RU2653443C2 - Bispecific anti-her2 / anti-her3 antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Bispecific antibody that specifically binds to human HER2 or Javanese macaque and HER3 is described, comprising: a VHH heavy chain variable domain that specifically binds to HER3, wherein the VHH heavy chain variable domain comprises amino acid sequences, at least 75 % homologous to the sequences of SEQ ID NO: 1–3, and an antibody or an antigen-binding domain thereof that specifically bind to human HER2 or Javanese macaque.

EFFECT: invention also relates to DNA encoding the said antibody corresponding to an expression vector and a production method, as well as to a method of treatment using the said bispecific antibody.

31 cl, 36 dwg, 27 ex, 7 tbl

Description

Technical field

The present invention relates to the field of biotechnology, namely to antibodies and the use of these antibodies. More specifically, the present invention relates to bispecific antibodies that specifically bind to HER2 and HER3. The invention also relates to a nucleic acid encoding a given antibody, an expression vector, a method for producing an antibody, and the use of an antibody for the treatment of diseases or disorders associated with HER2 and HER3.

State of the art

The group of human epidermal factor receptor (HER) receptors, also called ErbB receptors, belongs to the family of transmembrane receptor tyrosine kinases. This family includes the epidermal growth factor receptor (EGFR), also called ErbB-1 (or HER1), and the homologous receptors ErbB-2 (HER2), ErbB-3 (HER3) and ErbB-4 (HER4 ) These receptors are expressed in large numbers on the surface of epithelial cells (reviewed in Yarden and Pines 2012). Increased expression of HER receptors or their ligands, such as heregulin (HRG) or epidermal growth factor (EGF), is often observed in cancer patients (Wilson, Fridlyand et al. 2012).

The binding of the ligand to the extracellular domain of tyrosine kinases causes a receptor dimerization process, which can take place between two identical receptors (homodimerization) or between different receptors within the same family (heterodimerization). Dimerization activates the intracellular domains of tyrosine kinases and causes their autophosphorylation. This, in turn, triggers a series of downstream proliferative signaling cascades, including those mediated by mitogen-activated protein kinases, as well as the AKT signaling pathway aimed at cell survival (described in Yarden and Pines, 2012). Specific endogenous ligands could not be detected for ErbB-2, and it is assumed that normal activation of this receptor occurs through heterodimerization (Sergina, Rausch et al. 2007), while activation of the ErbB-3 receptor can occur with the participation of ligands. Such ligands include, among others, neuregulin (NRG) or heregulin (HRG).

Impaired regulation of ErbB receptors leads to the formation and growth of tumors (Yarden, Sliwkowski et al. 2001). A well-known example is the amplification and overexpression of the ErbB2 receptor, which is observed in 20-30% of breast and stomach cancers. Despite the absence of high affinity ligands, ErbB2 receptors successfully participate in the implementation of signaling pathways aimed at cell survival. This occurs through the formation of heterodimers with other receptors of the ErbB family, for example, with the ErbB-3 receptor.

Drugs targeting ErbB2 receptors (Baselga, Swain et al. 2009) have significantly improved treatment outcomes in many patients with tumors characterized by increased expression of ErbB2. However, in a significant proportion of patients, such treatment is ineffective, and those who initially responded to therapy develop resistance over time (Nahta, Yu et al. 2006). Activation of the ErbB3 receptor by its ligand can lead to resistance to ErbB-directed therapy (Huang, Gao et al. 2010; Sergina, Rausch et al. 2007; Gijsen, King et al. 2011). ErbB3 receptor expression is increased in trastuzumab resistant tumor cells (huMAb4D5-8, rhuMAb HER2, HERCEPTIN®; US Pat. No. 5,282,337, Narayan, Wilken et al. 2009), and is associated with an unfavorable prognosis in patients with breast cancer (Sassen , Rochon et al. 2008; Witton, Reeves et al. 2003). Despite the absence of an active kinase domain, ErbB3 forms heterodimers with other members of the ErbB family of receptors, which leads to phosphorylation of phosphoinositide-3-kinase (PI3K) and activation of powerful intracellular signaling cascades. The ErbB3 receptor is the preferred partner for ErbB2 during dimerization (Graus-Porta, Beerli et al. 1997; Tzahar, Waterman et al. 1996). The ErbB2 / ErbB3 heterodimer has been shown to be the most potent activator of the AKT cascade compared to other ErbB receptor dimers, with the inclusion of oncogenic signaling pathways strictly depending on the presence of ErbB3 (Holbro, Beerli et al. 2003; Lee-Hoeflich, Crocker et al. . 2008). However, since the ErbB3 receptor lacks an active kinase domain, and its amplification and significant overexpression do not occur in tumor cells, the ErbB2 receptor remained the main target for drugs attacking this heterodimeric unit.

Patent Document WO2009686825 describes a Nanobody specific for HER2.

Other HER2 antibodies with various properties are described in Tagliabue et al. Int. J. Cancer 47: 933-937 (1991); McKenzie et al. Oncogene 4: 543-548 (1989); Maier et al. Cancer Res. 51: 5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3: 350-362 (1990); Stancovski et al. PNAS (USA) 88: 8691-8695 (1991); Bacus et al. Cancer Research 52: 2580-2589 (1992); Xu et al. Int. J. Cancer 53: 401-408 (1993); WO 94/00136; Kasprzyk et al. Cancer Research 52: 2771-2776 (1992); Hancock et al. Cancer Res. 51: 4575-4580 (1991); Shawver et. al., Cancer Res. 54: 1367-1373 (1994); Arteaga et al. Cancer Res. 54: 3758-3765 (1994); Harwerth et al. J. Biol. Chem. 267: 15160-15167 (1992); U.S. Patent No. 5,783,186; and Klapper et al. Oncogene 14: 2099-2109 (1997); U.S. Patent No. 5,783,186; and Klapper et al. Oncogene 14: 2099-2109 (1997).

WO 97/35885 discloses HER3 antibodies. International application WO 2003/013602 describes inhibitors of HER activity, including HER antibodies. International publications WO 2007/077028 and WO 2008/100624 also describe HER3 antibodies.

The development of multispecific antibodies that specifically bind to HER family receptors is currently underway to reduce unwanted side effects and toxicity.

Patent EA 022201 B1 describes an agent capable of binding to a tumor cell and killing a tumor cell or inhibiting the proliferation of tumor cells containing a human serum albumin (HSA) linker that may be specific for HER2 / HER3.

Patent RU 2491294 C2 describes a dimeric antibody that is specific for HER2 / HER3.

Patent Document WO 2011143414 describes an isolated, recombinant antibody having binding affinity for an ErbB receptor signal molecule selected from the group consisting of at least one of EGFR, ErbB2, ErbB3 and ErbB4. The above antibody may be a monoclonal antibody, a polyclonal antibody, a single chain Fv antibody, a dimeric antibody, a trimeric antibody, a tetrameric antibody, a bispecific antibody, a mini antibody.

Patent Document WO 2012158818 describes a multispecific antibody specific for, in particular, HER2 and HER3.

In patent documents WO 2014182970, WO 2015066543, WO 2007084181, bispecific antibodies to HER2 / HER3 are described.

Patent documents WO 2008119353, WO 2015153765 and WO 2015173248 describe methods for producing a bispecific anti-HER2 / HER3 antibody.

EP 2727943 describes trispecific antibodies to HER2 / HER3 / EGFR.

An important discovery was the detection in the blood of representatives of the Camelidae family (camels, llamas, vicunas) in a significant number of special non-canonical antibodies with a simplified structure (Hamers Casterman C., Atarhouch T., Muyldermans S., Robinson G., Hamers C, Bajyana Songa E. , Bendahman N., Hamers R. 1993. Naturally occurring antibodies devoid of light chains. Nature. 363, 446-448). Such antibodies ("heavy chain antibody", HCAb) consist of a dimer of only one shortened heavy chain (without the CH1 domain), and there is no light chain. The antigen-recognizing site of HCAb is formed by only one heavy chain variable domain (VHH), which is directly connected through the hinge region to the Fc domain. Often, instead of VHH, the term “single domain antibody”, “nanobody”, “mini-antibody”, or “nanoantibody” is used. As it turned out, such a single-domain structure in an isolated form, in addition to small sizes (12-15 kDa), has several advantages over classical IgG antibodies, namely, aggregation, chemical and thermal stability. VHH antibodies can be efficiently cloned and expressed in bacteria and yeast. Possessing such properties, they received technological development both in the therapeutic direction developed by Ablynx, and in the field of laboratory and industrial chromatography with the CaptureSelect line of carriers.

Noncanonical antibodies (HCAb), consisting of an immunoglobulin heavy chain dimer only, were first detected by electrophoretic analysis of immunoglobulins in the blood serum of various members of the camelidae family (Hamers Casterman C, Atarhouch T., Muyldermans S., Robinson G., Hamers C, Bajyana Songa E ., Bendahman N., Hamers R. 1993. Naturally occurring antibodies devoid of light chains. Nature. 363, 446-448). The relative proportion of HCAb varies from about 15-25% (of all IgG) in llamas and vicunias to about 60-80% in camels (S.V. Tillib, “Camel Nanoantibodies” - an effective tool for research, diagnosis and therapy, Molecular Biology, 2011, Volume 45, No. 1, pp. 77-85).

Noncanonical antibodies (HCAb) are thought to be, at least in the case of camelids, the result of the relatively recent evolution of canonical antibody genes. The two constant domains of the heavy chains, CH2 and CH3, are highly conserved in the case of HCAb and classical antibodies. In the HCAb antibody composition, the domain corresponding to the first constant CH1 domain of classical antibodies is absent. The genome of a single-humped camel (Dromedary species) contains a cluster of about fifty VH and forty VHH gene, followed by multiple genes for D segments, J segments and genes for constant regions (Сμ, Сγ, Сε, Сα). Obviously, some of the Cγ genes are designed to form HCAb (mutation leads to the loss of the CH1 domain), while the rest are used to form classical antibodies (with a preserved CH1 domain). The same genes of segments D and J can randomly bind to either one of the VH or one of the VHH genes. This indicates that the VH and VHH genes are in the same locus (Nguyen VK, Hamers R., Wyns L., Muyldermans S. 1999. Loss of splice consensus signal is responsible for the removal of the entire CH1 domain of the functional camel IGG2A heavy-chain antibodies. Mol. Immunol. 36, 515-524; Woolven BP, Frenken L., van der Logt P., Nicholls PJ 1999. The structure of the llama heavy chain constant genes reveals a mechanism for heavy -chain antibody formation. Immunogenetics. 50, 98-101; Nguyen VK, Hamers R., Wyns L., Muyldermans S. 2000. Camel heavy-chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen-binding repertoire. EMBO J 19, 921-931; De Genst E., Saerens D., Muyldermans S., Conrath K. 2006. Antibody repertoire development in camelids. Develop. Comp. Immunol. 30, 187-198).

The organization of the variable domains of noncanonical antibodies (VHH) and the variable domains (VH) of classical antibodies is very similar, because in humans, the VH domains of the IgG3 subclass have a particularly pronounced homology with camelids VH and VHH. In both cases, V domains consist of four conserved FR framework regions that surround three complementarity determining complementarity regions, CDRs. In both cases, a spatial structure of two β-layers, typical of the immunoglobulin V domain, is formed, one of which consists of four amino acid chains and the second of five (Muyldermans S., Cambillau C., Wyns L. 2001. Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. TIBS. 26, 230-235; De Genst E., Silence K., Decanniere K., Loris R., Kinne J., Muyldermans S. 2006. Molecular basis for the preferential cleft recognition by dromedary heavy chain antibodies. Proc. Natl. Acad. Sci. USA. 103, 4586-4591). In this structure, all three hypervariable regions are clustered on one side of the V domain, where they participate in antigen recognition and are located in loops connecting β structures. However, there are important differences associated with the functioning of VHH in a single domain format. Thus, the hypervariable regions of CDR1 and CDR3 of VHH are markedly increased. Often, cysteine residues are immediately found in two regions in the hypervariable regions of VHH (most often in CDR1 and CDR3, less often in CDR2 and CDR3). When studying the crystal structures of VHH, it was shown that these cysteine residues form disulfide bonds, and this additionally stabilizes the structure of the loops of this antibody (De Genst E., Silence K., Decanniere K., Loris R., Kinne J., Muyldermans S. 2006. Molecular basis for the preferential cleft recognition by dromedary heavy chain antibodies. Proc. Natl. Acad. Sci. USA. 103, 4586-4591). The most obvious and reproducible distinguishing feature of VHH is the four substitutions of hydrophobic amino acid residues for hydrophilic ones in the second frame region (Val37Phe, Gly44Glu, Leu45Arg, Trp47Gly, according to the numbering of Kabat (Kabat) et al. (Kabat E., Wu T.T., Perry H M. M., Gottesman KS, Foeller S. 1991. Sequence of proteins of immunological interest. US Public Health Services. NIH, Bethesda, MD, Publication no. 91-3242)). This framework region at the VH domain is highly conserved, enriched in hydrophobic amino acid residues, and is especially important in the formation of a bond with the variable domain of the VL light chain. In this aspect, the VHH domain is very different: these substitutions of hydrophobic amino acids for hydrophilic ones make the association of VHH and VL impossible. These substitutions also explain the usually high solubility of VHH (nanoantibodies) when it is produced as a recombinant protein (Nguyen V.K., Desmyter A., Muyldermans S. 2001. Functional heavy-chain antibodies in camelidae. Adv. Immunol. 79, 261-296).

The repertoires of possible paratopes (antigen-binding structures of the antibody) of HCAb and classical antibodies, apparently, can differ markedly. Since these two types of antibodies coexist in one organism, it can be assumed that they do not compete, but mutually complement each other. Indeed, it has been repeatedly noted that both types of antibodies can occur in parallel, mutually exclusive, or in different proportions, with respect to different epitopes of antigenic material during immunization of the same animal. Despite the alleged lower diversity of possible paratopes in single-domain antibodies compared to classical two-domain antibodies, many authors have convincingly demonstrated that HCAb can be obtained against a wide variety of epitopes of a very wide range of antigens (Muyldermans S., Baral TN, Retamozzo VC, et al. 2009. Camelid immunoglobulins and nanobody technology. Vet. Immunol. Immunopathol. 128 (1-3), 178-183). Obviously, markedly increased hypervariable regions of CDR1 and CDR3 contribute to this. It should also be noted a surprisingly large (in comparison with the V-domains of classical antibodies) the number of somatic hypermutations in VHH that accumulate, apparently, during the affinity maturation of antibodies during immunization (de Genst E., Silence K., Decanniere K., Loris R., Kinne J., Wyns L., Muyldermans S. 2005. Strong in vivo maturation compensates for structurally restricted H3 loops in antibody repertoires. J. Biol. Chem. 280, 14114-14121). X-ray diffraction analysis showed that the VHH antigen-binding loop sites are capable of forming structures unusual for classical V domains (De Genst E., Silence K., Decanniere K., Loris R., Kinne J., Muyldermans S. 2006. Molecular basis for the preferential cleft recognition by dromedary heavy chain antibodies. Proc. Natl. Acad. Sci. USA. 103, 4586-4591; de Genst E., Silence K., Decanniere K., Loris R., Kinne J., Wyns L., Muyldermans S. 2005. Strong in vivo maturation compensates for structurally restricted H3 loops in antibody repertoires. J. Biol. Chem. 280, 14114-14121). If in the case of the VH and VL domains of classical antibodies, all six hypervariable regions make more or less the same contribution to antigen binding, then in the case of VHH, the CDR3 region is usually more important for paratope formation. It was shown that the CDR3 region in VHH (but not in VH or VL) can form unusual long finger-like protruding structures that can deepen into the antigen structure, in particular, recognize active centers of enzymes (De Genst E., Silence K., Decanniere K ., Loris R., Kinne J., Muyldermans S. 2006. Molecular basis for the preferential cleft recognition by dromedary heavy chain antibodies. Proc. Natl. Acad. Sci. USA. 103, 4586-4591). The small size of the antigen binding site (VHH) and its ability to form unusual protruding paratopes explains the possibility of producing HCAb capable of recognizing epitopes inaccessible to classical antibodies, for example, in the formation of antibodies that are effective enzyme inhibitors (Lauwereys M., Ghahroudi M., Desmyter A., Kinne J., Holzer W., De Genst E., Wyns L., Muyldermans S. 1998. Potent enzyme inhibitors derived from dromedary heavy chain antibodies. EMBO J. 17, 3512-3520).

