressed by microorganisms, for example, can be done by any conventional means, such as, for example, preparative chromatographic separations and immunological separations, such as those involving the use of antibodies monoclonal or polyclonal directed, for example, against a single chain bispecific antibody molecule tag of the present invention or, as described in the accompanying examples.
[000129] Conditions for culturing a host that allow expression are known in the art to depend on the host system and the expression/vector system used in such a process. The parameters to be modified in order to achieve conditions that allow expression of a recombinant polypeptide are known in the art. In this way, suitable conditions can be determined by one of ordinary skill in the art, in the absence of additional input to the present invention.
[000130] Once expressed, the single-chain bispecific antibody molecule of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like, see, Scopes, "Protein Purification", Springer-Verlag, NY (1982). Substantially pure polypeptides of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the bispecific single chain antibody molecule of the present invention can then be used therapeutically (including extracorporeally) or in the development and performance of assay procedures. Furthermore, examples of methods for recovering the bispecific single chain antibody molecule of the present invention from a culture are described in detail in the appended examples of WO 2008/119567 for other molecules of the same class. Recovery can also be achieved by a method for isolating the bispecific single chain antibody molecule of the present invention capable of binding to a human and non-chimpanzee non-primate CD3 epsilon epitope (CD3 ε), the method comprising the steps of : (a) contacting the polypeptide(s) with an N-terminal fragment of the extracellular domain of CD3 ε maximal 27 amino acids comprising the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly ( SEQ ID NO: 211) or Gln-Asp-Gly-Asn-Glu-Glu-Ile-Gly (SEQ ID NO: 212), attached via its C-terminal end to a solid phase; (b) eluting the bound polypeptide(s) from said fragment, and (c) isolating the polypeptide(s) from the eluate of (b).
[000131] It is preferred that the polypeptide(s) isolated by the above method of the present invention is (are) of human origin.
[000132] This method, or the isolation of the bispecific single chain antibody molecule of the present invention is understood as a method for the isolation of one or more different polypeptides, with the same specificity for the fragment of the extracellular domain of CD3 ε comprises in its N-terminal end of the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO: 211) or Gln-Asp-Gly-Asn-Glu-Glu-Ile-Gly (SEQ ID NO: 212) from a plurality of polypeptide candidates, as well as a method for purifying a polypeptide from a solution. A non-limiting example for the latter method for purifying a bispecific single chain antibody molecule from solution is, for example, the purification of a recombinantly expressed bispecific single chain antibody molecule from a supernatant of culture or from a preparation of such culture.
[000133] As stated above the fragment used in this method is an N-terminal fragment of the extracellular domain of the primate CD3ε molecule. The amino acid sequence of the extracellular domain of the CD3 ε molecule from different species is represented in SEQ ID NOs: 1, 3, 5 and 7. The two forms of the octamer at the N-terminus are shown in SEQ ID NOs: 211 and 212. It is preferable that this N-terminal end is freely available for binding the polypeptides to be identified by the method of the present invention. The term "freely available" is understood in the context of the present invention as without additional motifs such as a His tag. The interference of such a His tag with a binding molecule described in the present invention is described in WO 2008/119567.
[000134] According to this method, said fragment is fixed through its C-terminal end to a solid phase. The person skilled in the art will easily and without any invention elect a suitable solid phase support depending on the used embodiment of the method of the present invention. Examples for a solid support include, but are not limited to, matrices such as e.g. beads (granules, agarose beads, polystyrene sepharose beads, dextran beads), plates (culture plates or multi-well plates) as well. as known chips for example from Biacore ®. The selection of means and methods for fixation/immobilization of the fragment to said solid support will depend on the choice of solid support. A commonly used method for fixation/immobilization is a coupling via an N-hydroxysuccinimide (NHS). The coupling chemistry underlying this, such as alternative fixation/immobilization methods are known to the person skilled in the art, for example, from "Hermanson Bioconjugate Techniques", Academic Press, Inc. (1996). For the fixation of/for immobilization on chromatographic supports the following media are commonly used: NHS activated sepharose (eg HiTrap-NHS from GE Life Science-Amersham), CnBr activated sepharose (eg GE Life Science-Amersham) , NHS-activated dextran beads (Sigma) or activated polymethacrylate. These reagents can also be used in a batch approach. In addition, dextran spheres comprising iron oxide (eg available from Miltenyi) can be used in a batch approach. These spheres can be used in combination with a magnet to separate the spheres from a solution. Polypeptides can be immobilized on a Biacore chip (CM5 chips, for example) through the use of NHS-activated carboxymethyldextran. Other examples of a suitable solid support are amine reactive multi-well plates (eg Nunc ImmobilizerTM plates).
[000135] According to this method said fragment of the extracellular domain of CD3 epsilon can be directly coupled to the solid support or through a segment of amino acids, which can be a binding agent or other protein/polypeptide portion. Alternatively, the extracellular domain of CD3 epsilon can be indirectly coupled via one or more molecule adapter(s).
[000136] The means and methods for eluting a peptide or polypeptide linked to an immobilized epitope are well known in the art. The same is true for the methods of isolating the identified polypeptide(s) from the eluate.
[000137] A method for isolating one or more different single-chain bispecific antibody molecules with the same specificity for the extracellular domain fragment of CD3 ε comprises at its N-terminal end the amino acid sequence Gln-Asp -Gly-Asn- Glu-Glu-Gly-X (with X being Met or Ile) from a plurality of polypeptide candidates may comprise one or more steps of the following methods for selecting entity-specific antigens:
[000138] Specific CD3 ε binding domains can be selected from derived antibody repertoires. The phage display library can be built based on standard procedures, as for example described in "Display Phage: A Laboratory Manual"; Ed. Barbas, Burton, Scott &Silverman; Cold Spring Harbor Laboratory Press, 2001. The format of the antibody fragments in the scFv antibody library can be, but in general it can also be a Fab fragment or even a single domain antibody fragment. For the isolation of antibody fragments from libraries naive antibody fragments can be used. For the selection of potentially low immunogenic binding entities in later therapeutic use, human antibody fragment libraries may be favorable for direct selection of human antibody fragments. In some cases, they can form the basis for synthetic antibody libraries (Knappik et al. J Mol. Biol. 2000, 296:57 ff). The corresponding format can be Fab, scFv (as described below), or domain antibodies (DABS, as reviewed in Holt et al., Trends Biotechnol. 2003, 21:484 and ss.)
