RU2535992C2 - Bispecific binding agents with interspecific specificity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a polypeptide containing two binding fragments presented by antibodies; the first of them binds to CD3e(epsilon) chain epitope of a human or a primate, other than a chimpanzee, particularly Callithrix jacchus, Saguinus oedipus and Saimiri sciureus; the second one - to EGFR, Her2/neu or IgE of a human or a primate, other than a chimpanzee, with the above CD3e epitope containing the amino acid sequence Gln-Asp-Gly-Asn-Glu. There are also disclosed a coding sequence of the nucleic acid, a vector, a host cell and a method for preparing the above peptide, as well as a pharmaceutical composition and using the polypeptide in preventing, treating or relieving a proliferative disease, a malignant disease or an immunological disorder.

EFFECT: invention provides the clinical improvement of T-cell redistribution and the enhanced safety profile.

17 cl, 8 tbl, 26 dwg, 26 ex

Description

The present invention relates to a polypeptide containing a first human binding domain capable of binding to a CD3 epitope (from the English cluster of differentiation - human differentiation cluster) (epsilon) of a human and a non-chimpanzee primate, and a second binding domain capable of binding to EGFR (receptor epidermal growth factor), Her2 / neu (a member of the epidermal growth factor receptor family) or IgE (immunoglobulin E) of a human and / or non-chimpanzee primate, as well as a method for producing said polypeptide. In addition, the invention relates to nucleic acids encoding a given polypeptide, to vectors containing these nucleic acids, and to host cells containing this vector. In another aspect, the invention provides a pharmaceutical composition comprising said polypeptide, and provides medical uses for the polypeptide.

T-cell recognition is mediated by clonotypically distributed alpha-beta and gamma-delta T-cell receptors (TcRs) that interact with peptide-loaded molecules (complex) peptide-MHC (major histocompatibility complex) (pMHC) (Davis & Bjorkman, Nature 334 (1988), 395-402). Antigen-specific TcR chains do not contain signaling domains, but instead bind to the conserved CD3 multi-subunit signal transmission complex (Call, Cell 111 (2002), 967-979, Alarcon, Immunol. Rev. 191 (2003), 38-46, Malissen , Immunol. Rev. 191 (2003), 7-27). The mechanism by which TcR ligation is directly linked to the signal transducer remains a fundamental issue in T-cell biology (Alarcon, supra; Davis, Ceil 110 (2002), 285-287). It seems obvious that contacting with the coreceptor, TcR oligomerization, and the organization of higher-order TcR-pMHC complexes at the immunological synapse are involved in stable T-cell responses (Davis & van der Merwe, Curr. Biol. 11 (2001), R289-R291, Davis , Nat. Immunol. 4 (2003), 217-224). However, the earliest signal transduction via TcR occurs in the absence of these events and may involve a ligand-induced conformational change in the CP3 epsilon (Alarcon, supra, Davis (2002), supra, Gil, J. Biol. Chem. 276 (2001), 11174 11179, Gil, Cell, 109 (2002), 901-912). The epsilon, gamma, delta and zeta subunits of the signal complex are associated with each other to form CD3 epsilon-gamma heterodimer, CD3 epsilon-delta heterodimer and CD3 zeta-zeta homodimer (Call, above). Various studies have found that the presence of CD3 molecules is important for proper expression of TcR alpha-beta on the cell surface and normal development of T cells (Berkhout, J. Biol. Chem. 263 (1988), 8528-8536, Wang, J. Exp Med. 188 (1998), 1375-1380, Kappes, Curr. Opin. Immunol. 7 (1995), 441-447). Based on the allowed structure of the ectodomain fragments of mouse CD3 epsilon-gamma heterodimer, it was shown that both epsilon-gamma subunits are C2-like (C2-set) Ig domains that interact with each other with the formation of an unusual "side- to-side "(Sun, Cell 105 (2001), 913-923). Although the stalk enriched in cysteine residues appears to play an important role in triggering dimerization of CD3 (Su, supra, Borroto, J. Biol. Chem. 273 (1998), 12807-12816), interaction via the extracellular domains of CD3 Epsilon and CD3 gamma are sufficient to complex these proteins with TcR-beta (Manolios, Eur. J. Immunol. 24 (1994), 84-92, Manolios & Li, Immunol. Cell Biol. 73 (1995), 532 -536). Although this item is still controversial, the prevailing TcR stoichiometry most likely contains one alpha beta TcR, one CD3 epsilon-gamma heterodimer, one CD3 epsilon-delta heterodimer and one CD3 zeta-zeta homodimer (Call, above). The central role of CD3 epsilon-gamma heterodimer of a person in the immune response has become clear in connection with the recent establishment of the crystalline structure of this complex associated with the therapeutic antibody OCT3 (Kjer-Nielsen, PNAS, 101 (2004), 7675-7680).

Many therapeutic strategies modulate T cell immunity by targeting TcR signaling, in particular monoclonal antibodies (mAb) against human CD3, which are widely used in clinics for immunosuppressive regimens. CD3-specific murine mAb OKT3 was the first mAb approved for use in humans (Sgro, Toxicology 105 (1995), 23-29), and it is widely used in the clinic as an immunosuppressive agent for transplantation (Chatenoud, Clin. Transplant. 7 ( 1993), 422-430, Chatenoud, Nat. Rev. Immunol. 3 (2003), 123-132, Kumar, Transplant. Proc. 30 (1998), 1351-1352), type 1 diabetes (Chatenoud (2003), above ) and psoriasis (Utset, J. Rheumatol. 29 (2002), 1907-1913). Moreover, anti-CP3 mAb can induce partial T-cell signaling and clonal tolerance (Smith, J. Exp. Med. 185 (1997), 1413-1422). OCT3 has been described in the literature as a potent T-cell mitogen (Van Wauve, J. Immunol. 124 (1980), 2708-18), and also as a potent T-cell killer (Wong, Transplantation 50 (1990), 683- 9). OCT3 shows a time-dependent change in both of these activities: after early activation of T cells, leading to the release of cytokines, subsequent administration of OKT3 blocks all known T-cell functions. Due to this later blocking of T-cell function for OCT3, such widespread use as an immunosuppressant in treatment regimens has been shown to reduce or even eliminate tissue allograft rejection.

OCT3 reverses tissue allograft rejection most likely by blocking the function of all T cells that play a major role in acute rejection. OCT3 interacts with the CD3 complex and blocks the function of the CD3 complex in the membrane of human T cells, which is associated with the antigen-recognizing structure of T cells (TCR) and is important for signal transduction. Numerous studies have concerned which TCR / CD3 subunit binds to OCT3. However, some facts indicate that OCT3 is specific for the epsilon subunit of the TCR / CD3 complex (Tunnacliffe, Int. Immunol. 1 (1989), 546-50; Kjer-Nielsen, PNAS 101 (2004), 7675-7680). According to other data, it was shown that the binding of OKT3 to the TCR / CD3 complex requires the presence of other subunits of this complex (Salmeron, J. Immunol. 147 (1991), 3047-52).

Other well-known antibodies specific for the CD3 molecule are listed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50. As indicated above, such CD3-specific antibodies are capable of inducing various T-cell responses, such as lymphokine production (Von Wussow, J. Immunol. 127 (1981), 1197; Palacious, J. Immunol. 128 (1982), 337), proliferation (Van Wauve, J. Immunol. 124 (1980), 2708-18) and the induction of T-cell suppressors (Kunicka, in "Limphociting Typing II” 1 (1986), 223). That is, depending on the experimental conditions CD3- a specific monoclonal antibody can either 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, 94-106; Xu (2000), Cell Immunol. 200, 16-26; Kimball (1995), Transpl. Immunol. 3, 212-221).

Although many anti-CD3 antibodies described in the art are 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 TCR environment. Conformational epitopes are characterized by the presence of two or more separate amino acid residues that are spaced in the primary sequence but converge on the surface of the molecule when the polypeptide folds into a native protein / antigen (Sela (1969) Science 166, 1365 and Laver (1990) Cell 61, 553- 6). Conformational epitopes that bind to anti-CD3 epsilon antibodies described in the art can be divided into two groups. In the main group, these epitopes are formed by two CD3 subunits, for example, the CD3 epsilon chain and the CD3 gamma or CD3 delta chain. For example, several studies have found that the most widely used monoclonal antibodies against CD3 epsilon, namely, OCT3, WT31, UCHT1, 7D6 and Leu-4, did not bind to cells transfected with a single CD3 epsilon chain. However, these antibodies stained cells transfected with a double combination of CD3 epsilon plus or CD3 gamma or CD3 delta (Tunnacliffe, supra; Law, Int. Immunol. 14 (2002), 389-400; Salmeron, J. Immunol. 147 ( 1991), 3047-52; Coulie, Eur. J. Immunol. 21 (1991), 1703-9). In the second less numerous group, a conformational epitope is formed in the CD3 epsilon subunit itself. A member of this group is, for example, an APA 1/1 mAb, which is obtained against a denatured CD3 epsilon chain (Risueno, Blood 106 (2005), 601-8). Collectively, most anti-CD3 epsilon antibodies described in the art recognize conformational epitopes located on two or more CD3 subunits. Individual amino acid residues forming the three-dimensional structure of these epitopes can be localized either on the CD3 epsilon subunit itself, or on the CD3 epsilon subunit and other CD3 subunits, such as CD3 gamma or CD3 delta.

Another problem with anti-CD3 antibodies is that anti-CD3 antibodies have been found to be species-specific. Ahth-CD3 monoclonal antibodies, which is usually true for any other monoclonal antibodies, function by highly specific recognition of their target molecules. They recognize only one site, or epitope, on their target CD3 molecule. For example, one of the most widely used and best characterized monoclonal antibodies specific for the CD3 complex is OCT-3. This antibody interacts with chimpanzee CD3, but not with the CD3 homolog of other primates, such as macaques, or with CD3 dogs (Sandusky et al., J. Med. Primatol. 15 (1986), 441-451). The Ahth-CD3 monoclonal antibody UCHT-1 also interacts with CD3 from chimpanzees, but not with CD3 from macaque (own data). On the other hand, there are also examples of monoclonal antibodies that recognize macaque antigens, but not their human counterparts. One example of this group is the FN-18 monoclonal antibody directed to macaque CD3 (Uda et al., J. Med. Primatol. 30 (2001), 141-147). An interesting fact is found that peripheral lymphocytes from approximately 12% of cynomolgus monkeys do not interact with the monoclonal antibody against rhesus monkey CD3 (FN-18) due to polymorphism of the CD3 antigen in macaques. Uda et al. Described the replacement of two amino acids in the CD3 sequence of cynomolgus macaques, resulting in no interaction with FN-18 antibodies, compared to CD3 of other animals that interact with FN-18 antibodies (Uda et al., J Med. Primatol. 32 (2003), 105-10; Uda etal., J. Med. Primatol. 33 (2004), 34-7).

In addition, this selectivity, i.e. the species specificity inherent to anti-CD3 monoclonal antibodies and their fragments is a significant obstacle to their development as therapeutic agents for the treatment of human diseases. In order to obtain permission to sell, any new candidate drug must undergo rigorous testing. This testing can be divided into preclinical and clinical phases: while the latter, further subdivided into the well-known clinical phases I, II and III, is carried out on human patients, the previous one is performed on animals. The purpose of preclinical testing is to prove that the candidate drug has the desired activity and, most importantly, is safe. Only after establishing the safety and possible effectiveness of the candidate drug in preclinical testing on animals, this candidate drug will receive the approval of the relevant regulatory authority for conducting clinical testing in humans. Candidate medicines can be tested for animal safety in the following three ways: (1) in a relevant form, i.e. a form in which candidate drugs can recognize orthologic antigens, (2) on a transgenic animal containing human antigens, and (3) by using a candidate drug simulator that can bind to orthologic antigens present in the animal. Limitations on transgenic animals are that this technology is usually limited to rodents. There are significant differences in physiology between rodents and humans, and safety results cannot easily be extrapolated to humans. Limitations due to the simulator of the candidate drug are in a different composition of substances compared to the real drug candidate, and often used animals are rodents with the restriction discussed above. Thus, preclinical data obtained in rodents have limited prognostic ability in relation to the drug candidate. The approach with respect to the selection of safety testing is to use a relevant species, preferably a lower primacy. Currently, a limitation regarding CD3 binding molecules suitable for therapeutic intervention in humans and described in the art is that higher primates, in particular chimpanzees, are relevant species. Chimpanzees are considered an endangered species, and due to their humanoid nature, the use of such animals to test drug safety is prohibited in Europe and very limited elsewhere.

The present invention relates to a polypeptide comprising a first binding domain, preferably a human, capable of binding to an epitope of a human CD3ε (epsilon) chain and a non-chimpanzee primate, and a second binding domain capable of binding to EGFR, Her2 / neu or human IgE and / or a non-chimpanzee primate, said epitope being part of an amino acid sequence in the group consisting of SEQ ID NO: 2, 4, 6 or 8. The sequences shown in SEQ ID NO: 2, 4, 6 and 8 , and fragments thereof are context independent epitopes of CD3.

An advantage of the present invention is to provide a polypeptide comprising a binding domain, preferably a human, showing interspecific specificity for the human CD3ε (epsilon) chain and non-chimpanzee primate, which can be used as a preclinical assessment of safety, activity and / or pharmacokinetic profile of these binding domains, preferably of humans, on primates, and, in identical form, as a medicine in humans. In preclinical studies in animals, as well as in clinical studies in humans, the same molecule can be used. This leads to very comparable results and a significant prognostic ability of animal studies compared to species-specific simulator molecules. The present invention unexpectedly identified an N-terminal, containing amino acid residues 1-27, polypeptide fragment of the extracellular domain of CD3 epsilon, which, in contrast to all other known epitopes of CD3 epsilon described in the art, retains its three-dimensional structural integrity when removed from its native environment in the CD3 complex (and fusion with a heterologous amino acid sequence such as EpCAM (epithelial cell adhesion molecule) or immunoglobulin Fc region).

The phrase "environmental independence" of the CD3 epitope proposed in this invention corresponds to the first 27 N-terminal amino acids of CD3 epsilon or functional fragments of this 27 amino acid region. The phrase “environmentally independent” as used herein with respect to the CD3 epitope means that the binding of the binding / antibody molecules of the invention described herein does not alter or modify the conformation, sequence or structure surrounding the antigenic determinant or epitope. In contrast, the CD3 epitope recognized by a conventional CD3 binding molecule (e.g., as described in WO 99/54440 or WO 04/106380) is located in the C-terminal region of the CD3 epsilon chain relative to the N-terminal 1-27 amino acids independent of the environment epitope, and it takes on the proper conformation only if it is immersed in the rest of the epsilon chain and held in position by heterodimerization of the epsilon chain with either the gamma or delta CD3 chain.

Anti-CD3 binding molecules as part of the bispecific binding molecule that is proposed and designed (and directed) against the environmentally independent CD3 epitope provide an unexpected clinical improvement with respect to T cell redistribution and therefore a more suitable safety profile. Without being bound by theory, it is believed that since the CD3 epitope is independent of the environment, forming an autonomous self-sufficient subdomain without much influence on the rest of the CD3 complex, the CD3-binding molecules proposed in this application are less likely to induce allosteric changes in the CD3 conformation than traditional CD3 binding molecules (similar to those proposed in WO 99/54440) that recognize environmentally dependent CD3 epitopes.

The independence of the environment of the epitope of the CD3 binding molecules of the invention according to the invention as part of a bispecific binding molecule is associated with a smaller redistribution of T cells during the initial phase of treatment with the CD3 binding molecules of the invention, resulting in a better safety profile of the CD3 binding molecules of the invention compared to with traditional CD3 binding molecules known in the art that recognize environmentally dependent CD3 epitopes. In particular, since the redistribution of T cells during the initial phase of treatment with CD3-binding molecules represents a major risk factor for adverse CNS events, the CD3-binding molecules of the invention, due to recognition of an environmentally independent rather than environmental dependent CD3 epitope, have significant safety advantage over CD3 binding molecules known in the art. Patients with such adverse CNS events associated with T cell redistribution during the initial phase of treatment with traditional CD3 binding molecules usually suffer from confusion and disorientation, in some cases also from urinary incontinence. Confusion is a change in mental state in which the patient is unable to think with his or her usual level of clarity. Usually the patient has difficulty concentrating, and the thinking process becomes not only vague and unclear, but often slows down significantly. Patients with adverse CNS events associated with T cell redistribution during the initial phase of treatment with conventional CD3 binding molecules may also suffer from memory loss. Often confusion leads to a loss of the ability to distinguish between people and / or places or to name the time and date. Sensations of disorientation usually occur with confusion, and the ability to make decisions is impaired. Adverse events related to the central nervous system associated with the redistribution of T cells during the initial phase of treatment with traditional CD3-binding molecules may also include confused speech and / or difficulty in finding words. This disorder can impair both expressiveness and speech perception, as well as the process of reading and writing. In addition to urinary incontinence, vertigo and dizziness can also accompany CNS adverse events related to the redistribution of T cells during the initial phase of treatment with traditional CD3-binding molecules in some patients.

Maintaining a three-dimensional structure within the aforementioned 27-amino acid N-terminal polypeptide fragment of the CD3 epsilon can be used to create binding domains, preferably human, that bind to the N-terminal polypeptide fragment of the CD3 epsilon in vitro and with the native CD3 complex (CD3- the epsilon subunit of the CD3 complex) on T cells in vivo with the same binding affinity. These data strictly indicate that the N-terminal fragment, as described in this invention, forms a tertiary conformation, which is similar to its structure, normally existing in vivo. A very sensitive test was conducted on the importance of the structural integrity of amino acids 1-27 of the N-terminal polypeptide fragment of CD3 epsilon. Individual amino acids from amino acids 1-27 of the N-terminal polypeptide fragment of the CD3 epsilon were replaced with alanine (alanine scanning) to test the sensitivity of amino acids 1-27 of the N-terminal polypeptide fragment of the CD3 epsilon to minor disorders. CD3-specific antibody molecules as part of the bispecific binding molecule of the invention were used to test binding to alanine mutants of amino acids 1-27 of the N-terminal polypeptide fragment of CD3 epsilon (see the attached Example 5). Individual substitutions of the first five amino acid residues at the same N-terminal end of the fragment and two amino acids at positions 23 and 25 among amino acids 1-27 of the N-terminal polypeptide fragment of CD3 epsilon were critical for the binding of antibody molecules. Replacement of amino acid residues in positions 1-5 containing residues 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), on alanine, the binding of the binding molecules, preferably of a human, according to the invention to the indicated fragment was canceled. While, at least for some binding molecules, preferably a human, according to the invention, two amino acid residues at the C-terminus of said fragment T (threonine at position 23) and I (isoleucine at position 25) reduced the binding energy to the binding molecules, preferably a human, according to the invention.

Surprisingly, it was found that binding molecules thus isolated, preferably human, not only recognize the N-terminal fragment of human CD3 epsilon, but also the corresponding homologous CD3 epsilon fragments of various primates, including New World monkeys (Marmoset, common marmoset (Cailithrix jacchus); Oedipus tamarin (Saguinus oedipus); common squirrel monkey (Saimiri sciureus)) and Old World monkeys (long-tailed macaque (Masasa fascicularis), also known as Javanese macaque; or Masasa mulatta, also known as rhesus monkey). Thus, the specificity of the CD3 binding molecules of the invention was established for many primates (multi-primate specificity). Subsequent sequence analyzes confirmed that humans and primates share a common region with a highly homologous sequence at the N-terminus of the extracellular domain of CD3 epsilon.

The present invention has found that it is possible to create binding molecules, preferably humans, specific for CD3 epsilon, and the same molecule can be used in preclinical testing in animals, as well as in clinical studies in humans and even in human therapy. This is due to the unexpected identification of binding molecules, preferably humans, which, in addition to binding to human CD3 epsilon (and due to the likely genetic similarity to the chimpanzee equivalent), also bind to homologues of these non-chimpanzee primate antigens, including New World monkeys and Old monkeys Sveta. As shown in the following Examples, these CD3 epsilon-specific binding molecules, preferably human, can be integrated into bispecific single chain antibodies to create therapeutic agents against various diseases, including, but not limited to, cancer or immunological disorders. Thus, the need for the construction of a CD3 epsilon-binding domain-simulator or a bispecific single chain antibody, including it, for testing on a phylogenetically distant (from humans) form, disappears. As a result, in preclinical testing in animals, the same molecule can be used that is intended for administration to people in clinical testing, as well as for subsequent obtaining permission to sell and administration as a therapeutic drug. The ability to use the same molecule for preclinical testing in animals, as with later administration to humans, virtually eliminates or at least significantly reduces the risk that data obtained in preclinical testing in animals will have limited applicability in humans. Briefly, obtaining preclinical data on animal safety using the same molecule that will actually be introduced to humans greatly guarantees the applicability of these data to a scenario suitable for humans. Conversely, in traditional approaches using simulating molecules, these simulating molecules must be adapted at the molecular level to the animal test system used for preclinical safety assessment. Thus, the molecule to be used in human therapy actually differs in sequence and also, probably, in structure from the imitator molecule used in preclinical testing, in pharmacokinetic parameters and / or biological activity, as a result of which data obtained in preclinical animal testing have limited applicability / suitability in the case of humans. The use of simulation molecules requires the design, preparation, purification and characterization of a completely new design. This leads to additional costs and development time necessary to obtain such a molecule. In general, simulators must be developed separately in addition to the real drug that will be used in human therapy, so two development lines for two molecules must be drawn. Therefore, the main advantage of a human binding molecule or antibody-based constructs demonstrating the interspecific specificity described in this invention is that the same molecule can be used for human therapy and in preclinical testing in animals.

Preferably, the polypeptide of the invention, which is the first binding domain capable of binding to an epitope of a human and non-chimpanzee primate CD3 epsilon chain, is of human origin.

In addition, due to the human origin of the human binding molecules of the invention, the development of an immune response against said binding molecules is excluded as much as possible after the administration of these binding molecules to human patients.

Another major advantage of CD3 epsilon-specific binding molecules, preferably human, as part of the bispecific binding molecule of the invention is their applicability for preclinical testing on various primates. The behavior of the candidate drug in animals will ideally be an indicator of the expected behavior of the candidate drug after administration to humans. As a result, the data obtained on the basis of such preclinical testing, thus, will usually have high predictive ability in the case of humans. However, as it became clear from the tragic results of a recent clinical trial, phase I, on TGN1412 (monoclonal antibody CD28), the candidate drug can have different effects on the appearance of primates and on humans: despite the fact that during preclinical testing this antibody does not there are no or only limited adverse effects in animal studies conducted with cynomolgus monkeys, 6 human patients developed multiple organ failure after administration of the indicated antibody (Lancet 368 (2006), 22 06-7). The results of these undesirable negative events suggest that limiting preclinical testing to only one species (primates) may not be sufficient. The fact that molecules of the invention that specifically bind to human CD3 epsilon bind to CD3 of a number of New World and Old World monkeys can help overcome the problems encountered in the above case. Accordingly, the present invention provides means and methods for minimizing species differences in effects in the development and testing of drugs for human therapy.

In addition, when using the CD3 epsilon binding domain, preferably human, with interspecific specificity as part of the bispecific binding molecule of the invention, it is no longer necessary to adapt the test animal to a candidate drug intended for administration to humans, such as, for example, when creating transgenic animals. CD3 epsilon-specific binding molecules (or bispecific single chain antibodies containing them), preferably human, showing interspecific specificity according to the applications and methods of the invention, can be directly used in preclinical testing on non-chimpanzee primates without any genetic manipulation of animals. As is well known to those skilled in the art, approaches in which a test animal is adapted to a candidate drug always carries the risk that the results obtained in preclinical safety testing will be less representative and have less predictive value for humans due to animal modification. For example, in transgenic animals, proteins encoded by transgenes are often highly overexpressed to a high degree. Thus, the data obtained for the biological activity of antibodies against this protein antigen may be limited in their prognostic value for people in whom this protein is expressed at much lower, more physiological levels.

A further advantage of the use of CD3 epsilon-specific binding molecules (or bispecific single chain antibodies containing them), preferably human, showing interspecific specificity, is the fact that chimpanzees, as an endangered animal species, are excluded from animal testing. Chimpanzees are the closest relatives of humans and have recently been grouped into a hominid family based on genome sequencing data (Wildman et al., PNAS 100 (2003), 7181). Therefore, in the general case, it is believed that data obtained using chimpanzees have high predictive value for humans. However, due to its status as an endangered species, the number of chimpanzees that can be used for medical experiments is very limited. Therefore, as indicated above, providing chimpanzees for animal testing is both an expensive and an ethical problem. The use of CD3 epsilon-specific binding molecules, preferably humans, according to the invention (or bispecific single chain antibodies containing them) removes both ethical objections and financial difficulties during preclinical testing without compromising quality, i.e. the applicability of animal test results. In light of this, the use of CD3 epsilon-specific binding molecules (or bispecific single chain antibodies containing them), preferably human, provides a reasonable alternative to chimpanzee studies.

A further advantage of the CD3 epsilon-specific binding molecules, preferably humans, of the invention (or bispecific single chain antibodies containing them) is the ability to take multiple blood samples when used as part of preclinical animal testing, for example, in animal pharmacokinetic studies. Multiple blood samples can be obtained much more easily using a non-chimpanzee primate than more using lower animals such as mice. The selection of multiple blood samples allows for continuous testing of blood parameters to determine the biological effects induced by a binding molecule (or a bispecific single chain antibody), preferably a human, according to the invention. In addition, the selection of multiple blood samples allows the researcher to evaluate the pharmacokinetic profile of a binding molecule (or a bispecific single chain antibody), preferably a human, as defined in this application. In addition, possible side effects that can be induced by the specified binding molecule (or bispecific single chain antibody), preferably human, reflected in the blood parameters can be measured in various blood samples taken during the introduction of the indicated antibodies. This allows you to determine the possible toxicity profile of a binding molecule (or a bispecific single chain antibody), preferably a human, as defined in this application.

The advantages of binding molecules (or bispecific single chain antibodies), preferably human, as defined herein, showing interspecific specificity, can be summarized as follows.

First, the binding molecules (or bispecific single chain antibodies), preferably humans, as defined herein, used in preclinical testing, are the same as those used in human therapy. Thus, it is no longer necessary to develop two independent molecules, which may differ in their pharmacokinetic properties and biological activity. A huge advantage is that, for example, the pharmacokinetic results can be more directly transferred to the human environment (human setting) and applied to it than, for example, in traditional approaches using simulators.

Secondly, the use of binding molecules (or bispecific single chain antibodies), preferably human, as defined in this application, for the manufacture of therapeutic agents for humans is less expensive and time-consuming than approaches using simulators.

Thirdly, binding molecules (or bispecific single chain antibodies), preferably humans, as defined in this application, can be used for preclinical testing not only on one species of primates, but also on a number of other types of primates, thereby limiting the risk of species differences between primates and humans.

Fourth, chimpanzees, as an endangered animal species, are excluded from testing.

Fifth, for conducting intensive pharmacokinetic studies, it is possible to carry out the selection of many blood samples.

Sixth, due to the fact that the binding molecules, preferably humans, according to a preferred embodiment of the invention are of human origin, the development of an immune response against these binding molecules is minimized when administered to human patients. Induction of the immune response by antibodies specific to a candidate drug derived from a non-human species, such as a mouse, resulting in the production of human anti-mouse antibodies (NAMAs) against therapeutic molecules of mouse origin is excluded.

The term "protein" is well known in the art and describes biological compounds. Proteins contain one or more amino acid chains (polypeptides) in which amino acids are linked to each other via a peptide bond. The term "polypeptide", as used in this application, describes a group of molecules that consist of more than 30 amino acids. In accordance with the invention, this group of polypeptides contains “proteins”, provided that the proteins are composed of one polypeptide. In addition, in accordance with the definition, the term "polypeptide" describes fragments of proteins, provided that these fragments consist of more than 30 amino acids. Polypeptides can also form multimers, such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc., may be the same or not the same. Corresponding higher order structures of such multimers are denoted, therefore, homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which, in its naturally existing form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins in which the modification is carried out, for example, as a result of post-translational modifications such as glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.

As used in this application, “human” and “human” refers to the species Homo sapiens. Since the medical uses of these constructs described in this invention are discussed, human patients should be treated with the same molecule.

The term "human origin", as used in the context of the molecules of the invention, describes molecules obtained from human libraries or having a structure / sequence corresponding to the human equivalent. Accordingly, proteins having an amino acid sequence corresponding to a similar sequence in humans, for example, an antibody fragment having amino acid sequences in the framework corresponding to human germline sequences, are understood to be molecules of human origin.

As used in this application, the term "non-chimpanzee primate" or "non-chimpanzee primate." or its grammatical variants refer to any primacy different from chimpanzees, i.e. different from an animal belonging to the genus Pan and including species Pan paniscus (pygmy chimpanzee, bonobo) and Pan troglodytes (common chimpanzee), also known as Anthropopithecus troglodytes (common chimpanzee) or Simia satyrus (orangutan). The terms "primate", "species of primates", "primates" or their grammatical variants denote a detachment of placental mammals, divided into two suborders of semi-monkeys and anthropoids and including humans, anthropoids, monkeys and lemurs. Specifically, the term "primates," as used in this application, contains the suborder Strepsirrhini (wet-nosed monkeys) (semi-monkeys that are not tarsier pros (non-tarsier prosimians)), including the infraorder Lemuriformes (lemuriformes) (including the superfamilies Cheirogaleo lemera ( ) and Lemuroidea (lemurids)), the infraorder Chiromyiformes (arm-shaped) (including the family Daubentoniidae (arm-shaped)) and the infra-order Lohsiformes (arm-shaped) (including the families Lorisidae (loric) and Galagidae (galagic). The term "primates", as used in this application, also contains the suborder Haplorrhini (dry-nosed monkeys), including the Tarsiiformes (long-toed) infraorder (including the Tarsiidae (long-toed) family), and the Simiiformes (squid-like) infraorder (including the Platy monkeys) or New World monkeys, and Catarrhini (narrow-nosed monkeys), including Cercopithecidea (monkeys) or Old World monkeys)).