Pinto, Alexander Szyroki, Hans J. de Haard, Michael Saunders, Peter Vanlandschoot Inger Sandlie 2012, Selection of Nanobodies that Target Human Neonatal Fc Receptor Scientific Reports, 3, 1118). Однако технологичность и иммуногенность этих подходов остается пока неясной и находится только на стадии проверки применимости как в клинических исследованиях так и на более ранних стадиях исследований.For all its high potential, unique, in comparison with classical IgG antibodies, specificity, the use of single domain VHH for therapeutic use is limited in some cases due to the rapid elimination of this small molecule from the body. There are a number of solutions to improve the pharmacokinetics of VHH structures, which include the use of chemical conjugation with PEG (PEG) and a covalent compound with a polypeptide that mediates reduced clearance from the blood, such as human serum albumin (HSA) and the Fc fragment of the classic human antibody, in the form of hybrid proteins with a half-life in blood of up to three weeks (Kontermann RE. 2009 Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs .; 23 (2): 93-109; Ken Coppieters, Torsten Dreier, Karen Silence, Hans de Haard, Marc Lauwereys, Peter Casteels, Els Beirnaert, Heidi Jonckheere, 2006 Formatted anti-tumor necrosis f actor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis Arthritis & Rheumatology .; 54 (6): 1856-66; Gabrielle Richard, Ashley J Meyers, Michael D McLean, Mehdi Arbabi-Ghahroudi, Roger MacKenzie, J Christopher Hall 2013. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody. PLoS One 22; 8 (7): e69495). The successful use of small peptides that are coupled by genetic engineering to VHH and capable of high-affinity non-covalent interaction with the same components (HSA and IgG) in human blood (Jan Terje Andersen, Maria Gonzalez-Pajuelo, Stian Foss, Ole JB Landsverk,

Pinto, Alexander Szyroki, Hans J. de Haard, Michael Saunders, Peter Vanlandschoot Inger Sandlie 2012, Selection of Nanobodies that Target Human Neonatal Fc Receptor Scientific Reports, 3, 1118). However, the manufacturability and immunogenicity of these approaches is still unclear and is only at the stage of testing applicability both in clinical trials and at earlier stages of research.

In addition, aggregation and chemical stability, affinity and immunogenicity impose the greatest limitation in the use of antibodies as drugs for the treatment of various diseases. Because most monoclonal antibodies are currently derived from murine, the use of such antibodies in patients leads to the development of an immune response to antibody therapy, for example, allergic reactions. These types of immune responses can ultimately lead to a loss of effectiveness in the treatment of at least and, in the worst case, possible serious anaphylactic reactions. On the other hand, aggregation or chemically unstable therapeutic antibodies during storage of the drug worsen its therapeutic properties and can enhance immunogenicity when introduced into the patient's body.

In connection with the foregoing, it is relevant to create bispecific antibodies based on VHH antibodies that effectively act on the HER signaling system, in particular, on HER2 and / or HER3, and which would have, in comparison with previously known antibodies, improved functional and therapeutic properties, in particular, increased aggregation, chemical and thermal stability, and improved affinity, and at the same time would be distinguished by ease and ease of preparation, including and on an industrial scale.

The bispecific BCD090 molecule selectively binds to the ErbB2 / ErbB3 heterodimer, resulting in the formation of an inactive trimer. This molecule is a more effective inhibitor of the activation of ErbB3 and ErbB2 receptors than monoclonal antibodies against ErbB2 or ErbB3.

SUMMARY OF THE INVENTION

The present invention relates to bispecific binding molecules, in particular antibodies directed to binding to HER2 and HER3 receptors. Such antibodies can be used to treat a disease or disorder mediated by HER2 and HER3, for example, selected from the group: breast cancer (BC), malignant neoplasms of the stomach, non-small cell lung cancer, malignant neoplasms of the head and / or neck. Compared with current methods of treating such diseases, including antibody treatment, it is contemplated that bispecific binding molecules, in particular antibodies directed to bind to the HER2 and HER3 receptors of the present invention, in particular the BCD090 bispecific molecule, can provide the best clinical response, used both separately and in combination with other therapy.

In one aspect, the present invention relates to a bispecific antibody that specifically binds to HER2 and HER3. This antibody contains a VHH heavy chain variable domain specific for HER3, and an antibody or antigen binding domain thereof that specifically binds to HER2. In some embodiments of the invention, the VHH heavy chain variable domain specific for HER3 and an antibody or antigen binding domain thereof that specifically bind to HER2 are linked by a peptide linker.

In some embodiments, the bispecific antibody comprises a VHH heavy chain variable domain comprising an amino acid sequence at least 75% homologous to SEQ ID NO: 3. In some embodiments, the bispecific antibody comprises a VHH heavy chain variable domain that contains the amino acid sequence of SEQ ID NO: 3 .

In some embodiments, the bispecific antibody comprises an HER3 specific VHH heavy chain variable domain that contains amino acid sequences homologous to at least 75% of the sequences of SEQ ID NO: 1-3. In some embodiments of the invention, the VHH heavy chain variable domain specific for HER3 comprises amino acid sequences homologous to at least 80% of the sequences of SEQ ID NO: 1-3. In some embodiments, the HER3 specific VHH heavy chain variable domain comprises amino acid sequences homologous to at least 85% of SEQ ID NOS: 1-3. In some embodiments of the invention, the VHH heavy chain variable domain specific for HER3 comprises amino acid sequences homologous to at least 90% of the sequences of SEQ ID NO: 1-3. In some embodiments, the HER3 specific VHH heavy chain variable domain comprises amino acid sequences homologous to at least 95% of the sequences of SEQ ID NO: 1-3. In some embodiments, the VHH heavy chain variable domain specific for HER3 contains amino acid sequences homologous to at least 98% of the sequences of SEQ ID NO: 1-3. In some embodiments of the invention, the VHH heavy chain variable domain specific for HER3 comprises the amino acid sequences represented by SEQ ID NOS: 1-3.

In some embodiments, the bispecific antibody comprises a VHH heavy chain variable domain that competes for binding or binds to the same epitope as the binding domain containing the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the bispecific antibody comprises an HER3 specific VHH heavy chain variable domain sequence that is at least 90% homologous to the amino acid sequence of SEQ ID NO: 4. In some embodiments, an HER3 specific VHH heavy chain variable domain sequence, is at least 95% homologous to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the variable domain sequence t the HER3 specific VHH yellow chain is at least 98% homologous to the amino acid sequence of SEQ ID NO: 4. In a separate embodiment of the invention, the HER3 specific VHH heavy chain variable domain sequence consists of the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the bispecific antibody comprises an antibody specific for HER2, which is a full-length IgG antibody. In some embodiments, a full-length antibody specific for HER2 is of the human IgG1, IgG2, IgG3, IgG4 isotype. In some embodiments, a full-length HER2-specific antibody comprises heavy and light chains, which respectively comprise amino acid sequences that are at least 80% homologous to SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, of the invention, a full-length antibody specific for HER2 contains heavy and light chains, which respectively contain amino acid sequences that are at least 90% homologous to the sequences of SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments of the invention, a full-length antibody specific for HER2 contains heavy and light chains, which respectively contain amino acid sequences that are at least 95% homologous to the sequences of SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the full-length antibody an antibody specific for HER2 contains heavy and light chains, which respectively contain amino acid sequences that are at least 98% homologous to the sequences of SEQ ID NO: 5 and SEQ ID NO: 6. In a separate embodiment suschestvleniya invention full length antibody specific for HER2, comprising the heavy and light chains which respectively comprise the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 6.

In some embodiments, a bispecific antibody that specifically binds to HER2 and HER3 comprises an antigen binding domain that specifically binds to HER2.

In some embodiments, a bispecific antibody that specifically binds to HER2 and HER3 is an asymmetric antibody.

In some embodiments, the bispecific antibody comprises a VHH heavy chain variable domain specific for HER3 and an antibody or antigen binding domain thereof that specifically binds to HER2 which are linked by a peptide linker of more than five amino acids. In some embodiments, the amino acid residues of the peptide linker are selected from G, A, S, P, E, T, D, and K. The VHH heavy chain variable domain is attached to the N-terminus or C-terminus of the light or heavy chain of an anti-specific antibody HER2, via a peptide linker.

In some embodiments, the bispecific antibody has a dissociation constant of KD binding to human HER2 of not more than 10 ^-10 M and monkeys to not more than 10 ^-7 M, K _{D of} binding to human HER3 not more than 10 ^-9 M and monkeys not more than 10 ^-8 M .

In one aspect, the present invention provides a solution for inhibiting the biological activity of HER2 and / or HER3 in a subject, which comprises a bispecific antibody that specifically binds to HER2 and HER3, and one or more pharmaceutically acceptable excipients.

In some embodiments, the solution for inhibiting the biological activity of HER2 and / or HER3 in a subject has such aggregation stability that, at concentrations of more than 10 mg / ml and storage at t = 4 ° C for more than 6 months, the aggregate content does not increase by more than 5% of the initial content in the solution.

In some embodiments, the solution for inhibiting the biological activity of HER2 and / or HER3 in a subject has such aggregation stability that at concentrations of more than 10 mg / ml and when the temperature rises to 37 ° C for more than 2 weeks, the aggregate content does not increase by more than 5% of the initial content in the solution.

In some embodiments, the solution for inhibiting the biological activity of HER2 and / or HER3 in a subject has such aggregation stability that, at concentrations of more than 10 mg / ml and a temperature increase of up to 50 ° C for more than 6 hours, the aggregate content does not increase by more than 5% of the initial content in solution.

In one aspect, the present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence encoding any of the bispecific binding molecules described herein.

In one aspect, the present invention relates to an isolated nucleic acid molecule that contains a nucleotide sequence that encodes any VHH heavy chain variable domain described herein.

In one aspect, the present invention relates to an isolated nucleic acid molecule that contains a nucleotide sequence that encodes a heavy chain, light chain, or both chains of an antibody specific for HER2 described herein.

In one aspect, the present invention relates to an isolated nucleic acid molecule, which contains the following:

a) any nucleotide sequence that encodes the VHH heavy chain variable domain described herein;

b) any nucleotide sequence that encodes a heavy chain of an antibody specific for HER2 described herein;

c) any nucleotide sequence that encodes the light chain of an antibody specific for HER2 described herein; or

d) any combination of a) to c).

In one aspect, the present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence encoding any VHH heavy chain variable domain described herein and any nucleotide sequence encoding a heavy chain, light chain, or both chains of an antibody specific for HER2 described herein, in some cases, further comprises a nucleotide sequence encoding a peptide linker.

In one aspect, the present invention relates to an isolated nucleic acid molecule that encodes any of the amino acid sequences of SEQ ID NO: 1-6.

In one aspect, the present invention relates to an isolated nucleic acid molecule that contains a nucleotide sequence that encodes any of the bispecific antibodies that specifically bind to HER2 and HER3 described herein. In some embodiments, the isolated nucleic acid molecule comprises a DNA sequence that encodes any of the bispecific antibodies that specifically bind to the HER2 and HER3 described herein.

In one aspect, the present invention relates to a vector comprising any of the isolated nucleic acid molecules described above, said vector further comprising expression control sequences.

In one aspect, the present invention relates to a method for producing a host cell for producing any of the antibodies described above, wherein the method comprises transforming a cell with any of the above vectors.

The present invention also relates to a host cell containing any of the above nucleic acids.

In one aspect, the present invention relates to a method for producing any bispecific antibodies that specifically binds to HER2 and HER3 described herein, which comprises obtaining a host cell, as described above, culturing said host cell in a culture medium under conditions sufficient to obtain the specified antibodies, as well as, if necessary, followed by isolation and purification of the obtained antibodies.

In one aspect, the present invention relates to a pharmaceutical composition for the prophylaxis or treatment of a disease or disorder mediated by HER2 and HER3, which contains any bispecific antibody that specifically binds to HER2 and HER3 described herein in combination with one or more pharmaceutically acceptable excipients.

In some embodiments, a pharmaceutical composition that contains any bispecific antibody that specifically binds to HER2 and / or HER3 described herein is intended to prevent or treat a disease or disorder mediated by HER2 and HER3 selected from the group: breast cancer glands (BC), gastric malignancies, non-small cell lung cancer, head and / or neck malignancies.

In one aspect, the present invention relates to a method of inhibiting the biological activity of HER2 and / or HER3 in a subject in need of such inhibition, which comprises administering to the subject an effective amount of any bispecific antibody that specifically binds to HER2 and HER3 described herein.

In one aspect, the present invention relates to a method for treating a disease or disorder mediated by HER2 and / or HER3, which comprises administering to a subject in need of such treatment any bispecific antibody that specifically binds to HER2 and HER3 described herein, or any a solution described herein that contains any bispecific antibody that specifically binds to HER2 and HER3 described herein, or any pharmaceutical composition described herein m document, which contains any bespecifically antibody that specifically binds to HER2 and HER3 described herein in a therapeutically effective amount.

In some embodiments, the method of treatment is intended to treat a disease or disorder, wherein the disease or disorder is selected from the group: breast cancer (breast cancer), gastric malignancies, non-small cell lung cancer, head and / or neck malignancies.

In one aspect, the present invention relates to the use of any bispecific antibody that specifically binds to HER2 and HER3 described herein, or any solution described herein that contains any bispecific antibody that specifically binds to HER2 and HER3 described herein, or any pharmaceutical composition described herein that contains any bispecific antibody that specifically binds to HER2 and HER3 described herein Document, for treating a subject in need of such treatment, disease or disorder mediated HER2 and / or HER3.

In some embodiments, the use, for treating a subject in need of such treatment, of a disease or disorder mediated by HER2 and / or HER3, wherein the disease or disorder is selected from the group: breast cancer (BC), gastric malignancies, non-small cell lung cancer, malignant neoplasms of the head and / or neck.

Brief Description of the Drawings

FIG. 1. Scheme for the preparation of a bispecific anti-HER2 / anti-HER3 antibody.

FIG. 2. The sequence of the protein HER3-His6-FLAG (HER3HF).

FIG. 3. The sequence of the 3DHER3-His6-EPEA protein (3DHER3HE).

FIG. 4. The sequence of the protein HER3-Fc-His6 (HER3-Fc).

FIG. 5. The sequence of the control antibody ahti-HER3 AV203 with the published sequence (from AVEO, Biogen, WO 2011/136911 A2)

FIG. 6. Purified protein preparations HER3HF, 3DHER3HE, HER3-Fc.

FIG. 7. The purified preparation of a control antibody against HER3.

FIG. 8. Antibody titer against human HER3 obtained by immunization with Lama glama.

FIG. 9. Plasmid map for phage display of Fab antibody fragments.

FIG. 10. Plasmid map for secretion expression of Fab antibody fragments in E. coli.

FIG. 11. The graph of the dependence of the binding signal of the polyclonal phage with specific and non-specific antigens after selection of the immune phage library.

FIG. 12. An enzyme-linked immunosorbent assay for the interaction of a monospecific anti-HER3 antibody with HER3 and other antigens.

FIG. 13. ELISA binding of BCD-090 antibodies to various orthologous HER2 proteins.

FIG. 14. ELISA binding of BCD-090 antibodies to various orthologous HER3 proteins.

FIG. 15. Association / dissociation curves for the human HER3 / BCD090 complex obtained for two different analyte concentrations.

Fig.16. Association / dissociation curves for the HER3 Rhesus monkey / BCD090 complex obtained for two different analyte concentrations.

FIG. 17. Association / dissociation curves for the cynomolgus / BCD090 macaque HER2 complex, obtained for seven different analyte concentrations.

FIG. 18. Association / dissociation curves for the human HER2 / BCD090 complex.

FIG. 19. Sensogram for measuring the simultaneous binding of BCD090 with an antibody of both human ligands HER2 and HER3 on an OctetRED96 instrument.

FIG. 20. A graph of the percentage of living cells of the MCF7 line versus the concentration of antibodies in the medium after 72 hours in a CO ₂ incubator. Error bars represent the standard deviation calculated in three repetitions for each antibody concentration.

FIG. 21. A graph of the percentage of living cells of the BT474 line versus the concentration of antibodies in the medium after 72 hours in a CO ₂ incubator. Error bars represent the standard deviation calculated in three repetitions for each antibody concentration.

FIG. 22. A plot of the percentage of living cells of the A431NS line versus the concentration of antibodies in the medium after 72 hours in a CO ₂ incubator. Error bars represent the standard deviation calculated in three repetitions for each antibody concentration.

FIG. 23. A plot of the percentage of live SKBR-3 cells versus antibody concentration in the medium after 72 hours in a CO ₂ incubator. Error bars represent the standard deviation calculated in three repetitions for each antibody concentration.

FIG. 24. Determination of thermocolloidal stability by the point of protein aggregation by dynamic light scattering (DLS) for BCD-090 antibodies. The graph of the dependence of the average particle size (Z-average) on temperature in four buffer solutions.

Fig.25. Determination of thermal stability during prolonged exposure with the method of "Thermostress 50 ° C" for BCD090 antibodies in four different buffer solutions.

FIG. 26. Chromatograms reflecting the result of thermostress analysis of the bispecific antibody BCD090: red - intact, blue - 72 hours of incubation at 50 ° C. The wavelength is 220 nm.