[000139] It is also known in the art that in many cases there is no source of human immune antibody available against the target antigen. Therefore, animals are immunized with the target antigen and respective antibody libraries isolated from animal tissue, such as, for example, the spleen or PBMCs. The N-terminal fragment can be biotinylated or covalently linked to proteins such as KLH, or bovine serum albumin (BSA). According to common approaches, mice are used for immunization. Some of the repertoires of immune antibodies of non-human origin may be especially favorable for other reasons, for example, the presence of single domain antibodies (VHH) derived from cameloid species (as described in Muyldermans, J Biotechnol 74:277; De Genst et al Dev As Immunol 2006, 30:187 ff.). Therefore, a corresponding format of the antibody library can be Fab fragments, scFv (as described below), or single domain antibodies (VHH).
[000140] In one possible approach, ten-week-old F1 mice from Balb/cx C57black crosses can be immunized with whole cells, for example, expressing transmembrane EpCAM at the N-terminus exhibiting as translational fusion of the N-terminal amino acids of 1 the 27th of the mature CD3ε chain. Alternatively, mice can be immunized with the 1-27 CD3 epsilon-Fc fusion protein (a corresponding approach is described in the examples of WO 2008/119567). After booster(s) immunization, blood samples can be taken and the serum antibody titer against CD3-positive T cells can be tested, for example, in the FACS analysis. Typically, serum titers are significantly higher in immunized than in unimmunized animals.
[000141] Immunized animals can form the basis for the construction of immune antibody libraries. Examples of such libraries include phage display libraries. Such libraries can generally be built based on standard procedures, as for example described in "Display Phage: A Laboratory Manual"; Ed. Barbas, Burton, Scott &Silverman; Cold Spring Harbor Laboratory Press, 2001.
[000142] Non-human antibodies can also be humanized through phage display, due to the generation of more variable antibody libraries that can be further enriched for ligands during selection.
[000143] In a phage display approach any of the sets of phage displaying antibody libraries form a basis for selecting binding entities using the corresponding antigen as target molecule. The central step in which antigen-specific, antigen-bound phages are isolated is called panning. Due to the display of antibody fragments on the surface of phages, this general method is called phage display. A preferred method for selection is to use small proteins such as the N2 filamentous phage domain translationally fused to the N-terminus of the scFv displayed by the phage. Another display method known in the art that can be used to isolate binding entities is the ribosome display method (reviewed in Groves and Osbourn, Expert Opin Biol Ther 2005, 5:125 ff; Lipovsek & Pluckthun, J Immunol Methods 2004, 290: 52 et seq.) In order to demonstrate the binding of scFv phage particles to a 1-27 CD3 ε-Fc fusion protein from a cloned phage library carrying the scFv repertoire can be harvested from the supernatant of respective culture by PEG (polyethylene glycol). Phage ScFv particles can be incubated with the immobilized Fc CD3ε fusion protein. The immobilized CD3 ε Fc fusion protein can be coated onto a solid phase. Binding entities can be eluted and the eluate can be used to infect new, uninfected bacterial hosts. Successful bacterial hosts with a transduced phagemid copy, encoding a human scFv-fragment, can be reselected for carbenicillin resistance and subsequently infected with eg VCMS 13 helper phage to initiate the second round of antibody display and in vitro selection . A total of 4 to 5 cycles of selections is performed normally. Binding of isolated binding entities can be tested on CD3 epsilon positive Jurkat cells, HPBALL cells, PBMCs or transfected eukaryotic cells carrying the surface-displayed EpCAM-fused N-terminal CD3ε sequence using a flow cytometry assay (see, WO 2008 /119567).
[000144] Preferably, the above method may be a method, wherein the fragment of the extracellular domain of CD3 ε consists of one or more fragments of a polypeptide having an amino acid sequence of any one represented in SEQ ID NOs: 2, 4 , 6 or 8. More preferably, said fragment is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27 amino acid residues long.
[000145] This method of identifying a bispecific single chain antibody Molecule may be a method of screening a plurality of bispecific single chain antibody molecules comprising a crossover of specific binding domain-binding species to a CD3 epitope ε from human and non-chimpanzee primate. Alternatively, the method of identification is a method of purifying/isolation of a single-chain bispecific antibody molecule which comprises a species crossover of specific binding domain binding to a human and non-chimpanzee primate CD3ε epitope.
[000146] Furthermore, the present invention provides a composition comprising a bispecific single chain antibody Molecule of the present invention or a bispecific single chain antibody as produced by the process described above. Preferably, said composition is a pharmaceutical composition.
[000147] The present invention also provides a bispecific single chain antibody molecule as defined in the present invention or produced according to the process as defined in the present invention, wherein said bispecific single chain antibody molecule is for use in preventing, treating or ameliorating cancer. Preferably, said cancer is a solid tumor, more preferably a carcinoma or prostate cancer. It is preferred that the bispecific single chain antibody molecule further comprises the appropriate formulations of vehicles, stabilizers and/or excipients. Furthermore, it is preferable that said bispecific single chain antibody molecule is suitable to be administered in combination with an additional drug. Said drug may be a non-proteinaceous compound or a proteinaceous compound and may be administered simultaneously or non-concurrently with the Bispecific Single Chain Antibody Molecule as defined herein.
[000148] According to the present invention, the "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient. The particular preferred pharmaceutical composition of the present invention comprises bispecific single chain antibodies directed against and generated against context independent CD3 epitopes. Preferably, the pharmaceutical composition comprises suitable formulations of vehicles, stabilizers and/or excipients. In a preferred embodiment, the pharmaceutical composition comprises a composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or by direct injection into tissue. It is in particular envisaged that said composition is administered to a patient by infusion or injection. The administration of suitable compositions can be carried out by different routes, for example, subcutaneously, intravenously, intraperitoneally, intramuscularly, topically or intradermally. In particular, the present invention provides for continuous administration of the suitable composition. As a non-limiting example, without interruption, that is, continuous administration can be performed by a small pump system used by the patient to measure the flow of therapeutic agent into the patient's body. The pharmaceutical composition comprising the bispecific single chain antibodies directed against and generated against epitope-independent context-independent CD3 of the present invention can be administered using said pump systems. Pump systems of this type are generally known in the art, and generally rely on periodic exchange of cartridges containing the therapeutic agent to be infused. When changing the cartridge of such a pump system, a temporary interruption of the otherwise uninterrupted flow of the therapeutic agent into the patient's body may ensue. In such a case, the administration step before cartridge replacement and the next cartridge replacement administration step would still be considered, within the meaning of the pharmaceutical medium and methods of the present invention together, form a "continuous administration" of such a therapeutic agent. .