In the framework of the invention, a non-chimpanzee primate species can be understood as representing a lemur, tarsier, gibbon, marmoset (belonging to the New World monkeys of the Cebidae family (chain-tailed)) or the Old World monkey (belonging to the superfamily Cercopithecoidea (dog-like)).

As used in this application, the term "Old World monkey" includes any monkey that falls into the superfamily Cercopithecoidea, which itself is divided into families: Cercopithecinae (monkey monkeys), which are found mainly in Africa, but include a different genus of macaques, found in Asia and North Africa; and Colobinae (thin-bodied), which include most of the genera found in Asia, as well as African representatives of thick-bodied monkeys.

Specifically, within the subfamily Cercopithecinae, a preferred non-chimpanzee primate can be derived from the monkey tribe (Cercopithecini), within the genus Allenopithecus (Allen's marsh monkey, Allenopithecus nigrovihdis); within the genus Miopithecus (marmoset dwarf thalapoin, Miopithecus talapoin; Gabon thalapoin, Miopithecus ogouensis); within the genus Erythrocebus (common monkey hussar, Erythrocebus patas); within the genus Chlorocebus (monkey green, Chlorocebus sabaceus; monkey green grevet, Chlorocebus aethiops; monkey Gornobeyl vervet, Chlorocebus djamdjamensis; monkey tantalus, Chlorocebus tantalus; vervet, Chlorocebus malorussa, Chlorocebus pyrbusceb or within the genus Cercopithecus (dryas marmoset or Salongoy marmoset, Cercopithecus dryas; marmoset Diana, Cercopithecus diana; roloveka marmoset, Cercopithecus roloway; large white-marmoset monkey, Cercopithecus nictitans; blue marmoset, Cercopithecus marmoset, Cercopitcus mara; kandti; Syx's monkey, Cercopithecus albogularis; Mona's monkey, Cercopithecus mona; Mona Campbell's monkey, Cercopithecus campbelli; Mona Lowy's monkey, Cercopithecus lowei; Monkey's crested monkey, Cercopithecus pomonica montocheritopherontica monogolf Lesser White-Nosed, Cercopithecus petau rista; white-chinned monkey, Cercopithecus erythrogaster, Sclater's monkey, Cercopithecus sclateri; red-eared monkey, Cercopithecus erythrotis; blue-faced [moustached] monkey, Cercopithecus cephus; red-tailed monkey, Cercopithecus tuberculus, Cercopithecus erycus; sun-tailed monkey, Cercopithecus solatus; saffron monkey, Cercopithecus hamlyni; Marmoset de Brass, Cercopithecus neglectus).

Alternatively, a preferred non-chimpanzee primate, also within the subfamily Cercopithecinae, but within the tribe of the baboons (Papionini), may be from the macaque genus (Masasa) (barbarian monkey, Masasa sylvanus; macaque lion-tailed, Masasa silenus; macaque the southern porcupine (Beruk), Masasa nemesthna; Northern Pigtail Macaques, Masasa leonina; Pagay macaques or Bokkoi, Masasa pagensis; Siberu macaques, Masasa siberu; Moora macaques, Masasa maura; Shod macaques, Masasa ochreata; Tonkin macaque, Masasa gekaka, Macasa tonekana, Macaque macaque; macaque; hecki; Macaque of Gorontal, Masasa nigris cens; Celebs macaque, crested or black monkey, Masasa nigra; Cynomolgus macaques or Cynomolgus macaques, or Macaque long-tailed or Kera (Kera), Masasa fascicularis; Macaque short-tailed or bear macaques, Masasa arctoides; Rhesus macaque, Masasa mulatta; Macaque formosa , Masasa cyclopis; macaques japanese, masasa fuscata; macaque ceylon, masasa sinica; macaques indian, masasa radiata; tailless macaque, Masasa sylvanmus; Assamese macaques, Masasa assamensis; Tibetan macaques or macaques of Milne-Edwards, Masas thibetana; Arunachala macaques or "monkey from the depths of the forest", Masasa munzala); within the genus Lophocebus (gray-cheeked mangobey, Lophocebus albigena; Lophocebus albigena albigena; Lophocebus albigena osmani; Lophocebus albigena johnstoni; Black-crested mangobey, Lophocebus aterrimus; Opdenboha mangoba, Lophocebus ophthus, Lophocebus ophthus, Lophocebus ophthus) within the genus Papio (hamadryan baboon, Papio hamadryas; sphinx, or Guinean baboon, Papio papio; anubis baboon, Papio anubis; baboon baboon, or yellow baboon, Papio cynocephalus; chakma, or bear baboon, Papio ursinus); within the genus Theropithecus (gelada, Theropithecus gelada); within the genus Cercocebus (smoky mangobey, Cercocebus atys; Cercocebus atys atys; Cercocebus atys lunulatus; red-headed mangobey, Cercocebus torquatus; nimble mangobey, Cercocebus agilis; Golden-bellied mangobey, Cercoercebus sanjei); or within the genus Mandrillus (mandrill, Mandrillus sphinx; drill, Mandrillus leucophaeus).

Most preferred is Masasa fascicularis (also known as Javanese macaque, or crab beetle (Cynomolgus monkey) and therefore designated in the Examples as "Javanese") and Masasa mulatta (rhesus macaque designated as "rhesus").

Within the Colobinae subfamily, a preferred non-chimpanzee primacy may originate from the African group, within the genus Colobus (black colobus, Colobus satanas; Angolan colobus, Colobus angolensis; royal colobus, Colobus polykomos; bear colobus, Colobus vellerosus; black and white bean Colobus guereza); within the genus Piliocolobus (Western red colobus, Piliocolobus badius; Piliocolobus badius badius; Piliocolobus badius temminckii; Piliocolobus badius waldronae; Pennant colobus, Piliocolobus pennantii; Piliocolobus pennantii pennantii; Piliocolobus pennanti bollantii epienii piianti bii epi red folona, Piliocolobus tholloni; colobus red Central African, Piliocolobus foai; Piliocolobus foai foai; Piliocolobus foai ellioti; Piliocolobus foai oustaleti; Piliocolobus foai semlikiensis; Piliocolobus foai parmentierorum; colobus red columbolus columbolobus red Zanzibar, Piliocolobus kirkii; colobus red river (Tang), Piliocolobus rufomitratus); or within the genus Procolobus (olive colobus, Procolobus verus).

Within the subfamily Colobinae, the preferred non-chimpanzee primacy can alternatively be from the group of langurs (thinbodies), within the genus Semnopithecus (Langur gray Nepali, Semnopithecus schistaceus; Langur gray Kashmir, Semnopithecus ajax; Langur gray Taraysky langur, henor gray langur, henop , Semnopithecus entellus; Black-footed langur, Semnopithecus hypoleucos; South flat gray langur, Semnopithecus dussumieri; pubescent gray langur, Semnopithecus priam); within the group T. vetulus of the genus Trachypithecus (purple-faced langur, Trachypithecus vetulus; Nilgiri langur, Trachypithecus johnii); within the group T. cristatus of the genus Trachypithecus (Javanese langur, Trachypithecus auratus; silver thin body or silver langur, Trachypithecus cristatus; Indochinese langur, Trachypithecus germaini; tenasser langur, Trachypithecus barbel); within the group T. obscurus of the genus Trachypithecus (smoky or spectacled langur, Trachypithecus obscurus; Feira langur, Trachypithecus phayrei); within the group of T. pileatus of the species Trachypithecus (capillary langur, Trachypithecus pileatus; Shortridge langur, Trachypithecus shortridgei; golden langur, or Ghee trachypithecus, Trachypithecus geei); within the group T. francoisi of the genus Trachypithecus (Langur Francois, Trachypithecus francoisi; Langur Hathinkhen, Trachypithecus hatinhensis; Langur white-headed, Trachypithecus poliocephalus; Langur laotian, Tragicpithecus laotum; Langur black langur Trachypithecus laotum) or within the genus Presbytis (Sumatran shurili, Presbytis melalophos; striped thinbill, Presbytis femoralis; Sarabak thinbred, Presbytis chrysomelas; white-thinned body, Presbytis siamensis; white-breasted thinbill, Presbytis frontata; Tomotis tomellus Tomtis, Thomasby tonotel, Thomasbottom; , Presbytis hosei; chestnut thinbody, Presbytis rubicunda; Mentawai langur or Joja, Presbytis potenziani; Natun thinbody, Presbytis natunae).

Within the Colobinae subfamily, the preferred non-chimpanzee primate can alternatively be from the group of snub-nosed monkeys, within the genus Pygathrix (pygathrix nigripes, Pygathrix nigripes, Pygathrix nigripes, Pygathrix serigus; within the genus Rhinopithecus (golden snub-nosed monkey, Rhinopithecus roxellana; black snub-nosed monkey, Rhinopithecus bieti; gray snub-nosed monkey, Rhinopithecus brelichi; Tonkin snub langur, Rhinopithecus avunculus); within the genus Nasalis (common nosal, Nasalis larvatus); or within the genus Simias (Pigtail langur, Simias concolor).

As used in this application, the term “marmoset, or marmosette” means any New World monkey of the genus Callithrix, for example, belonging to the marmosets of the Atlantic coast of the subgenus Callithrix (so!) (Ordinary marmoset, or marmoset, Callithrix (Callithrix) jacchus; marmoset black-eared, Callithrix (Callithrix) penicillata; Warm's marmoset, Callithrix (Callithrix) kuhlii; white-headed marmoset, Callithrix (Callithrix) geoffroyi; yellow-headed marmoset, Callithrix (Callithrix) flaviceps; white-eared marmoset, Callithrix (Callithrix); Ammonia forests belonging to the marmosets of the subgenus Mico (silk marmosets) (marmoset Rio Akari, Callithrix (Mico) acariensis; marmoset Manicore, Callithrix (Mico) manicorensis; marmoset silver, Callithrix (Mico) argentata; white marmoset, Callithrix (Mico) leucippe; marmoset Emilia's (marmoset), Callithrix (Mico) emiliae; black marmoset, Callithrix (Mico) nigriceps; marmoset Mark (Marca's marmoset), Callithrix (Mico) marcai; black-tailed marmoset, Callithrix (Mico) melanura; marmoset ) humeralifera; marmoset Maués, Callithrix (Mico) mauesi; marmoset golden (gold-and-white marmoset), Callithrix (Mico) chrysoleuca; marmoset Hershkovitz (Hershkovitz's marmoset), Callithrix (Mico) interme dia; mate socket Satere, Callithrix (Mico) saterei); Roosmalens' Dwarf marmoset, belonging to the genus Callibella (Callithrix (Callibella) humilis); or a dwarf marmoset belonging to the subgenus Cebuella (Callithrix (Cebuella) pygmaea).

Other New World monkey genera include tamarins (or saguins) of the genus Saguinus (including the group S. oedipus, group S. midas, group S. nigricollis, group S. mystax, group S. bicolor and group S. inustus) and squirrel monkeys of the genus Saimiri (e.g. Saimiri sciureus, Saimiri oerstedii, Saimiri ustus, Saimiri boliviensis, Saimiri vanzolini).

The term "binding domain" characterizes, in accordance with the present invention, a domain of a polypeptide that specifically binds / interacts with a given target (s) structure / antigen / epitope. Thus, the binding domain is an “antigen interaction site”. The term "antigen interaction site" defines, in accordance with the present invention, a polypeptide motif that is capable of specifically interacting with a particular antigen or a specific group of antigens, for example, the same antigen in different species. It is also understood that said binding / interaction defines "specific recognition". The term “specifically recognizing” means in accordance with this invention that the antibody molecule is capable of specifically interacting and / or binding at least two, preferably at least three, more preferably at least four amino acids of an antigen, for example, human CD3 antigen, as defined in this application. Such binding can be illustrated by specificity in accordance with the "principle of lock and key." Thus, specific motifs in the amino acid sequence of the binding domain and antigen bind to each other due to their primary, secondary or tertiary structure, as well as due to secondary modifications of this structure. The specific interaction of this "site of interaction with the antigen" with its specific antigen can also simply lead to the binding of the specified site with the antigen. In addition, the specific interaction of a given “site of interaction with an antigen” with its specific antigen can alternatively cause signal initiation, for example, due to the induction of a change in antigen conformation, antigen oligomerization, etc. A preferred example of a binding domain in accordance with the present invention is an antibody. The binding domain may be a monoclonal or polyclonal antibody or derived from a monoclonal or polyclonal antibody.

The term “antibody” includes derivatives or functional fragments thereof that still retain binding specificity. Antibody production techniques are well known in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press , 1999. The term “antibody” also includes immunoglobulins (Ig) of different classes (ie, IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc.). These antibodies can be used, for example, for immunoprecipitation, affinity purification and immunolocalization of the polypeptides or fusion proteins of the invention, as well as for monitoring the presence and amount of such polypeptides, for example, in cultures of recombinant prokaryotes or eukaryotic cells or organisms.

The definition of the term “antibody” also includes such embodiments as chimeric, single chain, and humanized antibodies, as well as antibody fragments, such as, but not limited to, Fab fragments. Antibody fragments or derivatives also contain F (ab ') ₂ , Fv, scFv (single chain Fv) fragments or single domain antibodies, antibodies consisting of a single variable domain, or an immunoglobulin single variable domain containing only one variable domain, which may be VH or VL that specifically bind to an antigen or epitope independently of other V regions or β domains; see, for example, Harlow and Lane (1988) and (1999), above. Such an immunoglobulin single variable domain covers not only the isolated polypeptide of a single variable domain of an antibody, but also larger polypeptides that contain one or more monomers of a polypeptide sequence of a single variable domain of an antibody.

Various techniques are known in the art that can be used to produce such antibodies and / or fragments. So, these derivatives (antibodies) can be obtained using peptidomimetics. Further, the procedures described for the production of single chain antibodies (see, inter alia, US Pat. No. 4,946,778) can be adapted to produce single chain antibodies that are specific for the selected polypeptide (s). In addition, transgenic animals can be used to express humanized antibodies specific for the polypeptides and fusion proteins of this invention. To obtain monoclonal antibodies, you can use any technique that provides antibodies produced by cultures of stable cell lines. Examples of such techniques include a hybridoma technique (Köhler and Milstein, Nature, 256 (1975), 495-497), a triomeric technique, a hybridoma technique using human B cells (Kozbor, Immunology Today, 4 (1983), 72) and EBV- hybridoma technique (EBV - Epstein-Barr virus) for the production of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Surface plasmon resonance, which is used in the BIAcore system, can be used to increase the efficiency of phage antibodies that bind to the epitope of the target polypeptide, such as CD3 epsilon (Schier, Human Antibodies Hybridomas, 7 (1996), 97-105; Malmborg, J Immunol. Methods, 183 (1995), 7-13). In the context of the present invention, it is also contemplated that the term “antibody” includes antibody constructs that can be expressed in the host described herein below, for example, antibody constructs that can be transfected and / or transduced using, inter alia, viral or plasmid vectors.

The term “specific interaction” as used in accordance with the present invention means that the binding (domain) molecule does not have cross-reactivity or does not have significant cross-reactivity with polypeptides having a similar structure to the polypeptides with which a binding molecule forms a bond, and which could be expressed by the same cells as the polypeptide of interest. The cross-reactivity of a panel of test binding molecules can be tested, for example, by evaluating the binding of said panel of binding molecules under standard conditions (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999). Examples of specific interaction of a binding domain with a specific antigen include the specificity of the ligand to its receptor. The specified definition, in particular, includes the interaction of ligands that induce a signal after binding to its specific receptor. An example of this interaction, which, in particular, is also covered by this definition, is the interaction of an antigenic determinant (epitope) with a binding domain (antigen binding site) of an antibody.

The term “cross-species specificity” or “interspecific specificity” as used in this application means the binding of the binding domain described in this invention with the same target molecule in humans and non-chimpanzee primates. Thus, “cross-species specificity” or “interspecific specificity” should be understood as interspecific reactivity of the same X molecule expressed in different species, and not to a molecule different from X. Interspecific specificity of a monoclonal antibody that recognizes, for example, human CD3 epsilon , with respect to CD3 epsilon of a non-chimpanzee primate, for example CD3 epsilon of macaque, can be determined, for example, by FACS analysis (fluorescence-activated cell sorter). FACS analysis is carried out in such a way that the corresponding monoclonal antibody is tested for binding to human and non-chimpanzee primate cells, for example macaque cells expressing said human and non-chimpanzee CD3 epsilon antigens, respectively. A corresponding analysis is shown in the following examples.

As used in this application, the term “CD3 epsilon” means a molecule expressed as part of a T cell receptor and has the meaning usually assigned to it in the prior art. For humans, it encompasses an individual or independently combined form of all known CD3 subunits, for example CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta. The CD3 antigens of non-chimpanzee primates that are mentioned in this application are, for example, CD3 of Masas fascicularis and CD3 of Masas mulatta. As for Masas fascicularis, it covers CD3 epsilon FN-18 negative and CD3 epsilon FN-18 positive, CD3 gamma and CD3 delta. As for Masas mulatta, it covers CD3 epsilon, CD3 gamma and CD3 delta. Preferably, said CD3, as used herein, is CD3 epsilon.

The human CD3 epsilon is indicated in GenBank under accession number NM_000733 and contains SEQ ID NO: 1. The human CD3 gamma is indicated in the GenBank under accession number NM_000073. The human CD3 delta is listed on GenBank under accession number NM_000732.

The CD3 epsilon chain “FN-18 negative” by Masas fascicularis (ie, CD3 epsilon not recognized by the FN-18 monoclonal antibody due to polymorphism as described above) is indicated in GenBank Accession No. AB073994.

The CD3 epsilon chain “FN-18 positive” by Masas fascicularis (ie, CD3 epsilon recognized by the FN-18 monoclonal antibody) is indicated on GenBank Accession No. AB073993. Masas fascicularis CD3 gamma is listed on GenBank Accession No. AB073992. Masas CD3 delta fascicularis is listed on GenBank Accession No. AB073991.

Nucleic acid sequences and amino acid sequences of the corresponding Masas mulatta CD3 epsilon, gamma and delta homologs can be identified and isolated using recombinant techniques described in the art (Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3rd edition, 2001). This applies, subject to necessary changes, to the CD3 epsilon, gamma and delta homologues of other non-chimpanzee primates as defined in this application. The amino acid sequence identification of the common marmoset (Callithrix jacchus), the common squirrel monkey (Saimiri sciureus) and the oedipus marmoset (Saguinus oedipus) are described in the accompanying examples. The amino acid sequence of the extracellular domain of CD3 epsilon Callithrix jacchus is shown in SEQ ID NO: 3, the same sequence of Saguinus oedipus is shown in SEQ ID NO: 5, and the same sequence of Saimiri sciureus is shown in SEQ ID NO: 7.

In accordance with the above, the term "epitope" defines an antigenic determinant that specifically binds to / is identified by a binding molecule as defined above. Binding domains or molecules may specifically bind / interact with conformational or continuous epitopes that are unique to the target structure, for example, the CD3 epsilon chain of humans and non-chimpanzee primates. A hallmark of conformational or continuous epitopes is, for polypeptide antigens, the presence of two or more separate amino acid residues that are spaced in the primary sequence, but converge on the surface of the molecule when the polypeptide folds into a native protein / antigen (Sela, (1969) Science, 166, 1365 and Laver, (1990) Cell, 61, 553-6). Two or more individual amino acid residues contributing to this epitope are present in separately located regions of one or more than one polypeptide chain. These residues converge on the surface of the molecule when the polypeptide (s) chain (s) fold (s) into a three-dimensional structure with the formation of an epitope. Conversely, a continuous or linear epitope consists of two or more separate amino acid residues that are present in a single linear segment of the polypeptide chain. In the present invention, an "environment dependent" CD3 epitope refers to the conformation of said epitope. Such an environmentally dependent epitope located on the CD3 epsilon chain can fold into the correct conformation only if it is immersed in the remainder of the epsilon chain and held in position by heterodimerization of the epsilon chain with either the CD3 gamma or delta chain. Conversely, an environmentally independent CD3 epitope, as proposed in this application, refers to the N-terminal, containing amino acid residues 1-27, the polypeptide, or its functional fragment, CD3 epsilon. This N-terminal, containing amino acid residues 1-27, the polypeptide or its functional fragment retains its three-dimensional structural integrity and proper conformation when it is removed from the native environment in the CD3 complex. Independence from the environment of the N-terminal, containing amino acid residues 1-27, of the polypeptide or its functional fragment, which is part of the extracellular domain of CD3 epsilon, is thus an epitope that is completely different from the epitopes of CD3 epsilon described in WO 2004 / 106380 in connection with a method for producing human binding molecules. In this method, separately expressed recombinant CD3 epsilon was used. The conformation of this separately expressed recombinant CD3 epsilon was different from the conformation adapted to its natural form, i.e. a form in which the subunit of the CD3 epsilon of the TCR / CD3 complex exists as part of a non-covalent complex with either the subunit of the CD3 delta or the subunit of the CD3 gamma TCR / CD3 complex. If such a separately expressed recombinant CD3 epsilon protein is used as an antigen for selection of antibodies from an antibody library, then antibodies specific for this antigen are identified from this library, despite the fact that such a library does not contain antibodies specific for its own antigens / autoantigens. This is because the separately expressed recombinant CD3 epsilon protein does not exist in vivo; it is not an autoantigen. Therefore, subpopulations of B cells expressing antibodies specific for this protein were not depleted in vivo; an antibody library constructed from such B cells will contain genetic material for antibodies specific for this separately expressed recombinant protein CD3 epsilon.

However, since the environmentally independent N-terminal, containing amino acid residues 1-27, the polypeptide or its functional fragment is an epitope that folds into its native form, the binding domains of the present invention cannot be identified by methods based on the approach described in WO 04/106380. Therefore, it can be confirmed in tests that the binding molecules described in WO 04/106380 are not able to bind to the N-terminal amino acid residues 1-27 of the CD3 epsilon chain. Therefore, conventional anti-CD3 binding molecules or anti-CD3 antibody molecules (e.g., those described in WO 99/54440) bind to the CD3 epsilon chain at a position closer to the C-terminus than an environmentally independent N-terminal containing amino acid residues 1-27, a polypeptide or functional fragment, proposed in this application. The molecules of the OKT3 and UCHT-1 antibodies of the prior art are specific for the epsilon subunit of the TCR / CD3 complex between amino acid residues 35 and 85 and, accordingly, the epitope of these antibodies is also located closer to the C-terminus. In addition, UCHT-1 binds to the CD3 epsilon chain in the region between amino acid residues 43 and 77 (Tunnacliffe, Int. Immunol., 1 (1989), 546-50; Kjer-Nielsen, PNAS, 101 (2004), 7675-7680; Salmeron, J. Immunol., 147 (1991), 3047-52). Therefore, the anti-CD3 molecules of the prior art do not bind and are not directed against the N-terminal epitope (or its functional fragment), which is independent of the environment, N-terminal, containing amino acid residues 1-27.

To create a binding domain, preferably of a person, contained in a polypeptide according to the invention, for example in a bispecific single chain antibody, as defined in this application, for example, monoclonal antibodies that bind to CD3 epsilon of both human and non-human primate can be used chimpanzees (e.g. with CD3-epsilon macaque).

In a preferred embodiment of the polypeptide of the invention, the non-chimpanzee primate is an Old World monkey. In a more preferred embodiment of the polypeptide, the Old World monkey is a monkey of the genus Papio (baboons) or the genus of macaques. Most preferably, the monkey of the macaque genus is Assamese macaque (Masasa assamensis), barbarian monkey (Masasa sylvanus), Indian macaques, or bonnet (Masasa radiata), macaques shod (Masasa ochreata), celebs crested macasa (Masasa nigra), mountain macaques ( Masasa cyclopis), snow monkey or Japanese macaques (Masasa fuscata), Javanese macaques, or cynomolgus macaques, or long tailed macaques, or Javanese macaques (Masasa fascicularis), lion-tailed macaques (Masasa silenus), macaques southern ponytail (Masasa nemestrina), macaques Rhesus (Masasa mulatta), Macaque Tibetan Shorttail throated (Masas thibetana), Tonkin macaques (Masasa tonkeana), Ceylon macaques (Masasa sinica), short-tailed macaques, or red-faced macaques, or bear macaques (Masasa arctoides) or Moora macaques (Masasa maurus). Most preferably, the baboons monkey is Hamadryas Baboon, Papio hamadryas; Guinea Baboon, Papio papio; Olive Baboon, Papio anubis; Yellow Baboon, Papio cynocephalus; Chacma Baboon, Papio ursinus.

In an alternative preferred embodiment of the polypeptide of the invention, the non-chimpanzee primate is a New World monkey. In a more preferred embodiment of the polypeptide, the New World monkey is a monkey of the genus Callithrix (marmoset), a genus Saguinus (tamarins or saguins), or the genus Saimiri (squirrel monkeys). Most preferably, the monkey of the genus Callithrix is Callithrix jacchus (common marmoset), the monkey of the genus Saguinus is Saguinus oedipus (Oedipus tamarin), and the monkey of the genus Saimiri is Saimiri sciureus (common squirrel monkey).

As described in the present invention above, the polypeptide of the invention binds via the first binding domain to an epitope of the human CD3ε (epsilon) chain and a non-chimpanzee primate, the epitope being part of an amino acid sequence in the group of 27 amino acid residues, as presented in SEQ ID NO: 2, 4, 6, or 8.

In accordance with the present invention, for the polypeptide of the invention, it is preferable that said epitope be 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.

More preferably, said epitope contains at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (Q-D-G-N-E-D).

Within the framework of the present invention, a “functional fragment of N-terminal 1-27 amino acid residues” means that said functional fragment is still an environmentally independent epitope that retains its three-dimensional structural integrity when it is removed from its native environment in a CD3 complex ( and fused to a heterologous amino acid sequence, such as EpCAM or Fc region of immunoglobulin, for example, as shown in Example 3.1). Preserving the three-dimensional structure within the 27 amino acid N-terminal polypeptide, or its functional fragment, CD3 epsilon can be used to create binding domains that bind to the N-terminal polypeptide fragment of the CD3 epsilon in vitro and with the native CD3 complex (CD3- the epsilon subunit of the CD3 complex) on T cells in vivo with the same binding affinity. In the present invention, “a functional fragment of N-terminal 1-27 amino acid residues” means that the CD3-binding molecules proposed in this application can still bind to such functional fragments, and this does not depend on the environment. One skilled in the art is aware of epitope mapping techniques to determine which amino acid residues of an epitope are recognized by such anti-CD3 binding molecules (e.g., alanine scanning or pep-spot analysis).

In a preferred embodiment of the invention, the polypeptide of the invention comprises a (first) binding domain capable of binding to an epitope of a human CD3ε chain and a non-chimpanzee primate, and a second binding domain capable of binding to a cell surface antigen.

The term "cell surface antigen", as used in this application, means a molecule that is found on the surface of the cell. In most cases, this molecule will be located in or on the plasma membrane of the cell, so that at least a portion of this molecule remains accessible in its tertiary form outside the cell. A non-limiting example of a cell surface molecule that is localized in a plasma membrane is a transmembrane protein containing, in its tertiary conformation, a region of hydrophilicity and hydrophobicity. At the same time, at least one hydrophobic region makes it possible to insert or insert a cell surface molecule into the hydrophobic plasma membrane of the cell, while the hydrophilic regions are located on both sides of the plasma membrane in the cytoplasm and extracellular space, respectively. Non-limiting examples of cell surface molecules located on the plasma membrane are proteins that are modified at the cysteine residue to carry the palmitoyl group, proteins modified at the C-terminal cysteine residue to carry the farnesyl group, or proteins that are modified at C-terminus in order to carry a glycosyl phosphatidylinositol ("GPI") anchor. These groups allow covalent attachment of proteins to the outer surface of the plasma membrane, where they remain available for recognition by extracellular molecules, such as antibodies. Examples of cell surface antigens include EGFR, EGFRvlll (variant EGFR, type III), MCSP, carbonic anhydrase IX (CAIX), CD30, CD33, Her2 / neu, IgE, CD44v6 (variant CD44 type 6) and Mis-1. In addition, examples for corresponding cell surface antibodies contain antigens specific to a particular disease or ailment, i.e. cancer, autoimmune diseases or infectious diseases, including viral infections. Accordingly, the term “cell surface antigens” obviously includes viral proteins, such as native, non-processed viral proteins, exposed on the surface of infected cells (described, among others, for hepatitis B, C, and HIV-1 envelope proteins (human immunodeficiency virus )).

One of the protective functions of cytotoxic T cells is the destruction of virus-infected cells, therefore, the unique property of the bispecific binding molecules of the invention to activate and redirect cytotoxic T cells regardless of their autochthonous specificity has a strong effect on a wide range of chronic viral infections. For most of these infections, removal of persistently infected cells is the only chance of recovery. Adoptive therapies based on T cells against chronic infections of CMV (cytomegalovirus) and EBV (Rooney, S.M., et al., Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus- related lymphoproliferation. Lancet, 1995.345 (8941): p.9-13; Walter, EA, et al., Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T cells clones from the donor. N. Engl. J. Med., 1995.333 (16): p.1038-44).

Chronic hepatitis B infection (HBV) is undoubtedly one of the most interesting and noteworthy indications. Worldwide, 350 to 400 million people are infected with HBV. Current treatment for chronic HBV hepatitis is based on the use of γ-interferon and nucleoside or nucleotide analogues, long-term therapy with significant side effects, such as the induction of a sudden exacerbation of hepatitis, fever, myalgia, thrombocytopenia and depression. Despite the fact that there are currently more than 4 approved therapeutic regimens, the removal of the virus is rarely achieved. Persistent inflammation in chronic hepatitis B leads to liver cirrhosis and hepatocellular carcinoma in more than 25% of patients. In addition, up to 40% of patients with chronic hepatitis B die due to serious complications, which causes 0.6-1.0 million deaths per year worldwide.