Fig.27. Schemes of methods for detecting BCD090 in human serum.

FIG. 28. Openness of various concentrations of BCD090 in human serum before and after storage at 37 ° C.

Fig.29. The dependence of the number of dead cells of the VT474 line on the concentration of antibodies in the medium.

FIG. 30. Indicators of inhibition of tumor growth of the MCF7 line in the experimental groups, reflecting the antitumor activity of the drug.

FIG. 31. The growth dynamics of the MCF7 line tumor in the experimental groups.

FIG. 32. Indicators of inhibition of tumor growth line SK-BR-3 in the experimental groups, reflecting the antitumor activity of the drug.

FIG. 33. The growth dynamics of the tumor line SK-BR-3 in the experimental groups.

FIG. 34. Indices of inhibition of tumor growth of the VT474 line in the experimental groups, reflecting the antitumor activity of the drug.

FIG. 35. The growth dynamics of the tumor line VT474 in the experimental groups.

FIG. 36. Model of the complex HER3 (4LEO) and VHH-aHER3.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Methods

Unless otherwise specified herein, the scientific and technical terms used in connection with the present invention will have meanings that are commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in practice or in testing the present invention. All publications and other referenced materials mentioned herein are incorporated by reference in their entirety. In case of conflict, the present description, including definitions, will prevail. Although a number of documents are cited in this document, such citation is not an admission that any of these documents forms part of well-known knowledge in this field.

In addition, unless the context requires otherwise, the terms in the singular include the terms in the plural, and the terms in the plural include the terms in the singular. Typically, the classification and methods used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as protein and nucleic acid hybridization and chemistry described herein are well known. specialists and are widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is usually done in the art, or as described herein.

In this description and embodiments of the invention, the words “have” and “contain” or their variations, such as “has”, “having”, “contains” or “containing”, should be understood as including the specified whole or group of whole, but not an exception any other whole or group of whole.

Antibody Definitions

An “HER receptor” is a tyrosine protein kinase receptor that belongs to the HER receptor family and includes the EGFR, HER2, HER3 and HER4 receptors and other members of this family that will be identified in the future. The HER receptor, as a rule, may contain an extracellular domain that can bind the HER ligand; lipophilic transmembrane domain; conserved intracellular tyrosine kinase domain; and a signal transduction domain located at the carboxyl end that carries several tyrosine residues that can be phosphorylated. Preferably, the HER receptor is a human HER receptor with a native sequence.

The extracellular domain of HER2 contains four domains: domain I (amino acid residues from about 1 to 195), domain II (amino acid residues from about 196 to 319), domain III (amino acid residues from about 320 to 488) and domain IV (amino acid residues from about 489 to 630) (residue numbering without signal peptide). See Garrett et al. Mol. Cell. 11: 495-505 (2003), Cho et al. Nature 421: 756-760 (2003), Franklin et al. Cancer Cell 5: 317-328 (2004) and Plowman et al. Proc. Natl. Acad. Sci. 90: 1746-1750 (1993). Also see FIG. 1 in this description.

The terms “ErbB1”, “HER1”, “epidermal growth factor receptor” and “EGFR” are used interchangeably herein and refer to EGFR as described, for example, in Carpenter et al. Ann. Rev. Biochem. 56: 881-914 (1987), including its naturally occurring mutant forms (e.g., the deletion mutant EGFR, which is described in Humphrey et al. PNAS (USA) 87: 4207-4211 (1990)). ErbB1 refers to a gene encoding a protein product of EGFR.

The expressions “ErbB2” and “HER2” are used interchangeably herein and refer to the human HER2 protein described, for example, in Semba et al., PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al. Nature 319: 230-234 (1986) (access number in Genebank X03363). The term "erbB2" refers to a gene encoding human ErbB2, and "neu" refers to a gene encoding rat p185neu. Preferred HER2 is human HER2 with a native sequence.

“ErbB3” and “HER3” refer to a receptor polypeptide as described, for example, in US Pat. Nos. 5,183,884 and 5,480,968, as well as in Kraus et al. PNAS (USA) 86: 9193-9197 (1989).

The terms "ErbB4" and "HER4" in the present description refer to the receptor polypeptide, which is described, for example, in the application for the grant of patent EP No. 599274; Plowman et al., Proc. Natl. Acad. Sci. USA 90: 1746-1750 (1993); and Plowman et al., Nature, 366: 473-475 (1993), including its isoforms, which are described, for example, in WO 99/19488, published April 22, 1999.

By "HER ligand" is meant a polypeptide that binds and / or activates the HER receptor. The HER ligand of particular interest according to the present invention is a native sequence HER ligand such as epidermal growth factor (EGF) (Savage et al., J. Biol. Chem. 247: 7612-7621 (1972)); transforming growth factor alpha (TGF-α) (Marquardt et al., Science 223: 1079-1082 (1984)); amphiregulin, also known as autocrine growth factor of schwannoma and keratinocytes (Shoyab et al. Science 243: 1074-1076 (1989); Kimura et al. Nature 348: 257-260 (1990); and Cook et al. Mol. Cell. Biol 11: 2547-2557 (1991)); betacellululin (Shing et al., Science 259: 1604-1607 (1993); and Sasada et al. Biochem. Biophys. Res. Commun. 190: 1173 (1993)); heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al., Science 251: 936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem. 270: 7495-7500 (1995); and Komurasaki et al. Oncogene 15: 2841-2848 (1997)); heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al., Nature 387: 512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Natl. Acad. Sci. 94: 9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et al. Oncogene 18: 2681-89 (1999)); and cripto- (CR-1) (Kannan et al. J. Biol. Chem. 272 (6): 3330-3335 (1997)). HER ligands that bind EGFR include EGF, TGF-α, amphiregulin, betacellululin, HB-EGF and epiregulin. HER ligands that bind HER3 include heregulins. HER ligands capable of binding to HER4 include betacellululin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4, and heregulins.

As used herein, the terms “binding to human HER2” or “specifically binding to human HER2” or “anti-HER2 antibody” are used interchangeably and refer to an antibody that specifically binds to human HER2 antigen with binding affinity that matters KD 1 × 10 ⁻⁸ mol / L or lower at 25 ° C., in one embodiment, has a KD value of 1 × 10 ⁻⁹ mol / L or lower at 25 ° C. Binding affinity is determined in a standard binding assay at 25 ° C, such as a surface plasma resonance technique (BIAcore®, GE-Healthcare, Uppsala, Sweden). A method for determining the KD binding affinity value is described in Example 2b). Thus, the term “human HER2 binding antibody” as used herein refers to an antibody specifically binding to a human HER2 antigen with binding affinity with a KD value of 1 × 10 ⁻⁸ mol / L or lower (preferably 1 × 10 ^-8 mol / L - 1.0 × 10 ^-12 mol / L) at 25 ° C.

As used herein, the terms “binding to human HER3” or “specifically binding to human HER3” or “anti-HER3 antibody” are used interchangeably and refer to an antibody specifically binding to a human HER3 antigen with binding affinity that matters KD 1 × 10 ⁻⁸ mol / L or lower at 25 ° C., in one embodiment, has a KD value of 1 × 10 ⁻⁹ mol / L or lower at 25 ° C. Binding affinity is determined in a standard binding assay at 25 ° C, such as a surface plasma resonance technique (BIAcore®, GE-Healthcare, Uppsala, Sweden). A method for determining the KD binding affinity value is described in Example 2b). Thus, the term “human HER3 binding antibody” as used herein refers to an antibody specifically binding to a human HER3 antigen with binding affinity with a KD value of 1 × 10 ⁻⁸ mol / L or lower (preferably 1 × 10 ^-8 mol / L - 1.0 × 10 ^-12 mol / L) at 25 ° C.

Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, tyrosine kinase receptor erbB-3) encodes an epidermal growth factor receptor (EGFR), a member of the tyrosine kinase receptor family that also includes HER1 (also known as EGFR ), HER2 and HER4 (Kraus, M.N. et al, PNAS 86 (1989) 9193-9197; Plowman, GD et al, PNAS 87 (1990) 4905-4909; Kraus, MH et al, PNAS 90 (1993) 2900-2904). Like the prototype epidermal growth factor receptor, the HER3 transmembrane receptor consists of an extracellular ligand binding domain (ECD), an ECD dimerization domain, a transmembrane domain, an intracellular tyrosine kinase protein (TKD) domain, and a C-terminal phosphorylated domain. This membrane-bound protein has a heregulin (HRG) -binding domain of HER3 in the extracellular domain, but does not have an active kinase domain. Thus, it can bind this ligand, but does not transmit a signal into the cell through protein phosphorylation. However, it forms heterodimers with other members of the HER family that possess kinase activity. Heterodimerization leads to activation of the receptor-mediated signaling pathway and transphosphorylation of its intracellular domain. The formation of dimers between members of the HER family enhances the transmission of the HER3 signal and is a means of not only distributing the signal, but also amplifying the signal. For example, the HER2 / HER3 heterodimer induces one of the most important mitogenic signals through the PI3K and AKT pathways among members of the HER family (Sliwkowski M.Kh., et at, J. Biol. Chem. 269 (1994) 14661-14665; Alimandi M, et al, Oncogene. 10 (1995) 1813-1821; Hellyer, NJ, J. Biol. Chem. 276 (2001) 42153-4261; Singer, E., J. Biol. Chem. 276 (2001) 44266-44274; Schaefer , KL, Neoplasia 8 (2006) 613-622).

Amplification of this gene and / or overexpression of its protein was found in many cancers, including tumors of the prostate gland, bladder and breast. Alternative splicing transcriptional variants encoding various isoforms have been characterized. One isoform lacks an intermembrane region, and this isoform is secreted outside the cell. This form acts by modulating the activity of the form associated with the membrane. Additional splicing options were also reported, but they were not described in detail.

The term “antibody”, as used herein, includes whole antibodies and any antigen binding fragment (ie, “antigen binding part”) or its individual chains. The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding part. Each heavy chain contains a variable region of the heavy chain (abbreviated herein as VH) and a constant region of the heavy chain. The constant region of the heavy chain consists of three domains, CH1, CH2, and CH 3. Each light chain consists of a variable region of the light chain (abbreviated herein as VL) and a constant region of the light chain. The constant region of the light chain consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), scattered between regions that are more conservative, called framework regions (FR). Each VH and VL consists of three CDRs and four FRs located from the amino end to the carboxy end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system.

A “bispecific antibody” is an antibody containing an antigen binding domain or antigen binding domains that are capable of specifically binding to two different epitopes on one biological molecule or are capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having "double specificity" or as being an antibody with "double specificity".

By bispecific antibody BCD-090 is meant a bispecific antibody that specifically binds to HER2 and HER3. This antibody contains a VHH heavy chain variable domain specific for HER3, and an antibody or antigen binding domain thereof that specifically binds to HER2. In some embodiments of the invention, the VHH heavy chain variable domain specific for HER3 and an antibody or antigen binding domain thereof that specifically bind to HER2 are linked by a peptide linker.

The term "peptide linker" in this document means any peptide with the ability to join domains with a length depending on the domains that it binds to each other, containing any amino acid sequence. Preferably, the peptide linker has a length of more than 5 amino acids and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

HER2-HER3-binding antibodies also include heavy chain antibodies (HCAb). Exceptions to the structure of the H ₂ L ₂ conventional antibodies are found in certain immunoglobulin isotypes found in the Camelidae family (camels, dromedars (one-humped camels and llamas; Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al. , 1998 J. Mol. Biol. 275: 413), carpet sharks (wobbegongs) (Nuttal et al., Mol Immunol. 38: 313-26, 2001), baleen shark nannies (Greenberg et al., Nature 374: 164 -73, 1995; Roux et al., Proc. Natl. Acad. Sci. USA 95: 11804) and spotted chimeras (Nguyen et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," 2002 Immunogenetics 54 (1): 39-47)). Such antibodies can obviously form antigen binding regions using only the heavy chain variable region, so that functional antibodies are only heavy chain dimers (called "heavy chain antibodies" or designated as "HCAb"). Thus, some variants of these HER2-HER3-binding antibodies may be heavy chain antibodies (HCAb) that specifically bind to HER2 and HER3. For example, heavy chain antibodies, which are IgG class antibodies and lack light chains, are produced by animals of the Camelidae family, which includes camels, dromedars, and llamas (Hamers-Casterman et al., Nature 363: 446-448 (1993)). HCAb have a molecular weight of approximately 95 kDa instead of a molecular weight of approximately 160 kDa of conventional IgG antibodies. Their binding domains consist only of the variable domains of the heavy chain, often referred to in the present description as V _HH , to distinguish them from conventional V _H. Muyldermans et al., J. Mol. Recognit. 12: 131-140 (1999). The variable domain of heavy chain antibodies is sometimes referred to as the Nanobody (Cortez-Retamozo et al., Cancer Research 64: 2853-57, 2004). A library of nanobodies can be generated from immunized dromemedar as described by Conrath et al., Antimicrob Agents Chemother 45: 2807-12, 2001, or using recombinant methods.

Since the first constant domain (C _H1) is absent (spliced during mRNA processing due to loss of signal splice consensus) with the variable domain V _HH follows directly hinge region domains C _H2 and C _H3 (Nguyen et al, Mol Immunol 36...: 515-524 (1999); Woolven et al., Immunogenetics 50: 98-101 (1999)). _{VHH of} camelids is recombined with constant regions of IgG2 and IgG3, which contain a hinge region, CH2 and CH3 domains and lack the CH1 domain (Hamers-Casterman et al., Supra). For example, Llama IgG1 is a common antibody isotype (H ₂ L ₂ ) in which V _H recombines with a constant region that contains a hinge region, CH1, CH2 and CH3 domains, while Llama IgG2 and IgG3 are isotypes of the heavy chain only, which lacks CH1 domains and which does not contain light chains.

Although HCAb lacks light chains, they have an antigen binding repertoire. The mechanism for the genetic generation of HCAb is discussed in Nguyen et al., Adv. Immunol 79: 261-296 (2001) and Nguyen et al., Immunogenetics 54: 39-47 (2002). Sharks, including the whiskered nanny shark, exhibit similar individual monomeric V domains containing receptor antigen. Irving et al., J. Immunol. Methods 248: 31-45 (2001); Roux et al., Proc. Natl. Acad. Sci. USA 95: 11804 (1998).

V _HHs contain small intact antigen binding fragments (e.g., fragments that have a molecular weight of 15 kDa, 118-136 residues). It was found that the V _HH domains of camelids bind to the antigen with high affinity (Desmyter et al., J. Biol. Chem. 276: 26285-90, 2001), with V _HH affinity usually in the nanomolar range and comparable to the affinity of the fragments Fab and scFv. V _HHs are highly soluble and more stable than the corresponding derivatives of scFv and Fab fragments. The V _H fragments were relatively difficult to obtain in soluble form, but improvements in solubility and specific binding can be obtained when the framework residues are altered so that they are more V _HH-like . (See, for example, Reichman et al., J Immunol Methods 1999, 231: 25-38). V _HHs carry amino acid substitutions that make them more hydrophilic and prevent prolonged interaction with BiP (immunoglobulin heavy chain binding protein), which normally binds to the H chain in the endoplasmic reticulum (ER) during folding and assembly until it is displaced by L- chain. Due to the increased hydrophilicity of V _HH, secretion from ER improves.

Functional V _HH can be obtained by proteolytic cleavage of the HCAb of an immunized camelid animal family, direct cloning of V _HH genes from B cells of an immunized camel to produce recombinant V _HH , or from naive ("untrained") or synthetic libraries. V _HH with the desired antigenic specificity can also be obtained using the phage display method. The use of V _HH in phage display is much simpler and more efficient than Fab or scFv, since only one domain must be cloned and expressed to obtain a functional antigen binding fragment. Muyldermans, Biotechnol. 74: 277-302 (2001); Chahroudi et al., FEBS Lett. 441: 521-526 (1997) and van der Linden et al., J. Biotechnol. 80: 261-270 (2000). Methods for producing antibodies having camelid heavy chains are also described in US Pat. 20050136049 and 20050037421.

Ribosomal display methods can be used to identify and isolate scFv and / or V _HH molecules having the desired binding activity and affinity. Irving et al., J. Immunol. Methods 248: 31-45 (2001). Ribosomal display and selection has the potential to generate large display libraries (10 ¹⁴ ).

In other embodiments, provided are UNn-like molecules generated by the “camelization” method, by modifying non-camelid V _H , such as human V _HH , to improve their solubility and prevent non-specific binding. This is achieved by replacing the residues on the V _L side of the V _H V _HH-like residues, thereby simulating more soluble V _HH fragments. It is expected that “camelized” V _H fragments, in particular fragments based on the framework region of a human antibody, should exhibit a significantly reduced immune response when administered in vivo to a patient, and therefore are expected to have significant therapeutic benefits. applications. Davies et al., FEBS Lett. 339: 285-290 (1994); Davies et al., Protein Eng. 9: 531-537 (1996); Tanha et al., J. Biol. Chem. 276: 24774-24780 (2001) and Riechmann et al., Immunol. Methods 231: 25-38 (1999).