[000149] Continuous or uninterrupted administration of these bispecific single-chain antibodies directed against and generated against the context-independent CD3 epitopes of the present invention may be subcutaneous or intravenous via a fluid delivery device or small pump system including a delivery mechanism. fluid drive to direct fluid out of a reservoir and a drive mechanism to drive the drive mechanism. Pump systems for subcutaneous administration can include a needle or cannula for penetrating a patient's skin and delivering the appropriate composition into the patient's body. Said pump systems can be directly attached or attached to the patient's skin independently of an artery, vein or blood, which allows direct contact between the pump system and the patient's skin. The pump system can be attached to the patient's skin for 24 hours to several days. The pump system can be small in size, with a reservoir for small volumes. As a non-limiting example, the reservoir volume for the pharmaceutical composition suitable to be administered may be between 0.1 and 50 ml.
[000150] Continuous administration can be transdermal through a patch worn on the skin and replaced periodically. One skilled in the art is aware of adhesive drug delivery systems suitable for this purpose. It is noteworthy that transdermal administration is especially amenable to continuous administration, as the exchange of a spent first patch can advantageously be carried out simultaneously with the placement of a new, second patch, for example on the immediately adjacent skin surface. to the first exhausted adhesive and immediately prior to removing the first exhausted adhesive. Flow interruption or power outage issues do not arise.
[000151] The composition of the present invention, which especially comprises single-chain bispecific antibodies directed against and generated against context-independent CD3 epitopes, may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions such as oil/water, various types of wetting agent emulsions, sterile solutions, liposomes, etc. Compositions comprising such vehicles can be formulated by conventional, well-known methods. Formulations can include carbohydrates, buffer solutions, amino acids and/or surfactants. Carbohydrates can be non-reducing sugars, preferably sucrose, trehalose, sorbitol octasulfate or xylitol. Such formulations can be used for continuous administration, which can be intravenous or subcutaneous with and/or without pump systems. Amino acids can be charged amino acids, preferably lysine acetate, lysine, arginine, glutamate and/or histidine. The surface-active agents can be detergents, preferably with a molecular weight of > 1.2 KD and/or a polyether, preferably with a molecular weight of > 3 KD. Non-limiting examples for preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000 or PEG 5000. The buffer systems used herein. invention may have a preferred pH of 5-9, and may comprise citrate, succinate, phosphate, histidine and acetate. Compositions of the present invention can be administered to the subject at a suitable dose which can be determined, for example, by escalating dose studies by administering increasing doses of the bispecific single chain antibody molecule of the present invention exhibit species specificity herein described for non-chimpanzee primates, eg monkeys. As set out above, the bispecific single chain antibody molecule of the present invention exhibiting cross-species specificity described in the present invention can be advantageously used identically in preclinical testing in non-chimpanzee primates and as a drug in humans. These compositions can also be administered in combination with other proteinaceous and non-proteinaceous drugs. These drugs can be administered simultaneously with the composition comprising the bispecific single chain antibody molecule of the present invention as defined in the present invention, or separately, before or after administration of said polypeptide, in useful time defined intervals and doses. The dosing regimen will be determined by the treating physician and clinical factors. As is well known in the medical arts, dosages for any patient depend on many factors, including the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health , and other medications to be administered concomitantly. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous, emulsions or suspensions, including saline and buffered solutions. Parenteral vehicles include Sodium Chloride Solution, Ringer's Dextrose, Dextrose Sodium Chloride, Ringer's Lactate, or Fixed Oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. Furthermore, the composition of the present invention may comprise protein carriers such as, for example, serum albumin or immunoglobulin, preferably of human origin. It is anticipated that the composition of the present invention may comprise, in addition to the bispecific single chain antibody molecule of the present invention defined in the present invention, even more biologically active agents, depending on the intended use of the composition. Such agents can be drugs that act on the gastrointestinal system, drugs that act as cytostatic substances, drugs, drugs that prevent inhibitors of hyperuricemia immunoreactions (for example, corticosteroids), drugs that modulate the inflammatory response, drugs that act on the circulatory system and/ or agents, such as cytokines known in the art.
[000152] The biological activity of the pharmaceutical composition as defined in the present invention can be determined, for example, by cytotoxicity assays, as described in the examples below, in WO 99/54440 or by Schlereth et al. (Cancer Immunol Immunother 20 (2005), 1 to 12). "Efficacy" or "in vivo efficacy" as used in the present invention refers to response to treatment with the pharmaceutical composition of the present invention, for example, using standard NCI response criteria. The success or in vivo efficacy of therapy using the pharmaceutical composition of the present invention refers to the efficacy of the composition for its intended purpose, i.e., the ability of the composition to cause the desired effect, i.e., the depletion of pathological cells , for example, tumor cells. In vivo efficacy can be monitored using standard methods established for the respective disease entities, including but not limited to white blood cell count, differentials, fluorescence activated cell sorting, bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other standard established methods can be used. In addition, computer-aided tomography, X-ray, nuclear magnetic resonance tomography (eg for assessment of National Cancer Institute response baseline criteria [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group J Clin Oncol 1999 Apr;17(4):1244]), tomography by emission tomography, white blood cell count, differentials, classification of activated cells through fluorescence, bone marrow aspiration, lymph node biopsies/histologies, and various cancer-specific clinical chemistry parameters (eg, lactate dehydrogenase) and other established standard methods can be used.
[000153] Another major challenge for the development of drugs such as the pharmaceutical composition of the present invention is the predictable modulation of pharmacokinetic properties. To this end, the pharmacokinetic profile of the drug candidate, that is, a profile of pharmacokinetic parameters that has an effect on the ability of a particular drug to treat a given condition, is established. Drug pharmacokinetic parameters that influence a drug's ability to treat a particular disease entity include, but are not limited to: half-life, volume of distribution, first-pass hepatic metabolism, and degree of binding to serum blood. The effectiveness of a particular drug agent can be influenced by each of the aforementioned parameters.
[000154] The term "Half-life" means the time when 50% of an administered drug is eliminated through biological processes, eg metabolism, excretion, etc.