HBV, a prototype of hepatadaviruses, is an enveloped virus whose genome in relaxed ring (pk) form is back transcribed into an RNA pregen. After infection, rcDNA is imported into the hepatocyte nucleus, where it is converted into covalently closed circular DNA (cccDNA) containing four overlapping reading frames. It serves as a transcription matrix for pregenomic RNA and three subgenomic RNAs. The RNA pregene functions as an mRNA for translating viral core protein and viral polymerase. Infected cells continuously produce surface HBV protein (HBsAg) with ccDNA, even when HBV replication ceases. HBsAg consists of small surface (S) proteins with a very small proportion of medium and large (L) surface proteins. Both HBV S and L target the endoplasmic reticulum (ER) membrane, from where they are transported in membrane vesicles via trans-Golgi organelles to the plasma membrane (Gorelick, FS and C. Shugrue, Exiting the endoplasmic reticulum. Mol. Cell Endocrinol., 2001.177 (1-2): p.13-8). S and L proteins are constantly expressed on the surface of HBV-replicating hepatocytes, as recently shown (Chu, C.M. and YF Liaw, Membrane staining for hepatitis B surface antigen on hepatocytes: a sensitive and specific marker of active viral replication in hepatitis BJ Clin Pathol., 1995, 48 (5): p. 470-3).

The prototypes of viruses that expose enveloped proteins on the cell surface are hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV-1, which represent a heavy burden of disease in general. For HIV-1 virus, it has recently been shown that T cells modified with the FCR chimeric TCR construct directed to the gp120 envelope protein can kill HIV-1 infected target cells (Masiero, S., et al., T- cell engineering by a chimeric T-cell receptor with fntibody-type specificity for the HIV-1 gp120. Gene Ther., 2005, 12 (4): p. 299-310). Hepadnavirus-owned hepatitis B virus (HBV) expresses the HBsAg envelope protein complex, which is continuously produced from episomal cccDNA, even when HBV replication is reduced.

Expression of intact S and L HBV proteins on the cell surface makes them available for antibodies, which are a hallmark of seroconversion, when patients recover from the acute phase of infections and HBsAg circulating in the blood is replaced with anti-HBs. If seroconversion does not occur, then up to 30% of hepatocytes continue to express HBV S protein also after highly active antiviral therapy of long duration. Thus, in addition to T lymphocytes that specifically recognize processed intracellular HBV peptides and are presented by MHC molecules on the cell surface, other forms of T cell interaction may target an intact surface protein, such as S and L antigens, available on the outer cell membrane. Using single-chain fragments of antibodies that recognize small (S) and large (L) envelope proteins of hepatitis B virus, artificial T-cell receptors have been created that allow directing transplanted T-cells to infected hepatocytes and secrete cytokines after antigen-contact activation of these T-cells and destroy infected hepatocytes.

The limitation for this approach is that (1) there is a need for in vitro manipulation of T cells, (2) retroviruses used to transfer T cell receptors can lead to insertional mutagenesis in T cells, and that (3) after such transfer T cell cytotoxic response cannot be limited.

To overcome these limitations, bispecific single chain antibody molecules can be created containing the first domain with binding specificity for the human CD3 epsilon antigen and non-chimpanzee primate (as proposed in this application in the context of this invention), as well as a second domain with binding specificity for enveloped proteins of HBV or HCV of infected hepatocytes, and they are included in the scope of this invention.

It is also preferred according to the present invention that the second binding domain binds to a human cell surface antigen and / or originates from a non-chimpanzee primate, copies of human cell surface antigens selected from EGFR, Her2 / neu or IgE.

Monoclonal antibodies that bind to the corresponding cell surface antigens of both a human and / or non-chimpanzee primate can be used to form the second binding domain of the polypeptide of the invention, for example, bispecific single chain antibodies, as defined herein. Appropriate binding domains for a bispecific polypeptide, as defined herein, can be obtained, for example, from cross-species specific monoclonal antibodies in accordance with recombinant methods described in the art. A monoclonal antibody that binds to a human cell surface antigen and to a homolog of said non-chimpanzee primate cell surface antigen can be tested in the FACS assays described above. It will be apparent to those skilled in the art that antibodies with interspecific specificity can also be generated by the hybridoma methods described in the literature (Milstein and Köhler, Nature, 256 (1975), 495-7). For example, mice can be immunized alternately with human CD33 and non-chimpanzee primate. Hybridoma cells producing antibodies with interspecific specificity are isolated from these mice by hybridoma technology and analyzed by FACS as described above. The creation and analysis of bispecific polypeptides, such as bispecific single chain antibodies demonstrating interspecific specificity, as described in this invention, are shown in the following examples. The advantages of bispecific single chain antibodies exhibiting interspecific specificity include the items listed below.

Regarding the polypeptide of the invention, it is particularly preferred that the first binding domain capable of binding to an epitope of a human and non-chimpanzee CD3ε chain contains a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from :

(a) CDR-L1, as presented in SEQ ID NO: 27, CDR-L2, as presented in SEQ ID NO: 28, and CDR-L3, as presented in SEQ ID NO: 29;

(b) CDR-L1, as presented in SEQ ID NO: 117, CDR-L2, as presented in SEQ ID NO: 118, and CDR-L3, as presented in SEQ ID NO: 119; and

(c) CDR-L1, as presented in SEQ ID NO: 153, CDR-L2, as presented in SEQ ID NO: 154, and CDR-L3, as presented in SEQ ID NO: 155.

Variable areas, i.e. the variable light chain ("L" or "VL") and the variable heavy chain ("H" or "VH") are understood in the art to provide an antibody binding domain. These variable regions contain hypervariable regions.

The term "hypervariable region" (CDR) is well known in the art to determine the antigenic specificity of an antibody. The term "CDR-L" or "L CDR" refers to CDR in VL, while the term "CDR-H" or "H CDR" refers to CDR in VH.

In an alternative preferred embodiment of the polypeptide of the invention, a first binding domain capable of binding to an epitope of a human and non-chimpanzee CD3ε chain contains a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from:

(a) CDR-H1, as presented in SEQ ID NO: 12, CDR-H2, as presented in SEQ ID NO: 13, and CDR-H3, as presented in SEQ ID NO: 14;

(b) CDR-H1, as presented in SEQ ID NO: 30, CDR-H2, as presented in SEQ ID NO: 31, and CDR-H3, as presented in SEQ ID NO: 32;

(c) CDR-H1, as presented in SEQ ID NO: 48, CDR-H2, as presented in SEQ ID NO: 49, and CDR-H3, as presented in SEQ ID NO: 50;

(d) CDR-H1, as presented in SEQ ID NO: 66, CDR-H2, as presented in SEQ ID NO: 67, and CDR-H3, as presented in SEQ ID NO: 68;

(e) CDR-H1, as presented in SEQ ID NO: 84, CDR-H2, as presented in SEQ ID NO: 85, and CDR-H3, as presented in SEQ ID NO: 86;

(e) CDR-H1, as presented in SEQ ID NO: 102, CDR-H2, as presented in SEQ ID NO: 103, and CDR-H3, as presented in SEQ ID NO: 104;

(g) CDR-H1, as presented in SEQ ID NO: 120, CDR-H2, as presented in SEQ ID NO: 121, and CDR-H3, as presented in SEQ ID NO: 122;

(h) CDR-H1, as presented in SEQ ID NO: 138, CDR-H2, as presented in SEQ ID NO: 139, and CDR-H3, as presented in SEQ ID NO: 140;

(i) CDR-H1, as presented in SEQ ID NO: 156, CDR-H2, as represented in SEQ ID NO: 157, and CDR-H3, as represented in SEQ ID NO: 158; and

(k) CDR-H1, as presented in SEQ ID NO: 174, CDR-H2, as presented in SEQ ID NO: 175, and CDR-H3, as presented in SEQ ID NO: 176.

It is also preferred that the first binding domain capable of binding to an epitope of a human CD3ε chain and a non-chimpanzee primate contains a VL region selected from the group consisting of a VL region as shown in SEQ ID NO: 35, 39, 125 , 129, 161 or 165.

Alternatively, it is preferable that the first binding domain capable of binding to an epitope of a human CD3ε chain and a non-chimpanzee primate contains a VH region selected from the group consisting of a VH region as shown 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.

More preferably, the polypeptide of the invention is characterized by a first binding domain capable of binding to an epitope of the human CD3ε chain and a non-chimpanzee primate that contains a VL region and a VH region selected from the group consisting of:

(a) the VL region, as represented in SEQ ID NO: 17 or 21, and the VH region, which is represented in SEQ ID NO: 15 or 19;

(b) the VL region as represented in SEQ ID NO: 35 or 39, and the VH region as represented in SEQ ID NO: 33 or 37;

(c) the VL region as represented in SEQ ID NO: 53 or 57, and the VH region as represented in SEQ ID NO: 51 or 55;

(d) the VL region as represented in SEQ ID NO: 71 or 75, and the VH region as represented in SEQ ID NO: 69 or 73;

(e) the VL region as represented in SEQ ID NO: 89 or 93, and the VH region as represented in SEQ ID NO: 87 or 91;

(e) the VL region as represented in SEQ ID NO: 107 or 111, and the VH region as represented in SEQ ID NO: 105 or 109;

(g) the VL region as represented in SEQ ID NO: 125 or 129, and the VH region as represented in SEQ ID NO: 123 or 127;

(h) the VL region as represented in SEQ ID NO: 143 or 147, and the VH region as represented in SEQ ID NO: 141 or 145;

(i) the VL region as represented in SEQ ID NO: 161 or 165, and the VH region as represented in SEQ ID NO: 159 or 163; and

(k) the VL region as represented in SEQ ID NO: 179 or 183, and the VH region as represented in SEQ ID NO: 177 or 181.

According to a preferred embodiment of the polypeptide of the invention, pairs of VH regions and VL regions are in single chain antibody (scFv) format. VH and VL regions are organized in the following order: VH-VL or VL-VH. Preferably, the VH region is located at the N-terminus of the linker sequence. The VL region is located at the C-terminus of the linker sequence.

A preferred embodiment of the polypeptide of the invention described above is characterized by a first binding domain capable of binding to an epitope of a human and non-chimpanzee CD3ε chain containing an amino acid sequence selected 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.

The invention also relates to the polypeptide described above, wherein the second binding domain binds to a cell surface antigen, preferably a tumor antigen.

The term "tumor antigen", as used in this application, can be understood as such antigens that are present on the tumor cell. These antigens can be presented on the cell surface through the extracellular region, which is often combined with the transmembrane and cytoplasmic regions of the molecule. These antigens can sometimes be presented only by tumor cells and never by normal cells. Tumor antigens can be expressed exclusively on tumor cells or can be a tumor-specific mutation in comparison with normal cells. In this case, they are called tumor-specific antigens. More common are antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens can be overexpressed compared to normal cells or are available for binding of antibodies in tumor cells due to the less compact structure of tumor tissue compared to normal tissue. A non-limiting example of tumor antigens as used herein is EGFR (Liu, Br. J. Cancer, 82/12 (2000), 1991-1999; Bonner, Semin. Radiat. Oncol., 12 (2002), 11- 20; Kiyota, Oncology, 63/1 (2002), 92-98; Kuan, Brain Tumor Pathol., 17/2 (2000), 71-78).

EGFR (also known as c-erb1 or HER1) belongs to the erbB family of receptor tyrosine kinases. When activated by binding a ligand from the EGF family of growth factors, EGFR forms homodimers or heterodimers with a second EGFR or another member of the erbB receptor family, respectively, triggering a signaling cascade through mitogen-activated protein kinases and other transcription factors leading to proliferation, differentiation and repair ( Olayioye, EMBO J., 19 (2000), 3159-67). EGFR is overexpressed in many types of epithelial cancer, including colorectal cancer, breast, lung and head and neck cancer (Mendelsohn, J. Clin. Oncol., 21 (2003), 2787-99; Mendelsohn, J. Clin. Oncol., 20 (18, Suppl.) (2002), 1S-13S; Prewett, Clin. Cancer Res., 8 (2002), 994-1003). Overexpression and / or mutation of EGFR in malignant cells causes constitutive activation of kinase activity, which leads to proliferation, angiogenesis, invasion, metastasis and inhibition of apoptosis (Mendelsohn (2003, supra; Ciardiello, Clin. Cancer Res., 7 (2001), 2958- 70; Perez-Soler, Oncologist, 9 (2004), 58-67. Monoclonal antibodies directed to the extracellular ligand binding domain or the intracellular tyrosine kinase EGFR signaling cascade have been shown to be effective as an antitumor target (Laskin, Cancer Treat. Review 30 (2004), 1-17). For example, a humanized monoclone Cetuximab (Erbitux), an anti-EGFR antibody that competitively inhibits the extracellular domain of EGFR to inhibit receptor ligand activation, was approved by the Food and Drug Administration (FDA) in 2004 for the treatment of metastatic colon cancer in combinations with topoisomerase inhibitor irinotecan.

In a preferred embodiment of the invention, the polypeptide is a bispecific single chain antibody molecule.

The problems described above in this invention regarding the development of mimic molecules for preclinical studies are also exacerbated if the candidate drug is a bispecific antibody, for example a bispecific single chain antibody. Thus, a bispecific antibody requires that both recognized antigens have interspecific specificity for a given animal species in order to enable safety testing on such an animal.

As already noted in this application above, the present invention provides polypeptides comprising a first binding domain capable of binding to an epitope of a human CD3ε chain and a non-chimpanzee primate, and a second binding domain capable of binding to a cell surface antigen selected from EGFR, Her2 / neu or IgE, wherein the second binding domain also preferably binds to the cell surface antigen of a human and / or non-chimpanzee primate. The advantage of bispecific single chain antibody molecules as candidate drugs that satisfy the requirements for the preferred polypeptide of the invention is the use of such molecules in preclinical testing in animals, as well as in clinical studies and even for human therapy. In a preferred embodiment of the interspecific bispecific single chain antibodies of the invention, the second binding domain capable of binding to the cell surface antigen is of human origin. In a bispecific interspecific specificity molecule of the invention, a binding domain capable of binding to an epitope of the human and chimpanzee CD3 epsilon chain is located in the VH-VL or VL-VH order at the N-terminus or C-terminus of the bispecific molecule. Examples of interspecific bispecific molecules of the invention with different arrangement of the VH and VL chains in the first and second binding domain are described in the accompanying examples.

As used herein, “bispecific single chain antibody” means a single polypeptide chain containing two binding domains. Each binding domain contains one variable region from the antibody heavy chain (“VH region”), the VH region of the first binding domain specifically binding to the CD3ε molecule, and the VH region of the second binding domain specifically binding to the cell surface antigen, as defined in more detail below. The two binding domains are possibly linked to each other via a short polypeptide spacer. A non-limiting example of a polypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-S) and its repeats. Each binding domain may additionally contain one variable region from the light chain of the antibody ("VL region"), while the VH region and the VL region within each of the first and second binding domains are linked to each other via a polypeptide linker, for example, of the type described and claimed in EP 623679 B1, but in any case having a length sufficient to allow the VH regions and VL regions of the first binding domain and the VH region and VL regions of the second binding domain to pair with each other in this way, so that being VM ste, they were capable of specifically binding to respective first and second molecules.

According to a preferred embodiment of the invention, the bispecific single chain antibody molecule described above comprises a group of the following sequences, such as CDR H1, CDR H2, CDR NZ, CDR L1, CDR L2 and CDR L3 in the second binding domain selected from SEQ ID NO: 441-446 , SEQ ID NO: 453-458, SEQ ID NO: 463-468, SEQ ID NO: 481-486.

A particularly preferred embodiment of the invention relates to the polypeptide described above, wherein the bispecific single chain antibody molecule contains a sequence selected from:

(a) the amino acid sequence as represented in any of SEQ ID NO: 389, 391, 393, 395, 397, 399, 409, 411, 413, 415, 417, 419, 429, 431, 433, 435, 437, 439 447, 449, 451, 469, 471, 473, 475, 477, 479, 495, 497, 499, 501, 503 and 505; and

(b) the amino acid sequence encoded by the nucleic acid sequence, as represented in any of SEQ ID NO: 390, 392, 394, 396, 398, 400, 410, 412, 414, 416, 418, 420, 430, 432, 434, 436, 438, 440, 448, 450, 452, 470, 472, 474, 476, 478, 480, 496, 498, 500, 502, 504, and 506.

In a preferred embodiment of the invention, bispecific single chain antibodies have interspecific specificity for CD3 epsilon and for a tumor antigen recognized by their second binding domain.

In an alternative embodiment, the present invention provides a nucleic acid sequence encoding the polypeptide of the invention described above.

The present invention also relates to a vector comprising a nucleic acid molecule of the present invention.

Many suitable vectors are known to those skilled in molecular biology, the selection of which will depend on the desired function, and include plasmids, cosmids, viruses, bacteriophages and other vectors traditionally used in genetic engineering. Methods well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook et al. (above) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, polynucleotides and vectors of the invention can be incorporated into liposomes for delivery to target cells. As described in more detail below, a cloning vector was used to isolate individual DNA sequences. If expression of a particular polypeptide is necessary, relevant sequences can be transferred to expression vectors. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, pOR13CAT.

Preferably, said vector comprises a nucleic acid sequence, which is a regulatory sequence operably linked to said nucleic acid sequence as defined herein.

The term "regulatory sequence" refers to DNA sequences that are necessary to affect the expression of the coding sequences with which they are ligated. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences generally include a promoter, a ribosome binding site, and terminators. In eukaryotes, in general, regulatory sequences include promoters, terminators, and, in some cases, enhancers, transactivators, or transcription factors. The term "regulatory sequence" is intended to include at least all components whose presence is necessary for expression, and may also include additional preferred components.

The term “operably linked” refers to an adjacent position where the components described in this manner are interdependent, allowing them to function properly. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences. In the case where the regulatory sequence is a promoter, it will be apparent to those skilled in the art that double stranded nucleic acid is preferable.

Thus, said vector is preferably an expression vector. An “expression vector” is a construct that can be used to transform a selected host and allows expression of a coding sequence in a selected host. Expression vectors can be, for example, cloning vectors, binary vectors, or integrating vectors. Expression involves the transcription of a nucleic acid molecule, preferably into a translated (region) mRNA. Regulatory elements that provide expression in prokaryotes and / or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, they normally contain promoters that initiate transcription, and possibly poly-A signals that terminate transcription and stabilize this transcript. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, the P _L , lac, trp or tac promoter in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or promoters based on CMV, SV40 (monkey virus 40), RSV (Routh sarcoma virus), CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells.

In addition to the elements that are responsible for the initiation of transcription, such regulatory elements may also contain transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site located below this polynucleotide. In addition, depending on the expression system used, leader sequences capable of directing the polypeptide to the cell compartment or secreting it into the medium can be added to the coding sequence of the described nucleic acid sequence, and they are well known in the art; see also the attached Examples. The leader sequence (s) are assembled in the appropriate phase with translation initiation and termination sequences, and it is preferred that the leader sequence is capable of directing the secretion of the translated protein or its portion into the periplasmic space or extracellular medium. It is possible that the heterologous sequence can encode a fusion protein comprising an N-terminal identifying peptide that provides the desired characteristics, for example, stabilization or simplified purification of the expressed recombinant product; see above. In this context, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vector 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), 141-150) or pSPORT1 (GIBCO BRL).

Preferably, the expression regulatory sequences will be eukaryotic promoter systems in vectors capable of transforming transfected eukaryotic host cells, but regulatory sequences of prokaryotic hosts can also be used. After the vector has been included in the appropriate host, this host is maintained under conditions suitable for providing a high level of expression of the nucleotide sequences, and, if desired, collection and purification of the polypeptide of the invention may follow; see, for example, the attached Examples.

An alternative expression system that can be used to express a cell cycle active protein is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for the expression of foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The coding sequence of the described 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 the indicated coding sequence will inactivate the polyhedrin gene and result in a recombinant virus in which there is no envelope protein. These recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the protein of the invention is expressed (Smith, J. Virol. 46 (1983), 584; Engelhard, Proc. Nat. Acad. Sci. USA, 91 (1994) ), 3224-3227).

Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the vectors of the invention described above contain a selectable and / or comparative marker (scorable marker).

Selectable marker genes useful for breeding transformed cells and, for example, plant tissue and plants, are well known to those skilled in the art and give, for example, resistance to antimetabolites as a selection basis for dhfr, which provides resistance to methotrexate (Reiss, Plant Physiol . (Life Sci. Adv.) 13 (1994), 143-149); npt, which provides resistance to aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J., 2 (1983), 987-995); and hygro, which provides resistance to hygromycin (Marsh, Gene, 32 (1984), 481-485). Additional breeding genes are described, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA, 85 (1988), 8047); mannose-6-phosphatisomerase, which allows cells to utilize mannose (WO 94/20627); and ODC (ornithine decarboxylase), which provides resistance to the 2- (difluoromethyl) -DL-ornithine ornithine decarboxylase inhibitor (DFMO) (McConlogue, 1987, Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.); or deaminase from Aspergillus terreus, which provides resistance to blastidin S (Tamura, Biosci. Biotechnol. Biochem., 59 (1995), 2336-2338).

Useful comparative markers are also known to those skilled in the art and are commercially available. Preferably, said marker is a gene encoding luciferase (Giacomin, PI. Sci., 116 (1996), 59-72; Scikantha, J. Bact, 178 (1996), 121), a green fluorescent protein (Gerdes, FEBS Lett. , 389 (1996), 44-47) or (3-glucuronidase (Jefferson, EMBO J., 6 (1987), 3901-3907). This embodiment is particularly useful for simple and quick screening of cells, tissues and organisms, containing said vector.

As described above, said nucleic acid molecule can be used on its own or as part of a vector for expression of a polypeptide of the invention in cells, for example, for its purification, as well as for gene therapy tasks. Nucleic acid molecules or vectors containing DNA sequence (s) encoding (s) any of the polypeptides of the invention described above are introduced into cells, which in turn produce the polypeptide of interest. Gene therapy based on the introduction of therapeutic genes into cells using 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 are known to those skilled in the art; see, for example, Giordano, Nature Medicine, 2 (1996), 534-539; Schaper, Circ. Res., 79 (1996), 911-919; Anderson, Science, 256 (1992), 808-813; Verma, Nature, 389 (1994), 239; Isner, Lancet, 348 (1996), 370-374; Muhlhauser, Circ. Res., 77 (1995), 1077-1086; Onodera, Blood, 91 (1998), 30-36; Verma, Gene Then, 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci., 811 (1997), 289-292; Verzeletti, Hum. Gene Ther., 9 (1998), 2243-51; Wang, Nature Medicine, 2 (1996), 714-716; WO 94/29469; WO 97/00957, US 5580859; US 5589466; or Schaper, Current Opinion in Biotechnology, 7 (1996), 635-640. Said nucleic acid molecules and vectors can be designed for direct administration or for insertion via liposomes or viral vectors (e.g., adenovirus, retroviral) into a cell. Preferably, said cell is a germ line cell, embryonic cell or egg cell, or is derived from them, most preferably said cell is a stem cell. An example of an embryonic stem cell can be, inter alia, the stem cell, which is described in Nagy, Proc. Natl. Acad. Sci. USA, 90 (1993), 8424-8428.

In addition, according to the invention, a host is subjected to transformation or transfection with a vector according to the invention. The specified host can be obtained by introducing the above-described vector according to the invention or the above-described nucleic acid molecules of the 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 single chain antibody constructs described above.

The described nucleic acid molecule or vector according to the invention, which is introduced into the host, can either be integrated into the host genome or can be maintained extrachromosomally.

The host can be any prokaryotic or eukaryotic cell.

The term "prokaryote" is intended to include all bacteria that can be transformed or transfected with DNA or RNA molecules to express the protein of the invention. Prokaryotic hosts may include gram-negative as well as gram-positive bacteria, such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is intended to include cells of yeast, higher plants, insects, and preferably mammals. Depending on the host used in the recombinant production procedure, the protein encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated. Particularly preferred is the use of a plasmid or virus containing the coding sequence of a polypeptide of the invention genetically fused to an N-terminal FLAG tag and / or a C-terminal His tag. Preferably, the length of said FLAG tag is about 4-8 amino acids, most preferably 8 amino acids. The polynucleotide described above can be used to transform or transfect the host using any of the techniques generally recognized by those of ordinary skill in the art. In addition, methods for producing fused, functionally related genes and their expression, for example in mammalian cells and bacteria, are well known in the art (Sambrook, supra).

Preferably, said host is a bacterium or cell of insects, fungi, plants or animals.

In particular, it is contemplated that said host may be a mammalian cell. In particular, preferred host cells include CHO (Chinese Hamster Ovary) cells, COS (African green monkey fibroblast) cells, SP2 / 0 or NS / 0 type myeloma cells. As illustrated in the accompanying examples, CHO cells are particularly preferred as hosts.

More preferably, said host cell is a human cell or a human cell line, for example, per.c6 (Kroos, Biotechnol. Prog., 2003, 19: 163-168).

Thus, in a further embodiment, the present invention relates to a method for producing a polypeptide of the invention, comprising culturing a host of the invention under conditions conducive to expression of the polypeptide of the invention and recovering the obtained polypeptide from the culture.

Transformed hosts can be grown in fermenters and cultured in accordance with methods known in the art to achieve optimal cell growth. Then, the polypeptide of the invention can be isolated from growth medium, cell lysates, or cell membrane fractions. Isolation and purification, for example, of the polypeptides expressed by the microbes of the invention can be carried out by any conventional methods, such as, for example, preparative chromatographic separations and immunological separations, such as, for example, separations involving the use of monoclonal or polyclonal antibodies directed, for example, against the label ( tag) a polypeptide of the invention or as described in the accompanying examples.

The conditions of cultivation of the host, allowing expression, are known in the art as dependent on the host system and expression system / expression vector used in such a method. Parameters that must be changed to achieve conditions conducive to expression of the recombinant polypeptide are known in the art. Thus, suitable conditions can be determined by a person skilled in the art in the absence of additional inventive contribution.

After expression, the polypeptide of the invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982). For pharmaceutical applications, substantially pure polypeptides with a homogeneity of at least about 90-95%, and most preferred with a homogeneity of 98-99% or more, are preferred. After being purified, partially or homogeneously, if desired, the polypeptide of the invention can then be used for therapeutic purposes (including extracorporeal) or for the development and conduct of analytical procedures. In addition, the accompanying examples describe in detail examples of methods for extracting a polypeptide of the invention from a culture.

In addition, according to the present invention, there is provided a composition comprising a polypeptide of the invention or a polypeptide obtained by the method described above. Preferably, said composition is a pharmaceutical composition.

In accordance with the invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient. A particular preferred pharmaceutical composition of the invention comprises binding molecules directed against and created against environmentally independent CD3 epitopes. Preferably, the pharmaceutical composition contains suitable compositions of carriers, 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 administration by direct injection into the tissue. In particular, it is contemplated that said composition is administered to a patient by infusion or injection. The introduction of suitable compositions can be carried out in various ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, local or intradermal administration. In particular, the present invention provides continuous administration of a suitable composition. As a non-limiting example, continuous, i.e. continuous administration can be realized by means of a small pumping system driven by the patient to dose the therapeutic agent into the patient. A pharmaceutical composition comprising binding molecules directed against and created against environmentally independent CD3 epitopes of the invention can be administered using these pumping systems. Such pumping systems are generally known in the art and are typically designed to periodically replace cartridges containing an infusion-administered therapeutic agent. When replacing a cartridge in such a pumping system, a temporary cessation of the continuous flow of the therapeutic agent into the patient’s body may occur in other cases. In this case, the administration phase before replacing the cartridge and the administration phase after replacing the cartridge will nevertheless be considered together constituting the “continuous administration” of such a therapeutic agent in accordance with the meaning of the pharmaceutical agents and methods of the invention.

The continuous or continuous administration of these opposing binding molecules and created against the environmentally independent CD3 epitopes of this invention may be intravenous or subcutaneous using a fluid delivery device or small pump system, including a fluid supply mechanism for supplying fluid from a reservoir and a triggering mechanism to actuate the drive mechanism. Subcutaneous pumping systems may include a needle or cannula for piercing a patient's skin and delivering a suitable composition to the patient. These pumping systems can be directly fixed to the skin or attached to the patient’s skin in isolation from a vein, artery or blood vessel, thereby providing direct contact between the pumping system and the patient’s skin. The pumping system can be attached to the patient’s skin from 24 hours to several days, inclusive. The pump system can be small in size with a tank for small volumes. By way of non-limiting example, the reservoir volume for a suitable pharmaceutical composition to be administered may be from 0.1 to 50 ml.

Continuous administration may be transdermal using a patch applied to the skin and replaced at regular intervals. One of skill in the art is aware of drug delivery systems suitable for this task. It should be noted that transdermal administration is particularly suitable as a continuous administration, since the replacement of the first used patch can preferably be carried out simultaneously with the replacement with a new, second patch, for example, on the skin surface located in the immediate vicinity of the first used patch and immediately before the removal of the first used patch. There are no problems with interruption of flow or severe damage to cells.

The composition of the present invention, comprising, in particular, anti-binding and anti-environmentally binding CD3 epitopes, may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include solutions, for example phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, liposomes, etc. Compositions containing such carriers can be prepared by well-known conventional methods. Compositions may contain carbohydrates, buffers, amino acids and / or surfactants. The carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol. Such compositions may be used for continuous administrations, which may be intravenous or subcutaneous using and / or without pumping systems. Amino acids can be charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and / or histidine. Surfactants can be detergents, preferably with a molecular weight of more than 1.2 kDa, and / or a polyester, preferably with a molecular weight of more than 3 kDa. Non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80, or Tween 85. Non-limiting examples of a preferred polyester are PEG (polyethylene glycol) 3000, PEG 3350, PEG 4000, or PEG 5000. Preferred pH buffering systems used in the present the invention may be 5-9, and they may contain citrate, succinate, phosphate, histidine and acetate. The compositions of the present invention can be administered to a subject in a suitable dose, which can be determined, for example, from studies using increasing doses by administering increasing doses of the polypeptide of the invention showing the interspecific specificity described in this invention for non-chimpanzee primates, for example macaques. As indicated above, the polypeptide of the invention showing the interspecific specificity described in this invention can preferably be used in the same form in preclinical testing on non-chimpanzee primates and as a medicine in humans. These compositions can also be administered in combination with other protein and non-protein drugs. These drugs can be administered simultaneously with the composition containing the polypeptide of the invention as defined herein, or separately before or after administration of the polypeptide in accordance with certain time intervals and doses. The dosage regimen will be determined by the attending physician and clinical indicators. As is well known in the medical field, dosages for any patient depend on many factors, including patient size, body surface area, age, particular compound administered, gender, time and route of administration, general health, and other medications intended for co-administration. Compositions for parenteral administration include sterile aqueous or non-aqueous 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 carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral diluents include sodium chloride solution, dextrose in Ringer's solution, dextrose and sodium chloride, Ringer's solution with lactose, or fixed oils. Intravenous diluents include liquid and nutrient excipients, electrolyte excipients (for example, based on dextrose in Ringer's solution) and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like. In addition, the composition of the present invention may contain protein carriers, such as, for example, serum albumin or immunoglobulin, preferably of human origin. It is contemplated that the composition of the invention may contain, in addition to the polypeptide of the invention defined herein, additional biologically active agents depending on the intended use of the composition. Such agents may include drugs that act on the gastrointestinal system, drugs that act as cytostatic agents, drugs that prevent hyperuricemia, drugs that suppress immune responses (such as corticosteroids), drugs that modulate the inflammatory response, drugs acting on the circulatory system and / or agents such as cytokines known in the art.