The term "identity" or "homology" should be interpreted as meaning the percentage of amino acid residues in the candidate sequence that are identical to the residues of the corresponding sequence with which it is compared, after sequence comparison and the introduction of "gaps" if it is necessary to achieve the maximum percentage of identity for the full sequence and not considering any conservative substitutions as part of sequence identity. Neither the N- or C-terminal extension nor insertion segments should be construed as reducing identity or homology. Methods and computer programs for comparison are well known. Sequence identity can be determined using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software is suitable for such sequences by determining the degree of homology for a variety of substitutions, deletions (eliminations) and other modifications.

The term "identity" or "homology" in the context of nucleic acid or amino acid sequences, refers to residues in two sequences that are the same when aligned for maximum match. There are a number of different algorithms known in the art that can be used to measure the identity of nucleotide or amino acid sequences. For example, polynucleotide sequences can be compared using FASTA, Gap, or BESTFIT, which are programs in the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, for example, the FASTA2 and FASTA3 programs, provides alignment and percent sequence identity in the areas of best coverage between the requested and the desired sequences (Pearson, Methods Enzymol. 183: 63 98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258 (1996); Pearson, J. Mol. Biol. 276: 71-84 (1998)). Unless otherwise specified, the default parameters for a particular program or algorithm are used. For example, the percent sequence identity between nucleic acid sequences can be determined using FASTA with default parameters (word size 6 and NOPAM factor for the score matrix) or using Gap with default parameters, as provided in GCG version 6.1.

The phrase “homologous” with respect to the polypeptide sequence of an antibody should be interpreted as an antibody exhibiting at least 70%, preferably 80%, more preferably 90% and most preferably 95% sequence identity with respect to the polypeptide sequence . The term “nucleic acid sequence” should be interpreted as a nucleotide sequence exhibiting at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity with respect to the nucleic acid sequence.

Total homology is determined as follows:

(1) first, look for the sequence of the antibody or its fragment with the minimum editing distance according to Damerau-Levenshtein with respect to the sequence in relation to which the degree of homology is determined, then

(2) the specified Damerau-Levenshtein distance is normalized in such a way that the distance between identical sequences is taken as 100% homology, and the distance to a random sequence of the same length is taken as 0%,

(3) repeat the procedure with the remaining segments and

(4) determine the total homology as a weighted average over the length of the segments number.

Modification (s) of the amino acid sequences of the HER2-HER3 antibodies described in this publication is proposed. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the HER2-HER3 antibody are obtained by introducing corresponding nucleotide changes into the nucleic acid of the HER2-HER3 antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences of the HER2-HER3 antibody. Any combination of deletion, insertion, and substitution is performed to obtain the final construct, provided that the final construct possesses the required characteristics. Changes in amino acids can also alter post-translational processes in the HER2-HER3 antibody, such as changes in the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of an HER2-HER3 antibody that are preferred sites for mutagenesis is called "scanning alanine mutagenesis" as described by Cunningham and Wells Science, 244: 1081-1085 (1989). In this case, a residue or group of target residues is identified (e.g., charged residues such as arg, asp, his, lys and glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of amino acids with antigen HER2 and / or HER3. Those sections of amino acids that exhibit functional sensitivity to substitutions are then improved by introducing additional or other options into or instead of substitution sites. Thus, although the site of introduction of the variant amino acid sequence is pre-determined, there is no need for a preliminary determination of the nature of the mutation as such. For example, to analyze the effectiveness of a mutation at a given site, ala-scans or random mutagenesis are performed in the target codon or target region and the expressed variants of HER2-HER3 antibodies are screened for the desired activity.

Insertions in amino acid sequences include fusions with an amino and / or carboxyl terminus ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions within the sequence of one or more amino acid residues. Examples of terminal insertions include an HER2 antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertion variants of the HER2-HER3 antibody molecule include the fusion of the N- or C-terminus of the HER2-HER3 antibody with an enzyme (for example, in the case of ADEPT) or a polypeptide that increases the serum half-life of the antibody.

A variant of another type is the variant with amino acid substitutions. Such variants have at least one amino acid residue in the HER2-HER3 antibody molecule, replaced by another residue. Places of greatest interest for mutagenesis by substitutions include hypervariable regions or CDRs, but changes in the FR or Fc regions are also contemplated. Conservative substitutions are shown in table 1 under the heading of "preferred substitutions." If such substitutions result in a change in biological activity, then additional substantial changes can be introduced, called "examples of substitutions" in Table A, or changes additionally described below in the description of the classes of amino acids, and products can be screened.

Significant modifications of the biological properties of antibodies are carried out by selection of substitutions, which differ significantly in

 its effect on maintaining (a) the structure of the polypeptide backbone in the substitution region, for example, in the form of a layered or helical conformation, (b) the charge or hydrophobicity of the molecule in the target site, or (c) the size of the side chain. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, Biochemistry, second ed., Pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M),

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q),

(3) acidic: Asp (D), Glu (E),

(4) basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be divided into groups based on the common properties of the side chains:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail the replacement of a representative of one of these classes by a representative of another class.

Any cysteine residue not involved in maintaining the proper conformation of the HER2-HER3 antibody can also be replaced, usually with serine, to increase the oxidation stability of the molecule and prevent abnormal cross-linking. Conversely, a cysteine bond (s) can be added to the antibody to increase its stability (especially when the antibody is an antibody fragment, such as an Fv fragment).

A particularly preferred type of substitutional variant comprises replacing one or more residues of the hypervariable region of the parent antibody (e.g., a humanized antibody or a human antibody). In general, the resulting option (s) selected for further development will have improved biological properties compared to the original antibody from which they are made. A suitable way to create such substitutional variants is to mature affinity using a phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to create all possible amino acid substitutions at each site. Antibodies thus obtained are presented in a monovalent display on filamentous phage particles in the form of fusions with the product of gene III M13 packaged in each particle. The options presented in the phage display are then screened for their biological activity (e.g., binding affinity), as described in this publication. To identify sites suitable for modification of the hypervariable region, mutagenesis can be carried out by scanning with alanine, identifying residues of the hypervariable region that make a significant contribution to antigen binding. Alternatively or additionally, analysis of the crystal structure of the antigen-antibody complex may be useful to identify contact points between the antibody and human HER2 and / or HER3. Such residues in the contact area and adjacent residues are candidates for replacement by methods developed according to the invention. After creating such options, the options panel is screened as described in this publication, and antibodies with superior properties in one or more suitable assays can be selected for further development.

In another type of amino acid variant of the antibody, the initial glycosylation pattern of the antibody is altered. By change is meant the deletion of one or more carbohydrate residues found in the antibody and / or the addition of one or more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the binding of a carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic binding of the carbohydrate residue to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars of N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most often serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Adding glycosylation sites to an antibody is usually accomplished by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The change can also be done by adding or replacing one or more serine or threonine residues to the sequence of the parent antibody (for O-linked glycosylation sites).

In the case where the antibody contains an Fc region, the carbohydrate associated with it may be altered. For example, antibodies with a mature carbohydrate structure in which fucose bound to the Fc region of the antibody is lost are described in U.S. Patent Application No. US 2003/0157108 A1, Presta, L. See also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd). Branching N-acetylglucosamine (GlcNAc) antibodies in the oligosaccharide structure associated with the Fc region of an antibody are described in WO 03/011878, Jean-Mairet et al. and U.S. Pat. 6602684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide structure associated with the Fc region of an antibody are described in WO 97/30087, Patel et al. See also patent applications WO 98/58964 (Raju, S.) and WO 99/22764 (Raju, S.) for antibodies with altered carbohydrates linked to their Fc region. The present invention contemplates antibody compositions comprising a major type antibody with such carbohydrate structures linked to one or two heavy chains of the Fc region.

The term "antigen-binding part" of an antibody (or simply "part of an antibody"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-sized antibody. Examples of binding fragments included in the term “antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH 1 domains; (ii) P (ab ') 2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH 1 domains; (iv) an Fv fragment consisting of the VL and VH domains in a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341: 544-546), which consists of a VH / VHH domain; and (vi) a designated complementarity determining region (CDR). In addition, two regions of the Fv fragment, VL and VH, are encoded by different genes, they can be connected using recombinant methods using a synthetic linker, which makes it possible to obtain them as a single protein chain, in which the VL and VH regions mate to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879 -5883). It is contemplated that such single chain molecules are also included in the term “antigen binding portion” of an antibody. Such antibody fragments are prepared using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

Preferably, the CDR of the antigen binding site or the entire antigen binding site of the antibodies of the invention is from a mouse, llama, or human donor library, or essentially human, with certain amino acid residues altered, for example, substituted by different amino acid residues, in order to optimize specific properties of the antibody, for example KD , koff, IC50, EC50, ED50. Preferably, the antibody fragments of the invention are of human origin or essentially human origin (at least 80, 85, 90, 95, 96, 97, 98, or 99% human origin).

In other embodiments, the antigen binding site of an antibody of the invention may be derived from, but not limited to, other non-human species, including, but not limited to, a mouse, llama, rabbit, rat, or hamster. Alternatively, the antigen binding site may be derived from human species.

The term "variable" refers to the fact that certain segments of the variable domains vary widely in sequence among antibodies. Domain V mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, the variability is not evenly distributed across the 110-amino acid variable domain region. In contrast, V regions are composed of invariant fragments called framework regions (FR) of 15-30 amino acids, separated by shorter regions of extreme variability, called "hypervariable regions" or CDRs. Each variable domain of the native heavy and light chains contains four FRs, mainly taking the configuration of beta sheets connected by three hypervariable regions that form loops that bind, and in some cases are part of the beta-folded structure. The hypervariable regions in each chain are held together in close proximity by FR and, with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest. 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC, ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Typically, the hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR", and / or such residues from the "hypervariable loop".

"Frame regions" (FR) are variable domain residues other than CDR residues. Typically, each variable domain has four FRs, defined as FR1, FR2, FR3, and FR4. If CDRs are determined according to Kabat, FR light chain residues are located approximately in residual regions 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4), and heavy chain FR residues localized approximately in the region of residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4) in the heavy chain. If the CDR regions contain amino acid residues from hypervariable loops, FR light chain residues are located at approximately 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and 97-107 (LCFR4) in the light chain , a FR heavy chain residues are localized at about 1-25 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3) and 102-113 (HCFR4) in the heavy chain residues. In some examples, when the CDR contains amino acids from both the Kabat CDR and the amino acids from the hypervariable loop, the FR coo are adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, heavy chain FR1 residues are at positions 1-25, and FR2 residues are at positions 36-49.

An antibody of the invention that “binds” the target antigen is an antibody that binds the antigen with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent when targeting a protein or cell or tissue expressing the antigen, and slightly cross-reacts with other proteins. According to analytical methods: sorting of fluorescently activated cells (FACS), radioimmunoprecipitation (RIA) or ELISA (ELISA), in such embodiments of the invention the degree of binding of the antibody to a protein that is not a "target" (with a "non-target protein") is less than 10 % of the binding of the antibody to a specific target protein. With respect to the binding of an antibody to a target molecule, the term “specific binding” or the expressions “specifically binds” or “specific” to a particular polypeptide or epitope on a particular target polypeptide means a binding that is noticeably (measurably) different from a non-specific interaction (e.g. in the case of bH1-44 or bH1-81, the non-specific interaction is binding to bovine serum albumin, casein, fetal bovine serum or neutravidin). Specific binding can be quantified, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term “specific binding” or the expressions “specifically binds to” or “specific for” a particular polypeptide or epitope on a particular target polypeptide can be characterized by the example of a molecule having a Kd of at least about 200 nM to the target, or at least at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, and whether at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM or higher. In one embodiment of the invention, the term “specific binding” refers to a binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide.

The term "Ka", as used herein, refers to the rate of association of a particular antibody-antigen interaction, while the term or "Kd" refers to the dissociation rate of a specific antibody-antigen interaction.

“Binding affinity” generally refers to the strength of the cumulative non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, antigen). Unless otherwise specified, “binding affinity” refers to the intrinsic (characteristic, true) binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of molecule X for its partner Y can usually be represented by constant dissociation (Kd). Preferably, the Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less. Affinity can be measured by conventional methods known in the art, including those described herein. Low affinity antibodies usually bind slowly to the antigen and tend to readily dissociate, whereas high affinity antibodies usually bind to the antigen faster and tend to remain in the bound state for longer. Various methods for measuring binding affinity are known in the art, any of these methods can be used for the purposes of the present invention.

In one embodiment of the invention, the “Kd" or "Kd value" of this invention is measured by surface plasma resonance analysis with a BIAcore ™ -2000 or BIAcore ™ -3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25 ° C. using immobilized chips CM5 antigen at ~ 10 relative units (response units, RU). In short, carboxymethyldextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with a 10 mM sodium acetate solution, pH 4.8, to a concentration of 5 μg / ml (~ 0.2 μM), and then injected at a flow rate of 5 μl / min until approximately 10 relative units (RU) of the bound protein are reached. After administration of the antigen, a 1 M ethanolamine solution is administered to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., from 0.78 nM to 500 nM) are introduced into PBS with 0.05% Tween 20 (PBST) at 25 ° C at a flow rate of approximately 25 μl / min. The association rate (k _on ) and association rate (k _off ) values are calculated using a simple Langmuir model for one-plus-one binding (BIAcore Evaluation Software version 3.2), using simultaneous acquisition of association and dissociation sensograms. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off / k _on . See, for example, Chen, Y., et al. (1999) J. Mol. Biol. 293: 865-881. If, according to the above method of surface plasmon resonance, the association rate exceeds 10 ⁶ M ^-1 s ^-1 , then it can be determined by the fluorescence quenching method, which measures the increase or decrease in the intensity of fluorescence emission (excitation = 295 nm; emission (radiation) = 340 nm, band 16 nm) at 25 ° C of an anti-antigen antibody solution (Fab form) with a concentration of 20 nM in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured with a spectrometer such as a stopped flow spectrophotometer (Aviv I nstruments) or the SLM-Aminco spectrophotometer (ThermoSpectronie) of the 8000 series with a stirring cell.

The “on-rate” or “k _on ” of the present invention can also be determined by the same surface plasma resonance method as described above on a BIAcore ™ -2000 or BIAcore ™ -3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25 ° C using chips with immobilized CM5 antigen at ~ 10 relative units (response units, RU). In short, carboxymethyldextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N '- (3-dimethylamino propyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with a 10 mM sodium acetate solution, pH 4.8, to a concentration of 5 μg / ml (~ 0.2 μM), and then injected at a flow rate of 5 μl / min until approximately 10 relative units (RU) of the bound protein are reached. After administration of the antigen, a 1 M ethanolamine solution is administered to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., from 0.78 nM to 500 nM) are introduced into PBS with 0.05% Tween 20 (PBST) at 25 ° C at a flow rate of approximately 25 μl / min. Association rates (k _on ) and association rates (k _off ) are calculated using a simple Langmuir model for one-plus-one binding (BIAcore Evaluation Software version 3.2), while simultaneously receiving association and dissociation sensograms. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off / k _on . See, for example, Chen, Y., et al. (1999) J. Mol. Biol. 293: 865-881. However, if according to the above method of surface plasma resonance, the association rate exceeds 10 ⁶ M ^-1 sec ^-1 , then it can be determined by the fluorescence quenching method, which measures the increase or decrease in the intensity of fluorescence emission (excitation = 295 nm; emission (radiation) = 340 nm, band 16 nm) at 25 ° C anti-antigen antibody solution (Fab form) with a concentration of 20 nM in PBS, pH 7.2, in the presence of increasing concentrations of antigen, measured using a spectrometer such as a stopped sweat spectrophotometer eye (Aviv Instruments) or spectrophotometer SLM-Aminco (ThermoSpectronic) 8000 series with a cuvette with stirring.

Unless specifically indicated otherwise, the expressions "biologically active" and "biological activity" and "biological characteristics" with respect to the polypeptide of this invention mean having the ability to bind to a biological molecule.

The expression "biological molecule" refers to nucleic acid, protein, carbohydrate, lipid, and combinations thereof. In one embodiment of the invention, the biological molecule exists in nature.

Antibody sites, such as Fab and F (ab ') ₂ fragments, can be obtained from whole antibodies using traditional methods, such as papain or pepsin hydrolysis of whole antibodies. Moreover, antibodies, parts of antibodies and immunoadhesion molecules can be obtained using standard recombinant DTPS methods, for example, as described herein.

The term "recombinant antibody" means an antibody that is expressed from a cell or cell line containing a nucleotide sequence (nucleotide sequences) that encodes an antibody, wherein said nucleotide sequence (nucleotide sequences) is not associated with the cell in nature.