[000155] By "first-pass liver metabolism" is meant the propensity of a drug to be metabolized on first-time contact with the liver, that is, during its first pass through the liver.
[000156] The term "volume of distribution" means the degree of retention of a drug throughout the various compartments of the body, such as intracellular and extracellular spaces, tissues and organs, etc., and the distribution of the drug within these compartments.
[000157] The term "blood serum binding degree" means the propensity of a drug to interact with and bind to serum proteins, such as albumin, leading to a reduction or loss of the drug's biological activity.
[000158] Additional pharmacokinetic parameters include bioavailability, time delay (TLAG), Tmax, absorption rates, plus onset and/or Cmax for a given amount of drug administered.
[000159] The term "bioavailability" means the amount of a drug in the blood compartment.
[000160] The term "delay time" means the time interval between the administration of the drug and its detection and measurement capability in blood or plasma.
[000161] The term "Tmax" is the time after which the maximum blood concentration of the drug is reached, and "Cmax" is the maximum blood concentration achieved with a given drug. The time to reach a blood or tissue concentration of the drug that is necessary for its biological effect is influenced by all parameters. The pharmacokinetic parameters of bispecific single-chain antibodies exhibiting cross-species specificity, which can be determined in preclinical animal trials in non-chimpanzee primates as outlined above, are also presented, for example in the publication by Schlereth et al. (Cancer Immunol Immunother 20 (2005), 1 to 12).
[000162] The term "toxicity" as used in the present invention refers to the toxic effects of a drug manifested in adverse events or serious adverse events. These adverse events may refer to a lack of tolerability of the drug in general and/or a lack of local tolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.
[000163] The term "safety", "in vivo safety" or "tolerance" as used in the present invention defines the administration of a drug without inducing serious adverse effects directly after administration (local tolerance) and during a long period of application of the drug. "Safety", "in vivo safety" or "tolerance" can be assessed for example at regular intervals during treatment and the follow-up period. Measures include clinical assessment, eg organ manifestations, and screening for laboratory changes. Clinical evaluation can be performed and deviating to normal results recorded/coded according to NCI-CTC and/or MedDRA standards. Organ manifestations may include criteria such as allergy/blood/bone marrow immunology, cardiac arrhythmia, clotting and the like, as set out, for example, in the Common Terminology Criteria for Adverse Events v3.0 (CTCAE). Laboratory parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profile and urine analysis, and examinations of other bodily fluids, such as serum, plasma, spinal fluid or lymphoid liquor, and the like. Safety can therefore be assessed, for example, by physical examination, imaging techniques (ie, ultrasound, X-rays, computed tomography, magnetic resonance imaging (MRI), other measures with technical devices (electrocardiogram, for example), vital signs , measuring laboratory parameters and recording adverse events, for example, adverse events in non-chimpanzee primates in the uses and methods according to the present invention can be examined by means of histopathological and/or histochemical methods.
[000164] The term "effective and non-toxic dose" as used in the present invention refers to a tolerable dose of the single-chain bispecific antibody, as defined in the present invention, which is sufficiently high to cause depletion of pathological cells , tumor elimination, tumor shrinkage or disease stabilization without, or essentially without major toxic effects. Such effective and non-toxic doses can be determined, for example, in dose escalation studies described in the art and should be less than the dose inducing serious adverse events (dose limiting toxicity, DLT).
[000165] The above terms are also referred to, for example, in the preclinical safety assessment of pharmaceuticals derived from biotechnology S6; Tripartite Harmonized ICH Guidelines; Meeting of the ICH Steering Committee on 16 July 1997.
[000166] Furthermore, the present invention relates to a pharmaceutical composition comprising a single-chain bispecific antibody molecule of the present invention, or produced according to the process according to the present invention for the prevention, treatment or amelioration of cancer . Preferably said cancer is a solid tumor, preferably a carcinoma or cancer of the prostate. Preferably, said pharmaceutical composition further comprises suitable formulations of vehicles, stabilizers and/or excipients.
[000167] A further aspect of the present invention relates to a use of a bispecific single chain antibody molecule/polypeptide as defined in the present invention above, or produced according to a process as defined in the present invention above, for the preparing a pharmaceutical composition for preventing, treating or ameliorating a disease. Preferably, said disease is cancer. More preferably, said cancer is a solid tumor, preferably a carcinoma or prostate cancer.
[000168] In another preferred embodiment of the use of the bispecific single chain antibody molecule of the present invention said pharmaceutical composition is suitable to be administered in combination with an additional drug, i.e. as part of a cotherapy. In said cotherapy, an active agent may optionally be included in the same pharmaceutical composition as the bispecific single chain antibody molecule of the present invention, or may be included in a separate pharmaceutical composition. In the latter case, said separate pharmaceutical composition is suitable for administration before, simultaneously or after administration of said pharmaceutical composition comprising the bispecific single chain antibody molecule of the present invention. An additional drug or pharmaceutical composition can be a non-protein compound or a protein compound. In the case where the additional drug is a proteinaceous compound, it is advantageous that the proteinaceous compound is capable of providing an activation signal to the immune effector cells.
[000169] Preferably, said proteinaceous compound or non-proteinaceous compound may be administered simultaneously or non-concurrently with the bispecific single chain antibody molecule of the present invention, a nucleic acid molecule as defined hereinbefore, a vector as defined as defined above, or a host as defined as defined above herein.
[000170] Another aspect of the present invention relates to a method for the prevention, treatment or amelioration of a disease in an individual in need thereof, said method comprising the step of administering an effective amount of a pharmaceutical composition of the present invention. Preferably, said disease is cancer. Preferably said cancer is a solid tumor, preferably a carcinoma or cancer of the prostate.
[000171] In another preferred embodiment of the method of the present invention, said pharmaceutical composition is suitable to be administered in combination with an additional drug, that is, as part of a cotherapy. In said cotherapy, an active agent may optionally be included in the same pharmaceutical composition as the bispecific single chain antibody molecule of the present invention, or it may be included in a separate pharmaceutical composition. In the latter case, said separate pharmaceutical composition is suitable for administration before, simultaneously or after administration of said pharmaceutical composition comprising the bispecific single chain antibody molecule of the present invention. An additional drug or pharmaceutical composition can be a non-protein compound or a protein compound. In the case where the additional drug is a proteinaceous compound, it is advantageous that the proteinaceous compound is capable of providing an activation signal to the immune effector cells.