The biological activity of the pharmaceutical composition defined in this application can be determined, for example, based on cytotoxicity assays, as described in the following examples, in WO 99/54440, or Schlereth et al. (Cancer Immunol. Immunother., 20 (2005) , 1-12). "Efficacy" or "in vivo efficacy" as used herein refers to a response to therapy with a pharmaceutical composition of the invention obtained using, for example, standardized NCl response criteria (US National Cancer Institute). The success or effectiveness of in vivo therapy using the pharmaceutical composition of the invention relates to the effectiveness of the composition in relation to its intended purpose, i.e. to the ability of the composition to cause its desired effect, i.e. depletion of abnormal cells, such as tumor cells. In vivo efficacy can be monitored in accordance with approved standard methods for determining the appropriate nosological forms, including, but not limited to, determining the number of leukocytes, differential blood count, method for sorting fluorescence-activated cells, bone marrow aspiration. In addition to this, methods for determining clinical and chemical parameters specific to various diseases and other approved standard methods can be used. In addition, computed tomography, X-ray, nuclear magnetic resonance imaging (for example, to evaluate the response based on the criteria of the US National Cancer Institute [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister, can be used 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]), positron emission tomography scan, white blood cell count, differential analysis of cro vi, sorting of fluorescently activated cells, bone marrow aspiration, lymph node biopsy / histology, and determination of various lymphoma-specific clinical and chemical parameters (e.g., lactate dehydrogenase) and other approved standard methods.

Another major problem in the development of drugs such as the pharmaceutical composition of the invention is the predicted modulation of pharmacokinetic properties. To this end, the pharmacokinetic profile of the candidate drug is determined, i.e. a profile of pharmacokinetic parameters that affect the ability of a particular drug to treat a given condition. Pharmacokinetic parameters of a drug that affects the ability of a drug to treat a specific disease state include, but are not limited to: elimination half-life, volume of distribution, first passage through the liver, and degree of binding to blood serum. The effectiveness of a given drug can be influenced by each of the above parameters.

"Half-life" means the time during which 50% of the drug administered is excreted through biological processes, such as metabolism, excretion, etc.

By "first-pass metabolism through the liver" is meant the ability of a drug to be metabolized after first contact with the liver, i.e. during its first passage through the liver.

"Distribution volume" means the degree of drug retention in all the various compartments of the body, such as, for example, intracellular and extracellular spaces, tissues and organs, etc., and the distribution of the drug in these compartments.

“Degree of serum binding” means the ability of a drug to interact or bind to serum proteins, such as albumin, resulting in a decrease or loss in the biological activity of the drug.

Pharmacokinetic parameters also include bioavailability, latency (Tlag), Tmax, absorption rates, start time and / or Cmax for a given amount of drug administered.

"Bioavailability" means the amount of drug in the blood compartment.

"Latent period" means the time lag between the administration of the drug and its detection and the possibility of measurement in blood or plasma.

"Tmax" is the time after which the maximum concentration of the drug in the blood is reached, and "Cmax" is the maximum concentration in the blood obtained using this drug. All parameters affect the period of time to achieve the concentration of the drug in the blood or tissues, which is necessary for its biological effect. The pharmacokinetic parameters of the bispecific single chain antibodies, the preferred embodiment of the polypeptide of the invention, showing interspecific specificity, which can be determined in preclinical testing on non-chimpanzee primates listed above, are also given, for example, in Schlereth et al. (Cancer Immunol. Immunother., 20 (2005), 1-12).

The term "toxicity", as used in this application, refers to the toxic effects of a drug, expressed in adverse events or severe adverse events. These side effects could be attributed to the lack of tolerance of the drug as a whole and / or the lack of local tolerance after administration. Toxicity could also include teratogenic or carcinogenic effects caused by this drug.

The term "safety", "safety in vivo" or "tolerance", as used in this application, defines the administration of a drug without inducing severe adverse events immediately after administration (local tolerance) and for a longer use of the drug. “Safety”, “safety in vivo” or “tolerance” can be evaluated, for example, at regular intervals during treatment and follow-up. Measurements include clinical evaluation of, for example, organ manifestations and screening for laboratory abnormalities. In accordance with the NCI-CTC and / or MedDRA standards, a clinical evaluation can be performed and a deviation relative to normal data recorded / encoded. Organ manifestations may include criteria such as allergy / immunology, blood / bone marrow, cardiac arrhythmia, coagulation, and the like, which are given, 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 and urinalysis, and studies of other body fluids, such as serum, plasma, lymphoid or cerebrospinal fluid, cerebrospinal fluid, and the like. Thus, safety can be assessed, for example, by medical examination, using imaging methods (i.e., ultrasound, X-rays, CT scans, magnetic resonance imaging (MRI)), other measurements using technical devices (i.e. electrocardiogram), the main indicators of the state of the body, by measuring laboratory parameters and recording adverse events. For example, adverse events in non-chimpanzee primates in the applications and methods of the invention can be investigated by histopathological and / or histochemical methods.

The term "effective and non-toxic dose", as used in this application, refers to a tolerated dose of a bispecific single chain antibody, as defined in this application, which is high enough to cause depletion of pathological cells, elimination of the tumor, reduction of the tumor or stabilization of the disease without or essentially no major toxic effects. Such effective and non-toxic doses can be determined, for example, in studies of the effects of increasing doses described in the art and should be lower than the dose inducing severe adverse side effects (dose limiting toxicity (DLT)).

The above terms are also referenced, for example, in the Preclinical safety assessment of biotechnology-derived pharmaceuticals S6; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting on July 16, 1997.

The invention further relates to a pharmaceutical composition comprising a polypeptide of the invention (i.e., a polypeptide comprising at least one binding domain capable of binding to an epitope of the human CD3 epsilon chain and a non-chimpanzee primate, the epitope being part of the amino acid sequence included in the group consisting of SEQ ID NO: 2, 4, 6 or 8 according to this invention, or obtained in accordance with the method according to the invention) for the prevention, treatment or amelioration of the disease, select of a proliferative disease, tumor disease, or immunological disorder. Preferably, said pharmaceutical composition further comprises suitable carrier, stabilizing and / or excipient compositions.

A further aspect of the invention relates to the use of a polypeptide, as defined herein above or prepared according to the method defined herein, for the manufacture of a pharmaceutical composition for the prevention, treatment or amelioration of a disease. Preferably, said disease is a proliferative disease, tumor disease, or immunological disorder. It is also preferred that said tumorous disease is a malignant disease, preferably cancer.

In another preferred embodiment of the use of the polypeptide of the invention, said pharmaceutical composition is suitable for administration in combination with an additional drug, i.e. as part of combination therapy. In said combination therapy, the active agent may possibly be included in the same pharmaceutical composition as the polypeptide of the invention, or may be included in a separate pharmaceutical composition. In this latter case, said separate pharmaceutical composition is suitable for use before administration, simultaneously with or after administration of said pharmaceutical composition comprising a polypeptide of the invention. The additional drug or pharmaceutical composition may be a non-protein compound or a protein compound. In the event that the additional drug is a protein compound, it is preferable that this protein compound can provide an activation signal for immune effector cells.

Preferably, said protein compound or non-protein compound can be administered simultaneously or non-simultaneously with a polypeptide of the invention, a nucleic acid molecule as defined herein above, a vector as defined herein above, or a host as defined herein above .

Another aspect of the invention relates to a method for preventing, treating, or ameliorating a disease in a subject in need thereof, comprising the step of administering an effective amount of a pharmaceutical composition of the invention. Preferably, said disease is a proliferative disease, tumor disease, or immunological disorder. Even more preferably, said tumorous disease is a malignant disease, preferably cancer.

In another preferred embodiment of the method of the invention, said pharmaceutical composition is suitable for administration in combination with an additional drug, i.e. as part of combination therapy. In said combination therapy, the active agent may possibly be included in the same pharmaceutical composition as the polypeptide of the invention, or may be included in a separate pharmaceutical composition. In this latter case, said separate pharmaceutical composition is suitable for use before administration, simultaneously with or after administration of said pharmaceutical composition comprising a polypeptide of the invention. The additional drug or pharmaceutical composition may be a non-protein compound or a protein compound. In the event that the additional drug is a protein compound, it is preferable that this protein compound can provide an activation signal for the effector cells of the immune system.

Preferably, said protein compound or non-protein compound can be administered simultaneously or non-simultaneously with a polypeptide of the invention, a nucleic acid molecule as defined herein above, a vector as defined herein above, or a host as defined herein above .

Preferably, for the method of the invention described above, said subject is a human.

In a further aspect, the invention relates to a kit comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention or a host of the invention.

The present invention is further characterized by the following list of items.

Item 1. A method for identifying (a) a polypeptide (s) containing interspecific specificity binding domain capable of binding to the human and non-chimpanzee primate CD3 epsilon (CD3ε) epitope, the process comprising the steps of:

(a) contacting the polypeptide (s) with the N-terminal fragment of the extracellular domain of CD3ε consisting of a maximum of 27 amino acids containing the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO: 381) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO: 382), fixed by its C-terminus on the solid phase;

(b) eluting the bound (s) polypeptide (s) from said fragment; and

(c) isolation of the polypeptide (s) from the eluate (b).

Preferably, the polypeptide (s) identified by (a) the aforementioned method of the invention is of human origin.

The present "method for identifying (a) the polypeptide (s)" is understood as a method for isolating one or more different polypeptides with the same specificity for a CD3ε extracellular domain fragment containing the Gln-Asp-Gly-Asn-Glu amino acid sequence at its N-terminus -Glu-Met-Gly (SEQ ID NO: 381) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO: 382), from a variety of candidate polypeptides, as well as a method for purifying a polypeptide from a solution . Non-limiting embodiments of a method for isolating one or more different polypeptides with the same specificity for a CD3ε extracellular domain fragment include methods for selecting antigen-specific binding structures, for example, panning methods commonly used for hybridoma screening, screening for transiently / stably transfected clones of eukaryotic cells- hosts or in phage display methods. A non-limiting example of the latter method for purifying a polypeptide from a solution is, for example, purifying a recombinantly expressed polypeptide from a culture supernatant or preparation of such a culture.

As indicated above, the fragment used in the method according to the invention is an N-terminal fragment of the extracellular domain of a CD3ε primate molecule. The amino acid sequence of the extracellular domain of the CD3ε molecule of various primates is presented in SEQ ID NO: 1, 3, 5, and 7. Two forms of the N-terminal octamer are shown in SEQ ID NO: 381 and 382. Preferably, this N-terminus is available without limitation to binding of the polypeptides to be identified according to the method of the invention. The term "available without limitation" in the context of the invention is understood as not having additional motives, such as His-tag. The interference of such a His tag with a binding molecule identified by the method of the invention is described in the attached Example 6.

According to the method of the invention, said fragment is fixed through its C-terminus on a solid phase. One skilled in the art will easily and without any inventive difficulties choose a suitable solid phase substrate depending on the embodiment of the method of the invention used. Examples of a solid support include, but are not limited to, granule type matrices (e.g., agarose granules, Sepharose granules, polystyrene granules, dextran granules), plates (culture plates or multi-well plates), as well as chips known, for example, from Biacore®. The choice of means and methods for fixing / immobilizing a fragment on said solid support depends on the choice of solid support. A commonly used fixation / immobilization method is through N-hydroxysuccinimide (NHS) ester binding. The chemistry underlying this binding as well as alternative methods of fixation / immobilization are known to those skilled in the art, for example from Hermanson "Bioconjugate Techniques", Academic Press, Inc. (1996). The following agents are usually used for fixation / immobilization on chromatographic substrates: NHS-activated Sepharose (e.g., HiTrap-NHS from GE Life Science - Amersham), CnBr-activated Sepharose (e.g., GE Life Science - Amersham), NHS-activated dextran granules ( Sigma) or activated polymethacrylate. These reagents can also be used in a batch approach. In addition, dextran granules containing iron oxide (e.g., available from Miltenyi) may be used in a batch approach. These granules can be used in combination with a magnet to separate the granules from the solution. The polypeptides can be immobilized on a Biacore chip (e.g., CM5 chips) by using NHS-activated carboxymethyldextran. The following examples of suitable solid supports are multi-well plates with reactive amino groups (e.g., Nunc Immobilizer ™ plates).

According to the method of the invention, said CD3 epsilon extracellular domain fragment can be coupled to the solid support directly or via an amino acid site, which may be a linker or other protein / polypeptide moiety. Alternatively, the extracellular domain of a CD3 epsilon can be linked directly through one or more adapter molecules.

Means and methods for evaluating a peptide associated with an immobilized epitope are well known in the art. The same is true for methods for isolating identifiable polypeptide (s) from the eluate.

In accordance with the invention, a method for isolating from a plurality of candidate polypeptides one or more different polypeptides with the same specificity for a CD3e extracellular domain fragment containing at its N-terminus the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-X-Gly, may include one or more stages of the following methods for the selection of antigen-specific structures.

CD3ε-specific binding molecules can be selected from antibody repertoires. The phage display library can be constructed based on standard procedures that are described, for example, in "Phage Display: A Laboratory Manual"; Ed. Barbas, Burton, Scott &Silverman; Cold Spring Harbor Laboratory Press, 2001. The format of the antibody fragments in the antibody library may be scFv, but in the general case, there may also be a Fab fragment or even a single domain antibody fragment. Naive libraries of antibody fragments can be used to isolate antibody fragments. For the selection of binding structures with the lowest possible immunogenicity during their subsequent therapeutic use, libraries of fragments of human antibodies may be preferred for the direct selection of fragments of human antibodies. In some cases, they can form the basis for synthetic antibody libraries (Knappik et al. J. Mol. Biol., 2000, 296: 57). The appropriate format may be Fab, scFv (as described below) or domain antibodies (dAb, reviewed in Holt et al., Trends Biotechnol., 2003, 21: 484 ff).

It is also known in the art that in many cases there is no source of human immune antibodies useful against the target antigen. Therefore, animals are immunized with this target antigen, and appropriate antibody libraries are isolated from animal tissue, for example, spleen or PBMC (peripheral blood mononuclear cells). The N-terminal fragment may be biotinylated or covalently linked to proteins of the type KLH (cochlear lymphocytic hemocyanin) or bovine serum albumin (BSA). According to general approaches, rodents are used for immunization. Some repertoires of non-human immune antibodies may be particularly beneficial for other reasons, for example, due to the presence of single domain antibodies (VHH) originating from camelid species (as described in Muyldermans, J. Biotechnol., 74: 277 ; De Genst et al., Dev. Como. Immunol., 2006, 30: 187 ff). Thus, the appropriate antibody library format may be Fab, scFv (as described below) or single domain antibodies (VHH).

In one possible approach, 10-week-old F1 mice from balb / c x C57black crosses can be immunized with whole cells, for example, expressing transmembrane EpCAM, demonstrating N-terminal amino acids 1-27 of the mature CD3ε chain as a translational fusion . Alternatively, mice can be immunized with a 1-27 CD3-epsilon-PC fusion protein (a suitable approach is described in the attached Example 2). After booster immunization (s), blood samples can be taken and the serum titer of antibodies against CD3-positive T cells can be tested, for example, in a FACS analysis. Typically, serum titers are significantly higher in immunized than in non-immunized animals.

Immunized animals can be the basis for creating immune antibody libraries. Examples of such libraries include phage display libraries. Such libraries can generally be created based on standard techniques that are described, for example, in "Phage Display: A Laboratory Manual"; Ed. Barbas, Burton, Scott &Silverman; Cold Spring Harbor Laboratory Press, 2001.

In addition, non-human antibodies can be humanized through phage display by creating libraries of more variable antibodies, which can subsequently be enriched against binders during selection.

In a phage display approach, any of the phage pools that exposes antibody libraries forms the basis for the selection of binding structures using the appropriate antigen as the target molecule. The central stage in which antigen-specific, antigen-linked phages are isolated is referred to as panning. By exposing antibody fragments to the surface of phages, this general method is called phage display. One preferred selection method is the use of small proteins, such as the N2 domain of a filamentous phage, translationally fused to the N-terminus of scFv exposed by this phage. Another display method known in the art that can be used to isolate binding structures is the ribosomal display method (reviewed in Groves & Osbourn, Expert. Opin. Biol. Ther., 2005, 5: 125 ff; Lipovsek & Pluckthun, J. Immunol. Methods, 2004, 290: 52 ff).

To demonstrate the binding of scFv-containing phage particles to a 1-27 CD3ε-Fc fusion protein, a phage library carrying a cloned scFv repertoire can be obtained from the appropriate culture supernatant using PEG (polyethylene glycol). scFv-containing phage particles can be incubated with the immobilized CD3ε-Fc fusion protein. The immobilized CD3ε-Fc fusion protein can be coated onto the solid phase. Binding structures can be eluted and this eluate can be used to infect a fresh portion of uninfected bacterial hosts. Bacterial hosts successfully transduced with a phagmid copy encoding the human scFv fragment can be again selected for resistance to carbenicillin and then infected, for example, with the helper phage VCMS 13 to start the second round of antibody display and in vitro selection. Normally, a total of 4 to 5 selection cycles is carried out.

The binding of isolated binding structures can be tested on CD3 epsilon-positive Jurkat cells, HPBall cells, PBMCs or transfected eukaryotic cells that carry the N-terminal CD3ε sequence fused to the surface-exposed EpCAM using flow cytometric analysis (see attached Example 4) .

Paragraph 2. The method of paragraph 1, wherein the polypeptide (s) contains (at) an identified binding domain as a first binding domain and a second binding domain capable of binding to a cell surface antigen.

Monoclonal antibodies that bind to the corresponding cell surface antigens of both a human and a non-chimpanzee primate can be used to create a second binding domain of a polypeptide identified by the method of the invention, for example, bispecific single chain antibodies, as defined herein. Appropriate binding domains for a bispecific polypeptide, as defined herein, can be obtained, for example, from cross-species specific monoclonal antibodies using recombinant methods described in the art. A monoclonal antibody that binds to a human cell surface antigen and to a homolog of said non-chimpanzee primate cell surface antigen can be tested in the FACS assays described above. In addition, hybridoma techniques described in the literature (Milstein and Kohler, Nature, 256 (1975), 495-7) can be used to create antibodies with interspecific specificity. For example, mice can be immunized alternately with human CD33 and non-chimpanzee primate. Hybridoma cells producing antibodies with interspecific specificity are isolated from these mice by hybridoma technology and analyzed by FACS as described above. The creation and analysis of bispecific polypeptides, such as bispecific single chain antibodies demonstrating interspecific specificity, which are described in this invention are shown in the following Examples. The advantages of bispecific single chain antibodies exhibiting interspecific specificity include the items listed below.

Clause 3. The method of clause 2, wherein the second binding domain binds to a cell surface antigen of a human and / or non-chimpanzee primate.

Clause 4. The method according to any one of Claims 1-3, wherein the first binding domain is an antibody.

Paragraph 5. The method of paragraph 4, wherein the antibody is a single chain antibody.

Clause 6. The method according to any one of Claims 2-5, wherein the second binding domain is an antibody.

Clause 7. The method according to any one of Claims 1-6, wherein the CD3ε extracellular domain fragment consists of one or more polypeptide fragments having an amino acid sequence selected from any sequence shown in SEQ ID NO: 2, 4, 6, or 8.

Paragraph 8. The method according to paragraph 7, where the length of the specified 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.

Clause 9. The method according to any one of Claims 1-8, wherein the identification method is a screening method for a plurality of polypeptides containing a cross-species specific binding domain capable of binding to a human CD3ε epitope and a non-chimpanzee primate.

Paragraph 10. The method according to any one of paragraphs 1-8, wherein the identification method is a method for purification / isolation of a polypeptide comprising a cross-species specific binding domain capable of binding to a human CD3ε epitope and a non-chimpanzee primate.

Item 11. Use of an N-terminal fragment of the extracellular domain of CD3ε consisting of a maximum of 27 amino acids containing the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO: 381) or Gln-Asp-Gly- Asn-Glu-Glu-lle-Gly (SEQ ID NO: 382) to create a cross-species specific binding domain.

In accordance with the indicated use according to the invention, it is preferable that the created cross-species specific binding domain be of human origin.

Clause 12. The use of Claim 11, wherein the cross-species specific binding domain is an antibody.

Clause 13. The use of Claim 12, wherein the antibody is a single chain antibody.

Clause 14. The use of clauses 12-13, wherein the antibody is a bispecific antibody.

These and other embodiments are described and included in the description and Examples of the present invention. Recombinant techniques and immunological methods are described, 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; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual; Cold Spring Laboratory Press, 2002. Further literature regarding any of the antibodies, methods, uses, and compounds used in accordance with the present invention can be extracted from public libraries and databases using, for example, electronic devices. For example, you can use the shared Medline database, accessible via the Internet, for example, at http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Additional databases and addresses, such as http://www.ncbi.nlm.nih.gov/ or listed on the home page of the EMBL service (European Molecular Biology Laboratory) under the address http://www.embl.de/services/index .html are known to those skilled in the art and can also be obtained using, for example, http://www.google.com.

The graphic materials show the following.

Figure 1

A fusion construct of N-terminal amino acids 1-27 of primates CD3 epsilon with heterologous soluble protein.

Figure 2

In FIG. shows the average absorption values of duplicate samples measured in an ELISA (enzyme-linked immunosorbent assay), which determines the presence of a construct consisting of N-terminal amino acids 1-27 of the mature human CD3 epsilon chain fused to the hinge and Fc-gamma region of the human IgG1 and the C-terminal 6-histidine tag, in the supernatant (SN) of transiently transfected cells 293. The first column, labeled “27 aa huCD3E,” shows the average absorbance for the construct, and the second column, labeled “irrelevant. SN” , shows the average value for the supernatant of 293 cells transfected with an irrelevant construct as a negative control. A comparison of the values obtained for this construct with those obtained for the negative control clearly demonstrates the presence of a recombinant construct.

Figure 3

In FIG. shows the average absorbance values of samples repeated in four copies, measured in an ELISA assay that determines the binding of anti-CD3 binding molecules with interspecific specificity in the form of crude preparations of single chain antibodies expressed in the periplasmic space, with a construct containing N-terminal amino acids 1-27 mature human CD3 epsilon chain fused to the hinge and Fc gamma region of human IgG1 and His6 C-terminal tag. The columns show, from left to right, the average absorbance values for specificities designated as A2J HLP, I2C HLP, E2M HLP, F70 HLP, G4H HLP, H2C HLP, E1L HLP, F12Q HLP, F6A HLP and H1E HLP. The rightmost column labeled “Neg. Control.” Shows the average absorbance for a single-chain mouse anti-human CD3 antibody preparation as a negative control. Comparison of the values obtained for anti-CD3 specificities with those obtained for the negative control (Neg. Control) clearly demonstrates the strong binding of anti-CD3 specificities to the N-terminal amino acids 1-27 of the mature human CD3 epsilon chain.

Figure 4

The fused construct of N-terminal amino acids 1-27 of primates CD3 epsilon with a heterologous membrane-bound protein.

Figure 5

Overlapping histograms of different transfectants tested in the FACS analysis, determining the presence of recombinant transmembrane fusion proteins consisting of EpCAM cynomolgus monkey and N-terminal amino acids 1-27 epsilon chains of human CD3, marmoset, tamarin, squirrel monkey and domestic pig, respectively. The overlapping histograms, from left to right and from top to bottom, show results for transfectants expressing constructs containing 27-mer, 27-mer marmoset, 27-mer tamarin, 27-mer squirrel monkey and 27-mer pig, respectively. For individual overlaps, a thin line represents a sample incubated with PBS (phosphate buffered saline) containing 2% FCS (fetal calf serum) instead of an anti-Flag M2 antibody as a negative control, and a thick line shows a sample incubated with anti Flag M2 antibody. For each construct, histogram overlapping shows the binding of the anti-Flag M2 antibody to transfectants, which clearly demonstrates the expression of recombinant constructs in transfectants.

6

Overlapping histograms of different transfectants tested in the FACS analysis, which determines the binding of anti-CD3 binding molecules with interspecific specificity in the form of crude preparations of single-chain antibodies expressed in the periplasmic space, with N-terminal amino acids 1-27 of the human CD3 epsilon chain, marmosets, tamarine and squirrel monkeys, respectively, merged with EpCAM cynomolgus macaque.

Figa

Overlapping histograms, from left to right and from top to bottom, show results for transfectants expressing 1-27 CD3-EpCAM containing 27-mer person, tested using CD3-specific binding molecules designated as H2C HLP, F12Q HLP, E2M HLP and G4H HLP , respectively.

6B

Overlapping histograms, from left to right and from top to bottom, show the results for transfectants expressing 1-27 CD3-EpCAM containing 27 mer marmosets tested using CD3-specific binding molecules designated as H2C HLP, F12Q HLP, E2M HLP and G4H HLP , respectively.

6C

Overlapping histograms, from left to right and from top to bottom, show results for transfectants expressing 1-27 CD3-EpCAM containing 27-mer tamarin tested using CD3-specific binding molecules designated as H2C HLP, F12Q HLP, E2M HLP and G4H HLP , respectively.

Fig.6D

Overlapping histograms, from left to right and from top to bottom, show results for transfectants expressing 1-27 CD3-EpCAM containing 27-mer squirrel monkeys tested using CD3-specific binding molecules designated as H2C HLP, F12Q HLP, E2M HLP and G4H HLP, respectively.

6E

The histograms, from left to right and from top to bottom, show the results for transfectants expressing 1-27 CD3-EpCAM containing 27-mer pigs tested using CD3-specific binding molecules designated as H2C HLP, F12Q HLP, E2M HLP and G4H HLP , respectively.

For individual overlaps, a thin line represents a sample incubated with a single-chain mouse anti-human CD3 antibody preparation as a negative control, and a thick line shows a sample incubated with the corresponding indicated anti-CD3 binding molecules. Given the lack of binding to pig 27-mer transfectants and the expression levels of the constructs shown in Figure 5, histogram overlaps demonstrate specific and strong binding of the tested anti-CD3 specificities of human bispecific single chain antibodies with full interspecific specificity to cells expressing recombinant transmembrane fused proteins containing N-terminal amino acids 1-27 of the epsilon chain of human CD3, marmoset, tamarin and squirrel monkeys, respectively, fused to EpCAM javans macaque, and thus demonstrate the interspecific specificity of anti-CD3 binding molecules against many primates.

7

FACS analysis to determine human CD3 epsilon on transfected murine EL4 T cells. Graphical analysis demonstrates overlapping histograms. The bold line shows transfected cells incubated with UCHT-1 antibody against human CD3. The thin line represents cells incubated with the mouse control of the IgG1 isotype. The binding of the anti-CD3 UCHT1 antibody clearly demonstrates expression of the human CD3 epsilon chain on the cell surface of transfected murine EL4 T cells.

Fig. 8

Binding of interspecific specific anti-CD3 antibodies to alanine mutants in an alanine scanning experiment. The columns in separate graphic materials, from left to right and from top to bottom, show the binding values calculated in arbitrary units on a logarithmic scale for the wild-type transfectant (WT) and for all alanine mutants for position 1-27. The binding values are calculated using the following formula:

In this equation, value_Sample means the amount of binding in arbitrary units representing the degree of binding of a specific anti-CD3 antibody to a specific alanine mutant, as shown in this Fig., Sample means the geometric mean fluorescence obtained for a specific anti-CD3 antibody analyzed on specific alanine scanning transfectant, neg_Contr. means the geometric mean fluorescence obtained for the negative control analyzed on a specific alanine mutant, UCHT-1 means the geometric mean fluorescence obtained for the antibody UCHT-1 analyzed on a specific alanine mutant, WT means the geometric mean fluorescence obtained for a specific anti -CD3 antibodies analyzed on a wild-type transfectant, x indicates the corresponding transfectant, y indicates the corresponding an Ti-CD3 antibody, a wt indicates that the corresponding transfectant is of the wild type. The individual positions of the alanine mutants are labeled with the wild letter amino acid code and position number.

Figa

In FIG. shows the results for the anti-CD3 antibody A2J HLP with interspecific specificity expressed as a chimeric IgG molecule. Reduced binding activity is observed for mutations with substitution for alanine at position 4 (asparagine), at position 23 (threonine) and at position 25 (isoleucine). A complete loss of binding is observed for mutations with substitution for alanine at position 1 (glutamine), at position 2 (aspartate), at position 3 (glycine) and at position 5 (glutamate).

Figv

On Fig. Shows the results for anti-CD3 antibodies E2M HLP with interspecific specificity expressed as a chimeric IgG molecule. Reduced binding activity is observed for mutations with substitution for alanine at position 4 (asparagine), at position 23 (threonine) and at position 25 (isoleucine). A complete loss of binding is observed for mutations with substitution for alanine at position 1 (glutamine), at position 2 (aspartate), at position 3 (glycine) and at position 5 (glutamate).