The term "variant" antibody, as used herein, refers to an antibody having an amino acid sequence that differs from the amino acid sequence of its "parent" antibody by adding, removing and / or replacing one or more amino acid residues relative to the sequence of the parent antibody. In a preferred embodiment, the variant antibody comprises at least one or more (e.g., one to twelve, e.g., two, three, four, five, six, seven, eight or nine, ten, eleven or twelve; and in some embodiments one to about ten) additions, deletions and / or substitutions of amino acids relative to the parent antibody. In some embodiments, additions, deletions, and / or substitutions are made at the CDR regions of a variant antibody. Identity or homology with respect to the sequence of the variant antibody is defined herein as the percentage of amino acid residues in the sequence of the variant antibody that are identical to the residues of the parent antibody, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. A variant antibody retains the ability to bind to the same antigen, and preferably the epitope, to which the parent antibody binds, and in some embodiments, the at least one property or biological activity exceeds that of the parent antibody. For example, a variant antibody may have, for example, a more pronounced binding affinity, a longer half-life, a lower IC50 value or an increased ability to suppress the biological activity of the antigen compared to the parent antibody. Of particular interest in this document is a variant antibody showing a biological activity greater than at least 2 times (preferably at least 5 times, 10 times or 20 times) the biological activity of the parent antibody.

The term "chimeric antibody" refers in a broad sense to an antibody that contains one or more regions of one antibody, and one or more regions of one or more other antibodies, typically an antibody, partially human or partially non-human, that is, obtained partially from a non-human animal, such as a mouse, rat, or other rodent or camelid, such as a llama or alpaca. Chimeric antibodies are preferred over non-human antibodies in order to reduce the risk of an immune response directed against antibodies in humans, for example, a response directed against murine bodies in humans in the case of a mouse antibody. An example of a typical chimeric antibody is one in which the variable region sequences are murine, while the constant region sequences are human. In the case of a chimeric antibody, non-human parts can be further modified to humanize the antibody.

The term “humanization” refers to the fact that when an antibody is wholly or partially non-human, for example, a mouse or llama antibody obtained by immunizing mice or llamas with an antigen of interest, respectively, or is a chimeric antibody based on such a mouse or llama antibody , you can replace some amino acids, in particular, in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize the immune response in humans. The specificity of the interaction of the antibody with the target antigen is inherent mainly in amino acid residues located in six CDR regions of the heavy and light chains. Therefore, the amino acid sequences within the CDR regions are much more variable between the individual antibodies compared to sequences outside the CDR regions. Since CDR sequence sequences are responsible for most antibody-antigen interactions, recombinant antibodies can be expressed that mimic the properties of a specific natural antibody, or more generally any specific antibody with a given amino acid sequence, for example, by constructing expression vectors that express sequences CDR plots from a specific antibody to the framework sequences of another antibody. As a result, a non-human antibody can be “humanized” and the binding specificity and affinity of the original antibody can be largely maintained. Although it is not possible to accurately predict the immunogenicity and thereby the immune response directed against an antibody in humans to a particular antibody, non-human antibodies are generally more immunogenic than human antibodies. Chimeric antibodies in which foreign (e.g., rodent or camel) constant regions have been replaced by sequences of human origin have shown generally lower immunogenicity than antibodies of completely foreign origin, and there is a tendency to use humanized or fully human antibodies in therapeutic antibodies. Chimeric antibodies or other non-human antibodies can thus be humanized to reduce the risk of an anti-antibody immune response in humans.

For chimeric antibodies, humanization usually involves modifying the frame regions of the variable region sequences. Amino acid residues that are part of the complementarity determining regions (CDRs) will most often not change due to humanization, although in some cases it may be desirable to modify individual amino acid residues of the CDRs, for example, to remove the glycosylation site , a deamidation site, an aspartate isomerization site, or an undesired cysteine or methionine residue. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in the tripeptide sequence of Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of the N-glycosylation site can be achieved by mutating the Asn or Ser / Thr residue with another residue, preferably by conservative substitution. The deamidation of asparagine and glutamine residues may occur depending on factors such as pH and exposure of the surface. Asparagine residues are particularly susceptible to deamidation, especially if they are present in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences, such as Asn-Ala. In the presence of such a deamidated region, in particular Asn-Gly, in the sequence of the CDR region, it may be preferable to remove this region, usually by conservative substitution, to remove one of the residues involved.

Numerous methods for humanizing an antibody sequence are known in the art; see, for example, a review by Almagro & Fransson, Front Biosci. 13: 1619-1633 (2008). One of the most commonly used methods is transplantation of CDR regions, for example, when chimeric antibodies of mouse origin involve the identification of human germline gene equivalents to murine variable region genes and the transplantation of sequences of murine CDR regions into this framework. CDR site transplantation may be based on Kabat CDR site definitions, although a later publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64: 210-225 (2007)) suggests that IMGT® determination (the international ImMunoGeneTics information system®, www.imgt.org) can improve the result of humanization (see Lefranc et al., Dev. Comp Immunol. 27: 55-77 (2003)). In some cases, transplantation of a CDR region can reduce the specificity and affinity of binding, and therefore biological activity, of a CDR transplanted non-human antibody, compared to the parent antibody from which the CDR regions are derived. Reverse mutations (sometimes referred to as “frame repair”) can be applied at selected positions of a CDR transplanted antibody, typically in frame sites, in order to restore the specificity and affinity of binding of the parent antibody. The determination of positions for possible reverse mutations can be performed using information available in the literature and in the antibody databases. Amino acid residues that are candidates for reverse mutations are typically located on the surface of an antibody molecule, while residues that are deepened or have a low degree of exposure to the surface will usually not be affected. The humanization method, an alternative to transplantation of the CDR region and reverse mutation, is a surface change in which residues of non-human origin unexposed on the surface are retained, while residues exposed on the surface are transformed into human residues.

In some cases, it may also be preferable to change one or more amino acid residues of the CDR regions in order to increase the binding affinity of the target epitope. This is known as “affinity maturation” and in some cases can be performed in connection with humanization, for example, in situations where humanization of an antibody leads to a decrease in binding specificity or affinity, and it is not possible to sufficiently improve binding specificity or affinity by using only inverse mutations. Various affinity maturation methods are known in the art, for example, the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94: 412-417 (1997), and the in vitro stepwise affinity maturation method proposed by Wu et al., Proc Natl Acad Sci USA 95: 6037-6042 (1998).

The term "isolated" ("isolated"), used to describe the various antibodies described herein, means an antibody identified and isolated and / or regenerated from the cell or cell culture in which it is expressed. Impurities (contaminants) from the natural environment are materials that, as a rule, interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments of the invention, the antibody is purified (1) to a degree sufficient to produce at least 15 residues of the N-terminal or internal amino acid sequence using a rotating glass cup sequencer (Edman sequencer), or (2) until homogeneous by SDS-PAGE in non-reducing or reducing conditions using Coomassie staining with brilliant blue or, preferably, silver. An isolated antibody includes antibodies in situ within recombinant cells, since at least one component of the natural environment of the polypeptide is absent. Typically, an isolated polypeptide is obtained from at least one purification step.

The term “epitope” as used herein refers to a part (determinant) of an antigen that specifically binds to a binding molecule (for example, an antibody or a related molecule, such as a bispecific binding molecule). Epitope determinants usually consist of chemically active surface groups of molecules, such as amino acids or carbohydrates, or sugar side chains, and, as a rule, have specific three-dimensional structural characteristics, as well as specific characteristics of charges. An epitope can be “linear” or “conformational.” In a linear epitope, all points of interaction between a protein (eg, an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, interaction points occur through amino acid residues on a protein that are separated from each other in the primary amino acid sequence. When the desired antigen epitope is determined, antibodies to this epitope can be generated using techniques well known in the art. In addition, the generation and characterization of antibodies or other binding molecules can shed light on information about the desired epitopes. Based on this information, binding molecules can then be competitively screened for binding to the same or similar epitopes, for example, by conducting competition studies to find binding molecules that compete for binding to antigen.

You can determine whether an antibody or other binding molecule binds to the same epitope or cross-competes for binding to Her2 and Her3 with the bispecific binding molecule of the present invention using methods well known in the art. In one embodiment of the invention, it is possible to bind the bispecific binding molecule of the invention to Her2 and Her3 under saturation conditions, followed by measuring the ability of the test antibody to bind to the target antigen. If the test antibody is capable of binding to the target antigen at the same time as the reference bispecific binding molecule, the test antibody binds to an epitope other than the epitope of the reference bispecific binding molecule. However, if the test antibody is unable to bind to the target antigen at the same time, then the test antibody binds to the same epitope that overlaps with the epitope or epitope that is in close proximity to the epitope associated with the bispecific binding molecule. This experiment can be performed using ELISA, RIA, analysis of intermolecular interactions on BIACORE ™, biolayer interferometry or flow cytometry. In order to check whether the bispecific binding molecule of the present invention cross-competes with another binding molecule, the two-way competitive assay described above can be used, i.e., determine whether a known binding molecule blocks the test binding molecule, and vice versa. Such cross-competition experiments can be performed, for example, using an IBIS MX96 SPR instrument or an Octet ™ system.

In one embodiment, the binding molecule of the invention is a monoclonal antibody. As used herein, the abbreviation "mAb" refers to a monoclonal antibody, that is, an antibody that is synthesized and isolated by a separate clonal cell population. A clonal population may be a clonal population of immortalized cells. In some embodiments, immortalized cells in a clonal population are hybrid cells, hybridomas that are usually obtained by fusing individual B lymphocytes from immunized animals with individual lymphocyte tumor cells. Hybridomas are a type of engineered cell and are not found in nature.

The class (isotype) and subclass of antibodies can be determined by any method known in the art. In general, the class and subclass of an antibody can be determined using antibodies specific to a particular class and subclass of antibodies. Such antibodies are commercially available. The class and subclass can be determined using ELISA, Western blot analysis, as well as other methods. In another embodiment, the class and subclass can be determined by sequencing all or part of the constant domains of the heavy and / or light chains of antibodies, comparing their amino acid sequences with known amino acid sequences of different classes and subclasses of immunoglobulins, and determining the class and subclass of antibodies.

"Antibody-dependent cell mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which non-specific cytotoxic cells that express Fc receptors (FcR) (e.g., natural killer cells (NK), neutrophils and macrophages) recognize a bound antibody on the target cell and then cause lysis of the target cell. Primary cells to mediate ADCC, NK cells, express FcγRIII only, while monocytes express FcγRI, FcγRII and FcγRIII. The expression of FcR on hematopoietic cells is summarized in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). In vitro ADCC assays, such as the assays described in US Pat. . Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model, such as the model described in Clynes et al. PNAS (USA) 95: 652-656 (1998).

"Human effector cells" are white blood cells that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer cells (NKs), monocytes, cytotoxic T cells, and neutrophils; RVMS and NK cells are preferred. Effector cells can be isolated from their natural source, for example from blood or PBMC, as described in this publication.

, Annu. Rev. Immunol. 15: 203-234 (1997)). Обзор, посвященный FcR, представлен в Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); и de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Другие FcR, включая FcR, которые будут идентифицированы в будущем, включены в настоящем описании в термин "FcR". Термин также включает неонатальный рецептор, FcRn, который отвечает за перенос материнских IgG в плод (Guyer et al., J. Immunol. 117: 587 (1976) и Kim et al., J. Immunol. 24: 249 (1994)).The terms “Fc receptor” and “FcR” are used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a human FcR with a native sequence. In addition, a preferred FcR is FcR, which binds an IgG antibody (gamma receptor), and preferred receptors include receptors of the subclasses FcγRI, FcγRII and FcγRIII, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains in its cytoplasmic domain a tyrosine-based immunoreceptor inhibition motif (ITIM) (see review in

Annu. Rev. Immunol. 15: 203-234 (1997)). A review of FcR is presented in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including FcRs, which will be identified in the future, are included in the present description in the term "FcR". The term also includes a neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)).

"Complement dependent cytotoxicity" and "CDC" refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) in complex with its antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

"Native antibodies" are usually heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains varies in different immunoglobulin isotypes. Each heavy and light chain also has evenly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the variable domains of the light chain and the heavy chain.

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. In some embodiments, the vector is a plasmid, i.e. a circular double-stranded portion of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors having a bacterial replication origin and episomal mammalian vectors). In other embodiments, vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicate together with the host gene. Moreover, some vectors are capable of directing the expression of the genes with which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).

The term “recombinant host cell” (or simply “host cell”) as used herein means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. The present invention also relates to host cells, which include, for example, a nucleotide sequence encoding a heavy chain or its antigen binding parts, a nucleotide sequence encoding a light chain or its antigen binding parts, or both, a full length antibody and / or a nucleotide sequence encoding the variable domain of the heavy chain of VHH, bispecific antibodies of this invention. It should be understood that “recombinant host cell” and “host cell” mean not only the specific cell claimed, but also the progeny of such a cell. Since modifications can occur in subsequent generations due to mutations or environmental influences, such offspring may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein.

Nucleic acid molecules and vectors

The present invention also relates to nucleic acid molecules, and sequences encoding the bispecific binding molecules of the invention described herein. In some embodiments, various nucleic acid molecules encode the first domain and an antibody or second domain of the amino acid sequence of the bispecific binding molecule. Where the first domain is VHH and the second domain contains a heavy chain and / or light chain, in some embodiments, various nucleic acids encode the heavy chain and amino acid sequences of the light chain. In other embodiments, the same nucleic acid molecule encodes a sequence of the heavy chain and light chain amino acids. In a specific embodiment, the nucleic acid molecule can encode the amino acid sequence of the first VHH binding domain and the light chain amino acid sequence of the second binding domain, optionally including any sequence of the peptide linker connecting them.

A reference to a nucleotide sequence covers its compliment, unless otherwise indicated. Thus, a reference to a nucleic acid having a specific sequence should be understood as encompassing its complementary chain with its complementary sequence. The term “polynucleotide” as used herein means a polymeric form of nucleotides at least 10 bases in length, or ribonucleotides, or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single and double chain forms.

The present invention also relates to nucleotide sequences that are at least 75%, 80%>, 85%), 90%>, 95%, 97%, 98% or 99% homologous or identical to one or more of the aforementioned nucleotide sequences or a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3. In certain embodiments, the nucleotide sequences of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are homologous or identical to the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4. The present invention also relates to nucleotide sequences that are at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more of the aforementioned nucleotide sequences or a nucleotide sequence encoding amino acid sequence selected from the group consisting of SEQ ID NO: 5-6.

In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-6. The nucleic acid molecule may also contain any combination of these nucleotide sequences. In one embodiment of the invention, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 3. In another embodiment, the nucleic acid molecule comprises nucleotide sequences encoding SEQ ID NO: 1-3. In one embodiment, the nucleic acid molecule contains a nucleotide sequence encoding SEQ ID NO: 4. In another embodiment, the nucleic acid molecule contains nucleotide sequences encoding SEQ ID NO: 4 and 5. In another embodiment, the nucleic acid molecule contains nucleotide sequences encoding SEQ ID NOs: 4 and 6. In another embodiment, the nucleic acid molecule contains nucleotide sequences encoding ue SEQ ID NO: 4, 5 and 6.

In any of the above embodiments, the nucleic acid molecules may be isolated.

In another aspect, the present invention relates to a vector suitable for expression of any of the nucleotide sequences described herein. The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. In some embodiments, the vector is a plasmid, i.e. a circular double-stranded portion of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors having a bacterial replication origin and episomal mammalian vectors). In other embodiments, vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicate together with the host gene. Moreover, some vectors are capable of directing the expression of the genes with which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).

The present invention relates to vectors containing nucleic acid molecules that encode any of the amino acid sequences of bispecific binding molecules or parts thereof (e.g., heavy chain sequences of the first and / or heavy and / or light chain of the second binding domain) as described herein. The present invention further relates to vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments.

The nucleic acid molecule of this invention can be isolated from any source that produces a bispecific binding molecule or part thereof. In certain embodiments, a nucleic acid molecule of the invention may be synthesized rather than isolated.

In some embodiments, the nucleic acid molecule of the invention may comprise a nucleotide sequence encoding the VHH of the first domain and / or the VH of the second domain of the bispecific binding molecule of this invention, coupled as read to a nucleotide sequence encoding the constant domain of a heavy chain from any source. Similarly, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding the VL domain of a second region of the bispecific binding molecule of the invention combined in reading with a nucleotide sequence encoding the constant domain of a light chain from any source.