[000172] Preferably, said proteinaceous compound or non-proteinaceous compound may be administered simultaneously or non-concurrently with the bispecific single chain antibody molecule of the present invention, a nucleic acid molecule as defined hereinbefore, a vector as defined as defined above, or a host as defined as defined above herein.
[000173] It is preferable that the above-described method of the present invention that said subject be a human being.
[000174] In a further aspect, the present invention relates to a kit comprising a bispecific single chain antibody molecule of the present invention, a nucleic acid molecule of the present invention, a vector of the present invention, or a host of the present invention.
[000175] These and other embodiments are described and encompassed in the description and examples of the present invention. Recombinant techniques and methods are described in immunology for example in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3rd edition, 2001; Lefkovits; Immunology Methods Manual; The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemies; Protein-protein Interactions: A Molecular Cloning Manual; Cold Spring Laboratory Press, 2002. Additional literature on any of the antibodies, methods, uses and compounds to be used in accordance with the present invention can be retrieved from public libraries and databases, using for example electronic devices. For example, the public database "Medline", available on the Internet, can be used, for example, under http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Other databases and addresses, such as http://www.ncbi.nlm.nih.gov/, or included on the EMBL-services homepage under http://www.embl.de/services/index.html are known of the skilled artisan and may also be obtained using, for example, http://www. google.com.
[000176] The figures show: Figure 1
[000177] FACS binding analysis of the so-called interspecies bispecific single-chain constructs specific for CHO cells transfected with human PSMA, human T cell line HPB-ALL CD3+, CHO cells transfected with a monkey PSMA and T cell line 4119 LnPx. Marking for FACS is performed as described in Example 2.1. The thick line represents cells incubated with cell culture supernatant which are then incubated with anti-His antibody and PE tagged detection antibody. The thin histogram line reflects the negative control: cells incubated only with anti-His antibody and detection antibody. Figure 2
[000178] Cytotoxic activity induced by designating specific bispecific single-chain constructs redirected to indicated target cell lines. Stimulated human CD4/CD56 T cells are used as effector cells, CHO cells transfected with human PSMA as target cells. The test is carried out as described in Example 2.2.
[000179] The present invention is further described by means of the following non-limiting illustrative examples which provide a better understanding of the present invention and its many advantages. EXAMPLES 1. Generation and Characterization of PSMA and Species-Specific Crossed CD3 Single Chain Bispecific Antibody Molecules 1.1 Cloning and Expression of Human PSMA Antigen in CHO Cells
[000180] The human PSMA antigen sequence ('AY101595', Prostate-specific membrane antigen mRNA Homo sapiens, complete cds, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/entrez) was used to obtain a synthetic molecule through gene synthesis according to standard protocols. The gene synthesis fragment was also designed to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and end of the DNA. XbaI restriction sites introduced at the 5' end and SalI at the 3' end were used during the cloning step for expression of plasmid pEFDHFR designated as described in Mack et al. (Mack M et al, Proc Natl Acad Sci USA 1995, 92:7021-5 and Raum et al Cancer Immunol Immunother (2001) 50 (3)). After checking the plasmid sequence it was used to transfect CHO/dhfr- cells as follows. A verified plasmid sequence was used to transfect CHO/dhfr- cells (ATCC No. CRL 9096; cultured in RPMI 1640 with stabilized glutamine obtained from Biochrom AG of Berlin, Germany, supplemented with 10% FCS, 1% penicillin/streptomycin all obtained from Biochrom AG of Berlin, Germany, from nucleosides of a stock solution of cell culture grade reagents obtained from Chemie GmbH Sigma-Aldrich, Taufkirchen, Germany, to a final concentration of 10 ug/ml of adenosine, 10 ug/ml deoxyadenosine and 10 ug/ml thymidine, in an incubator at 37°C, 95% humidity and 7% CO2). Transfection was performed using PolyFect transfection reagent (Qiagen GmbH, Hilden, Germany) and 5 ug plasmid DNA according to the manufacturer's protocol. After a 24 hour culture, cells were washed once with PBS and again cultured in cell culture medium mentioned above, except that the medium used was not supplemented with dialyzed FCS and nucleosides (obtained from Biochrom AG Berlin , Germany). Thus, the cell culture medium did not contain nucleosides and thus the selection was applied to the transfected cells. Approximately 14 days after transfection the growth of resistant cells was observed. After an additional 7 to 14 days the transfectants were tested positive for construct expression by FACS. Expression of eukaryotic protein in DHFR-deficient CHO cells is performed as described by Kaufmann RJ (1990) Methods Enzymol. 185, 537 to 566. Chimera gene amplification is induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to MTX at 20 nM 1.2 Cloning and expression of monkey PSMA antigen in CHO cells
[000181] The cDNA sequence of macaque PSMA (cynomolgus) was obtained by a set of five PCRs in cDNA from the macaque monkey prostate prepared according to standard protocols. The following reaction conditions were used: 1 cycle at 94°C for 2 minutes, followed by 40 cycles with 94°C for 1 minute, 52°C for 1 minute and 72°C for 1.5 minutes, followed by one cycle terminal 72°C for 3 minutes, and the following primers were used: • forward primer: 5'-catgtggcccaggttcgagg-3' (SEQ ID NO: 213) • reverse primer: 5'-gacataccacacaaattcaatacgg-3' (SEQ ID NO: 214) • forward primer: 5'-gctctgctcgcgccgagatgtgg-3' (SEQ ID NO: 215) • reverse primer: 5'-acgctggacaccacctccagg-3' (SEQ ID NO: 216) • forward primer: 5'-ggttctatgagtgggcagagg-3 ' (SEQ ID NO: 217) • reverse primer: 5'-attgttgtggctgcttggagc-3' (SEQ ID NO: 218) • forward primer: 5'-gggtgaagtcctatccagatgg-3' (SEQ ID NO: 219) • reverse primer: 5' -gtgctctgcctgaagcaattcc-3' (SEQ ID NO: 220) • forward primer: 5'-ctcggcttcctcttcgggtgg-3' (SEQ ID NO: 221) • reverse primer: 5'-gcatattcatttgctgggtaacctgg-3' (SEQ ID NO: 222)
[000182] These PCRs generated five overlapping fragments, which were isolated and sequenced according to conventional protocols, using PCR primers and, in this way, provided to a portion of the cDNA sequence encoding the macaque codon PSMA 3 to the last codon of the mature protein. To generate a construct for the expression of monkey PSMA a cDNA fragment was obtained by gene synthesis according to standard protocols (the cDNA and amino acid sequence of the construct is listed in SEQ ID 223 and 224). In this construct, the monkey PSMA coding sequence from amino acid 3 to the last amino acid of the mature PSMA protein followed by a stop codon was fused in frame to the coding sequence of the first two amino acids of the human PSMA protein. The gene synthesis fragment was also designed to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and end of the cDNA-containing fragment. The XbaI at the 5' end and the SalI at the 3' end were used in the following cloning procedures. The gene synthesis fragment was cloned via XbaI and SalI from a plasmid designated the following standard pEF-DHFR protocols. The above-mentioned procedures were performed according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). A clone with the verified nucleotide sequence sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Expression of eukaryotic protein in DHFR-deficient CHO cells was performed as described by Kaufmann RJ (1990) Methods Enzymol. 185, 537 to 566. Amplification of the chimera genus was induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to 20 nM MTX. Example 2 2.1 Flow of Analysis by PSMA Binding Cytometry and Cross Species-Specific Bispecific CD3 Antibodies
[000183] In order to test the functionality of the cross-species specific bispecific antibody constructs with respect to the binding capacity to human and monkey PSMA and human and monkey CD3 in a FACS analysis was performed. For this purpose, CHO cells transfected with human PSMA and human CD3 positive from HPB-ALL leukemia cell line T cells (DSMZ, Braunschweig, ACC483) were used to verify binding to human antigens. Monkey antigen-binding reactivity was tested using the generated macaque transfectant PSMA and a 4119LnPx macaque T cell line (kindly provided by Prof Fickenscher, Institute of Hygiene, Virology, Erlangen-Nuernberg, published in Knappe A, et al, and H Fickenscher., Blood 2000, 95, 3256 to 61).