Figs

In Fig. Shows the results for anti-CD3 antibodies H2C HLP with interspecific specificity expressed as a chimeric IgG molecule. Reduced binding activity is observed for mutations with substitution for alanine at position 4 (asparagine). Complete loss of binding is observed for mutations on alanine glutamine at position 1 (glutamine), at position 2 (aspartate), at position 3 (glycine) and at position 5 (glutamate).

Fig.8D

shows the results for the cross-species specific F12Q HLP anti-CD3 antibody tested as a periplasmatically expressed single chain antibody. A complete loss of binding is observed for mutations with substitution for alanine at position 1 (glutamine), at position 2 (aspartate), at position 3 (glycine) and at position 5 (glutamate).

Fig.9

FACS analysis, which determines the binding of the anti-CD3 binding H2C HLP molecule with interspecific specificity to human CD3 containing and not containing an N-terminal His6-tag.

Overlapping histograms is carried out on an EL4 cell line transfected with a wild-type human CD3 epsilon chain (left histogram) or a human CD3 epsilon chain containing an N-terminal His6 tag (right histogram) tested in a FACS analysis that determines the binding of the H2C binding molecule HLP with interspecific specificity. Samples are incubated with the control of the corresponding isotype as a negative control (thin line), UCHT-1 anti-human CD3 antibody as a positive control (dashed line), and interspecific specific anti-CD3 antibody H2C HLP in the form of a chimeric IgG molecule (bold line) .

Overlapping histograms show comparable binding of the UCHT-1 antibody to both transfectants compared to the isotypic control demonstrating the expression of both recombinant constructs. Overlapping histograms also demonstrate the binding of the anti-CD3 binding H2C HLP molecule only to the wild-type human CD3 epsilon chain, but not to the human His6-CD3 epsilon chain. These results demonstrate that the presence of a free N-terminus is essential for the binding of the interspecific specificity of the anti-CD3 binding H2C HLP molecule.

Figure 10

Saturated binding of EGFR-21-63 LH x H2C to CD3 positive human PBMCs to determine KD CD3 binding values on cells using FACS analysis. The analysis is carried out as described in Example 7.

11

FACS analysis of the binding of these interspecific bispecific single chain constructs to CHO cells transfected with human EGFR, CDB + human HPB-ALL T cell line, CHO cells transfected with cynomolgus EGFR, and 4119 LnPx macaque T cell line. FACS staining is carried out as described in Example 12. The bold line represents cells incubated with purified protein at a concentration of 2 μg / ml, which are then incubated with anti-his-antibody and PE-labeled detection antibody. The thin line in the histograms reflects the negative control: cells incubated only with anti-his-antibody and a detecting antibody.

12

Cytotoxic activity induced by these cross-species specific EGFR-specific single chain constructs redirected to the indicated target cell lines. A) and B): stimulated human CD4- / CD56-PBMCs are used as effector cells, CHO cells transfected with human EGFR as target cells. The analysis is carried out as described in Example 13.

Fig.13

Cytotoxic activity induced by these cross-species specific EGFR-specific single chain constructs redirected to the indicated target cell lines. A) and B): The macaque T-cell line 4119 LnPx is used as effector cells, CHO cells transfected with cynomolgus EGFR as target cells. The analysis is carried out as described in Example 13.

Fig.14

FACS analysis of the binding of these interspecific bispecific single chain constructs to CHO cells transfected with human MCSP D3, CDB + human T-cell line HPB-ALL, CHO cells transfected with MCSP D3 cynomolgus macaques, and Macaque 4119 T-cell line LnPx. FACS staining was carried out as described in Example 17. The bold line represents cells incubated with purified protein at a concentration of 2 μg / ml, which are then incubated with anti-his antibody and PE-labeled detection antibody. The thin line in the histograms reflects the negative control: cells incubated only with anti-his-antibody and a detecting antibody.

Fig.15

FACS analysis of the binding of these bispecific single chain constructs with interspecific specificity to CHO cells transfected with human MCSP D3, HPB-ALL CD3 + human T-cell line, CHO cells transfected with cynomolgus MCSP D3 Macaque, and 4119 LnPx macaque T-cell line . FACS staining was carried out as described in Example 17. The bold line represents cells incubated with purified protein at a concentration of 2 μg / ml, which are then incubated with anti-his antibody and PE-labeled detection antibody. The thin line in the histograms reflects the negative control: cells incubated only with anti-his-antibody and a detecting antibody.

Fig.16

FACS analysis of the binding of these interspecific bispecific single chain constructs to CHO cells transfected with human MCSP D3, CDB + human T-cell line HPB-ALL, CHO cells transfected with MCSP D3 cynomolgus macaques, and Macaque 4119 T-cell line LnPx. FACS staining was carried out as described in Example 17. The bold line represents cells incubated with purified protein at a concentration of 2 μg / ml, which are then incubated with anti-his antibody and PE-labeled detection antibody. The thin line in the histograms reflects the negative control: cells incubated only with anti-his-antibody and a detecting antibody.

Fig.17

Cytotoxic activity induced by these cross-species specific MCSP-specific single chain constructs redirected to the indicated target cell lines. A): stimulated human CD4- / CD56-PBMCs are used as effector cells, CHO cells transfected with human MCSP D3 as target cells. C): T-cell line of macaque 4119 LnPx used as effector cells, cells SNO, transfected with MCSP D3 cynomolgus macaque, as target cells. The analysis is carried out as described in Example 18.

Fig. 18

Cytotoxic activity induced by these cross-species specific MCSP-specific single chain constructs redirected to the indicated target cell lines. A) and B): The T-cell line of macaque 4119 LnPx is used as effector cells, CHO cells transfected with MCSP D3 of cynomolgus macaque as target cells. The analysis is carried out as described in Example 18.

Fig.19

Cytotoxic activity induced by these cross-species specific MCSP-specific single chain constructs redirected to the indicated target cell lines.

A) and B): stimulated human CD4- / CD56-PBMCs are used as effector cells, CHO cells transfected with human MCSP D3 as target cells. The analysis is carried out as described in Example 18.

Fig.20

Cytotoxic activity induced by these cross-species specific MCSP-specific single chain constructs redirected to the indicated target cell lines. A): stimulated human CD4- / CD56-PBMCs are used as effector cells, CHO cells transfected with human MCSP D3 as target cells. C): T-cell line of macaque 4119 LnPx used as effector cells, cells SNO, transfected with MCSP D3 cynomolgus macaque, as target cells. The analysis is carried out as described in Example 18.

Fig.21

Cytotoxic activity induced by these cross-species specific MCSP-specific single chain constructs redirected to the indicated target cell lines. A): stimulated human CD4- / CD56-PBMCs are used as effector cells, CHO cells transfected with human MCSP D3 as target cells. C): T-cell line of macaque 4119 LnPx used as effector cells, cells SNO, transfected with MCSP D3 cynomolgus macaque, as target cells. The analysis is carried out as described in Example 18.

Fig.22

Plasma stability of interspecific specific MCSP and CD3 bispecific single chain antibodies tested by measuring the cytotoxic activity induced by samples of these single chain constructs incubated in the presence of 50% human plasma at 37 ° C and 4 ° C for 24 hours, respectively, or with the addition of 50% of human plasma immediately before the cytotoxicity test or without the addition of plasma. CHO cells transfected with human MCSP are used as target cell lines, and human CD4- / CD56-PBMC-stimulated cells are used as effector cells. The analysis is carried out as described in Example 19.

Fig.23

FACS analysis of the binding of these interspecific bispecific single chain constructs with CHO cells transfected with human HER2, with CD3 + human HPB-ALL T cell line, with CHO cells transfected with macaque HER2 and macaque T cell line 4119 LnPx, respectively. FACS staining is carried out as described in Example 23.4. Bold lines represent cells incubated with a purified bispecific single chain construct at a concentration of 2 μg / ml. Thin lines reflect negative controls. PBS with 2% FCS was used as a negative control. For each interspecific specific bispecific single chain construct, histogram overlapping demonstrates the specific binding of this construct to human and macaque HER2 and human and macaque CD3.

Fig.24

The diagrams show the results of chromium release assays that measure the cytotoxic activity induced by these HEP2-specific single chain constructs with interspecific specificity redirected to the indicated target cell lines. The effector cells used are also indicated. Assays are carried out as described in Example 23.5. The diagrams clearly demonstrate for each shown construct a strong increase in the cytotoxic activity of human and macaque effector cells against CHO cells transfected with human and macaque HER2, respectively.

Fig.25

CD3-specific ELISA analysis of periplasmic preparations containing Flag-labeled protein scFv fragments from selected clones. The periplasmic preparations of soluble scFv protein fragments were added to the wells of an ELISA plate, which were coated with a soluble human [CD3 epsilon (aa 1-27)] -Fc (aa amino acid) fusion protein and were further blocked with PBS with 3% BSA. Determination was performed using a monoclonal anti-Flag-biotin-labeled antibody, then conjugated to streptavidin peroxidase. The ELISA was developed with an ABTS substrate solution (2,2'-azinobis (3-ethylbenzothiozolin-6-sulfonate). OD (optical density) values (axis y) were measured at 405 nm using an ELISA reader. Clone names are shown on the x axis.

Fig.26

ELISA analysis of periplasmic preparations containing Flag-labeled protein scFv fragments from selected clones. The same periplasmic preparations of soluble scFv protein fragments as in FIG. 25 were added to the wells of an ELISA plate that were not coated with a soluble [CD3 epsilon (aa 1-27) human] -Fc fusion protein but were coated with IgG1 human (Sigma) and blocked with 3% BSA in PBS.

Determination was performed using a monoclonal anti-Flag-biotin-labeled antibody, then conjugated to streptavidin peroxidase. ELISA showed an ABTS substrate solution. OD values (y axis) were measured at 405 nm using an ELISA reader. The names of the clones are presented on the x axis.

The present invention is further described by the following non-limiting illustrative examples, which provide a better understanding of the present invention and many of its advantages.

EXAMPLES

1. Identification of CD3 epsilon sequences from non-human primate blood samples

Blood samples of the following non-human primates were used to identify CD3 epsilon: Callithrix jacchus, Saguinus oedipus, and Saimiris ciureus. To isolate total cellular RNA, fresh heparin-treated whole blood samples were prepared according to the manufacturer's protocol (QIAamp RNA Blood Mini Kit, Qiagen). Transcribed mRNA was transcribed into cDNA in accordance with published protocols. Briefly, 10 μl of precipitated RNA was incubated with 1.2 μl of a 10-fold hexanucleotide mixture (Roche) at 70 ° C. for 10 minutes and stored on ice. A reaction mixture was added containing 4 μl of 5x superscript II buffer, 0.2 μl of 0.1 M dithiothreitol, 0.8 μl of superscript II (Invitrogen), 1.2 μl of deoxyribonucleoside triphosphates (25 μM), 0.8 μl of an RNase inhibitor (Roche) and 1.8 μl of water containing no DNases and RNases (Roth). The reaction mixture was incubated at room temperature for 10 minutes, followed by incubation at 42 ° C for 50 minutes and at 90 ° C for 5 minutes. The reaction mixture was allowed to cool on ice, then 0.8 μl of RNase H (1 unit / μl, Roche) was added and incubated for 20 minutes at 37 ° C.

The first strand cDNA from each sample was subjected to 35 separate polymerase chain reaction cycles using Tag (Sigma) DNA polymerase and the following primer combination constructed from the database: direct primer 5'-AGAGTTCTGGGCCTCTGC-3 '(SEQ ID NO: 377) ; reverse primer 5'-CGGATGGGCTCATAGTCTG-3 '(SEQ ID NO: 378). Amplified zones 550 bp isolated from the gel (Gel Extraction Kit, Qiagen) and sequenced (Sequiserve, Vaterstetten / Germany, see sequence listing).

CD3 epsilon Callithrix iacchus

Nucleotides

CAGGACGGTAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACATGAAATAAAATGGCTCGTAAATAGTCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAGGACTTTTCGGAAATGGAGCAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACTCCCGCAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGTGAGAACTGCGTGGAGGTGGAT

Amino acids

QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPAEEASHYLYLKARVCENCVEVD

CD3 epsilon Saguinus oedipus

Nucleotides

CAGGACGGTAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACATGAAATAAAATGGCTTGTAAATAGTCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAGGATTTTTCGGAAATGGAGCAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACTCCCGCAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGTGAGAACTGCGTGGAGGTGGAT

Amino acids

QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPAEEASHYLYLKARVCENCVEVD

CD3 epsilon Saimiri sciureus

Nucleotides

CAGGACGGTAATGAAGAGATTGGTGATACTACCCAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACAGGAAATAAAATGGCTCGTAAATGATCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAAGATTTTTCAGAAATGGAACAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACCCCCACAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGT GAGAACTGCGTGGAGGTGGAT

Amino acids

QDGNEEIGDTTQNPYKVSISGTTVTLTCPRYDGQEIKWLVNDQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPTEEASHYLYLKARVCENCVEVD

2. The creation of single-chain fragments of antibodies (scFv) with interspecific specificity, binding to the N-terminal amino acids 1-27 of human CD3 epsilon and various non-chimpanzee primates

2.1. Immunization of mice using the N-terminus of a CD3 epsilon isolated from its native CD3 environment by fusion with a heterologous soluble protein

Ten-week-old F1 mice from balb / c x C57black crosses were immunized with a CD3 epsilon-Fc fusion protein carrying the majority of the N-terminal amino acids (1-27) of the mature human CD3 (1-27 CD3-Fc) epsilon chain and / or an ordinary squirrel monkey. For this, 40 μg of fused protein 1-27 CD3-Fc together with 10 nmol of a thioate-modified CpG oligonucleotide (5'-tccatgacgttcctgatgct-3 ') in 300 μl of PBS per mouse was injected by intraperitoneal injection. Mice received booster immunizations after 21, 42, and possibly 63 days in the same way. Ten days after the first booster immunization, blood samples were taken and the serum antibody titer against the 1-27 CD3-FC fusion protein was tested using ELISA. Additionally, the titer against the CD3-positive human HPBall T cell line was tested using flow cytometry according to standard protocols. Serum titers were significantly higher in immunized than in non-immunized animals.

2.2. Creation of a murine antibody scFv immune library: constructing a combinatorial antibody library and phage display

Three days after the last injection, mouse spleen cells were harvested to obtain total RNA according to standard protocols.

Using RT-PCR (reverse transcription polymerase chain reaction) on mouse spleen RNA using VK- and VH-specific primers, a library of DNA fragments of the variable region of the light chain (kappa) (VK) and the variable region of the heavy chain (VH) of immunoglobulins were constructed (Ig) mice. cDNA was synthesized according to standard protocols.

The primers were designed to create a recognition site for 5'-Xhol and 3'-BstEII for amplified heavy chain V fragments and a recognition site for 5'-Sacl and 3'-Spel for amplified VK DNA fragments. For PCR amplification of VH DNA fragments, each of eight different primers specific for the 5'-VH family (MVH1 (GC) AG GTG CAG STC GAG GAG TCA GGA CCT; MVH2 GAG GTC CAG CTC GAG CAG TCT GGA CCT; MVH3 CAG GTC CAA STS GAG CAG CCT GGG GCT; MVH4 GAG GTT CAG CTC GAG CAG TCT GGG GCA; MVH5 GA (AG) GTG AAG CTC GAG GAG TCT GGA GGA; MVH6 GAG GTG AAG CTT CTC GAG TCT GGA GGT; MVH7 GAA GTG AAG GAG GAG TCT GGG GGA; MVH8 GAG GTT CAG CTC GAG CAG TCT GGA GCT) were combined with one 3'-VH primer (3'MuVHBstEII tga gga gac ggt gac cgt ggt ccc ttg gcc cca g); for PCR amplification of fragments of the VK chain, each of seven different primers specific for the 5'-VK family (MUVK1 CCA GTT CCG AGC TCG TTG TGA STS AGG AAT ST; MUVK2 CCA GTT CCG AGC TCG TGT TGA CGC AGC CGC CC; MUVK3 CCA GTT CCG AGC TCG TGC TCA VCC AGT STS CA; MUVK4 CCA GTT CCG AGC TSS AGA TGA CCC AGT STS CA; MUVK5 CCA GAT GTG AGC TCG TGA TGA CCC AGA STS CA; MUVK6 CCA GAT GTG AGC TCG TCA TGA TGA ; MUVK7 CCA GTT CCG AGC TCG TGA TGA CAC AGT CTC CA) were combined with one 3'-VK primer (3'MuVkHindIII / BsiW1 tgg tgc act agt cgt acg ttt gat ctc aag ctt ggt ccc).

The following PCR program was used for amplification: denaturation at 94 ° C for 20 s; annealing of the primers at 52 ° C for 50 s and elongation of the primers at 72 ° C for 60 s; and 40 cycles, then final extension for 10 min at 72 ° C.

450 ng of kappa light chain fragments (Sacl-Spel cleaved) were ligated with 1400 ng of pComb3H5Bhis phagemid (Sacl-Spel cleaved; large fragment). The resulting combinatorial antibody library was then transformed into 300 μl of Escherichia coli XL1 Blue electrocompetent cells by electroporation (2.5 kV; cuvette with 0.2 cm slit; 25 μF; 200 Ohm, Biorad gene-pulser), resulting in a library of size More than 107 independent clones. After one hour of phenotypic expression, positive transformants were selected for carbenicillin resistance encoded by the pComb3H5BHis vector in 100 ml liquid super (broth) culture overnight. The cells were then harvested by centrifugation and the plasmid was obtained using a commercially available plasmid preparation kit (Qiagen).

2800 ng of this plasmid DNA containing the VK library (Xhol-BstEll cleaved; large fragment) was ligated with 900 ng heavy chain V fragments (Xhol-BstEll cleaved), and again transformed into two aliquots of 300 μl of E. competent cells. coli XL1 Blue using electroporation (2.5 kV; cuvette with a slit of 0.2 cm; 25 μF, 200 Ohm), resulting in a library of VH-VK scFv (single chain variable fragment) with a total size of more than 107 independent clones.

After phenotypic expression and slow adaptation to carbenicillin, E. coli cells containing the antibody library were transferred to SB-carbenicillin (50 μg / ml) selective medium. E. coli cells containing the antibody library were then infected with an infectious dose equal to 1012 particles of the helper phage VCSM13, leading to the production and secretion of filamentous phage M13, where the phage particle contains a single-stranded pComb3H5BHis-flHK that encodes a murine scFv fragment corresponding to scFv, and protein in the form of translational fusion with the envelope protein of the III phage. This pool of phages exhibiting an antibody library was then used to select antigen binding structures.

2.3. Selection of CD3-specific binding agents based on phage display

A phage library carrying the cloned scFv repertoire was obtained from the corresponding culture supernatant by precipitation with PEG8000 / NaCl and centrifugation. Approximately 10 ¹¹ to 10 ¹² scFv phage particles were resuspended in 0.4 ml PBS / 0.1% BSA and incubated with 105-107 Jurkat cells (CD3 positive human T cell line) for 1 hour on ice with weak stirring. These Jurkat cells were previously grown in RPMI medium enriched in fetal calf serum (10%), glutamine and penicillin / streptomycin, collected by centrifugation, washed in PBS and resuspended in PBS / 1% FCS (containing sodium azide). scFv-carrying phages that did not specifically bind to Jurkat cells were removed through five washing steps using PBS / 1% FCS (containing sodium azide). After washing, the bound structures were eluted from the cells by resuspending the cells in HCl-glycine pH 2.2 buffer (10 min incubation followed by vigorous stirring) and after neutralization with 2 M Tris pH 12 eluate was used to infect a fresh uninfected E. coli XL1 Blue culture (OD600 is greater than 0.5). An E. coli culture containing E. coli cells successfully transduced with a phagmid copy encoding a human scFv fragment was again selected for carbenicillin resistance and then infected with the helper phage VCMS 13 in order to start the second round of antibody exposure and in vitro selection . Usually, a total of 4 to 5 selection cycles was carried out.

2.4. Screening for CD3-specific binding agents

Plasmid DNA corresponding to 4-5 panning cycles was isolated from E. coli cultures after selection. To obtain soluble scFv protein, VH-VL-DNA fragments were excised from plasmids (Xhol-Spel). These fragments were cloned through the same restriction sites into the pComb3H5BFlag / His plasmid, which differs from the original pComb3H5BHis in that the expressed construct (e.g. scFv) includes a Flag tag (TGD YKDDDDK) between the scFv and His6 tag and additional phage proteins are deleted. After ligation, each pool (various rounds of panning) of plasmid DNA was transformed into 100 μl of heat-shock competent E. coli TG1 or XLI blue and plated on carbenicillin-LB agar. Single colonies were transferred by chipping in 100 μl of LB-carb (50 μg / ml).

E. coli transformed with pComb3H5BHis containing the VL and VH segments produce significant soluble scFv after excision of the gene III fragment and induction with 1 mM IPTG (isopropylthiogalactopyranoside). Thanks to a suitable signal sequence, the scFv chain is exported to the periplasm, where it folds into a functional conformation.

Single E. coli TG1 bacterial colonies from transformation plates were selected to obtain small amounts of periplasm and grown on SB medium (e.g. 10 ml) supplemented with 20 mM MgCl ₂ and carbenicillin (50 μg / ml) and resuspended in PBS (e.g. , 1 ml) after collection. As a result of four cycles of freezing at -70 ° C and thawing at 37 ° C, the outer membrane of bacteria was destroyed by temperature shock and soluble periplasmic proteins, including scFv, were released into the supernatant. After removal of the intact cells and cell debris by centrifugation, the supernatant containing human scFv against human CD3 was collected and used for further research.

2.5. Identification of CD3-specific binding agents

The binding of isolated scFvs was tested by flow cytometry on eukaryotic cells expressing a heterologous protein on its surface, representing at its N-terminus the first 27 N-terminal amino acids of CD3 epsilon.

As described in Example 4, the first amino acids 1-27 of the N-terminal sequence of the mature epsilon chain of the CD3 human T cell receptor complex (amino acid sequence: QDGNEEMGGITQTPYKVSISGTTVILT) were fused to the N-terminus of the EpCAM transmembrane protein so that the N-terminus was located on the outside of the cell. In addition, a FLAG epitope was inserted between the N-terminal 1-27 CD3 epsilon sequence and the EpCAM sequence. This fusion product was expressed in human embryonic kidney cells (HEK) and Chinese hamster ovary cells (CHO).

Eukaryotic cells exhibiting most of the 27 N-terminal amino acids of mature CD3 epsilon of other primates were obtained in the same way for Saimiri sciureus (common squirrel monkey) (CD3 epsilon N-terminal amino acid sequence: QDGNEEIGDTTQNPYKVSISGTTVTl3-, for epsilon N-terminal amino acid sequence: QPGNEEMGPTTQNPYKVSISGTTVTLT) and for Saguinus oedipus (CD3 epsilon N-terminal amino acid sequence: QPGNEEMGPTTQNPYKVSISGTTVTLT).

For analysis by flow cytometry, 2.5 × 10 ⁵ cells are incubated with 50 μl of the supernatant or with 5 μg / ml of purified constructs in 50 μl of PBS with 2% FCS. Structural binding was determined using an anti-His antibody (Penta-His antibody not containing BSA, Qiagen GmbH, Hilden, FRG) at a concentration of 2 μg / ml in 50 μl of PBS with 2% FCS. As a reagent for the second stage, R-phycoerythrin conjugated affinity purified F (ab ') ₂ fragment of goat anti-mouse IgG antibody (specific for the Fc gamma fragment) diluted 1: 100 in 50 μl PBS with 2% FCS was used (Pianova, Hamburg, FRG). Samples were measured on FACSscan (BP biosciences, Heidelberg, FRG).

Binding has always been confirmed by flow cytometry, as described in the previous paragraph, on primary T cells of humans and various primates (e.g., common squirrel monkey (Saimiri sciureus), common marmoset (Callithrix jacchus); oedipus tamarin (Saguinus oedipus).

2.6. Creation of human / humanized scFv equivalents specific for non-human CD3 epsilon

The VH region of murine anti-CD3 scFv was aligned with the amino acid sequences of a human germline antibody. The VH sequence of the human germline antibody was selected which had the greatest homology with the VH of a non-human species, and the alignment of these two amino acid sequences was directly performed. There were a number of framework residues for the non-human VH species that differed from the human VH framework regions (“different positions in the framework”). Some of these residues may be involved in the binding of the antibody to its target and the activity of the antibody against its target.

To construct a library that contains murine CDRs and which in each frame position differs from both variants of the selected human VH sequence (human and maternal murine amino acid residue), degenerate oligonucleotides were synthesized. The composition of these oligonucleotides includes in different positions a “human” residue with a probability of 75% and a murine residue with a probability of 25%. For one human VH, for example, it was necessary to synthesize six such oligonucleotides that span the terminal portion of approximately 20 nucleotides. For this, every second primer was an antisense primer. The restriction sites necessary for subsequent cloning into oligonucleotides were deleted.

These primers can have a length of 60 to 90 nucleotides, depending on the number of primers that are required to overlap throughout the V sequence.

These primers, for example six primers, were mixed in equal amounts (for example, 1 μl of each primer (concentrated primer solution: 20-100 μM) with 20 μl of PCR reaction) and added to the PCR mixture consisting of PCR buffer, nucleotides and Taq polymerase. This mixture was incubated at 94 ° C for 3 minutes, at 65 ° C for 1 minute, at 62 ° C for 1 minute, at 59 ° C for 1 minute, at 56 ° C for 1 minute, at 52 ° C for 1 minute, at 50 ° C for 1 minute, and at 72 ° C for 10 minutes in a PCR cycle. After that, the product was subjected to agarose gel electrophoresis and the product, size 200 to 400, was isolated from the gel according to standard methods.

This PCR product was then used as a template for the standard PCR reaction using primers that introduced the N-terminal and C-terminal restriction sites suitable for cloning. A DNA fragment of the correct size (for VH approximately 350 nucleotides) was isolated by agarose gel electrophoresis according to standard methods. In this way, a sufficient VH DNA fragment was amplified. This VH fragment was now a pool of VH fragments, each of which had a different amount of “human” and “mouse” residues at corresponding different positions in the framework (pool of humanized VH). A similar procedure was carried out for the VL region of murine anti-CD3 scFv (humanized VL pool).

The humanized VH pool was then combined with the humanized VL pool in the pComb3H5Bhis phage display vector to form a functional scFv library, from which anti-CD3 binding agents were selected using a filamentous phage, screened, identified and confirmed as described above for non-parent -human (mouse) anti-CD3 scFv. Single clones were then analyzed for suitable properties and amino acid sequence. Those scFvs whose amino acid sequence is most homologous to human germline V-segments are preferred, in particular those that have at least one CDR among CDR I and II VH and CDR I and II of VL-kappa or CDR I and II VL-lambda demonstrate more than 80% amino acid sequence identity with the closest corresponding CDR of all human germline V-segments. Ahth-CD3 scFv was converted to recombinant bispecific single chain antibodies, as described in the following Examples 10 and 16, and further characterized.

3. Creation of a recombinant fusion protein consisting of N-terminal amino acids 1-27 of the human CD3 epsilon chain fused to the IgG1 Fc region (1-27 CD3-Fc)

3.1. Cloning and expression of 1-27 CD3-Fc

The coding sequence of the 1-27 N-terminal amino acids of the human CD3 epsilon chain fused to the hinge region and the Fc gamma region of human IgG1 immunoglobulin, as well as to the label consisting of 6 histidine residues, was obtained by gene synthesis according to standard protocols ( the cDNA sequence and the amino acid sequence of the recombinant fusion protein are presented in SEQ ID NO: 350 and 349). The gene synthesis fragment was designed so that it contained first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, encoding the sequence of the first 27 amino acids of the extracellular region of the mature human CD3 epsilon chain, then a reading frame encoding the sequence of the hinge region and the Fc-gamma region of human IgG1, then, in a reading frame, encoding a sequence of 6 histidine residues a tag and a stop codon (Figure 1). The gene synthesis fragment was also constructed in such a way as to introduce restriction sites at the beginning and at the end of the cDNA encoding this fusion protein. The introduced restriction sites, EcoRI at the 5'-end and Sall at the 3'-end, are used in subsequent cloning procedures. A gene synthesis fragment was cloned via EcoRI and Sall into a plasmid designated pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) following standard protocols. A confirmed sequence plasmid was used for transfection in the FreeStyle 293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's protocol. After 3 days, supernatants of transfectant cell cultures were collected and tested using ELISA for the presence of a recombinant construct. The goat antibody specific for the Fc gamma fragment of human IgG (obtained from Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK) was diluted in PBS to a concentration of 5 μg / ml and coated (100 μl per well) in the wells of a 96-well MaxiSorp plate for ELISA (Nunc GmbH & Co. KG, Wiesbaden, Germany) overnight at 4 ° C. Wells were washed with PBS with 0.05% Tween 20 (PBS / Tween) and blocked with 3% BSA in PBS (bovine albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) for 60 minutes at room temperature (CT ) After this, the wells were washed again with PBS / Tween and then incubated with cell culture supernatants for 60 minutes at RT. After washing the wells, incubation was performed with peroxidase-conjugated anti-His6 antibody (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) diluted 1: 500 in PBS with 1% BSA for 60 minutes at RT. The wells were then washed with 200 μl PBS / Tween and 100 μl of a SIGMAFAST OPD substrate solution (SIGMAFAST OPD [o-phenylenediamine dihydrochloride]) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was added according to the manufacturer's protocol. The reaction was stopped by adding 100 μl of 1 M H ₂ SO ₄ . The intensity of the resulting color was measured on a PowerWaveX microplate spectrophotometer (BioTek Instruments, Inc., Winooski, Vermont, USA) at 490 nm with subtraction of the background absorbance at 620 nm. As shown in FIG. 2, the presence of the construct compared to the irrelevant supernatant of the imaginary translated (mock-transfected) HEK 293 cells used as a negative control was clearly defined.