In yet another aspect of the present invention, nucleic acid molecules encoding a heavy chain variable domain (VHH) of a first binding domain and / or a heavy chain (VH) and / or light chain variable domain (VL) of a second binding domain can be "transformed" along the entire gene length antibodies. In one embodiment of the invention, nucleic acid molecules encoding the VHH and / or VH and / or VL domains are transformed into antibody genes along the entire length by insertion into an expression vector already encoding the constant domains of the heavy chain (CH) or light chain (CL), respectively so that the VHH / VH segment is operatively connected to the CH segment (s) in the vector and / or the VL segment is operatively connected to the CL segment in the vector. In another embodiment, the nucleic acid molecules encoding the VHH / VH and / or VL domains are transformed into genes along the entire length of the antibody by connecting, for example, ligation, a nucleic acid molecule encoding the VHH / VH and / or VL domains to a nucleic acid molecule encoding CH and / or CL domains using standard molecular biological methods. Nucleic acid molecules encoding the entire length of the heavy and / or light chains can then be expressed from the cell into which they were introduced.

Nucleic acid molecules can be used to express a large number of recombinant bispecific binding molecules. Nucleic acid molecules can be used to produce human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives, as described herein.

In another embodiment, nucleic acid molecules and vectors can be used to make mutated bispecific binding molecules. Antibodies can mutate in the variable domains of the heavy chain of the first and / or heavy and / or light chains of the second binding domain, for example, to change the binding ability of the bispecific binding molecule. For example, a mutation can occur at one or more CDR sites to increase or decrease the K _{D of the} bispecific binding molecule, to increase or decrease k _off, or to change the binding specificity of the antibody for HER2 and / or HER3. In another embodiment, one or more mutations are subjected to an amino acid residue that is known to be altered from the germ line in the antibody corresponding to the first or second binding domain of the bispecific binding molecule of this invention. These mutations can be made in the CDR region or the framework region of the variable domain, or in the constant domain. In a preferred embodiment, the mutations are made in the variable domain. In another embodiment, the amino acid residue is known to undergo one or more mutations, which is known to be altered from the germ line in the CDR region or frame region of the variable domain of the bispecific binding molecule of this invention.

In another embodiment, the framework region (s) are mutated so that the resulting framework region (s) has the amino acid sequence of the corresponding germ line gene. Mutations can occur in the framework region or constant domain to increase the half-life of the bispecific binding molecule. See, for example, WO 00/09560. Mutation in the framework region or constant domain can also be made to alter the immunogenicity of the bispecific binding molecule and / or provide a region for covalent or non-covalent binding to another molecule. In accordance with the invention, the bispecific binding molecule may have mutations in any one or more of the CDR regions or framework regions of the variable domain or in the constant domain.

In some embodiments, the bispecific binding molecules of the invention are expressed by inserting a DNA that encodes partially or fully the sequence of the first and second binding domain (e.g., heavy / VHH or heavy and light chain sequences, where the binding domain contains heavy / VHH or heavy and light chain) obtained, as described above, in expression vectors so that the genes are functionally linked to the desired sequences, control expression, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YAC, EBV derived episomas and the like. DNA molecules can be ligated into a vector so that sequences that control transcription and translation in the vector fulfill the intended function of regulating the transcription and translation of DNA. The expression vector and expression control sequences can be selected so as to be compatible with the expression host cell used. DNA molecules encoding partially or along the entire length of the sequence of the first and second binding domains (e.g., heavy and light chain sequences, where the binding domain contains a heavy and light chain sequence) can be introduced into separate vectors. In one embodiment of the invention, any combination of the above DNA molecules is introduced into the same expression vector. DNA molecules can be introduced into the expression vector by standard methods (for example, by ligation of complementary restriction sites on a fragment of an antibody and vector gene or by ligation of blunt ends if there are no restriction sites).

A suitable vector is one that encodes functionally complete human immunoglobulin CH or CL sequences to construct an appropriate restriction site such that any VHH and / or VH and / or VL sequence can be easily incorporated and expressed as described above. NS and LC coding of genes in such vectors may contain intron sequences, which leads to a general increase in the protein products of antibodies by stabilization of the corresponding mRNA. The intron sequence is surrounded by a splice donor and a splice acceptor sites that determine where RNA splicing will occur. The arrangement of intron sequences can be either in the variable or constant regions of the antibody chains, or in both variable and constant regions when several introns are used. Termination of polyadenylation and transcription may occur downstream of the native chromosome site of the encoded regions. The recombinant expression vector can also encode a signal peptide that facilitates the production of an antibody chain by a host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the reading frame of the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide of a non-immunoglobulin nature protein).

In addition to the chain of antibody genes, the recombinant expression of the vectors of this invention can carry regulatory sequences that control the expression of antibody chain genes in the host cell. Those skilled in the art will understand that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as selection of the host cell for transformation, expression level of the desired protein, etc. Preferred regulatory sequences for an expressing mammalian host cell include viral elements providing a high level of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTR, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), monkey virus 40 ( SV40) (e.g. SV40 promoter / enhancer), adenovirus (e.g. Late promoter adenovirus (AdMLP)), polyoma virus, as well as strong mammalian promoters such as native immunoglob Uline and actin promoters. For further description of viral regulatory elements and their sequences, see, for example, US patents 5,168,062, 4,510,245 and 4,968,615. Methods for expressing binding molecules, such as plant antibodies, including a description of promoters and vectors, as well as plant transformation, are known in the art. See, for example, US Pat. No. 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, for example, yeast cells, are also well known in the art.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., replication origin) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US patents 4,399,216, 4,634,665 and 5,179,017). For example, usually a selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, to the host cell into which the vector is introduced. For example, breeding marker genes include the dihydrofolate reductase gene (DHFR) (for use in dhfr host cells for the selection / amplification of methotrexate), the neo gene (for G418 selection), and the glutamate synthetase gene.

The term “expression control sequence” as used herein means polynucleotide sequences that are necessary to affect the expression and processing of the coding sequences to which they are ligated. Expression control sequences include corresponding transcription, termination, promoter and enhancer initiation sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize the cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter of the ribosomal binding site, as well as transcription termination sequences; in eukaryotes, typically, such control sequences include promoters and transcription termination sequences. The term “control sequences” includes at least all components whose presence is important for expression and processing, and may also include additional components whose presence is useful, for example, lead sequences and sequences of fused cells.

Host cells and methods for producing a bispecific binding molecule

An additional aspect of the present invention relates to methods for producing bispecific binding molecules of the present invention. One embodiment of the invention relates to a method for producing bispecific binding molecules, as defined herein, comprising administering a recombinant host cell capable of expressing a bispecific binding molecule, culturing said host cell under conditions suitable for expression of the bispecific binding molecule, and isolating the resulting bispecific binding molecule. binding molecule. The bispecific binding molecules obtained by such expression in such recombinant host cells are referred to herein as “recombinant bispecific binding molecules”. Where bispecific binding molecules are antibodies, they are called "recombinant antibodies." The invention also relates to progeny of cells of such host cells and bispecific binding molecules obtained in a similar manner.

The term “recombinant host cell” (or simply “host cell”) as used herein means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. The present invention also relates to host cells, which include, for example, a nucleotide sequence encoding a heavy chain or its antigen binding parts or VHH, a nucleotide sequence encoding a light chain or its antigen binding parts, or both of them, a first binding domain or a second binding domain or a full-sized antibody that is part of the bispecific binding molecule of this invention. It should be understood that “recombinant host cell” and “host cell” mean not only the specific cell claimed, but also the progeny of such a cell. Since modifications can occur in subsequent generations due to mutations or environmental influences, such offspring may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein.

Nucleic acid molecules encoding the bispecific binding molecules of the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or its cell, plant or its cell, bacterial or yeast host cell. The conversion can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, precipitation of calcium phosphate, polybren-mediated transfection, fusion of protoplasts, encapsulation of polynucleotide (s) into liposomes and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Cell transformation methods are well known in the art. See, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461 and 4,959,455. Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, in particular, Chinese hamster ovary cells (CHO), NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster kidney cells (BHK), kidney cells African green monkeys (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines of particular preference are selected by determining which cell lines have high levels of expression. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors encoding bispecific binding molecules are introduced into mammalian host cells, the binding molecules are produced by culturing the host cells for a time sufficient to express the binding molecule in the host cells or, preferably, isolating the binding molecule in a nutrient medium, which hosts cells are grown. Bispecific binding molecules can be recovered from the culture medium using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potatoes, etc. Host bacterial cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

In addition, the expression of the bispecific binding molecules of the invention from a producing cell line can be enhanced by a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is common enough to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with the patents EP 0216846, 0256055, 0323997 and 0338841.

It is likely that the bispecific binding molecules of different cell lines or transgenic animals will differ from each other by a glycosylation profile. However, all the bispecific binding molecules encoded by the nucleic acid molecules described herein or containing the amino acid sequences described herein are part of this invention, regardless of the glycosylation state of the binding molecules and in general, regardless of the presence or absence of post-translational modifications.

In some embodiments, the binding domains of the bispecific binding molecule can be paired together using an innovative linker molecule designed to protect against the proteolytic degradation of the binding molecule. Such a linker is usually devoid of a proteolytic cleavage site.

Production method

Methods for producing bispecific antibodies are known in the art. The traditional preparation of full-sized bispecific antibodies is based on coexpression of two pairs of the heavy chain-light chain of immunoglobulin, and the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Due to the random selection of immunoglobulin heavy and light chains, such hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually carried out using affinity chromatography steps, is rather cumbersome and product yields are low. Similar methods are described in WO 93/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

According to another method, the variable domains of antibodies with the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. The fusion is preferably carried out with the constant domain of the heavy chain of immunoglobulin containing at least part of the hinge region, regions of CH2 and CHS. It is preferable to have a first heavy chain constant region (C _H 1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding immunoglobulin heavy chain fusions and, if necessary, immunoglobulin light chains are inserted into separate expression vectors and cotransfected into a suitable host organism. This gives great flexibility in adjusting the mutual ratios of the three polypeptide fragments in such cases when unequal ratios of the three polypeptide chains used in the construction give optimal yields. However, coding sequences for two or all three polypeptide chains can be inserted into one expression vector in the case when the expression of at least two polypeptide chains in equal proportions gives high yields or when the ratios are not significant.

In a preferred embodiment of this method, the VHH heavy chain variable domains that specifically bind to HER3 are fused to a full length antibody or antigen binding domain thereof that specifically binds to HER2.

In a preferred embodiment of the method, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, for example, VHH, and an immunoglobulin heavy chain-light chain hybrid pair (providing a second binding specificity) in the other arm. It has been found that such an asymmetric structure facilitates the separation of the desired bispecific compound from undesired combinations of immunoglobulin chains, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a simple separation method. Such a method is described in WO 94/04690. For a more detailed description of the creation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

Bespecifically antibodies include cross-linked antibodies or "heteroconjugates" of antibodies. For example, one of the antibodies in the heteroconjugate may be associated with avidin, the other with biotin. Such antibodies, for example, have been proposed for directing immune cells to unwanted cells (US Pat. No. 4,669,980) and for treating HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are described in US Pat.

Methods for creating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be obtained using chemical binding. Brennan et al. (Science, 229: 81 (1985)) describe a method in which intact antibodies are proteolytically cleaved to create F (ab ') ₂ fragments. The obtained fragments are reduced in the presence of an agent that forms complexes of dithiol, sodium arsenite in order to stabilize neighboring dithiols and prevent the formation of intermolecular disulfide. Then the created Fab'-fragments are converted into derivatives of thionitrobenzoate (TNB). One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction using mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The obtained bispecific antibodies can be used as agents for the selective immobilization of enzymes.

Recent advances have facilitated the direct extraction of Fab'-SH fragments from E. coli, which may be chemically coupled to form bispecific antibodies. In Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describes the preparation of a fully humanized molecule of a bispecific antibody based on F (ab ') ₂ . Each Fab'fragment was separately secreted from E. coli cells, and it was chemically coupled in vitro to form a bispecific antibody. The bispecific antibody thus formed is capable of binding to cells overexpressing the HER2 and / or HER3 receptor and normal human T cells, as well as triggering the lytic activity of human cytotoxic lymphocytes against tumor targets of the human mammary gland.

Antibodies with two valencies, as well as with more than two valencies, are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991), as well as four specific antibodies.

Pharmaceutical Compositions

Another aspect of the invention is a pharmaceutical composition comprising, as the active ingredient (or as the sole active ingredient), the bispecific binding molecule of the invention that is specific for HER2 and HER3. The pharmaceutical composition may include any of the bispecific binding molecules, as described herein. In some embodiments, the compositions are intended to improve, prevent, or treat disorders that may be associated with HER2 and / or HER3 activity.

Typically, the bispecific binding molecules of this invention are suitable for use in dosage forms in combination with one or more pharmaceutically acceptable excipients, for example, as described below.

The pharmaceutical compositions of this invention may contain at least one bispecific binding molecule and one or more additional binding molecules (eg, antibodies) that target one or more corresponding surface receptors.

The pharmaceutical composition is "sterile" if it is aseptic, i.e. free from microorganisms and their spores.

The pharmaceutical composition is “stable” if the active agent retains its physical stability and / or chemical stability and / or biological activity during the entire shelf life at a storage temperature, for example, at a temperature of 2-8 ° C. Preferably, the active agent retains both physical and chemical stability as well as biological activity. The storage period is selected based on the results of the study of stability during accelerated and natural storage.

The term “excipient” is used herein to describe any ingredient other than the compound (s) of this invention. The choice of an inert excipient will largely depend on factors such as the particular route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, a “pharmaceutically acceptable excipient” includes, without exception, all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and similar physiologically compatible substances. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffer, dextrose, glycerin, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Additional examples of pharmaceutically acceptable substances are wetting agents or a small amount of excipients, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness of the antibody.

By “buffering agent” is meant a solution capable of maintaining a pH value due to the interaction of acidic and alkaline components included in its composition. In general, pH values of the pharmaceutical composition of 4.5 to 7.0 are preferred. As buffering agents, for example, acetate, phosphate, citrate, histidine, succinate and the like can be used. buffer solutions, but not limited to them.

By "isotonic agents" is meant an auxiliary substance or a mixture of two or more auxiliary substances that provide the isotonic osmotic pressure of the solution. "Isotonic" is a solution that creates an osmotic pressure of about 250 to 350 mOsm / kg. As isotonic agents, polyols, mono- and disaccharides, amino acids, metal salts, for example, sodium chloride, and the like, can be used, but not limited to.

By “stabilizer” is meant an auxiliary substance or a mixture of two or more auxiliary substances that provide the physical and / or chemical stability of the active agent. As stabilizers, amino acids can be used, for example, but not limited to arginine, histidine, glycine, lysine, glutamine, proline; surfactants, for example, polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxamer, Pluronic), sodium dodecyl sulfate (SDS ), but not limited to them; antioxidants, for example, methionine, acetylcysteine, ascorbic acid, monothioglycerol, salts of seric acids, and the like, but not limited to; chelating agents, for example, but not limited to EDTA, DTPA, sodium citrate, and the like.

The pharmaceutical compositions of the present invention and methods for their manufacture will be undeniably apparent to those skilled in the art. Such compositions and methods for their manufacture can be found, for example, in Remington, The Science and Practice of Pharmacy, 21th Edition, Troy, Beringer., Lippincott Williams and Wilkins., Philadelphia, PA 2006. The manufacture of pharmaceutical compositions should preferably comply with GMP (appropriate field trip).

The pharmaceutical composition of this invention can be manufactured, packaged or sold widely, in the form of a single unit dose or many single unit doses. As used herein, the term “unit dosage unit” means a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is usually equal to the dosage of the active ingredient that will be administered to the subject, or a convenient portion of such a dosage, for example half or a third of such a dosage.

Any method of administering peptides, proteins, or antibodies adopted in the art can be suitably used for the bispecific binding molecule of the invention.

The pharmaceutical compositions of the present invention are generally suitable for parenteral administration. As used herein, the term “parenteral administration” of a pharmaceutical composition includes any route of administration that is characterized by a physical violation of the integrity of the tissue of the subject and the administration of the pharmaceutical composition through a violation in the tissue, which usually leads to direct entry into the bloodstream, muscle or internal organ. Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injecting the composition by administering the composition through a surgical incision, by applying the composition with a non-surgical wound penetrating the tissue, and the like. In particular, it is contemplated that parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intraarticular injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, for example, intratumoral injection, may also be useful. Regional perfusion is also provided. Preferred embodiments of the invention include intravenous and subcutaneous routes.