[000184] Flow cytometry analysis was performed as follows:
[000185] 200,000 cells of the respective cell lines were incubated for 30 min on ice with 50 µl of the purified protein from the specific cross-species bispecific antibody constructs (2 µg/ml) or cell culture supernatants from transfected cells expressing the constructs of specific cross-species bispecific antibodies. Cells were washed twice in PBS with 2% FCS and construct binding was detected with a murine anti-His antibody (Penta Sua antibody; Qiagen; diluted 1:20 in 50 ul PBS with 2% FCS). After washing, bound anti-His antibodies were detected with a specific Fc-gamma antibody (Dianova) conjugated to phycoerythrin, diluted 1:100 in PBS with 2% FCS. Supernatant from untransfected CHO cells was used as a negative control for binding to the T cell lines. The single strand construct with irrelevant target specificity was used as a negative control for binding to the PSMA transfected CHO cells.
[000186] Flow cytometry was performed in a FACS-Calibur apparatus, CellQuest software was used for data acquisition and analysis (Becton Dickinson Biosciences, Heidelberg). FACS staining and measurement of fluorescence intensity were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).
[000187] The bispecific binding of single-chain molecules, which are cross-species specific for PSMA and cross-species specific for human and monkey CD3 was clearly detectable, as shown in figure 1. In FACS analysis showed all binding constructs for CD3 and PSMA compared to the respective negative controls. Bispecific antibodies to cross-species human and monkey CD3 and human and macaque PSMA antigens have been demonstrated. 2.2 Bioactivity of PSMA and cross-species specific bispecific CD3 single chain antibodies
[000188] The bioactivity of the generated bispecific single chain antibodies was analyzed by chromium 51 (51Cr) release in in vitro cytotoxicity assays using human PSMA positive cells of the CHO cell lineage. PBMC depleted human CD4/CD56 stimulated effector cells are used.
[000189] The generation of stimulated CD4/CD56 depleted PBMC was performed as follows:
[000190] Coating of a Petri dish (145 mm in diameter, Greiner bio-one GmbH, Frickenhausen) was performed with a commercially available anti-CD3 specific antibody (eg OKT3, Othoclone) at a final concentration of 1 ug/ mL for 1 hour at 37°C. Unbound protein was removed by a PBS wash step. PBMC were isolated from peripheral blood vials (30 to 50 mL of human blood) by Ficoll gradient centrifugation according to standard protocols. 3-5 x 107 PBMC were added to the Petri dish pre-coated in 120 ml of RPMI 1640 with stabilized glutamine/10% FCS/ 20 U/ml IL-2 (Proleukin, Chiron) and stimulated for 2 days. On the third day, cells were harvested and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and cells were cultured again for one day in the same cell culture medium as described above. By depleting CD4 + T cells and CD56 + NK cells according to conventional protocols CD8 + cytotoxic T lymphocytes (CTL) were enriched.
[000191] The target cells were washed twice with PBS and labeled with 51Cr 11.1 MBq, in a final volume of 100 µl of RPMI medium with 50% FCS for 45 minutes at 37°C. Subsequently, the labeled target cells were washed 3 times with 5 ml of RPMI medium and then used in the cytotoxicity assay. The assay was performed in a 96-well plate in a total volume of 250 µL RPMI supplemented (as above) with an E:T ratio of 10:1. 1 µg/ml of the bispecific trans-species specific single chain antibody molecules and 20 triple dilutions of it were applied. Assay time was 18 hours and cytotoxicity was measured as relative values of chromium released in the supernatant related to the difference in maximum lysis (addition of Triton-X) and spontaneous lysis (no effector cells). All measurements were performed in quadruplicate. Measurement of chromium activity in supernatants was performed with a Wizard 3'' gamma counter (Perkin Elmer Life Sciences GmbH, Cologne, Germany). The analysis of experimental data was performed with Prism 5, for Windows (version 5.01, GraphPad Software Inc., San Diego, California, USA). Sigmoidal dose-response curves typically have R2 values > 0.90, as determined by the software. EC50 values calculated by the analysis program were used to compare bioactivity.