3.2. Cross-species specific single chain antibody binding assay for 1-27 CD3-Fc

The binding of crude preparations of interspecific specificity expressing periplasm of single chain antibodies specific for CD3 epsilon to 1-27 CD3-Fc was tested in an ELISA assay. Goat anti-human IgG antibody specific for the Fc gamma fragment (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK) was diluted in PBS to a concentration of 5 μg / ml and coated as 100 μl per well in 96 well ELISA plate MaxiSorp (Nunc GmbH & Co. KG, Wiesbaden, Germany) overnight at 4 ° C. Wells were washed with PBS with 0.05% Tween 20 (PBS / Tween) and blocked with PBS with 3% BSA (bovine albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) for 60 minutes at RT. After this, the wells were washed with PBS / Tween and incubated with supernatants of cells expressing the 1-27 CD3-FC construct for 60 minutes at RT. The wells were washed with PBS / Tween and incubated with crude preparations of periplasm-expressing single chain antibodies with interspecific specificity, as described above, for 60 minutes at room temperature. After washing with PBS / Tween, the wells were incubated with peroxidase conjugated to anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) diluted 1: 10000 in PBS with 1% BSA for 60 minutes at RT. The wells were washed with PBS / Tween and incubated with 100 μl of a solution of SIGMAFAST OPD substrate (OPD [o-phenylenediamine dihydrochloride]) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) according to the manufacturer's protocol. Color development was stopped using 100 μl of 1 M H ₂ SO ₄ and measurements were made on a PowerWaveX microplate spectrophotometer (BioTek Instruments, Inc., Winooski, Vermont, USA) at 490 nm with subtraction of background absorption at 620 nm. Strong binding of cross-species specific human single chain antibodies specific for CD3 epsilon with a 1-27 CD3-fc construct compared to murine anti-CD3 single chain antibody was observed (Figure 3).

4. Creation of recombinant transmembrane fusion proteins from N-terminal amino acids of 1-27 CD3 epsilon of various non-chimpanzee primates fused to cynomolgus monkey EpCAM (1-27 CD3-EpCAM)

4.1. Cloning and expression of 1-27 CD3-EpCAM

CD3 epsilon was isolated from various non-chimpanzee primates (marmosets, tamarines, squirrel monkeys) and pigs. Sequences encoding N-terminal amino acids 1-27 of the mature human CD3 epsilon chain, common marmoset (Callithrix jacchus), oedipus tamarin (Saguinus oedipus), common squirrel monkey (Saimiri sciureus) and domestic pig (Sus scrota; used as a negative control ) fused to the N-terminus of Flag-labeled EpCAM cynomolgus macaque were obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of recombinant fusion proteins are presented in SEQ ID NOs: 351-360). Gene synthesis fragments were designed so that they first contain a BsrGI site that allows fusion in the correct reading frame with the coding sequence of the 19 amino acid immunoglobulin leader peptide already in the target expression vector, then, in the reading frame, the coding sequence 1-27 N-terminal amino acids of the extracellular region of mature CD3 epsilon chains, then, in the reading frame, encoding the sequence of the Flag tag and then, in the reading frame, encoding the the ability of the mature transmembrane protein EpCAM cynomolgus macaque (Figure 4). Gene synthesis fragments were also designed to introduce a restriction site at the end of the cDNA encoding this fusion protein. The introduced restriction sites, BsrGI at the 5'-end and Sall at the 3'-end, were used in subsequent cloning procedures. Gene synthesis fragments were then cloned via BsrGI and Sall, following standard protocols, into a derivative of the plasmid designated pEF DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025), which already contained the coding sequence of an immunoglobulin leader peptide of 19 amino acids. Sequenced plasmids were used for transient transfection of HEK 293 cells using MATra-A reagent (IBA GmbH, Gottingen, Germany) and 12 μg of plasmid DNA for attached HEK 293 cells in 175 ml cell culture vials according to the manufacturer's protocol. After 3 days of cell culture, transfectants were tested for expression on the cell surface of a recombinant transmembrane protein using FACS analysis according to standard protocols. To this end, 2.5 × 10 ⁵ cells were incubated with anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) at a concentration of 5 μg / ml in PBS with 2% FCS. Bound antibody was determined using R-phycoerythrin-conjugated, affinity purified F (ab ') ₂ goat anti-mouse IgG antibody fragment (specific for the Fc gamma fragment) diluted 1: 100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany). The expression of Flag-labeled recombinant transmembrane fusion proteins consisting of EpCAM cynomolgus monkey and N-terminal amino acids 1-27 of the epsilon chain of human CD3, marmoset, tamarin, squirrel monkey and pig, respectively, was clearly determined on transfected cells (Figure 5).

4.2. The binding of single-chain anti-CD3 antibodies with interspecific specificity with 1-27 CD3-EpCAM

Binding of crude preparations of interspecific specific single chain anti-CD3 antibodies expressed in periplasm with N-terminal amino acids of 1-27 human CD3, marmoset, tamarin and squirrel monkey epsilon chains, respectively, fused to the EP-CAM of cynomolgus monkey, was tested using FACS -analysis according to standard protocols. To this end, 2.5 × 10 ⁵ cells were incubated with crude preparations of periplasm-expressing single-chain interspecific anti-CD3 antibodies (preparation was performed as described above and according to standard protocols) and single-chain mouse anti-human CD3 antibody as a negative control . A Penta-His antibody (Qiagen GmbH, Hildesheim, Germany) at a concentration of 5 μg / ml in 50 μl of PBS with 2% FCS was used as a secondary antibody. Antibody binding was determined using R-phycoerythrin-conjugated, affinity purified F (ab ') ₂ goat anti-mouse IgG antibody fragment (specific for the Fc gamma fragment) diluted 1: 100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany). As shown in FIG. 6 (AE), binding of single chain antibodies to transfectants expressing recombinant transmembrane fusion proteins consisting of 1-27 N-terminal amino acids of human CD3 epsilon, marmoset, tamarin or squirrel monkey fused to Javanese EpCAM was observed. toque. No binding of interspecific specificity single-chain antibodies to a fusion protein consisting of 1-27 N-terminal amino acids of pig CD3 epsilon fused to the cynomolgus monkey EPCAM, which was used as a negative control, was not observed. The interspecific specificity of single chain anti-CD3 antibodies has been shown for many primates. Signals obtained using anti-Flag M2 antibodies and interspecific specificity single chain antibodies were comparable, indicating strong binding activity of interspecific specificity single chain antibodies with N-terminal amino acids 1-27 of CD3 epsilon.

5. Analysis of the binding of single-chain anti-CD3 antibodies with interspecific specificity by alanine scanning of mouse cells transfected with the epsilon chain of human CD3 and its alanine mutants

5.1. Cloning and expression of human wild-type CD3 epsilon chain

The human CD3 epsilon chain coding sequence was obtained by gene synthesis according to standard protocols (the cDNA sequence and amino acid sequence of the human CD3 epsilon chain are shown in SEQ ID NOs: 362 and 361). The gene synthesis fragment was designed so that it contained a Kozak site for eukaryotic expression of this construct and restriction sites at the beginning and at the end of the cDNA encoding human CD3 epsilon. The introduced restriction sites, EcoRI at the 5'-end and Sall at the 3'-end, were used in subsequent cloning procedures. The gene synthesis fragment was then cloned via EcoRI and Sall into the plasmid designated pEF NEO following standard protocols. pEF NEO was obtained from pEF DHFR (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) by replacing DHFR cDNA with neomycin resistance cDNA (gene) using conventional molecular cloning. A confirmed sequence plasmid was used to transfect the EL4 mouse T cell line (ATCC No. TIB-39) cultured in RPMI with a stabilizing L-glutamine supplement supplemented with 10% FCS, 1% penicillin / streptomycin, 1% HEPES (N-2- hydroxyethyl piperazine-N-2-ethanesulfonic acid), 1% pyruvate, 1% non-essential amino acids (all Biochrom AG Berlin, Germany) at 37 ° C, 95% humidity and 7% CO ₂ . Transfection was carried out using SuperFect transfection reagent (Qiagen GmbH, Hilden, Germany) and 2 μg of plasmid DNA in accordance with the manufacturer's protocol. After 24 hours, the cells were washed with PBS and cultured again in the above cell culture medium with 600 μg / ml G418 for selection (PAA Laboratories GmbH, Pasching, Austria). 16-20 days after transfection, growth of resistant cells was observed. After another 7-14 days, the cells were tested for expression of human CD3 epsilon using FACS analysis according to standard protocols. 2.5 × 10 ⁵ cells were incubated with UCHT-1 antibody against human CD3 (BD biosciences, Heidelberg, Germany) at a concentration of 5 μg / ml in PBS with 2% FCS. Antibody binding was determined using R-phycoerythrin-conjugated, affinity purified F (ab ') ₂ goat anti-mouse IgG antibody fragment (specific for the Fc gamma fragment) diluted 1: 100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACSCalibur (BD biosciences, Heidelberg, Germany). Expression of wild-type human CD3 epsilon chain in transfected EL4 cells is shown in FIG. 7.

5.2. Cloning and expression of single chain anti-interspecific anti-CD3 antibodies as IgG1 antibodies

To provide improved means of determining the binding of single chain anti-CD3 antibodies with interspecific specificity, H2C HLP, A2J HLP and E2M HLP were converted to IgG1 antibodies containing murine IgG1 and human lambda constant regions. The cDNA sequences encoding the heavy and light chains of the corresponding IgG antibodies were obtained by gene synthesis according to standard protocols. Gene synthesis fragments for each specificity were designed so that they contained first the Kozak site for eukaryotic expression of this construct, then the immunoglobulin leader peptide of 19 amino acids (SEQ ID NO: 364 and 363), then, in the reading frame, the coding sequence of the corresponding variable region the heavy chain or the corresponding variable region of the light chain, then, in the reading frame, encoding the sequence of the constant region of the heavy chain of mouse IgG1 (SEQ ID NO: 366 and 365) or encoding the sequence of the constant region of the light chain of human lambda (SEQ ID N0: 368 and 367), respectively. Restriction sites were introduced at the beginning and at the end of the cDNA encoding this fusion protein. Restriction sites, EcoRI at the 5'-end and Sall at the 3'-end, were used for subsequent cloning procedures. Gene synthesis fragments were cloned via EcoRI and Sall into a plasmid designated pEF DHFR (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) for heavy chain constructs and pEF ADA (pEF ADA described in Raum et al., Cancer Immunol. Immunother., 50 (3), (2001), 141-50) for light chain constructs, according to standard protocols. Sequenced plasmids were used for cotransfection of the respective light and heavy chain constructs in the Freestyle 293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's protocol. After 3 days, the supernatants of the transfectant cell cultures were collected and used for the alanine scanning experiment.

5.3. Cloning and expression of alanine mutants human CD3 epsilon for alanine scanning

27 cDNA fragments encoding a human CD3 epsilon chain, replacing one codon in the wild-type sequence of human CD3 epsilon with an alanine coding codon (GCC), for each amino acid among amino acids 1-27 of the extracellular domain of the mature human CD3 epsilon chain, respectively were obtained by gene synthesis. With the exception of the replaced codon, the cDNA fragments were identical to the aforementioned wild-type human CD3 cDNA fragment. Only one codon was replaced in each construct compared to the wild-type human CD3 cDNA fragment described above. The restriction sites EcoRI and Sall were introduced into cDNA fragments at positions identical to wild-type constructs. All alanine scanned constructs were cloned into pEF NEO and confirmed sequence plasmids were transfected into EL4 cells. Transfection and selection of transfectants was carried out as described above. As a result, a panel of expressed constructs was obtained in which the first amino acid of the human CD3 epsilon chain, glutamine (Q, GIn) at position 1, was replaced with alanine. The last amino acid replaced by alanine was threonine (T, Thr) at position 27 of mature human wild-type CD3 epsilon. For each amino acid between glutamine 1 and threonine 27, respectively, transfectants were obtained with the replacement of wild-type amino acids with alanine.

5.4. Alanine Scanning Experiment

Chimeric IgG antibodies as described in 2) and single chain antibodies with interspecific specificity specific for CD3 epsilon were tested in an alanine scanning experiment. Antibody binding to EL4 cell lines transfected with alanine mutant human CD3 epsilon constructs as described in 3) was tested using FACS analysis according to standard protocols. 2.5 × 10 ⁵ cells of the respective transfectants were incubated with 50 μl of cell culture supernatant containing chimeric IgG antibodies, or with 50 μl of crude preparations of single chain antibodies expressed in periplasm. For samples incubated with crude preparations of periplasm expressing single chain antibodies, an anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was used as a secondary antibody at a concentration of 5 μg / ml in 50 μl PBS with 2% FCS. For samples incubated with chimeric IgG antibodies, there was no need for a secondary antibody. For all samples, the binding of antibody molecules was determined using R-phycoerythrin-conjugated affinity purified F (ab ') ₂ fragment of goat anti-mouse IgG antibody (specific for the Fc-gamma fragment) diluted 1: 100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACSCalibur (BP biosciences, Heidelberg, Germany). Differential binding of chimeric IgG molecules or single chain antibodies with interspecific specificity was determined with EL4 cell lines transfected with human CD3 epsilon alanine mutants. As a negative control, either an isotypic control or a crude preparation of a single-chain antibody of irrelevant specificity expressed in the periplasm was used, respectively. The UCHT-1 antibody was used as a positive control of the expression level of alanine mutants of human CD3 epsilon. EL4 cell lines transfected with alanine mutants of tyrosine amino acids at position 15, valine at position 17, isoleucine at position 19, valine at position 24, or leucine at position 26 of the mature CD3 epsilon chain were not evaluated due to very low expression levels (data not shown) shown). The binding of interspecific specificity single chain antibodies and chimeric IgG single chain antibodies to EL4 cell lines transfected with human CD3 epsilon alanine mutants is shown in Fig. 8 (AD) as relative binding in arbitrary units to geometric mean fluorescence values of the corresponding negative controls, subtracted from all corresponding geometric mean fluorescence values for the samples. To compensate for different expression levels, all values for samples for a particular transfectant were then divided by the geometric mean fluorescence of the UCHT-1 antibody for the corresponding transfectant. For comparison with the specificity value for the wild-type sample, all values of the corresponding specificities for the samples were finally divided by the value for the wild-type sample, while the value for the wild-type sample was taken as 1 conventional unit of binding.

The calculations used are represented in detail by the following formula:

In this equation, value_Sample means the amount of binding in arbitrary units representing the degree of binding of a specific anti-CD3 antibody to a specific alanine mutant, as shown in Fig. 8 (A-D); Sample means the geometric mean fluorescence obtained for a specific anti-CD3 antibody analyzed on a specific alanine-scanning transfectant; neg_Contr. means the geometric mean fluorescence obtained for the negative control, analyzed on a specific alanine mutant; UCHT-1 means the geometric mean fluorescence obtained for the UCHT-1 antibody analyzed on a specific alanine mutant; WT means the geometric mean fluorescence obtained for a specific anti-CD3 antibody analyzed on a wild-type transfectant; x indicates the corresponding transfectant; y indicates the corresponding anti-CD3 antibody, and wt indicates that the corresponding transfectant is of the wild type.

As can be seen from Fig. 8 (A-D), the A2J HLP IgG antibody showed a pronounced loss of binding for amino acids: asparagine at position 4, threonine at position 23, and isoleucine at position 25 of the mature CD3 epsilon chain. A complete loss of binding of the IgG antibody A2J HLP was observed for amino acids: glutamine at position 1, aspartate at position 2, glycine at position 3 and glutamate at position 5 of the mature CD3 epsilon chain. The IgG antibody E2M HLP showed a pronounced loss of binding for amino acids: asparagine at position 4, threonine at position 23, and isoleucine at position 25 of the mature epsilon chain CD3. IgG antibody E2M HLP showed complete loss of amino acid binding: glutamine at position 1, aspartate at position 2, glycine at position 3, and glutamate at position 5 of the mature CD3 epsilon chain. The H2L HLP IgG antibody showed an intermediate loss of binding for the asparagine amino acid at position 4 of the mature CD3 epsilon chain and it showed a complete loss of binding for the amino acids: glutamine at position 1, aspartate at position 2, glycine at position 3 and glutamate at position 5 of mature epsilon chain CD3. The single chain F12Q HLP antibody showed a substantially complete loss of binding for amino acids: glutamine at position 1, aspartate at position 2, glycine at position 3 of the mature CD3 epsilon chain and glutamate at position 5 of the mature CD3 epsilon chain.

6. Analysis of the binding of the anti-CD3 binding H2C HLP molecule with interspecific specificity to human CD3 epsilon chain with and without N-terminal His6 tag, which was transfected into the murine EL4 T cell line

6.1. Cloning and expression of a human CD3 epsilon chain with an N-terminal hexahistidine tag (His6 tag)

A cDNA fragment encoding a human CD3 epsilon chain with an N-terminal His6 tag was obtained by gene synthesis. The gene synthesis fragment was designed so that it contained first the Kozak site for eukaryotic expression of this construct, then, in the reading frame, an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, encoding the sequence of the His6 tag, then, in the reading frame, the coding sequence of a mature human CD3 epsilon chain (the cDNA sequence and amino acid sequence of this construct are presented in SEQ ID NO: 380 and 379). The gene synthesis fragment was also designed so that it contained restriction sites at the beginning and end of cDNA. The introduced restriction sites, EcoRI at the 5'-end and Sall at the 3'-end, were used in subsequent cloning procedures. The gene synthesis fragment was then cloned via EcoRI and Sall into the plasmid designated pEF-NEO (as described above) following standard protocols. A confirmed sequence plasmid was used to transfect the EL4 murine T cell line. Transfection and selection of transfectants was carried out as described above. After 34 days of cell culture, transfectants were used in the assay described below.

6.2. Interspecific binding of the anti-CD3 binding H2C HLP molecule with human CD3 epsilon chain with and without N-terminal His6 tag

Chimeric IgG antibody H2C HLP with binding specificity specific for CD3 epsilon was tested for binding to human CD3 epsilon with and without N-terminal His6 tag. The binding of this antibody to EL4 cell lines transfected with human His6-CD3 epsilon and wild-type human CD3 epsilon, respectively, was tested using FACS analysis according to standard protocols. 2.5 × 105 transfectant cells were incubated with 50 μl of cell culture supernatant containing a chimeric IgG antibody or 50 μl of the corresponding control antibodies at a concentration of 5 μg / ml in PBS with 2% FCS. The corresponding isotypic control was used as a negative control, and the Biacore construct expression was used as a positive control. Unbound protein was removed by vigorous washing followed by blocking of the unreacted remaining NHS-activated carboxy groups by adding ethanolamine solution. Successful protein attachment was confirmed by a higher signal value, measured as Response Units, compared to the signal prior to attachment. A comparison cell was prepared as described, but without the addition of a protein solution.

The purified bispecific antibody EGFR-21-63 LH × H2C HLP was vigorously dialyzed against an HBS-EP buffer (Biacore, Uppsala, Sweden) in a Slide-A-Lyzer® mini-dialysis cell (Pierce, Rockford-II, USA). The protein concentration after dialysis was determined by absorbance at 280 nm, obtaining a concentration of 43 μg / ml.

The protein solution was transferred to a 96-well plate and diluted sequentially with HBS-EP buffer at a ratio of 1: 1 for 10 subsequent wells.

Surface plasmon resonance measurements were carried out by separately adding samples to all 11 wells. Between measurements, the flow cell was regenerated with acetate buffer to release bound protein.

Binding signals of bispecific antibody molecules were obtained by subtracting the signal of the comparison cell from the signal of the measured cell conjugated to protein 1-27 CD3-Fc. Association and dissociation curves were measured as response units and recorded. Binding constants were calculated using Biacore® curve fitting software based on the Langmuir model.

The calculated value of the KD binding constant for the first five concentrations was 1.52 × 10 ^-7 M.

7.2. Determination of the binding constant of CD3 in the measurement using FACS

To test the affinity of interspecific specificity bispecific antibody molecules for binding strength to native human CD3, an additional saturation binding assay was performed using FACS. The selected molecule of the bispecific antibody EGFR-21-63 LH × H2C HLP was used to establish a series of dilutions with a ratio of 1: 1.5 and an initial concentration of 63.3 μg / ml. Used CD3-specific antibody UCHT-1, respectively. Antibody binding was determined using an R-phycoerythrin-conjugated, affinity-purified P (ab ') 2 fragment of a goat anti-mouse IgG antibody (specific for the Fc gamma fragment) diluted 1: 100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACSCalibur (BD biosciences, Heidelberg, Germany). Compared to the EL4 cell line transfected with wild-type human CD3 epsilon, the apparent loss of binding of chimeric IgG with binding specificity — H2C HLP — to human CD3 epsilon with an N-terminal His6 tag was determined. These results showed that the free N-terminus of CD3 epsilon is essential for the binding of a specific interspecific specific anti-CD3 binding H2C HLP molecule to human CD3 epsilon chain (Figure 9).

7. Determination of the KD binding constant of a bispecific single chain antibody with interspecific specificity for EGFR primates and CD3 primates (EGFR LH × H2C HLP), with fusion protein 1-27 CD3-Fc, measured by surface plasmon resonance, in comparison with binding to CD3- expressing PBMC measured using fluorescence activated cell sorter (FACS)

7.1. Surface plasmon resonance measurement

In order to determine the binding affinity of the bispecific single chain antibody EGFR-21-63 LH × H2C HLP with full interspecific specificity with amino acids 1-27 of the N-terminus of the human CD3 epsilon chain, surface plasmon resonance measurement was performed with a recombinant fusion protein consisting of N-terminal amino acids 1-27 mature human CD3 epsilon chains fused to the Fc region of human IgG1 (1-27 CD3-Fc). For this, a Biacore CM5 carboxymethyl-dextran chip (Biacore, Uppsala, Sweden) was placed in a Biacore 2000® system (Biacore, Uppsala, Sweden). One flow cell was activated with a solution of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide / N-hydroxysuccinimide hydrochloride in accordance with standard procedures. Then a solution of fused protein 1-27 CD3-Fc was added, which led to the formation of a stable covalent bond between the protein and the dextran layer of the chip. Each of the bispecific antibody molecules was incubated at these different concentrations with 1.25 × 10 ⁵ human PBMCs for 1 hour at 4 ° C followed by two stages of washing with PBS at 4 ° C. The binding of the bispecific antibody molecules was determined using a Penta-His antibody (Qiagen GmbH, Hildesheim, Germany) at a concentration of 5 μg / ml in 50 μl of PBS with 2% FCS. After 45 minutes incubation at 4 ° C and two washing steps, Penta-His antibody binding was determined using R-phycoerythrin-conjugated, affinity purified F (ab ') ₂ fragment of goat anti-mouse IgG antibody (specific for Fc-gamma fragment) diluted 1: 100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Flow cytometry was performed on a FACS-Canto II apparatus; FACS Diva software (Becton Dickinson biosciences, Heidelberg) was used to obtain and analyze data. FACS staining and fluorescence intensity measurements were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnterscience, 2002). We plotted the obtained average fluorescence intensity values versus the concentration of molecules of the bispecific antibody used and analyzed using the Prism biomathematical software in a one side binding analysis (hyperbola). Using software, the corresponding KD value was calculated that describes the binding of the ligand (bispecific antibody molecule) to the receptor (CD3-positive subfraction of PBMC) in accordance with the law of mass action. The underlying formula is: Y = Bmax × X / (Kd + X), where Bmax is the maximum binding. KD is the ligand concentration necessary to achieve half-maximal binding. FACS staining was performed in duplicate; R ² values were greater than 0.95.

According to the results of the determination, the half-maximum binding for the molecule of the bispecific antibody EGFR-21-63 LH × Н2С HLP was achieved at a concentration of 8472 ng / ml, which corresponds to 154 nM (1.54 × 10 ^-7 M) at a set molecular weight of 55,000 daltons (Fig. 10).

Thus, it was proved that the affinity of EGFR-21-63 LH × H2C HLP for the N-terminal amino acids 1-27 of the human CD3 epsilon chain isolated from its native CD3 environment is equal to the affinity of EGFR-21-63 LH × H2C HLP for native CD3 on intact T cells.

8. Obtaining CHO cells transfected with human EGFR

A cell line positive for human EGFR, A431 (epidermoid carcinoma cell line, CRL-1555, American Type Culture Collection, Rockville, MD) was used to generate total RNA, which was isolated according to the kit instructions (Qiagen, RNeasy Mini Kit , Hilden, Germany). The resulting RNA was used for cDNA synthesis by reverse transcription polymerase chain reaction with random primers. The following oligonucleotides were used to clone the full length sequence of the human EGFR antigen:

5 'EGFR AG Xbal 5'-GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG-3';

3 'EGFR AG Sall 5'-TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT-3'.

The coding sequence was amplified by PCR (denaturation at 94 ° C for 5 min, annealing at 58 ° C for 1 min, elongation at 72 ° C for 2 min during the first cycle; denaturation at 94 ° C for 1 min , annealing at 58 ° C for 1 min, elongation at 72 ° C for 2 min for 30 cycles; final elongation at 72 ° C for 5 min). The PCR product was then digested with Xbal and Sall, ligated into a suitably cleaved pEF-DHFR expression vector (Raum et al., Cancer Immunol. Immunother. 2001; 50: 141-150) and transformed into E. coli. The above procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). A confirmed nucleotide sequence clone (SEQ ID 370, amino acid sequence SEQ ID 369) was transfected into CHF defective CHO cells for eukaryotic expression of this construct. The expression of the eukaryotic protein in DHFR-defective CHO cells was carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of 20 nM MTX, inclusive.

9. Obtaining CHO cells expressing the extracellular domain of cynomolgus EGFR

The cDNA sequence of the extracellular domain of EGFR of cynomolgus macaque was obtained as a result of a series of two PCRs on cDNA from the colon of cynomolgus macaque (catalog number C1534090-Su-BC; obtained from BioCat GmbH, Heidelberg, Germany) using the following reaction conditions: 1 cycle at 94 ° C for 3 minutes, then 35 cycles at 94 ° C for 1 minute, at 53 ° C for 1 minute and at 72 ° C for 2 minutes, then the final cycle at 72 ° C for 3 minutes. The following primers were used:

1) direct primer: 5'-CGCTCTGCCCGGCGAGTCGGGC-3 ',

reverse primer: 5'-CCGTCTTCCTCCATCTCATAGC-3 ';

2) direct primer: 5'-ACATCCGGAGGTGACAGATCACGGCTCGTGC-3,

reverse primer: 5'-CAGGATATCCGAACGATGTGGCGCCTTCGC-3 '.

As a result of these PCRs, two overlapping fragments (A: 1-869, B: 848-1923) were obtained, which were isolated and sequenced according to standard protocols using PCR primers, and thus a 1923 nb Macaque EGFR cDNA sequence region was obtained. about. from the third nucleotide in the +1 codon of the mature protein to the 21st codon of the transmembrane domain. In order to create a construct for the expression of cynomolgus EGFR, a cDNA fragment was obtained by gene synthesis according to standard protocols (the cDNA sequence and amino acid sequence of this construct are presented in SEQ ID NO: 372 and 371). In this construct, the cynomolgus EGFR coding sequence from amino acids +2 to +641 of the mature EGFR protein is included in the human EGFR coding sequence, replacing the coding sequence of amino acids +2 to +641. The gene synthesis fragment was also designed in such a way as to contain the Kozak site for eukaryotic expression of this construct and restriction sites at the beginning and at the end of the cDNA encoding the essentially extracellular domain of cynomolgus EGFR fused to the transmembrane and intracellular domains of human EGFR. In addition, a conservative mutation was introduced at the position corresponding to amino acid 627 (4th amino acid of the transmembrane domain), replacing valine with leucine to create a restriction site (Sphl) for cloning. The introduced restriction sites, Xbal at the 5'-end and Sall at the 3'-end, were used in subsequent cloning procedures. The gene synthesis fragment was then cloned via Xbal and Sall into a plasmid designated pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA, 92 (1995) 7021-7025). A confirmed sequence clone of this plasmid was used to transfect CHO / dhfr- cells as described above.

10. Creation of EGFR and CD3 bispecific single chain molecules with interspecific specificity

10.1. Cloning binding molecules with interspecific specificity

As a rule, molecules of bispecific single chain antibodies, each of which contains a domain with binding specificity, demonstrating interspecific specificity for human and non-chimpanzee CD3 epsilon, as well as a domain with specificity for binding, interspecific specificity for human and non-chimpanzee EGFR chimpanzees were constructed as shown in the following Table 1.

Table 1 Molecular Formats of Anti-CD3 and Anti-EGFR Bispecific Single-Chain Antibodies with Interspecific Specificity SEQ ID (nucleon / white) Formats of protein constructs (N → C) 294/293 EGFR-21-63 LH × H2C HL 296/295 EGFR-21-63 LH × H2C HLP 302/301 EGFR-21-63 LH × A2J HLP 298/297 EGFR-21-63 LH × H1E HLP 306/305 EGFR-21-63 LH × E2M HLP 308/307 EGFR-21-63 LH × F70 HLP 390/389 EGFR1 HL × I2C HL 392/391 EGFR1 LH × I2C HL 394/393 EGFR1 HL × F12Q HL 396/395 EGFR1 LH × F12Q HL 398/397 EGFR1 HL × H2C HL 400/399 EGFR1 LH × H2C HL 448/447 EGFR1 HL × H2C HL 450/449 EGFR1 HL × F12Q LH 452/451 EGFR1 HL × I2C HL 410/409 EGFR2 HL × I2C HL 412/411 EGFR2 LH × I2C HL 414/413 EGFR2 HL × F12Q HL 416/415 EGFR2 LH × F12Q HL 418/417 EGFR2 HL × H2C HL 420/419 EGFR2 LH × H2C HL

The above constructions containing light chain variable domains (L) and heavy chain variable domains (H) with interspecific specificity for human and cynomolgus EGFRs were obtained by gene synthesis. Gene synthesis fragments were designed so that they contained first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, the coding sequence of the corresponding molecule of the bispecific single chain antibody, then, in the reading frame, the coding sequence consisting of of 6 histidine tag residues and a stop codon. The gene synthesis fragment was also designed to introduce suitable N- and C-terminal restriction sites. A gene synthesis fragment was cloned through these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150) 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 a confirmed nucleotide sequence was transfected into dihydrofolate reductase (DHFR) defective Chinese hamster ovary (CHO) cells for eukaryotic expression of this construct.

These constructs were transfected stably or temporarily into DHFR defective CHO cells (ATCC No. CRL 9096) by electroporation or, alternatively, temporarily transfected into HEK 293 (human embryonic kidney cells, ATCC number: CRL-1573) according to standard protocols.