Dosage forms of pharmaceutical compositions suitable for parenteral administration usually contain the active ingredient in combination with a pharmaceutically acceptable carrier / excipient, for example, sterile water or a sterile isotonic solution. Such dosage forms can be manufactured, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable dosage forms can be manufactured, packaged, or sold in unit dosage form, for example, in ampoules, or in multi-dose containers containing a preservative. Dosage forms for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like. Such dosage forms may also contain one or more additional ingredients, including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of the composition for parenteral administration, the active ingredient is provided in dry form (i.e., powder or granules) for dissolution with a suitable base (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral dosage forms also include aqueous solutions that may contain excipients, such as salts, carbohydrates and buffering agents (preferably pH 3 to 9, even more preferably pH 4.5 to 7), but for some uses a more suitable dosage form the form may be a sterile non-aqueous solution or a dry form for use in combination with a suitable base, such as sterile pyrogen-free water. An example of a form for parenteral administration are solutions or suspensions in sterile aqueous solutions, for example, aqueous solutions of propylene glycol or dextrose. Such dosage forms, if necessary, may be buffered. Other suitable parenteral dosage forms may include those that contain the active ingredient in microcrystalline form or in a liposome preparation. Dosage forms for parenteral administration can be performed for immediate and / or modified release. Modified release dosage forms include delayed, delayed, pulsating, controlled, targeted and programmed release.

For example, in one aspect, sterile injectable solutions can be prepared by incorporating the bispecific binding molecule in the required amount into an appropriate solvent with one or a combination of the ingredients listed above, as necessary, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound in a sterile solvent that contains a basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders to obtain sterile injectable solutions, the preparation methods are freeze-drying (lyophilization), which gives the powder of the active ingredient plus any additional desired ingredient from its pre-sterilized filtration solution. The proper fluidity of the solution can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable compositions can be accomplished by incorporating into the composition an agent that delays absorption, for example, monostearates and gelatin, and / or by modified release coatings (eg, slow release coatings).

The bispecific binding molecules of this invention can also be administered intranasally or inhaled, usually in the form of a dry powder (alone, as a mixture or in the form of particles with mixed components, for example, mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, such as aerosol container under pressure, pump, spray, atomizer (preferably an atomizer that uses the principle of electrohydrodynamics to obtain fine mist) or a nebulizer, in torus or no use suitable propellant, or as nasal drops.

A pressurized container, pump, spray, nebulizer or nebulizer typically contains a solution or suspension of the binding molecule of this invention, including, for example, a suitable substance for dispersing, dissolving or prolonging the release of the active substance, a propellant as a solvent.

Prior to use as a dry powder or suspension, the drug is usually micronized to a size suitable for delivery by inhalation (usually less than 5 microns). This can be achieved by any suitable grinding method, such as grinding in a spiral jet mill, grinding in a fluidized bed jet mill, supercritical cleaning of a liquid to form nanoparticles, high pressure homogenization, or spray drying.

Capsules, blisters, and cartridges for use in an inhaler or insufflator may be configured to contain a powder mixture of a compound of this invention, a suitable powder base, and an activity modifier.

A suitable solution formula for use in a nebulizer that utilizes the electrohydrodynamic principle to produce fine mist may contain a suitable dose of the bispecific binding molecule of the present invention with one press, and the volume per press may vary, for example, from 1 μl to 200 μl, more preferably from 1 μl to 100 μl.

Suitable flavoring agents, such as menthol and levomenthol, or sweeteners, such as saccharin or sodium saccharin, may be added to the dosage forms of this invention for inhalation / intranasal administration.

Dosage forms for parenteral administration can be performed for immediate and / or modified release. Modified release dosage forms include delayed, delayed, pulsating, controlled, targeted and programmed release.

In the case of powder inhalers and aerosols, the dosage unit is set by means of a valve that delivers a metered amount. Units in accordance with this invention are usually set for the introduction of a measured dose or "injection" of a binding molecule according to this invention. The total daily dose will usually be administered as a single dose or more often as divided doses throughout the day.

The bispecific molecules of this invention may also be formulated for oral administration. Oral administration may include swallowing, so that the compound enters the gastrointestinal tract and / or buccally, lingually or sublingually enters the bloodstream directly from the oral cavity.

Dosage forms suitable for oral administration include solid, semi-solid and liquid systems, such as tablets; soft or hard capsules containing multi- or nanoparticles, liquids or powders; lozenges (including those filled with liquid); chewing forms; gels; rapidly soluble dosage forms; films; suppositories; Sprays and buccal / mucoadhesive adhesives.

Liquid dosage forms include suspensions, solutions, syrups and elixirs. Such dosage forms can be used as fillers in soft or hard capsules (for example, gelatin or hydroxypropyl methylcellulose) and usually contain a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methyl cellulose or a suitable oil and one or more emulsifiers and / or suspending agents . Liquid dosage forms can also be made by reconstituting a solid, for example, from a sachet.

In one embodiment, the bispecific binding molecule of this invention is part of a composition comprising histidine and acetate in a concentration of 1 to 100 mM in a buffer with a pH of from 5.0 to 6.5.

Therapeutic Use of the Bispecific Binding Molecule of the Invention

In one aspect, the bispecific binding molecule of the invention is used in the treatment of disorders that are associated with the activity of HER2 and HER3.

The term "violation" means any condition that can be improved as a result of treatment of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that cause a mammal's predisposition to the occurrence of this violation. Non-limiting examples of diseases to be treated include benign and malignant tumors; leukemia and lymphoid malignancies, in particular cancer of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid gland, pancreas, prostate or bladder; neural, glial, astrocytal, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocellular disorders; inflammatory, angiogenic and immunological disorders. A preferred disorder to be treated according to the invention is a malignant tumor.

The terms "cancer" or "cancerous" refer to a physiological state or describe the physiological state in mammals, which is usually characterized by unregulated (/ -) cell growth / proliferation. This definition covers benign and malignant cancers. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatic cell carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial and uterine carcinoma, salivary carcinoma, kidney cancer (renal cell carcinoma), Yelnia prostate (prostate cancer), cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile, melanoma, and various types of head and neck cancer.

“Treat,” “treatment,” and “therapy” refer to a method of alleviating or eliminating a biological disorder and / or at least one of its accompanying symptoms. As used herein to “alleviate” a disease, disease, or condition, it means reducing the severity and / or frequency of symptoms of the disease, disorder, or condition. In addition, references herein to “treatment” include references to therapeutic, palliative, and preventive therapy.

By the expression “reduces or inhibits” is meant the ability to cause a general decrease, preferably by 20% or more, more preferably by 50% or more, and most preferably by 75%, 85%, 90%), 95% or more. “Reduces or inhibits” may refer to the presence or size of metastases, the size of the primary tumor, or the size or number of blood vessels in angiogenic disorders.

In one aspect, the subject of treatment or the patient is a mammal, preferably a human subject. The above subject may be male or female and of any age.

The term “therapeutically effective amount” refers to the amount of an antibody or antibody fragment for treating a disease or disorder in a subject. In the case of a tumor (eg, cancer), a therapeutically effective amount of an antibody or antibody fragment (eg, an antibody or antibody fragment that specifically binds HER2 and HER3) can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (i.e., to some extent slow down and, preferably, stop) cancer cell infiltration of peripheral organs; inhibit (i.e., to some extent slow down and, preferably, stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or alleviate, to some extent, one or more symptoms associated with the disorder. An antibody or antibody fragment can, to some extent, prevent the growth and / or kill existing cancer cells, it can cause a cytostatic and / or cytotoxic effect. In cancer therapy, in vivo efficacy can be determined, for example, by assessing life expectancy, time to disease progression (TTR), tumor response rate (RR), response duration and / or quality of life.

As used herein, the terms “co-administration,” “co-administration,” and “in conjunction with” referring to bispecific binding molecules with one or more other therapeutic agents are intended to mean, refer to, or include:

1) the simultaneous administration of such a combination of the bispecific binding molecule of this invention and a therapeutic agent to a patient who needs treatment when such components are formulated together in one dosage form from which these components are released almost simultaneously to the specified patient,

2) the simultaneous administration of such a combination of the bispecific binding molecule according to this invention and a therapeutic agent to a patient who needs treatment when such components are formulated separately in different dosage forms, the administration of which occurs almost at the same time to the specified patient, after which these components are released almost simultaneously to the specified patient,

3) the sequential administration of such a combination of the bispecific binding molecule of the present invention and a therapeutic agent to a patient who needs treatment when such components are formulated separately from each other in separate dosage forms that are taken sequentially by the indicated patient with a significant time interval between each administration after which these components are released at substantially different times to the specified patient; as well as

4) sequential administration of such a combination of the bispecific binding molecule of this invention and a therapeutic agent to a patient who needs treatment when such components are formulated together in a single dosage form from which the release of these components occurs in a controlled manner, after which they are simultaneously, sequentially or jointly released at the same time and / or different times to the specified patient, where each part can be entered in one or different ways.

The bispecific binding molecules of this invention may be administered without additional therapeutic treatment, i.e. as an independent therapy. In addition, treatment with the bispecific binding molecules of the invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the bispecific binding molecule may be co-administered or formulated with another cancer medication / preparation.

The term "cytotoxic agent" as used in the present description refers to a substance that inhibits or prevents the functioning of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and radioactive isotopes Lu), chemotherapeutic agents and toxins, such as low molecular weight toxins or enzymatically active toxins originating from bacteria, fungi, plants or animals, including fragments and / or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in the treatment of a malignant tumor. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ^®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethyl melamine; acetogenins (in particular, bullatacin and bullatacinon); delta-9-tetrahydrocannabinol (dronabinol, MARINOL ^® ); beta lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue of topotecan (HYCAMTIN ^® ), CPT-11 (irinotecan, CAMPTOSAR ^® ), acetyl camptothecin, scopolectin and 9-aminocamptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogs, adozelesin, carzelesin and biselezin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodiktiin; spongistatin; nitrogen mustards, such as chlorambucil, chlorofazine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhine, phenesterol, prednimustine, trophosphamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediin antibiotics (e.g. calicheamicin, in particular calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynamin, including dynemycin A; esperamycin; as well as the neocarcinostatin chromophore and related chromophores of the chromoprotein enediin antibiotics, aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carabicin, carcinomycin, carzinofinin-dichrominocinin, 5-chrominomycin, dichrominocinin, 5 chromin L-norleucine, doxorubicin (including ADRIAMICIN ^®, orfolinodoksorubitsin, tsianomorfolinodoksorubitsin, 2-pyrrolino-doxorubicin HCl in injectable liposomes (DOXOL ^®), liposomal doxorubicin TLC D-99 (MYOCET ^®), pegylated liposomal doxorubicin (CAELYX ^®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, martsellomitsin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR ^® ), tegafur (UFTORAL ^® ), capecitabine (XELODA ^® ), epothilone and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimerexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, phloxuridine; adrenal suppressants, such as aminoglutethimide, mitotane, trilostane; a folic acid compensator such as folinic acid; Aceglaton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcin; diazikhon; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pyrarubicin; losoxantrone; 2-ethyl hydrazide; procarbazine; PSK ^® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spiro germanium; tenuazonic acid; triaziquon; 2,2 ', 2''-trichlorotriethylamine; trichotecenes (in particular, T-2 toxin, verracurin A, roridin A and anguidin); urethane; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside ("ara-C");thiotepa; a taxoid, for example paclitaxel (TAXOL ^® ), a paclitaxel preparation based on engineered albumin-linked nanoparticles (ABRAXANETM) and docetaxel (TAXOTERE ^® ); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based products such as cisplatin, oxaliplatin and carboplatin; vinca alkaloids that prevent the polymerization of tubulin from the resulting microtubules, including vinblastine (VELBAN ^® ), vincristine (ONCOVIN ^® ) vindesine (ELDISINE ^® , FILDESIN ^® ) and vinorelbine (NAVELBINE ^® ); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantron; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN ^® ); bisphosphonates such as clodronate (for example, BONEFOS ^® or OSTAC ^®), etidronate (DIDROCAL ^®), NE-58095, zoledronic acid / zoledronate (ZOMETA ^®), alendronate (FOSAMAX ^®), pamidronate (AREDIA ^®), tiludronate (SKELID ^® ) or risendronate (ACTONEL ^® ); troxacitabine (1,3-dioxolane nucleoside analog of cytosine); antisense oligonucleotides, in particular oligonucleotides that inhibit gene expression in signaling pathways involved in the proliferation of aberrant cells, such as, for example, PKC alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE ^® vaccine and vaccines for gene therapy, for example ALLOVECTIN ^® vaccine, LEUVECTIN ^® vaccine and VAXID ^® vaccine; topoisomerase 1 inhibitor (e.g. LURTOTECAN ^® ); rmRH (e.g., ABARELIX ^®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosin, a COX-2 inhibitor (e.g., celecoxib or etoricoxib), a proteosome inhibitor (e.g., PS341); bortezomib (VELCADE ^® ); CCI-779; tipifarnib (811577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE ^® ); pixantron; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, short name for combination therapy with cyclophosphamide, doxorubicin, vincristine and prednisone, and FOLFOX, short name for oxaliplatin treatment regimen (ELOXATINTM) in combination with 5-FU and leucovorin.

Also included in this definition are anti-hormonal drugs that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX ^® ), 4-hydroxy tamoxifen, trioxifen, toremifene (FARESTON ^® ); idoxifene, droloxifene, raloxifene (EVISTA ^®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERM), such as SERM3; pure antiestrogens without agonistic properties, such as fulvestrant (FASLODEX ^® ) and EM800 (such agents can block estrogen receptor dimerization (ER), inhibit DNA binding, enhance ER metabolism and / or reduce ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN ^® ), and non-steroidal aromatase inhibitors such as anastrazole (ARIMIDEX ^® ), letrozole (FEMARA ^® ) and aminoglutetimide, and other aromatase inhibitors, including Vorozol (RIVISOR ^® ) megestrol acetate (MEGASE ^® ), fadrozole, imidazole; agonists releasing hormone luteinizing hormone, including leuprolide (LUPRON ^® and ELIGARD ^®), goserelin, buserelin, and tripterelin; sex steroids, including progestins, such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin and androgens / retinoids such as fluoxymesterone, fully transretinoic acid and phenretinide; shepriston; antiprogesterones; estrogen receptor lowering regulators (ERD); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

It is understood that the bispecific binding molecules of the present invention can be used in treatment methods as described above, can be used in treatment as described above, and / or can be used in the manufacture of medicaments for treatment as described above.

Doses and routes of administration

The bispecific binding molecules of this invention will be administered in an amount effective to treat the condition in question, i.e. in doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the patient’s age, gender, and weight, and whether the administration of the bispecific binding molecules is an independent treatment or is it combined with one or more additional anti- autoimmune or anti-inflammatory treatments.

Drug regimens can be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the severity of the therapeutic situation. Particularly useful is the manufacture of parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. The unit dosage form, as used herein, refers to physically discrete units suitable as unit doses for patients / subjects to be treated; each unit contains a predetermined amount of the active compound, calculated to obtain the desired therapeutic effect in combination with the desired pharmaceutical carrier. The specification for the unit dosage forms of the present invention is generally dictated and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the compounding technique of such an active compound for treatment of sensitivity in subjects.

Thus, qualified specialists understand, based on the disclosure presented in this document, that the dose and dosage regimen are adjusted in accordance with methods well known in the therapeutic field. This means that the maximum tolerated dose can be easily set and an effective amount can be determined that provides a detectable therapeutic effect for the patient, as well as the time requirements for the administration of each agent to achieve a visible therapeutic effect for the patient. Thus, although some dosages and regimen regimens are given as examples herein, these examples do not in any way limit the dosages and regimens that may be necessary for the patient to practice the present invention.

It should be noted that dosage values may vary, depending on the type and severity of the condition, which should be alleviated, and may include one or more doses. In addition, it is necessary to understand that for any particular patient, specific administration schedules should be adjusted after some time according to individual needs and at the discretion of the medical professional who administers or controls the administration of the compositions, and that the concentration ranges given in this description are only given as an example and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen with the compositions of this invention can be based on various factors, including the type of disease, age, weight, gender, health status of the patient, severity of the condition, route of administration, and the particular bispecific binding molecule used. Thus, the dosage regimen can vary widely, but can be determined regularly using standard methods. For example, doses may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects, such as toxic effects or laboratory values. Thus, the present invention encompasses an individual dose increase, which is determined by a qualified specialist. Determination of the required dose and modes are well known in the relevant field of technology and will be clear to a person skilled in the art after familiarization with the ideas disclosed herein.

Examples of suitable routes of administration are provided above.

It is contemplated that a suitable dose of the bispecific binding molecule of the present invention will be in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5-50 mg / kg, for example, about 1-20 mg / kg. A bispecific binding molecule can be introduced, for example, at a dose of at least 0.25 mg / kg, for example at least 0.5 mg / kg, including at least 1 mg / kg, for example at least 1, 5 mg / kg, for example, as well as at least 2 mg / kg, for example at least 3 mg / kg, including at least 4 mg / kg, for example, at least 5 mg / kg; and for example up to a maximum of 50 mg / kg, including up to a maximum of 30 mg / kg, for example, up to a maximum of 20 mg / kg, including up to a maximum of 15 mg / kg. The administration will usually be repeated at suitable intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long as it is deemed appropriate by the responsible physician, who may in some cases increase or reduce the dose if necessary.