[000192] As shown in figure 2, all bispecific single chain antibody constructs generated between specific species demonstrate cytotoxic activity against stimulated human PSMA positive target cells induced by depletion of human CD4/CD56 PBMC. Example 3: Binding analysis of scFvs against various cell lines 3.1. Expression of Single Chain Antibody Constructs in E. coli
[000193] The scFv molecules EpCAM 4-7 (WO 99/25818), PM74-G3, PM52-H3,-C3 PM52, PM75 and PM91-A10-B6 are expressed by using plasmid pComb3H5BFlag/His in which the expression constructs (eg scFv ) include the tag (DYKDDDDK) and the His6 tag. Plasmid DNA from each scFv molecule is transformed into 100 μl heat shock competent E. coli TG1 cells and plated on LB-carbenicillin agar. E. coli transformed with a pComb3H5BFlag/His containing scFv VL- and soluble VH-segment produce in sufficient amounts after induction with 1 mM IPTG. Due to an adequate signal sequence, the scFv-chain is exported to the periplasm, where it folds into a functional conformation.
[000194] A single colony of E. Coli TG1 bacteria from the transformation plates is chosen for small-scale periplasmic preparations and grown in SB medium (eg 10 mL) supplemented with 20 mM MgCl2 and 50μg/ μl carbenicillin (and re-dissolved in PBS (eg 1 μl) after harvesting. For four cycles of freezing at -70 °C and thawing at 37 °C, the outer membrane of bacteria is destroyed by heat shock and proteins The periplasmic cells, including the soluble scFvs are released into the supernatant After removing the intact cells and the cell debris by means of centrifugation, the supernatant containing the anti-PSMA scFv is collected and used for the determination of binding of the different cell lines. 3.2. Flow cytometry binding analysis of single-chain antibody constructs to various cell lines
[000195] Periplasmic preparations of E. coli clones producing scFv molecules EpCAM 4-7, PM74-G3, PM52-H3,-C3 PM52, PM75 and PM91-A10-B6 are used to examine specific binding to Human PSMA or human EpCAM from the transfected cell lines. As a negative control, untransfected CHO cells are used.
[000196] For flow cytometry 2.5 x105 cells are incubated with 50 μl of scFv periplasmic preparation. Binding of scFv to cells is detected with an anti-His antibody (Penta-His Antibody, free BSA, Qiagen GmbH, Hilden, FRG) at 2 µg/ml, in 50 µl PBS with 2% FCS. As a second-step reagent of F(ab')2 affinity purified conjugated R-Phycoerythrin, the goat anti-mouse IgG fragment (Fc-gamma specific fragment), diluted 1:100 in 50 μl PBS with FCS 2% (Dianova, Hamburg, FRG) is used. Samples are measured on a FACSscan (BD Biosciences, Heidelberg, FRG).
[000197] Flow cytometry is performed in a FACS-Calibur apparatus, CellQuest software is used for data acquisition and analysis (Becton Dickinson Biosciences, Heidelberg). FACS staining and measurement of fluorescence intensity are performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).
[000198] scFv EpCAM 4-7 showed strong binding to human EpCAM from the transfected CHO cell line, but no significant binding to PSMA transfected or non-transfected human cells. In contrast scFvs PM74-G3, PM52-H3,-C3 PM52, PM75 and PM91-A10-B6 showed strong binding to human PSMA cells transfected CHO cells, but not to human EpCAM transfected or CHO transfected. (The periplasmic cell binding results of the extracts of the respective scFvs in the different transfected cell lines are shown in Table 1). Table 1: FACS analysis results: '+' indicates binding signal, "-" indicates no significant binding signal











































































































TABLE LEGEND: • human = human • VL of = VL of .... • VH of = VH of • squirrel monkey = Squirrel monkey • Swine = Swine • Human CD3 epsilon chain • 19 amino acid immunoglobulin leader peptide = • Peptide 19 amino acid immunoglobulin leader • murine igG1 heavy chain constant region = • murine igG1 heavy chain constant region • human lambdia light chain constant region • human lambda light chain constant region • Leader = Leader • humanCD3 (N-terminus) • Human CD3 (N-terminal) • forward primer = Advanced primer • reverse primer = Reverse primer • Cynomolgus = Cynomolgus

权利要求:
Claims (10)
[0001]
1. Single-chain bispecific antibody molecule characterized by the fact that it comprises a first binding domain that is an antigen interaction site capable of binding to an epitope of the human CD3ε (epsilon) chain and Callithrix jacchus, Saguinis oedipus or Saimiri sciureus, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs: 2, 4, 6 or 8, and comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (QDGNE), and wherein the first binding domain comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from: (a) CDR-L1 as described in SEQ ID NO: 27, CDR -L2 as set forth in SEQ ID NO:28 and CDR-L3 as set forth in SEQ ID NO:29; (b) CDR-L1 as set out in SEQ ID NO: 117, CDR-L2 as set out in SEQ ID NO: 118 and CDR-L3 as set out in SEQ ID NO: 119; and (c) CDR-L1 as set forth in SEQ ID NO: 153, CDR-L2 as set forth in SEQ ID NO: 154 and CDR-L3 as set forth in SEQ ID NO: 155, and wherein the first domain of binding comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from: (a) CDR-H1 as described in SEQ ID NO: 12, CDR-H2 as described in SEQ ID NO: 13 and CDR -H3 as described in SEQ ID NO: 14; (b) CDR-H1 as set out in SEQ ID NO: 30, CDR-H2 as set out in SEQ ID NO: 31 and CDR-H3 as set out in SEQ ID NO: 32; (c) CDR-H1 as set out in SEQ ID NO: 48, CDR-H2 as set out in SEQ ID NO: 49 and CDR-H3 as set out in SEQ ID NO: 50; (d) CDR-H1 as set out in SEQ ID NO: 66, CDR-H2 as set out in SEQ ID NO: 67 and CDR-H3 as set out in SEQ ID NO: 68; (e) CDR-H1 as set out in SEQ ID NO: 84, CDR-H2 as set out in SEQ ID NO: 85 and CDR-H3 as set out in SEQ ID NO: 86; (f) CDR-H1 as set out in SEQ ID NO: 102, CDR-H2 as set out in SEQ ID NO: 103 and CDR-H3 as set out in SEQ ID NO: 104; (g) CDR-H1 as set out in SEQ ID NO: 120, CDR-H2 as set out in SEQ ID NO: 121 and CDR-H3 as set out in SEQ ID NO: 122; (h) CDR-H1 as set out in SEQ ID NO: 138, CDR-H2 as set out in SEQ ID NO: 139 and CDR-H3 as set out in SEQ ID NO: 140; (i) CDR-H1 as set out in SEQ ID NO: 156, CDR-H2 as set out in SEQ ID NO: 157 and CDR-H3 as set out in SEQ ID NO: 158; and (j) CDR-H1 as set out in SEQ ID NO: 174, CDR-H2 as set out in SEQ ID NO: 175 and CDR-H3 as set out in SEQ ID NO: 176; and a second binding domain capable of binding prostate specific membrane antigen (PSMA), wherein the second binding domain comprises a group of the following sequences as CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and L3 CDR in the second binding domain selected from: a) H1-3 CDR of SEQ ID NO: 254-256 and L1-3 CDR of SEQ ID NO: 259-261; b) H1-3 CDR of SEQ ID NO: 268-270 and L1-3 CDR of SEQ ID NO: 273-275; c) H1-3 CDR of SEQ ID NO: 618-620 and L1-3 CDR of SEQ ID NO: 623-625; d) H1-3 CDR of SEQ ID NO: 282-284 and L1-3 CDR of SEQ ID NO: 287-289; e) H1-3 CDR of SEQ ID NO: 296-298 and L1-3 CDR of SEQ ID NO: 301-303; f) H1-3 CDR of SEQ ID NO: 310-312 and L1-3 CDR of SEQ ID NO: 315-317; g) H1-3 CDR of SEQ ID NO: 324-326 and L1-3 CDR of SEQ ID NO: 329-331; h) H1-3 CDR of SEQ ID NO: 338-340 and L1-3 CDR of SEQ ID NO: 343-345; i) H1-3 CDR of SEQ ID NO: 352-354 and L1-3 CDR of SEQ ID NO: 357-359; and j) H1-3 CDR of SEQ ID NO: 366-368 and L1-3 CDR of SEQ ID NO: 371-373.