10.2. Expression and purification of bispecific single chain antibody molecules

Bispecific single chain antibody molecules were expressed in Chinese hamster ovary (CHO) cells. The expression of the eukaryotic protein in DHFR-defective CHO cells was carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by increasing the final MTX concentration to 20 nM inclusive. After two passages of the stationary culture, the cells were grown in roller bottles in liquid soybean medium HyQ PF CHO containing no nucleosides (with 4.0 mM L-glutamine, with 0.1% Pluronic F-68; HyClone), for 7 days before collection . Cells were isolated by centrifugation and the supernatant containing the expressed protein was stored at -20 ° C. Alternatively, constructs were temporarily expressed in HEK 293 cells. Transfection was performed using 293fectin reagent (Invitrogen, No. 12347-019) in accordance with the manufacturer's protocol.

For chromatography, Akta® Explorer (GE Health Systems) and Unicorn® software were used. Immobilized metal affinity chromatography ("IMAC") was carried out using Fractogel EMD chelate® (Merck), which was loaded with ZnCl2 according to the protocol proposed by the manufacturer. The column was equilibrated with buffer A (20 mM sodium phosphate buffer pH 7.2; 0.1 M NaCl) and the cell culture supernatant (500 ml) was applied to the column (10 ml) at a flow rate of 3 ml / min. The column was washed with buffer A to remove unbound sample. The bound protein was eluted using a two-step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2; 0.1 M NaCl; 0.5 M imidazole) in accordance with the following protocol:

stage 1: 20% buffer B in 6 column volumes;

Stage 2: 100% buffer B in 6 column volumes.

The eluted protein fractions from step 2 were combined for further purification. All chemicals had a purity suitable for research, and were purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration was performed on a HiLoad 16/60 Superdex 200 preparative column (GE / Amersham) equilibrated with Equi buffer (25 mM citrate; 200 mM lysine; 5% glycerol; pH 7.2). The eluted protein samples (flow rate 1 ml / min) were then subjected to standard SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) and Western blot for detection. Before cleaning, the column was calibrated to determine molecular weight (molecular weight marker set, Sigma MW GF-200). Protein concentration was determined using OD at 280 nm.

The purified protein, a bispecific single chain antibody, was analyzed in SDS-PAGE under reducing conditions and pre-filled with 4-12% bis-Tris gels (Invitrogen). Preparation and application of samples was carried out according to the protocol proposed by the manufacturer. Molecular weight was determined using the MultiMark protein standard (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen protocol). The purity of the isolated protein was over 95% as determined by SDS-PAGE.

The bispecific single chain antibody has a molecular weight of about 52 kDa under native conditions, as determined by gel filtration in PBS. All designs were cleaned according to this method.

Western blotting was performed using an Optitran® BA-S83 membrane in an Invitrogen blotter in accordance with the protocol proposed by the manufacturer. The antibodies used were directed against His tag (Penta-His, Qiagen) and goat antibodies against mouse alkaline phosphatase (AP) labeled Ig (Sigma) and BCIP / NBT (5-bromo-4-chloro-3-indolylphosphate) / nitrotetrazolium blue) (Sigma) as a substrate. A single band at 52 kDa was determined, which corresponded to a purified bispecific single chain antibody.

11. Determination of the binding constant KD bispecific single-chain antibodies with full interspecific specificity, with the fused protein 1-27 CD3-Fc, measured by surface plasmon resonance

To determine the binding affinities of the bispecific single chain antibody molecules with interspecific specificity for EGFR primates and CD3 primates with amino acids 1-27 of the N-terminus of the mature human CD3 epsilon chain, surface plasmon resonance measurements were carried out with a recombinant fusion protein consisting of N-terminal amino acids 1- 27 epsilon chains of human CD3 fused to the Fc region of human IgG1 (1-27 CD3-Fc). For this, a Biacore carboxymethyl-dextran CM5 chip Biacore (Biacore, Uppsala, Sweden) was installed in the Biacore 2000® system (Biacore, Uppsala, Sweden). The flow cell was activated with a N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide / N-hydroxysuccinimide hydrochloride solution in accordance with standard procedures. Then a solution of fusion protein 1-27 CD3-Fc was added, which led to the formation of a stable covalent bond between the protein and the dextran layer of the Biacore chip. Unbound protein was removed by vigorous washing followed by blocking of the remaining unreacted NHS-activated carboxy groups by adding ethanolamine solution. Successful protein addition was confirmed by a higher signal value, measured as response units, compared to the signal prior to addition. A comparison cell was prepared as described, but without the addition of a protein solution.

The concentration of purified bispecific single chain antibodies shown below was adjusted to 5 μg / ml with HBS-EP buffer (Biacore, Uppsala, Sweden) and transferred to a 96-well plate, each in a volume of 150 μl.

Surface plasmon resonance measurements were performed for all samples and flow cells were regenerated between measurements with acetate buffer to release bound protein (all according to standard protocols).

Bispecific single chain antibody binding signals were obtained by subtracting the signal from the reference cell from the signal from the measured cell conjugated to protein 1-27 CD3-Fc.

Association and dissociation curves were measured as response units and recorded. Binding constants were calculated using Biacore® curve fitting software based on the Langmuir model. The calculated affinity values for the tested bispecific single chain molecules with full interspecific specificity for the N-terminal amino acids 1-27 of human CD3 epsilon are given in the form of KD values below and range from 2.54 × 10 ^-6 M to 2.49 × 10 ^{- 7} M. "LH" refers to the organization of variable domains in the order of VL-VH. "HL" refers to the organization of variable domains in the order of VH-VL. G4H, F70, A2J, E1L, E2M, H1E and F6A belong to various interspecific specific CD3-binding molecules.

Bispecific Antibody Molecule KD (M) EGFR LH × F70 HLP 1.01 × 10 ^-6 EGFR LH × A2J HLP 2.49 × 10 ^-7 EGFR LH × E2M HLP 2.46 × 10 ^-6

EGFR LH × H1E HLP 2.54 × 10 ^-6

12. Flow cytometric analysis of the binding of EGFR and CD3 bispecific antibodies with interspecific specificity

In order to test the functionality of interspecific specificity bispecific antibody constructs with respect to the ability to bind to human EGFR and CD3 and cynomolgus macaques, respectively, FACS analysis was performed. For this, CHO cells transfected with human EGFR as described in Example 8 and a CD3-positive human T-cell leukemia HPB-ALL cell line (DSMZ, Braunschweig, ACC483) were used to test binding to human antigens. The ability to bind to cynomolgus antigens was tested using the generated transfectant with EGFR of cynomolgus macaque described in Example 9 and the T-cell line of macaque 4119 LnPx (kindly provided by Prof. Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200,000 cells of the corresponding cell population were incubated for 30 min on ice with 50 μl of purified interspecific specific bispecific antibody construct protein (2 μg / ml). Alternatively, a cell culture supernatant with temporarily produced proteins was used. Cells were washed twice in PBS and the binding of this construct was determined using a mouse Penta-His antibody (Qiagen; diluted 1:20 in 50 μl of PBS with 2% FCS). After washing, the bound anti-His antibodies were determined using an Fc-gamma-specific antibody (Dianova) conjugated with phycoerythrin diluted 1: 100 in PBS with 2% FCS. Fresh culture medium was used as a negative control.

Flow cytometry was performed on a FACS-Calibur apparatus; CellQuest software (Becton Dickinson biosciences, Heidelberg) was used to obtain and analyze data. FACS staining and fluorescence intensity measurements were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).

The binding ability of some bispecific single chain molecules specific for EGFR and having interspecific specificity for human CD3 and non-chimpanzee primate was clearly defined, as shown in FIG. 11. In the FACS analysis, all constructs showed binding to CD3 and EGFR compared to the culture medium and the first and second detecting antibodies as a negative control. The interspecific specificity of the bispecific antibody to human and cynomolgus macaque CD3 and EGFR antigens was demonstrated.

13. Biological activity of EGFR and CD3 bispecific single chain antibodies with interspecific specificity

The biological activity of the created bispecific single chain antibodies was analyzed by the release of chromium 51 ( ⁵¹ Cr) in in vitro cytotoxicity assays using the EGFR-positive cell lines described in Examples 8 and 9. As the effector cells used stimulated human CD8-positive T cells or T the macaque cell line 4119 LnPx, respectively.

Obtaining stimulated CD8 + T cells was carried out as follows.

A commercially available anti-CD3-specific antibody was pre-applied to a Petri dish (145 mm diameter; Greiner) at a final concentration of 1 μg / ml for 1 hour at 37 ° C. Unbound protein was removed in one washing step using PBS. Fresh PBMCs were isolated from peripheral blood (30-50 ml of human blood) by ficoll gradient centrifugation according to standard protocols. 3-5 × 10 ⁷ PBMCs were added to a precoated Petri dish in 120 ml of RPMI 1640/10% FCS / IL-2 (interleukin-2) 20 units / ml (proleukin, Chiron) and stimulated for 2 days. On the third day, cells were harvested, washed once with RPMI 1640. IL-2 was added to a final concentration of 20 units / ml and cultured again for one day. CD8 + cytotoxic T lymphocytes (CTL) were isolated by depletion of CD4 + T cells and CD56 + NK cells.

Target cells were washed twice with PBS and labeled with 11.1 MBq ⁵¹ Cr in a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37 ° C. After this, the labeled target cells were washed 3 times with 5 ml RPMI and then used in the analysis of cytotoxicity. The analysis was carried out in a 96-well plate in a total volume of 250 μl of RPMI supplemented medium (as above) with an E: T ratio of 10: 1. Put 1 μg / ml molecules of bespecifically single-chain antibodies with interspecific specificity and 20 triple dilutions thereof. Alternatively, the cell culture supernatant of temporarily produced proteins was subsequently diluted 1: 2 in stages. The analysis time is 18 hours, and cytotoxicity was measured in relative values of the released chromium in the supernatant, related to the difference in maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements were performed in four replicates. Measurement of chromium activity in supernatants was performed using a Wizard 3 "gamma counter (Perkin Elmer Life Sciences GmbH, Köln, Germany). Experimental data were analyzed using Prism 4 for Windows (version 4.02, GraphPad Software Inc., San Diego, California, USA) Sigmoidal dose-response curves typically had R ² values greater than 0.90, as determined using software, and the EC ₅₀ values calculated by this assay program were used to compare biological activity.

As shown in FIGS. 12 and 13, all cross-species specific bispecific single chain antibody constructs created showed cytotoxic activity against target cells positive for human EGFR induced by human CD8 + cells and target cells positive for EGFR cynomolgus monkey induced by the T-cell line of macaque 4119 LnPx. A bispecific single chain antibody with a different target specificity was used as a negative control.

14. Cloning and expression of C-terminal, transmembrane and truncated extracellular domains of human MCSP

The coding sequence of the C-terminal, transmembrane and shortened extracellular domain of the human MCSP (amino acids 1538-2322) was obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the recombinant construct for expression of the C-terminal, transmembrane and shortened extracellular domain of the human MCSP (designated as D3 human) are presented in SEQ ID NO: 374 and 373). The gene synthesis fragment was designed so that it contained first the Kozak site for eukaryotic expression of this construct, then the coding sequence of the immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, a FLAG tag, then, in the reading frame, a sequence containing several sites restriction for cloning purposes and encoding an artificial amino acid linker of 9 amino acids (SRTRSGSQL), then, in a reading frame, encoding a sequence of C-terminal, transmembrane and shortened th extracellular domain of human MCSP and a stop codon. Restriction sites were introduced at the beginning and at the end of this DNA fragment. Restriction sites, EcoRI at the 5'-end and Sall at the 3'-end, were used in subsequent cloning procedures. The fragment was digested with EcoRI and Sall and cloned into pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) following standard protocols. A confirmed sequence plasmid was used to transfect CHO / dhfr- cells (ATCC No. CRL 9096). Cells were cultured in RPMI 1640 medium with a stabilizing glutamine supplement supplemented with 10% FCS, 1% penicillin / streptomycin (all purchased from Biochrom AG Berlin, Germany) and nucleosides from a concentrated cell culture reagent solution (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of adenosine 10 μg / ml, deoxyadenosine 10 μg / ml and thymidine 10 μg / ml, in an incubator at 37 ° C, 95% humidity and 7% CO ₂ . Transfection was performed using a PolyFect transfection reagent (Qiagen GmbH, Hilden, Germany) and 5 μg of plasmid DNA according to the manufacturer's protocol. After culturing for 24 hours, the cells were washed once with PBS and cultured again in RPMI 1640 with a stabilizing addition of glutamine and 1% penicillin / streptomycin. Thus, the cell culture medium did not contain nucleosides, and therefore, selection was carried out on transfected cells. Approximately 14 days after transfection, growth of resistant cells was observed. After another 7-14 days, transfectants were tested for expression of the construct using FACS analysis. 2.5 × 10 ⁵ cells were incubated with 50 μl of anti-Flag-M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) diluted to a concentration of 5 μg / ml in PBS with 2% FCS. Antibody binding was determined using R-phycoerythrin-conjugated affinity purified F (ab ') ₂ goat anti-mouse IgG antibody fragment (specific for the Fc gamma fragment) diluted 1: 100 in PBS with 2% FCS (ImmunoResearch Europe Ltd ., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).

15. Cloning and expression of C-terminal, transmembrane and truncated extracellular domains of macaque MCSP

The DNA sequence of the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP (designated as macaque D3) was obtained as a result of a series of three PCRs on macaque skin cDNA (catalog number C1534218-Su-BC; BioCat GmbH, Heidelberg, Germany) with using the following reaction conditions: 1 cycle at 94 ° C for 3 min, 40 cycles at 94 ° C for 0.5 min, at 52 ° C for 0.5 min and at 72 ° C for 1.75 min , then the final cycle at 72 ° C for 3 minutes The following primers were used:

forward primer: 5'-GATCTGGTCTACACCATCGAGC-3 ';

reverse primer: 5'-GGAGCTGCTGCTGGCTCAGTGAGG-3 ';

forward primer: 5'-TTCCAGCTGAGCATGTCTGATGG-3 ';

reverse primer: 5'-CGATCAGCATCTGGGCCCAGG-3 ';

forward primer: 5'-GTGGAGCAGTTCACTCAGCAGGACC-3 ';

reverse primer: 5'-GCCTTCACACCCAGTACTGGCC-3 '.

As a result of these PCRs, three overlapping fragments (A: 1-1329, B: 1229-2428, C: 1782-2547) were obtained, which were isolated and sequenced according to standard protocols using these PCR primers, and thus a portion of the cDNA sequence was obtained MCSP macaque 2547 bp (the cDNA sequence and amino acid sequence of this macaque MCSP region are shown in SEQ ID NOs: 376 and 375) from 74 bp above the sequence encoding the C-terminal domain to 121 bp below the stop codon. Another PCR using the following reaction conditions: 1 cycle at 94 ° C for 3 min, 10 cycles at 94 ° C for 1 min, at 52 ° C for 1 min and at 72 ° C for 2.5 min, the final cycle at 72 ° C for 3 min, was used to merge the PCR products A and B of the above reactions. The following primers were used:

forward primer: 5'-tcccgtacgagatctggatcccaattggatggcggactcgtgctgttctcacacagagg-3 '; reverse primer: 5'-agtgggtcgactcacacccagtactggccattcttaagggcaggg-3 '.

Primers for this PCR were designed to introduce restriction sites at the beginning and end of a cDNA fragment encoding the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP. The introduced restriction sites, Mfel at the 5'-end and Sall at the 3'-end, were used in subsequent cloning procedures. The PCR fragment was then cloned via Mfel and Sall into a Bluescript plasmid containing the EcoRI / Mfel fragment of the aforementioned pEF-DHFR plasmid (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150), -terminal, transmembrane and shortened extracellular domains of human MCSP. The gene synthesis fragment contained the coding sequences of the immunoglobulin leader peptide and Flag tag, as well as an artificial linker (SRTRSGSQL), in the reading frame, with the 5'-end of the cDNA fragment encoding the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP. This vector was used to transfect CHO / dhfr- cells (ATCC No. CRL 9096). Cells were cultured in RPMI 1640 with a stabilizing glutamine supplement supplemented with 10% FCS, 1% penicillin / streptomycin (all from Biochrom AG Berlin, Germany) and nucleosides from a concentrated cell culture reagent solution (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of adenosine 10 μg / ml, deoxyadenosine 10 μg / ml and thymidine 10 μg / ml, in an incubator at 37 ° C, 95% humidity and 7% CO ₂ . Transfection was performed using a PolyFect transfection reagent (Qiagen GmbH, Hilden, Germany) and 5 μg of plasmid DNA according to the manufacturer's protocol. After culturing for 24 hours, the cells were washed once with PBS and cultured again in RPMI 1640 with a stabilizing addition of glutamine and 1% penicillin / streptomycin. Thus, the cell culture medium did not contain nucleosides, and therefore, selection was carried out on transfected cells. Approximately 14 days after transfection, growth of resistant cells was observed. After another 7-14 days, transfectants were tested for expression of the recombinant construct using FACS analysis. 2.5 × 10 ⁵ cells were incubated with 50 μl of anti-Flag-M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) diluted to a concentration of 5 μg / ml in PBS with 2% FCS. Antibody binding was determined using R-phycoerythrin-conjugated affinity purified F (ab ') ₂ goat anti-mouse IgG antibody fragment (specific for the Fc gamma fragment) diluted 1: 100 in PBS with 2% FCS (ImmunoResearch Europe Ltd ., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).

16. Creation and characterization of MCSP- and CD3-bispecific single-chain molecules with interspecific specificity

Bispecific single chain antibody molecules, each containing a binding domain with interspecific specificity for human and non-chimpanzee CD3 epsilon primates, as well as a binding domain with interspecific specificity for human and primitive non-chimpanzee MCSPs, were constructed as follows. Table 2.

table 2 Cross-species specific MCSP and CD3 bispecific single chain antibodies formats SEQ ID (nucleon / white) Formats of protein constructs (N → C) 310/309 MCSP-G4 HL × H2C HL 312/311 MCSP-G4 HL × F12Q HL 314/313 MCSP-G4 HL × I2C HL 316/315 MCSP-G4 HLP × F6A HLP 318/317 MCSP-G4 HLP × H2C HLP 322/321 MCSP-G4 HLP × G4H HLP 326/325 MCSP-G4 HLP × E1L HLP 328/327 MCSP-G4 HLP × E2M HLP 332/331 MCSP-G4 HLP × F12Q HL 334/333 MCSP-G4 HLP × I2C HL 336/335 MCSP-D2 HL × H2C HL 338/337 MCSP-D2 HL × F12Q HL 340/339 MCSP-D2 HL × I2C HL 342/341 MCSP-D2 HLP × H2C HLP 344/343 MCSP-F9 HL × H2C HL 346/345 MCSP-F9 HLP × H2C HLP 348/347 MCSP-F9 HLP × G4H HLP

The above constructs containing the heavy chain variable domains (VH) and light chain variable domains (VL) with interspecific specificity for human and macaque MC3 D3, and the VH and VL domains with interspecific specificity for human and macaque CD3, were obtained by gene synthesis. Gene synthesis fragments were designed so that they contained first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, encoding the sequence of the corresponding molecule of the bispecific single chain antibody, then, in the reading frame, encoding the histidine sequence ₆ tags and stop codon. The gene synthesis fragment was also designed to introduce suitable N- and C-terminal restriction sites. A gene synthesis fragment was cloned through these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150) according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). These constructs were transfected stably or temporarily into DHFR defective CHO cells (ATCC No. CRL 9096) by electroporation or, alternatively, temporarily transfected into HEK 293 (human embryonic kidney cells, ATCC number: CRL-1573) according to standard protocols.

The expression of the eukaryotic protein in DHFR-defective CHO cells was carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by adding increasing concentrations of methotrexate (MTX) to a final concentration of 20 nM MTX, inclusive. After two passages of the stationary culture, the cells were grown in roller bottles in liquid soybean medium HyQ PF CHO without nucleosides (with 4.0 mM L-glutamine, with 0.1% Pluronic F-68; HyClone) for 7 days before collection. Cells were isolated by centrifugation and the supernatant containing the expressed protein was stored at -20 ° C.

For chromatography, Äkta® Explorer (GE Health Systems) and Unicorn® software were used. Immobilized metal affinity chromatography ("IMAC") was performed using Fractogel EMD chelate® (Merck), which was loaded with ZnCl ₂ according to the protocol proposed by the manufacturer. The column was equilibrated with buffer A (20 mM sodium phosphate buffer pH 7.2; 0.1 M NaCl), and the cell culture supernatant (500 ml) was applied to the column (10 ml) at a flow rate of 3 ml / min. The column was washed with buffer A to remove unbound sample. The bound protein was eluted using a two-step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2; 0.1 M NaCl; 0.5 M imidazole) in accordance with the following protocol:

stage 1: 20% buffer B in 6 column volumes;

Stage 2: 100% buffer B in 6 column volumes.

The eluted protein fractions from step 2 were combined for further purification. All chemicals had a purity suitable for research, and were purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration was performed on a HiLoad 16/60 Superdex 200 preparative column (GE / Amersham) equilibrated with Equi buffer (25 mM citrate; 200 mM lysine; 5% glycerol; pH 7.2). Eluted protein samples (flow rate 1 ml / min) were subjected to standard SDS-PAGE and Western blotting for detection. Before cleaning, the column was calibrated to determine molecular weight (molecular weight marker set, Sigma MW GF-200). Protein concentration was determined using OD at 280 nm.

The purified protein, a bispecific single chain antibody, was analyzed using SDS-PAGE under reducing conditions and pre-filled with 4-12% gels in bis-Tris buffer (Invitrogen). Preparation and application of samples was carried out according to the protocol proposed by the manufacturer. Molecular weight was determined using the MultiMark protein standard (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen protocol). The purity of the isolated protein was over 95% as determined by SDS-PAGE.

A bispecific single chain antibody has a molecular weight of approximately 52 kDa under native conditions as determined by gel filtration in phosphate buffered saline (PBS). All designs were cleaned according to this method.

Western blotting was performed using an Optitran® BA-S83 membrane in an Invitrogen blotter in accordance with the protocol proposed by the manufacturer. An anti-His tag antibody (Penta-His, Qiagen) was used to determine the protein of the bispecific single chain antibody. The goat anti-mouse Ig antibody labeled with alkaline phosphatase (AP) (Sigma) was used as a secondary antibody, and BClP / NBT (Sigma) as a substrate. A single band at 52 kDa was determined, which corresponded to a purified bispecific single chain antibody.

Alternatively, constructs were temporarily expressed in CHF defective CHO cells. Briefly, 4 × 10 ⁵ cells per construct were cultured in 3 ml of complete RPMI 1640 medium supplemented with a stabilizing glutamine supplement supplemented with 10% fetal calf serum, 1% penicillin / streptomycin and nucleosides from a concentrated cell culture reagent solution (cell culture grade) ( Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of adenosine 10 μg / ml, deoxyadenosine 10 μg / ml and thymidine 10 μg / ml, in an incubator at 37 ° C, 95% humidity and 7% CO ₂ one day before transfection. Transfection was carried out using a Fugene 6 transfection reagent (Roche, No. 111815091001) in accordance with the manufacturer's protocol. 94 μl of OptiMEM medium (Invitrogen) and 6 μl of Fugene 6 are mixed and incubated for 5 minutes at room temperature. After that, 1.5 μg of DNA per structure was added, mixed and incubated for 15 minutes at room temperature. At the same time, DHFR defective CHO cells were washed with 1x PBS and resuspended in 1.5 ml of complete RPMI 1640 medium. The transfection mixture was diluted with 600 μl of full RPMI 1640 medium, added to the cells and incubated overnight at 37 ° C, 95% humidity and 7% CO ₂ . One day after transfection, the incubated volume for each approach was increased to 5 ml by adding complete RPMI 1640 medium. The supernatant was collected after 3 days of incubation.

17. Flow cytometric analysis of the binding of MCSP- and CD3-bispecific antibodies with interspecific specificity

In order to test the functionality of interspecific specificity bispecific antibody constructs with respect to the ability to bind to human and macaque MCSP D3 and CD3, respectively, FACS analysis was performed. For this, CHO cells transfected with human MCSP D3 (as described in Example 14) and a CD3-positive human T-cell leukemia HPB-ALL cell line (DSMZ, Braunschweig, ACC483) were used to test binding to human antigens. The ability to bind to macaque antigens was tested using the created transfectant with MCSP D3 macaque (described in Example 15) and the T-cell line of macaque 4119 LnPx (kindly provided by Prof. Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200,000 cells of the corresponding cell population were incubated for 30 min on ice with 50 μl of purified interspecific specific bispecific antibody construct protein (2 μg / ml) or with a cell culture supernatant of transfected cells expressing these interspecific specific bispecific antibody constructs. Cells were washed twice in PBS with 2% FCS and the binding of this construct was determined using a mouse anti-His antibody (Penta-His antibody; Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS). After washing, the bound anti-His antibodies were determined using an Fc-gamma-specific antibody (Dianova) conjugated with phycoerythrin diluted 1: 100 in PBS with 2% FCS. As a negative control, the supernatant of untransfected CHO cells was used to bind to T cell lines. A single chain construct with irrelevant target specificity was used as a negative control for binding to MCSP-D3 transfected CHO cells.

Flow cytometry was performed on a FACS-Calibur apparatus; CellQuest software (Becton Dickinson biosciences, Heidelberg) was used to obtain and analyze data. FACS staining and fluorescence intensity measurements were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).

The bispecific binding of the single chain molecules above that exhibit interspecific specificity for MCSP D3 and interspecific specificity for human and macaque CD3 was clearly defined as shown in Figs. 14, 15, 16. In the FACS analysis, all constructs showed binding to CD3 and MCSP D3 compared to negative controls. The interspecific specificity of bispecific antibodies to human and macaque CD3 and MCSP D3 antigens has been demonstrated.

18. Biological activity of MCSP- and CD3-bispecific single chain antibodies with interspecific specificity

As shown in FIGS. 17-21, all cross-species specific bispecific single chain antibody constructs exhibited cytotoxic activity against MCSP-positive human target cells induced by CD8 + human cells and MCSP-positive target cells of cynomolgus macaque induced by the T cell line Macaque 4119 LnPx. A bispecific single chain antibody with a different target specificity is used as a negative control.

19. Plasma stability of cross-species specific MCSP and CD3 bispecific single chain antibodies

The stability of the generated bispecific single chain antibodies in human plasma was analyzed by incubating the bispecific single chain antibodies in 50% human plasma at 37 ° C and 4 ° C for 24 hours and subsequent testing of biological activity. Biological activity was investigated in an in vitro analysis of cytotoxicity for chromium release 51 ( ⁵¹ Cr) using the MCSP-positive CHO cell line (expressing MCSP cloned in accordance with Example 14 or 15) as target and stimulated human CD8-positive T cells as effector cells.

EC ₅₀ values calculated using the assay program described above are used to compare the biological activity of bispecific single chain antibodies incubated with 50% human plasma for 24 hours at 37 ° C and 4 ° C, respectively, with bispecific single chain antibodies without adding plasma or mixed with the same amount of plasma immediately before analysis.

As shown in FIG. 22 and Table 3, the biological activity of the bispecific antibodies G4 HL × I2C HL, G4 HL × H2C HL and G4 HL × F12Q HL was slightly reduced compared to controls without plasma or with plasma added immediately before biological testing activity.

Table 3 The biological activity of bespecifically antibodies without or with the addition of plasma Design Plasma free With plasma Plasma 37 ° C Plasma 4 ° C G4 H-L × I2C H-L 300 796 902 867 G4 H-L × H2C H-L 496 575 2363 1449 G4 H-L × F12Q H-L 493 358 1521 1040

20. Creation of bispecific single chain molecules with interspecific specificity for human EGFR and CD3

Bispecific single chain antibody molecules with a binding domain exhibiting interspecific specificity for human and cynomolgus macaque CD3 epsilon, as well as a binding domain exhibiting interspecific specificity for human EGFR, were constructed as shown in Table 4 below.

Table 4 Formats of EGFR and CD3 bispecific single chain antibodies with interspecific specificity SEQ ID (nucleon / white) Formats of protein constructs (N → C) 390/389 EGFR H-L × I2C HL 392/391 EGFR L-H × I2C HL 394/393 EGFR H-L × F12Q HL 396/395 EGFR L-H × F12Q HL 398/397 EGFR H-L × H2C HL 400/399 EGFR L-H × H2C HL 448/447 EGFR HL × H2C HL 450/449 EGFR HL × F12Q LH 452/451 EGFR HL × I2C HL

The above constructions containing light chain variable domains (L) and heavy chain variable domains (H) with interspecific specificity for human and cynomolgus EGFRs were obtained by gene synthesis. Gene synthesis fragments were designed so that they contained first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, the coding sequence of the corresponding molecule of the bispecific single chain antibody, then, in the reading frame, the coding sequence consisting of of 6 histidine tag residues and a stop codon.

The gene synthesis fragment was also designed to introduce suitable N- and C-terminal restriction sites. A gene synthesis fragment was cloned through these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150) according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). These constructs were stably transfected into CHF defective CHO cells (ATCC No. CRL 9096), and also obtained and purified as described in Example 10.

21. The creation of bespecifically single-chain molecules with interspecific specificity for human EGFR and CD3

Bispecific single chain antibody molecules with a binding domain exhibiting interspecific specificity for human and cynomolgus macaque CD3 epsilon, as well as a binding domain exhibiting interspecific specificity for human EGFR, were constructed as shown in Table 5 below.

Table 5 Formats of EGFR and CD3 bispecific single chain antibodies with interspecific specificity SEQ ID (nucleon / white) Formats of protein constructs (N → C) 410/4099 EGFR H-L × I2C HL 412/411 EGFR L-H × I2C HL 414/413 EGFR H-L × F12Q HL 416/415 EGFR L-H × F12Q HL 418/417 EGFR H-L × H2C HL 420/419 EGFR L-H × H2C HL

The above constructions containing light chain variable domains (L) and heavy chain variable domains (H) with interspecific specificity for human and cynomolgus EGFRs were obtained by gene synthesis. Gene synthesis fragments were designed so that they contained first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, the coding sequence of the corresponding molecule of the bispecific single chain antibody, then, in the reading frame, the coding sequence consisting of of 6 histidine tag residues and a stop codon.