[0002]
2. Single-chain bispecific antibody molecule according to claim 1, characterized in that the first binding domain capable of binding an epitope of the human and non-chimpanzee primate CD3ε chain comprises a VL region selected from from the group consisting of a VL region as described in SEQ ID NO: 35, 39, 125, 129, 161 or 165.
[0003]
3. Single-chain bispecific antibody molecule according to claim 1 or 2, characterized in that the first binding domain capable of binding to an epitope of the human and non-chimpanzee primate CD3ε chain comprises a VH region selected from the group consisting of a VH region as described in SEQ ID NO: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181.
[0004]
4. Bispecific single chain antibody molecule according to any one of claims 1 to 3, characterized in that the first binding domain capable of binding to an epitope of the human and non-chimpanzee primate CD3ε chain comprises a region of VL and a VH region selected from the group consisting of: (a) a VL region as set forth in SEQ ID NO: 17 or 21 and a VH region as set forth in SEQ ID NO: 15 or 19; (b) a VL region as set out in SEQ ID NO: 35 or 39 and a VH region as set out in SEQ ID NO: 33 or 37; (c) a VL region as set out in SEQ ID NO: 53 or 57 and a VH region as set out in SEQ ID NO: 51 or 55; (d) a VL region as set out in SEQ ID NO: 71 or 75 and a VH region as set out in SEQ ID NO: 69 or 73; (e) a VL region as set out in SEQ ID NO: 89 or 93 and a VH region as set out in SEQ ID NO: 87 or 91; (f) a VL region as set out in SEQ ID NO: 107 or 111 and a VH region as set out in SEQ ID NO: 105 or 109; (g) a VL region as set out in SEQ ID NO: 125 or 129 and a VH region as set out in SEQ ID NO: 123 or 127; (h) a VL region as set forth in SEQ ID NO: 143 or 147 and a VH region as set forth in SEQ ID NO: 141 or 145; (i) a VL region as set out in SEQ ID NO: 161 or 165 and a VH region as set out in SEQ ID NO: 159 or 163; and (j) a VL region as set forth in SEQ ID NO: 179 or 183 and a VH region as set forth in SEQ ID NO: 177 or 181.
[0005]
5. Single-chain bispecific antibody molecule according to claim 4, characterized in that the first binding domain capable of binding an epitope of the human and non-chimpanzee primate CD3ε chain comprises an amino acid sequence selected from from the group consisting of SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187.
[0006]
6. Bispecific single chain antibody molecule according to claim 1, characterized in that it comprises a sequence selected from: (A) an amino acid sequence as described in any one of SEQ ID NOs: 237, 251 , 265, 279, 629, 293, 307, 321, 335, 349, 363, 377, 391, 405, 419, 433, 447, 461, 475, 489, 503, 517, 531, 545, 559, 573, 587 , 601 or 615; and (B) an amino acid sequence encoded by a nucleic acid sequence as described in any one of SEQ ID NOs: 238, 252, 266, 280, 630, 294, 308, 322, 336, 350, 364, 378, 392, 406, 420, 434, 448, 462, 476, 490, 504, 518, 532, 546, 560, 574, 588, 602 or 616.
[0007]
7. Single-chain bispecific antibody molecule according to any one of claims 1 to 6, characterized in that it contains a tag, preferably a C-terminal His-tag.
[0008]
8. Process for the production of a bispecific single-chain antibody molecule, as defined in any one of claims 1 to 7, characterized in that it comprises the cultivation of a host cell transformed or transfected with a vector comprising an acid sequence nucleic encoding the bispecific single chain antibody molecule as defined in any one of claims 1 to 7 under conditions which permit expression of the bispecific single chain antibody molecule, and recovery of the polypeptide produced from the culture.
[0009]
9. Pharmaceutical composition, characterized in that it comprises a single-chain bispecific antibody molecule, as defined in any one of claims 1 to 7, or produced according to the process as defined in claim 8.
[0010]
10. Use of a single-chain bispecific antibody molecule as defined in any one of claims 1 to 7, or produced according to the process as defined in claim 8, characterized in that it is in the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of solid tumor cancer, preferably a prostate cancer or carcinoma.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-11-19| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-03-03| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-11-03| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-03-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-25| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/04/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US32005210P| true| 2010-04-01|2010-04-01|

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US61/320,052|2010-04-01|

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PCT/EP2011/055104|WO2011121110A1|2010-04-01|2011-04-01|CROSS-SPECIES-SPECIFIC PSMAxCD3 BISPECIFIC SINGLE CHAIN ANTIBODY|

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