The gene synthesis fragment was also designed to introduce suitable N- and C-terminal restriction sites. A gene synthesis fragment was cloned through these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150) according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). These constructs were stably transfected into CHF defective CHO cells (ATCC No. CRL 9096), and also obtained and purified as described in Example 10.

22. The creation of bespecifically single-chain molecules with interspecific specificity for Her2 / neu and human CD3

Bispecific single chain antibody molecules with a binding domain exhibiting interspecific specificity for human and cynomolgus monkey CD3 epsilon, as well as a binding domain exhibiting interspecific specificity for human Her2 / neu, were constructed as shown in the following Table 6.

Table 6 Formats of Her2 / neu- and CD3-bispecific single chain antibodies with interspecific specificity SEQ ID (nucleon / white) Formats of protein constructs (N → C) 430/439 HER2 / neu VH-VL × I2C VH VL 432/431 HER2 / neu VL-VH × I2C VH VL 434/433 HER2 / neu VH-VL × F12Q VH VL 436/435 HER2 / neu VL-VH × F12Q VH VL 438/437 HER2 / neu VH-VL × H2C VH VL 440/439 HER2 / neu VL-VH × H2C VH VL

The aforementioned constructs containing light chain variable domains (L) and heavy chain variable domains (H) with interspecific specificity for human Her2 / neu and cynomolgus monkey were obtained by gene synthesis. Gene synthesis fragments were designed so that they contained first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, the coding sequence of the corresponding molecule of the bispecific single chain antibody, then, in the reading frame, the coding sequence consisting of of 6 histidine tag residues and a stop codon.

The gene synthesis fragment was also designed to introduce suitable N- and C-terminal restriction sites. A gene synthesis fragment was cloned through these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150) according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). These constructs were stably transfected into CHF defective CHO cells (ATCC No. CRL 9096), and also obtained and purified as described in Example 10.

23.1. Obtaining CHO cells expressing human HER2

The human HER2 coding sequence, which is published in GenBank (accession number X03363), is obtained by gene synthesis according to standard protocols. A gene synthesis fragment is designed to contain the human HER2 protein coding sequence including its leader peptide (the cDNA sequence and amino acid sequence of this construct are presented in SEQ ID NOs: 459 and 460). A gene synthesis fragment is also designed to introduce restriction sites at the beginning and end of the fragment. The introduced restriction sites, Xbal at the 5'-end and Sall at the 3'-end, are used in subsequent cloning procedures. A gene synthesis fragment is cloned using Xbal and Sall into a plasmid designated pEFDHFR (pEFDHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150) following standard protocols. The above procedures are carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). A confirmed nucleotide sequence clone is used to transfect DHFR defective CHO cells for eukaryotic expression of this construct. Expression of the eukaryotic protein in CHF defective CHO cells is carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct is induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX inclusive.

23.2. Obtaining CHO cells expressing the extracellular domain of Macaque Her2

The coding sequence of human Ner2 described above is modified to include amino acids 123-1038 of macaque Ner2 protein, as published in GenBank (accession number XP_001090430). The coding sequence of this chimeric protein is obtained by gene synthesis according to standard protocols (the cDNA sequence and amino acid sequence of this construct are presented in SEQ ID NO: 461 and 462). The gene synthesis fragment is also designed so that it contains the Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and end of the fragment. The introduced restriction sites, Xbal at the 5'-end and Sall at the 3'-end, are used in subsequent cloning procedures. The gene synthesis fragment is then cloned using Xbal and Sall into a plasmid designated pEFDHFR (pEFDHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150). A confirmed sequence clone of this plasmid is used to transfect CHO / dhfr- cells as described above.

23.3. Creation of HER2 and CD3 bispecific single chain molecules with interspecific specificity

3.1. Cloning binding molecules with interspecific specificity

As a rule, molecules of bispecific single chain antibodies, each of which contains a binding specificity domain demonstrating interspecific specificity for human and macaque CD3 epsilon, as well as a binding specificity domain showing human and macaque interspecific specificity for HER2, are constructed as shown in the following below Table 7.

Table 7 Formats of Molecules of Anti-CD3 and Anti-HER2 Bispecific Single Chain Antibodies with Interspecific Specificity SEQ ID (nucleon / white) Formats of protein constructs (N → C) 432/431 Her2 LH × I2C HL 436/435 Her2 LH × F12Q HL 440/439 Her2 LH × H2C HL 430/429 Her2 HL × I2C HL 434/433 Her2 HL × F12Q HL 438/437 Ner2 HL × H2C HL 480/479 I2C HL × Her2 LH 478/477 F12Q HL × Her2 LH 476/475 H2C HL × Her2 LH

The above constructs containing light chain variable domains (L) and heavy chain variable domains (H) with interspecific specificity for human and macaque HER2, and CD3-specific VH and VL combinations with interspecific specificity for human and macaque CD3, are obtained by gene synthesis . Fragments of gene synthesis are designed so that they contain first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, the coding sequence of the corresponding molecule of the bispecific single chain antibody, then, in the reading frame, the coding sequence consisting of of 6 histidine tag residues and a stop codon. A gene synthesis fragment is also designed to introduce suitable restriction sites at the beginning and end of the fragment. The introduced restriction sites are used in subsequent cloning procedures. A gene synthesis fragment is cloned using these restriction sites into a plasmid designated pEFDHFR (pEFDHFR is described in Raum et al. Cancer Immunol. Immunother. 50 (2001) 141-150) following standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). A confirmed nucleotide sequence clone is transfected into CHF defective CHO cells for eukaryotic expression of this construct. Expression of the eukaryotic protein in CHF defective CHO cells is carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct is induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX inclusive.

3.2. Expression and purification of bispecific single chain antibody molecules

Bispecific single chain antibody molecules are expressed in Chinese hamster ovary (CHO) cells. Expression of the eukaryotic protein in CHF defective CHO cells is carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of constructs is induced by adding increasing concentrations of MTX to final concentrations of 20 nM MTX, inclusive. After two passages of stationary cell culture, cells were grown in roller bottles in liquid soybean medium HyQ PF CHO without nucleosides (with 4.0 mM L-glutamine, with 0.1% Pluronic F-68; HyClone) for 7 days before harvesting. Cells were isolated by centrifugation and the supernatant containing the expressed protein was stored at -80 ° C. Transfection is carried out using 293fectin reagent (Invitrogen, No. 12347-019) in accordance with the manufacturer's protocol.

For chromatography, the Äkta® Explorer (GE Health Systems) and Unicorn® software are used. Immobilized Metal Affinity Chromatography ("IMAC") was performed using Fractogel EMD chelate® (Merck), which was loaded with ZnCb according to the protocol proposed by the manufacturer. The column was equilibrated with buffer A (20 mM sodium phosphate buffer pH 7.2; 0.1 M NaCl) and the cell culture supernatant (500 ml) was applied to the column (10 ml) at a flow rate of 3 ml / min. The column is washed with buffer A to remove unbound sample. The bound protein is eluted using a two-step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2; 0.1 M NaCl; 0.5 M imidazole) in accordance with the following protocol:

stage 1: 20% buffer B in 6 column volumes;

Stage 2: 100% buffer B in 6 column volumes.

The eluted protein fractions from step 2 are combined for further purification. All chemicals are of a purity suitable for research and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration was performed on a HiLoad 16/60 Superdex 200 preparative column (GE / Amersham) equilibrated with Equi buffer (25 mM citrate; 200 mM lysine; 5% glycerol; pH 7.2). Eluted protein samples (flow rate 1 ml / min) are then subjected to standard SDS-PAGE and Western blotting for detection. Before cleaning, the column is calibrated to determine molecular weight (molecular weight marker set, Sigma MW GF-200). Protein concentration is determined using an OD of 280 nm.

The purified protein, a bispecific single chain antibody, is analyzed using SDS-PAGE under reducing conditions and pre-filled with 4-12% gels in bis-Tris buffer (Invitrogen). Preparation and application of samples is carried out in accordance with the protocol proposed by the manufacturer. Molecular weight is determined using the MultiMark protein standard (Invitrogen). The gel is stained with colloidal Coomassie (Invitrogen protocol). The purity of the isolated protein is more than 95%, as determined by SDS-PAGE.

The bispecific single chain antibody has a molecular weight of about 52 kDa under native conditions, as determined by gel filtration in PBS. All designs are cleaned according to this method.

Western blotting is performed using an Optitran® BA-S83 membrane in an Invitrogen blotter in accordance with a protocol proposed by the manufacturer. An anti-His-tag antibody (Penta-His, Qiagen) is used to determine the protein of a bispecific single chain antibody. The goat anti-mouse Ig antibody labeled with alkaline phosphatase (AP) (Sigma) is used as a secondary antibody, and BCIP / NBT (Sigma) as a substrate. A single band at 52 kDa was determined, which corresponds to a purified bispecific single chain antibody.

23.4. Flow cytometric analysis of the binding of HER2 and CD3 bispecific single chain antibodies with interspecific specificity

In order to test the functionality of interspecific specificity bispecific antibody constructs with respect to the ability to bind to human and macaque HER2 and CD3, respectively, FACS analysis is performed. For this, CHO cells transfected with human HER2 as described in Example 23.1 and a CD3-positive human T-cell leukemia HPB-ALL cell line (DSMZ, Braunschweig, ACC483) are used to test binding to human antigens. The ability to bind to macaque antigens is tested using the created transfectant with HER2 macaque described in Example 23.2 and the T-cell line of macaque 4119 LnPx (kindly provided by Prof. Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; published in Knappe A, et al al. ., and Fickenscher H., Blood 2000, 95, 3256-61). 200,000 cells of the corresponding cell lines are incubated for 30 min on ice with 50 μl of purified interspecific specific bispecific antibody construct protein (2 μg / ml). Cells were washed twice in PBS with 2% FCS and the binding of this construct was determined using a mouse anti-His antibody (Penta-His antibody; Qiagen; diluted 1:20 in 50 μl of PBS with 2% FCS). After washing, the bound anti-His antibodies are determined using an Fc-gamma-specific antibody (Dianova) conjugated with phycoerythrin diluted 1: 100 in PBS with 2% FCS. PBS with 2% FCS is used as a negative control for binding to T cell lines, as well as to HER2 transfected CHO cells.

Flow cytometry was performed on a FACS-Calibur apparatus; CellQuest software (Becton Dickinson biosciences, Heidelberg) is used to obtain and analyze data. FACS staining and measurement of fluorescence intensity is carried out as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).

The bispecific binding of the single chain molecules shown above that exhibit interspecific specificity for HER2 and interspecific specificity for CD3 of a human and non-chimpanzee primate was clearly defined as shown in FIG. 23. In the FACS analysis, all constructs show binding to CD3 and HER2 when compared with the corresponding negative controls. The interspecific specificity of bispecific antibodies to human and macaque CD3 and HER2 antigens was demonstrated.

23.5. The biological activity of HER2 and CD3 bispecific single chain antibodies with interspecific specificity

The biological activity of the generated bispecific single chain antibodies is analyzed by chromium 51 ( ⁵¹ Cr) release in in vitro cytotoxicity assays using the HER2-positive cell lines described in Examples 23.1 and 23.2. As effector cells, stimulated human CD4 / CD56-depleted PBMCs or the macaque T-cell line 4119 LnPx are used, as shown in the corresponding graphic materials.

Obtaining stimulated CD4 / CD56-depleted PBMC is as follows.

A commercially available anti-CD3-specific antibody (e.g., OCT3, Othoclone) is applied to a Petri dish (diameter 85 mm; Nunc) at a final concentration of 1 μg / ml for 1 hour at 37 ° C. Unbound protein is removed in one wash step using PBS. Fresh PBMCs are isolated from peripheral blood (30-50 ml of human blood) by ficoll gradient centrifugation according to standard protocols. 3-5 × 10 ⁷ PBMCs are added to a precoated Petri dish in 50 ml RPMI 1640 with a stabilizing additive of glutamine / 10% FCS / IL-2 20 units / ml (Proleukin, Chiron) and stimulated for 2 days. On the third day, the cells are harvested and washed once with RPMI 1640. IL-2 is added to a final concentration of 20 units / ml and the cells are cultured again for one day in the same cell culture medium as mentioned above. By depleting CD4 + T cells and CD56 + NK cells, CD8 + cytotoxic T lymphocytes (CTL) are enriched according to standard protocols.

Target cells were washed twice with PBS and labeled with 11.1 MBq ⁵¹ Cr in a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37 ° C. After this, the labeled target cells are washed 3 times with 5 ml RPMI and then used in the analysis of cytotoxicity. The analysis is carried out in a 96-well plate in a total volume of 250 μl of RPMI supplemented medium (as above) with E: T ratios of 1: 1 or 10: 1, which are indicated in the corresponding graphic materials. Put 1 μg / ml molecules of bespecifically single-chain antibodies with interspecific specificity and 15-21 of their five-fold dilutions. The analysis time is 18 hours, and cytotoxicity is measured in relative values of the released chromium in the supernatant, corresponding to the difference between the maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements are carried out in four replicates. Measurement of chromium radioactivity in supernatants is carried out using a Wizard 3 "gamma counter (Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of experimental data is carried out using Prism 4 for Windows (version 4.02, GraphPad Software Inc., San Diego, California, USA) Sigmoidal dose response curves typically have R ² values greater than 0.90 as determined using this software, and EC ₅₀ values calculated using this assay program are used to compare biological activity.

As shown in FIG. 24, cross-species specific bispecific single chain antibody constructs exhibit cytotoxic activity against human HER2 positive cells induced by stimulated CD4 / CD56-depleted human PBMCs and against macaque HER2 positive cells, induced by the T-cell line of macaque 4119 LnPx.

Example 24. Cloning and expression of membrane-bound forms of human IgE and macaque

The murine cell line J558L (obtained from Interlab Project, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy, ECACC 88032902), a variant of the myeloma cell line with spontaneous loss of the heavy chain that synthesizes and secretes the lambda light chain, was used to complement the membrane-bound variant human and macaque IgE chains, respectively. To create such constructs, synthetic molecules were obtained by gene synthesis according to standard protocols (nucleotide sequences of the constructs are presented in SEQ ID NO: 507 and 508). In these constructs, the human and macaque epsilon chain coding sequence was fused to the transmembrane region of human IgE, respectively. A built-in function in the specificity of the VH chain is directed against hapten ((4-hydroxy-3-nitro-phenyl) acetyl) (NP). The gene synthesis fragment was also designed so that it contained the Kozak site for eukaryotic expression of this construct, the immunoglobulin leader peptide and restriction sites at the beginning and end of the DNA. The introduced restriction sites, EcoRI at the 5'-end and Sall at the 3'-end, were used during the cloning step into the expression plasmid designated pEFDHFR. After confirming the sequence (macaque: XM_001116734: epsilon C-region Ig Rhesus monkey (Masas mulatta), mRNA; human: NC_000014: chromosome 14 Homo sapiens, complete sequence, National Center for Biotechnological Information, http: //www.ncbi.nlm .nih.gov / entrez) these plasmids were used to transfect CHO / dhfr- cells as described above. Expression of the eukaryotic protein in CHF defective CHO cells is carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct is induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX inclusive.

Example 25. The creation of IgE - and CD3-bespecifically single-chain molecules with interspecific specificity

Typically, molecules of bispecific single chain antibodies, each of which contains a domain with binding specificity for the CD3 antigen of human and cynomolgus macaque, as well as a domain with specificity of binding to the IgE antigen of human and macaque, were constructed as shown in Table 8 below.

Table 8 Formats of Molecules of Anti-CD3 and Anti-IgE Bispecific Single Chain Antibodies with Interspecific Specificity SEQ ID (nucleon / white) Formats of protein constructs (N → C) 496/495 IgE HL × H2C HL 498/497 IgE HL × F12Q HL 500/499 IgE HL × I2C HL 502/501 IgE LH × H2C HL 504/503 IgE LH × F12Q HL 506/505 IgE LH × I2C HL

The aforementioned constructs containing light chain variable domains (L) and heavy chain variable domains (H) with interspecific specificity for human and macaque IgE and CD3-specific VH and VL combinations with interspecific specificity for human and macaque CD3 are obtained by gene synthesis. Fragments of gene synthesis are designed so that they contain first the Kozak site for eukaryotic expression of this construct, then an immunoglobulin leader peptide of 19 amino acids, then, in the reading frame, the coding sequence of the corresponding molecule of the bispecific single chain antibody, then, in the reading frame, the coding sequence consisting of of 6 histidine tag residues and a stop codon. A gene synthesis fragment is also designed to introduce suitable restriction sites at the beginning and end of the fragment. The introduced restriction sites are used in subsequent cloning procedures. A gene synthesis fragment is cloned using these restriction sites into a plasmid designated pEFDHFR following standard protocols. A confirmed nucleotide sequence clone is transfected into CHF defective CHO cells for eukaryotic expression of this construct. Expression of the eukaryotic protein in CHF defective CHO cells is carried out as described in Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct is induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX inclusive. Alternatively, the constructs are used for transient transfection of DHFR defective CHO cells according to standard protocols.

To assess the ability to bind to human IgE, binding experiments were performed using FACS on a J558L cell line transfected with human IgE. Interspecific specificity for macaque IgE positive cells was tested using J558L cells transfected with macaque IgE. The same changes in cell lines are used in cytotoxicity assays performed with IgE and CD3 bispecific single chain antibodies with interspecific specificity. Other than this, analyzes were performed as described in Examples 4 and 5.

As shown in Fig. 23, the created IgE and CD3 bispecific single chain antibodies with interspecific specificity demonstrated binding to both human and cynomolgus macaque antigens, which confirmed their complete interspecific specificity.

As shown in FIG. 24, all of the cross-species specific bispecific single chain antibody constructs created showed cytotoxic activity against target cells positive for human IgE induced by CD8 + cells and target cells positive for macaque IgE induced by T- macaque cell line 4119 LnPx. An irrelevant bispecific single chain antibody was used as a negative control.

Example 26. Specific binding of scFv clones to the N-terminus of human CD3 epsilon

26.1. Bacterial expression of scFv constructs in E. coli XL1 Blue

As mentioned above, E. coli XL1 Blue cells transformed with pComb3H5Bhis / Flag containing VL and VH segments produce sufficient soluble scFv after excision of gene III fragment and induction with 1 mM IPTG. The scFv chain is exported to the periplasm, where it folds into a functional conformation.

The following scFv clones were selected for this experiment:

1) scFv4-10, 3-106, 3-114, 3-148, 4-48, 3-190 and 3-271, which are described in WO 2004/106380,

2) scFv from the H2C, F12Q and I2C clones that bind human anti-CD3 epsilon, which are described in this invention.

To obtain periplasmic preparations, bacterial cells transformed with corresponding scFv-containing plasmids that allow periplasmic expression were grown in SB medium supplemented with 20 mM MgCl ₂ and carbenicillin (50 μg / ml) and resuspended in PBS after collection. As a result of four cycles of freezing at -70 ° C and thawing at 37 ° C, the outer membrane of bacteria was destroyed by osmotic shock and soluble periplasmic proteins, including scFv, were released into the supernatant. After removal of the intact cells and cell debris by centrifugation, the supernatant containing human scFv against human CD3 was collected and used for further research. These crude scFv-containing supernatants will hereinafter be referred to as periplasmic preparations (PPP).

26.2. The binding of scFv to the fusion protein [CD3-epsilon (aa 1-27) person] -Fc

ELISA experiments were carried out by coating the wells of 96-well plastic plates (Nunc, maxisorb) with human [CD3 epsilon (aa 1-27)] -Fc fusion protein, usually at 4 ° C. overnight. The coating antigen solution was then removed, the wells were washed once with PBS / 0.05% Tween 20, and then blocked with PBS / 3% BSA for at least one hour. After removing the blocking solution, PPP and control solutions were added to the wells and were usually incubated for one hour at room temperature. The wells were then washed three times with PBS / 0.05% Tween 20. Determination of scFv bound to the immobilized antigen was carried out using biotin-labeled anti-FLAG-tag antibody (M2 anti Flag-Bio, Sigma, usually at a final concentration of 1 μg / ml PBS ), and was determined using peroxidase-labeled streptavidin (Dianova, 1 μg / ml PBS). The signal developed when an ABTS substrate solution was added and measured at a wavelength of 405 nm. Nonspecific binding of test samples to a blocking agent and / or a portion of a human IgG1 from a human [CD3 epsilon (aa 1-27)] -Fc fusion protein was investigated by performing identical analysis with identical reagents and identical timing on ELISA plates that were coated with IgG1 human (Sigma). As a negative control, PBS was used.

As shown in FIG. 25, scFv H2C, F12Q, and I2C show strong binding signals on the human [CD3 epsilon (aa 1-27)] -Fc fusion protein. human scFv 3-106, 3-114, 3-148, 3-190, 3-271, 4-10 and 4-48 (which are described in WO 2004/106380) do not show any significant binding that exceeds the level of negative control .

To exclude the possibility that the positive binding of scFv H2C, F12Q, and I2C to the wells coated with the human [CD3 epsilon (aa 1-27)] -Fc fusion protein may be due to binding to BSA (used as a blocking agent) and / or the Fc gamma region of human IgG1 from the fusion protein [human CD3 epsilon (aa 1-27)] -Fc, a second ELISA experiment was carried out in parallel. In this second ELISA experiment, all parameters were identical to those in the first ELISA experiment, except that in the second ELISA experiment, human IgG1 (Sigma) was applied instead of the fusion protein [human CD3 epsilon (aa 1-27)] -Fc. As shown in FIG. 26, none of the scFvs tested showed any significant binding to human BSA and / or IgG1 above the background level.

Taken together, these results allow us to conclude that scFv 4-10, 3-271, 3-148, 3-190, 4-48, 3-106 and 3-114 do not specifically bind to (aa 1-27) -region of human CD3 epsilon, while scFv H2C, F12Q, and I2C clearly show specific binding to the N-terminal 27 amino acids of human CD3 epsilon.

Claims (17)

1. The polypeptide containing the first binding domain, which is an antibody capable of specifically binding to the epitope of the human CD3ε chain and Callithrix jacchus, Saguinus oedipus or Saimirí sciureus , where the epitope is part of an amino acid sequence that is part of the group consisting of SEQ ID NO: 2, 4, 6, or 8, and a second binding domain, which is an antibody capable of specifically binding to EGFR (epidermal growth factor receptor), Her2 / neu (member of the epidermal growth factor receptor family), or IgE (immuno lobulin E) human and / or primate, non-chimpanzee, wherein the first binding domain comprises:
CDR (hypervariable region) -L1 selected from the group consisting of SEQ ID NO: 27, 117 and 153;
CDR-L2 selected from the group consisting of SEQ ID NO: 28, 118 and 154;
CDR-L3 selected from the group consisting of SEQ ID NO: 29, 119 and 155;
CDR-H1 selected from the group consisting of SEQ ID NO: 12, 30, 48, 66, 84, 102, 120, 138, 156 and 174;
CDR-H2 selected from the group consisting of SEQ ID NO: 13, 31, 49, 67, 85, 103, 121, 139, 157 and 175; and
CDR-H3 selected from the group consisting of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158 and 176. 2. The polypeptide according to claim 1, where the specified epitope is a part of the amino acid sequence included in the group consisting of SEQ ID NO: 2, 4, 6 or 8, and contains at least the amino acid sequence Gln-Asp-Gly-Asn- Glu. 3. The polypeptide according to claim 1, where the first binding domain contains a VL region (variable region of the light chain) containing CDR-L1, CDR-L2 and CDR-L3 selected from:
(a) CDR-L1, as presented in SEQ ID NO: 27, CDR-L2, as presented in SEQ ID NO: 28, and CDR-L3, as presented in SEQ ID NO: 29;
(b) CDR-L1, as presented in SEQ ID NO: 117, CDR-L2, as presented in SEQ ID NO: 118, and CDR-L3, as presented in SEQ ID NO: 119; and
(c) CDR-L1, as presented in SEQ ID NO: 153, CDR-L2, as presented in SEQ ID NO: 154, and CDR-L3, as presented in SEQ ID NO: 155. 4. The polypeptide according to claim 1, where the first binding domain contains a VH region (variable region of the heavy chain) containing CDR-H1, CDR-H2 and CDR-H3 selected from:
(a) CDR-H1, as presented in SEQ ID NO: 12, CDR-H2, as presented in SEQ ID NO: 13, and CDR-H3, as presented in SEQ ID NO: 14;
(b) CDR-H1, as presented in SEQ ID NO: 30, CDR-H2, as presented in SEQ ID NO: 31, and CDR-H3, as presented in SEQ ID NO: 32;
(c) CDR-H1, as presented in SEQ ID NO: 48, CDR-H2, as presented in SEQ ID NO: 49, and CDR-H3, as presented in SEQ ID NO: 50;
(d) CDR-H1, as presented in SEQ ID NO: 66, CDR-H2, as presented in SEQ ID NO: 67, and CDR-H3, as presented in SEQ ID NO: 68;
(e) CDR-H1, as presented in SEQ ID NO: 84, CDR-H2, as presented in SEQ ID NO: 85, and CDR-H3, as presented in SEQ ID NO: 86;
(e) CDR-H1, as presented in SEQ ID NO: 102, CDR-H2, as presented in SEQ ID NO: 103, and CDR-H3, as presented in SEQ ID NO: 104;
(g) CDR-H1, as presented in SEQ ID NO: 120, CDR-H2, as presented in SEQ ID NO: 121, and CDR-H3, as presented in SEQ ID NO: 122;
(h) CDR-H1, as presented in SEQ ID NO: 138, CDR-H2, as presented in SEQ ID NO: 139, and CDR-H3, as presented in SEQ ID NO: 140;
(i) CDR-H1, as presented in SEQ ID NO: 156, CDR-H2, as represented in SEQ ID NO: 157, and CDR-H3, as represented in SEQ ID NO: 158; and
(k) CDR-H1, as presented in SEQ ID NO: 174, CDR-H2, as presented in SEQ ID NO: 175, and CDR-H3, as presented in SEQ ID NO: 176. 5. The polypeptide according to claim 1, where the first binding domain contains a VL region selected from the group consisting of a VL region, as represented in SEQ ID NO: 35, 39, 125, 129, 161 or 165. 6. The polypeptide according to claim 1, where the first binding domain contains a VH region selected from the group consisting of a VH region, as represented 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. 7. The polypeptide according to claim 1, where the first binding domain contains a VL region and a VH region selected from the group consisting of:
(a) the VL region as represented in SEQ ID NO: 17 or 21, and the VH region as represented in SEQ ID NO: 15 or 19;
(b) the VL region as represented in SEQ ID NO: 35 or 39, and the VH region as represented in SEQ ID NO: 33 or 37;
(c) the VL region as represented in SEQ ID NO: 53 or 57, and the VH region as represented in SEQ ID NO: 51 or 55;
(d) the VL region as represented in SEQ ID NO: 71 or 75, and the VH region as represented in SEQ ID NO: 69 or 73;
(e) the VL region as represented in SEQ ID NO: 89 or 93, and the VH region as represented in SEQ ID NO: 87 or 91;
(e) the VL region as represented in SEQ ID NO: 107 or 111, and the VH region as represented in SEQ ID NO: 105 or 109;
(g) the VL region as represented in SEQ ID NO: 125 or 129, and the VH region as represented in SEQ ID NO: 123 or 127;
(h) the VL region as represented in SEQ ID NO: 143 or 147, and the VH region as represented in SEQ ID NO: 141 or 145;
(i) the VL region as represented in SEQ ID NO: 161 or 165, and the VH region as represented in SEQ ID NO: 159 or 163; and
(k) the VL region as represented in SEQ ID NO: 179 or 183, and the VH region as represented in SEQ ID NO: 177 or 181. 8. The polypeptide according to claim 7, where the first binding domain contains an amino acid sequence selected 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. 9. The polypeptide according to any one of claims 1 to 8, wherein said polypeptide is a bispecific single chain antibody molecule. 10. The polypeptide according to claim 9, where the bispecific single chain antibody molecule contains a group of the following sequences, such as CDR H1, CDR H2, CDR H3, CDR L1, CDR L2 and CDR L3, in a second binding domain selected from SEQ ID NO: 441 -446, SEQ ID NO: 453-458, SEQ ID NO: 463-468, SEQ ID NO: 481-486. 11. The polypeptide according to claim 9, where the bispecific single chain antibody molecule contains a sequence selected from:
(a) the amino acid sequence as represented in any of SEQ ID NO: 389, 391, 393, 395, 397, 399, 409, 411, 413, 415, 417, 419, 429, 431, 433, 435, 437, 439 447, 449, 451, 469, 471, 473, 475, 477, 479, 495, 497, 499, 501, 503 and 505; and
(b) the amino acid sequence encoded by the nucleic acid sequence, as represented in any of SEQ ID NO: 390, 392, 394, 396, 398, 400, 410, 412, 414, 416, 418, 420, 430, 432, 434, 436, 438, 440, 448, 450, 452, 470, 472, 474, 476, 478, 480, 496, 498, 500, 502, 504, and 506. 12. A nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 11. 13. A vector containing a nucleic acid sequence, as defined in clause 12, to obtain a polypeptide, as defined in any one of claims 1 to 11. 14. A host cell transformed or transfected with a vector as defined in clause 13 to produce a polypeptide as defined in any one of claims 1-11. 15. A method of producing a polypeptide according to any one of claims 1 to 11, comprising culturing a host cell as defined in 14, under conditions conducive to the expression of said polypeptide, and recovering said obtained polypeptide from the culture. 16. A pharmaceutical composition comprising an effective amount of a polypeptide according to any one of claims 1 to 11 or obtained according to the method of claim 15, for preventing, treating, or ameliorating a disease selected from a proliferative disease, tumor disease, or immunological disorder. 17. The use of the polypeptide according to any one of claims 1 to 11 or obtained according to the method according to clause 15 in the prevention, treatment or amelioration of a disease selected from a proliferative disease, tumor disease or immunological disorder.

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