Diagnostic Use and Compositions

The bispecific binding molecules of the present invention are also used in diagnostic processes (e.g., in vitro, ex vivo). For example, bispecific binding molecules can be used to detect or measure HER2 and / or HER3 levels in samples obtained from a patient (for example, a tissue sample or a sample of body fluid such as inflammatory exudate, blood, blood serum, intestinal fluid, saliva, or urine) . Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescent analysis, radioimmunoassay and immunohistology. The invention further includes kits (e.g., diagnostic kits) containing the bispecific binding molecules described herein.

For a better understanding of the invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents and patent applications referred to in this specification are incorporated herein by reference. Although the aforementioned invention has been described in some detail by way of illustration and example in order to avoid ambiguous interpretation, it will be readily understood by those skilled in the art that certain changes and modifications may be made without departing from the spirit and scope of the enclosed embodiments. the implementation of the invention.

Examples

Example 1

The scheme for bispecific antibodies antiHER2 / antiHER3 (BCD090) antibodies

In FIG. 1 is a schematic diagram of the creation and optimization of a bispecific antiHER2 / antiHER3 antibody.

Lama was immunized with the HER3HF recombinant antigen. Then a fraction of monunuclear cells was isolated, from which total RNA was subsequently isolated. After that, a series of genetic engineering procedures were carried out aimed at creating a phage Fab library. All of the above procedures are described in more detail in Example 3.

Then spent several rounds of selection of the obtained library (Example 4) and screenings (Examples 5, 6, 7). After that, sequences of selected clones were sequenced. To carry out a functional analysis of the selected candidates using genetic engineering procedures, they were transferred to the following format: full-sized IgG, where instead of the VH domain there is a VHH domain. After the choice of the most affine and functionally active cadadate, a genetic engineering construct was created in which the anti-HER3 VHH Fab gene was cloned to the N-terminus of the aHER2 IgG HER2 light chain, separated from the light chain by a special flexible amicacid linker necessary for the correct independent folding of both polypeptides into the composition of the fusion molecule and independent binding to the HER2 and HER3 antigens (Example 17).

Example 2

Obtaining recombinant antigens and antibodies in a suspension culture of mammalian cells

Antibodies and antigens were produced in cells of a constant cell line derived from Chinese hamster ovary cells (line CHO-K1) according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91 (6): 670-677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun; 28 (11): 843-848; Biotechnol Bioeng. 2003 Nov 5; 84 (3): 332-342]. We used cells constitutively expressing the EBNA1 protein gene (Epstein-Barrvirus nuclear antigen 1). Suspension cultivation was carried out in flasks on an orbital shaker using serum-free media manufactured by Life Technologies Corporation, according to the manufacturer's instructions. For transient expression, cells at a concentration of 2E + 6 cells / ml were transfected with linear polyethyleneimine (PEI "MAX", "Polysciences"). The ratio of DNA / PEI was 1: 3-1: 10. 5-7 days after transfection, the culture medium was centrifuged at 2000 g for 20 minutes and filtered through a 0.22 μm filter. Target proteins were isolated from the culture fluid by affinity chromatography.

HER3 recombinant proteins containing six histidine amino acids at the C-terminus of the protein (Figs. 2, 3) were isolated and purified from the culture fluid using a Profinity IMAC Ni-charged resin (Bio-Rad). Prior to purification, NiCl _{2 was} added to the culture liquid to a concentration of 1 mM. Next, Profinity IMAC Ni-charged in a ratio of 1 to 200 was added to the culture fluid and stirred for 1 hour at room temperature. The sorbent was transferred onto a 5 ml Thermo scientific Polypropylene columns column and washed with phosphate-buffered saline (PBS). The bound protein was eluted with a solution of 0.3 M imidazole pH8 with 0.3 M NaCl. The protein was then transferred to PBS (pH 7.4) using dialysis using SnakeSkin Dialysis Tubing, filtered (0.22 μm), transferred to tubes and stored at -70 ° C. The purity of the resulting protein solution was evaluated using SDS gel electrophoresis (Fig. 6).

The chimeric recombinant HER3 protein containing 2 immunoglobulin Fc domains at the C-terminus of the protein (Fig. 4) was isolated and purified from the culture fluid using affinity chromatography on a 5 ml HiTrap rProtein A Sepharose FF column (GE Healthcare). The clarified culture fluid was passed through a column that was equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with PBS to wash the non-specific binding components. The bound antigen was eluted with 0.1 M glycine pH3 buffer. The major elution protein peak was collected and adjusted to neutral pH with 1M TrisHCl (pH 8). All purification steps were performed on an AKTA prime plus chromatographic system at a flow rate of 110 cm / h. Next, the protein was transferred to PBS using dialysis using SnakeSkin Dialysis Tubing, filtered (0.22 μm), transferred to tubes and stored at -70 ° C. The purity of the resulting protein solution was evaluated using SDS gel electrophoresis (Fig. 6).

The test and control IgG1 antibodies (Fig. 5.) were purified on a 1 ml HiTrapr ProteinAFF column (GE Healthcare) according to the procedure described above for the HER3-Fc antigen. The purity of the resulting protein solution was evaluated using SDS gel electrophoresis (Fig. 7).

Example 3

Immunization of human LER HER3 and creation of a Fab library of phage llama antibodies

The Lama Glama animal was sequentially immunized 5 times by subcutaneous administration of antigenic material mixed with an equal volume of Freund's adjuvant (with the first injection) or incomplete (with the remaining injections). A mixture of recombinant proteins (0.1 mg of each of the proteins per 1 injection) was used as antigen, one of which was human HER3HF. Injections of antigens were performed on the following dates: 0, 2, 4, 5, 8 weeks. Blood sampling (50 ml) was performed 5 days after each injection, starting with the third. 3.8% sodium citrate in a ratio of 1: 9 was used as an anticoagulant. The selected blood of the llama was diluted 2 times with sterile saline. 30 ml of diluted blood solution was layered on Lymphoprep ™ medium (Axis-Shield, Norway) with a density of 1.077 g / ml and a volume of 15 ml and centrifuged for 20 min at 800 g. Mononuclear cells (lymphocytes and monocytes) were selected from the interphase plasma / Lymphoprep medium, and then washed with a sterile PBS solution.

The obtained titer of serum immunoglobulin against HER3, evaluated according to the standard protocol, was not less than 1/8000 (Fig. 8.), which is satisfactory for the preparation of an antibody library.

Total RNA of llama mononuclear cells was isolated using the RNeasy Mini Kit according to the proposed protocol (QIAGEN). RNA concentration was determined using Nanovue (GE Healthcare) and the quality of the isolated RNA was checked by electrophoresis on a 1.5% agarose gel.

The reverse transcription reaction was performed using the MMLV RT kit (from Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexamer oligonucleotides as a seed.

Reverse transcription products were used as a template in a two-step polymerase chain reaction to obtain variable domain genes flanked by restriction sites using a set of oligonucleotides and the protocols of the authors [J Biol Chem. 1999 Jun 25; 274 (26): 18218-30]. Further, combining the genes of the variable domains of light and heavy chains into one fragment was carried out by sequential restriction, ligation and amplification reactions. The heavy chain genes were linked to the kappa genes and separately to the light chain lambda genes. Moreover, the calculated number of matrix molecules in all reactions was not less than 1011. The obtained DNA preparation VL-CK-VH was treated with restriction enzymes NheI / Eco91I and ligated into the original phagemid pH5. The structure of the obtained phagemid is shown in (Fig. 9). Ligation products were transformed into electrocompetent cells of strain SS320, obtained according to the protocols [Methods Enzymol. 2000; 328: 333-63.]. The repertoire of the derivative Kappa Fab library was 1.3E + 7 and the lambda Fab library was 8.4E + 7, respectively. The preparation of phage libraries was prepared according to the procedure described previously [J Mol Biol. 1991 Dec 5; 222 (3): 581-97].

Example 4

Selection of Fab libraries of phage antibodies

Specific phage Fab antibodies against HER3 were obtained from a phage Fab display library obtained by immunizing a llama. Selection was performed on a recombinant 3DHER3HE protein as previously described [Nat Biotechnol. 1996 Mar; 14 (3): 309-14; J Mol Biol. 1991 Dec 5; 222 (3): 581-97]. For panning selection, 3DHER3HE protein (in 50 mM carbonate buffer (pH 9.5) was adsorbed overnight at 4 ° C on the surface of EIA / RIA high binding (Greineer bio-one) sorbing tubes. The tubes were washed several times with PBST ( PBS pH 7.4 and 0.1% Tween 20 v / V), and then the vacant protein binding sites on the surface of the tubes were blocked with a skim milk solution (0.5% m / V in PBST pH 7.4). The skim milk solution was incubated in tubes for 1 h. Next, the tubes were washed with PBST solution. Then the phage library was diluted in 2-4 ml of PBST solution with 0.5% fat free milk to a final concentration of 1013 phage particles per ml. The prepared library was added to tubes with surface-bound antigen and incubated for 1 h with stirring. Unbound phages were removed during a series of washing the tubes with PBST solution. The number of washes was increased from the first round to the third round: 20-30-40 times, respectively, The remaining phage particles bound were eluted from a plastic tube with a 0.1 M Gly-HCl solution (pH 2.2) for 15 minutes with stirring. The solution was then transferred to clean plastic tubes and neutralized with 1 M Tris-HCl (pH 8) 5: 1. Bacteria of E. coli TG1 strain were infected with the obtained phages, the phages were amplified and used in the next selection cycle.

After the second and third round of selection for 3DHER3HE, an ELISA analysis of the polyclonal phage preparation showed significant enrichment (Fig. 11). The resulting pool of clones enriched in human Fab specific against 3DHER3HE was recloned into the pLL expression plasmid (FIG. 10) containing myc-tag and His6 tag at the C-terminus of the heavy chain CH1 gene. The cells of the E. coli Bl21Gold expression strain were transformed with a clone library and plated on solid medium containing ampicillin (100 μg / ml) and kanamycin (30 μg / ml). Cultivated for 15 hours at 30 ° C. Single colonies were transferred with a sterile automatic pipette tip into the wells of 96-well plates containing 100 μl of 2TY medium with ampicillin (100 μg / ml), kanamycin (30 μg / ml) and glucose (0.4%). The plates were sealed with a breathable Breathseal film and cultured for 16 hours on an orbital shaker at 30 ° C, 900 rpm. Then the cells were besieged by centrifugation of the plates at 2500 rpm for 20 minutes, and supernatants were used to analyze the Fab fragments of antibodies contained in them.

Example 5

Analysis of specific binding of Fab to human HER3

ELISA was used to measure the binding of the studied Fab fragments to human HER3. As a positive control used Fab antibodies AV203 with the published sequence (WO 2011/136911 A2). For analysis of specific binding, well plates (from Nunc ImmunoMaxisorp) were coated with 50 μl (0.5 μg / ml in 1X carbonate coating buffer) 3DHER3HE, sealed and incubated overnight at 4 ° C. All subsequent steps were performed according to the standard ELISA protocol using a high-performance automated platform based on GenetixQ-pix2xt robotic systems (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). In order to block non-specific binding, BB blocking buffer (200 μl of 0.5% nonfat milk in PBS) was added to the wells of the plates. The plates were incubated on a shaker for one hour at room temperature. After washing with phosphate-buffered saline containing Tween-20 (PBST), 50 μl per well of test cell supernatant containing the studied Fab was added. The plates were again incubated by shaking for one hour at room temperature, after which each well of the plates was washed five times with PBST. After washing, 50 μl / well of an anti-human Fab HRP-conjugated secondary antibody (from Pierce-ThermoScientific) was added in a ratio of 1: 7500 in PBS-Tween. The plates were shaken on a rotary shaker (50 min, room temperature) and washed five times with PBST. The colorimetric signal was developed by adding TMB (50 μl / well) to saturation (average 10-12 min), then further development was stopped by adding a stop solution (25 μl / well, 1% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The degree of antibody binding was proportional to the production of a color signal. Clones in which the color signal exceeded the background more than 5 times were tested in a competitive ELISA to detect antagonistic Fabs that block the interaction of the ligand and receptor.

Example 6

Competitive ELISA analysis of the blocking interaction of rhHRG1-β1ECD ligand and Her3 receptor

Competitive ELISA was used to test the antagonistic ability of previously selected specific Fabs against human HER3. Fab antibodies AV203 (WO 2011/136911 A2) were used as a positive antagonist control. The ligand rhHRG1-β1ECD (from R&D Systems) was immobilized in the wells of plates (from Nunc Immuno Maxisorp) at 50 μl with a concentration of 1 μg / ml in 1X carbonate buffer and incubated overnight at 4 ° C. All subsequent steps were carried out according to standard ELISA protocols using a high-performance automated platform based on GenetixQ-pix2xt robotic systems (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). In order to block non-specific binding, BB blocking buffer (200 μl of 0.5% nonfat milk in PBS) was added to the wells of the plates. The plates were incubated on a shaker for one hour at room temperature.

In parallel, 40 μl of the test cell supernatant containing the test Fab was mixed in a non-binding 96-well plate with 40 μl of HER3-Fc at a concentration of 2 μg / ml in 0.5% milk on PBS-Tween. Incubated for 1 hour at room temperature with stirring at 500 rpm.

After washing from the blocking buffer into a plate containing the rhHRG1-β1ECD ligand, the reaction mixture Fab and HER3-Fc receptor described above was transferred at a rate of 80 μl per well. The plates were again incubated by shaking for 45 minutes at room temperature, after which each well of the plates was washed five times with PBST. 50 μl / well of anti-human Fab HRP-conjugated secondary antibody (from Pierce-ThermoScientific) was added in a ratio of 1: 7500 in PBST. The plates were shaken on a rotary shaker for 45 minutes at room temperature and washed 5 times with PBST. The colorimetric signal was developed by adding TMB (50 μl / well) to saturation (average 10-12 min), then further development was stopped by adding a stop solution (25 μl / well, 1% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The degree of antibody binding was proportional to the production of a color signal.

Clones that showed blocking at the level of the control Fab antibody AV203 were noted as positive and were used in further analyzes. The genes for the variable domains of positive clones were sequenced according to standard protocols on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems).

Example 7

Comparative screening of anti-Her3 Fab human candidates by koff

Koff screening was performed using a Pall Forte Bio Octet QK 96 instrument. Anti FABCH1 biosensors were rehydrated for 30 minutes in working buffer containing 10 mM PBS, pH 7.2-7.4, 0.1% Tween-20, 0.1% BSA. In the test samples of E. coli supernatants, 10 × working buffer was added to a final concentration of 1 ×. Then, anti-FABCH1 biosensors were immersed in E. coli supernatants containing Fab fragments of antibody candidates for 12 hours at 4 ° C. Sensors with Fab fragments immobilized on the surface were transferred to wells with a working buffer, where the baseline was recorded (for 60 sec). The sensors were then transferred to wells with an analyte solution (3DHER3HE, 30 μg / ml) for association of the antigen-Fab complex (within 300 sec). Then the sensors were returned to the wells containing the working buffer for the subsequent stage of dissociation (within 600 sec). After each experiment, the sensors used were regenerated by placing them in the buffer for regeneration three times (Gly-HCl, pH 1.7) and then they could be used in the next experiment. The analysis of the obtained curves was carried out using Octet Data Analysis software (version 7.0) according to the standard procedure using a 1: 1 interaction model. As a control was used Fab reproduced antibodies AV203 (WO 2011/136911 A2). The results are presented in Table 1. Candidates MP3: H3 (1), MP3: A4 (2), MP3: B4 (3) have a similar or better Koff value compared to the Koff Fab fragment of the reproduced AV203 antibody.

Example 8

Construction of a library of VHH-aHER3 mutant monodomain antibodies specific for HER3

To create libraries of VHH-aHER3 mutant single domain antibodies specific for HER3, a pool of genes encoding the sequence of VHH-aHER3 having four neighboring randomized NNK triplets in the region of each CDR region was synthesized (Table 2). The obtained DNA of the randomized gene was cloned into a phage plasmid pH6 described in [FEBS Lett. 1997 Sep 15; 414 (3): 521-6], according to the protocol described in the work, for display in the composition of the gpIII capsid protein. The transformation of these structures into the SS320 strain yielded from the 2E + 8 independent transformants for the library, according to the procedure [Methods Enzymol. 2000; 328: 333-63]. Phage preparations of the mutant VHH-aHER3 libraries were prepared according to the procedure described previously [Mol Biol. 1991 Dec 5; 222 (3): 581-97].

The selection of the obtained phage mutant VHH-aHER3 libraries was carried out under the conditions similar to those described above (Example 4).

After the third round of selection on a recombinant human HER3 preparation, ELISA analysis of polyclonal phage preparations showed significant enrichment and an excess of more than 10 times over the background of non-specific binding. The gene pools from the VHH-aHER3 enriched phage libraries of mutant single domain antibodies specific for human HER3 were transferred to the Novagen expression plasmid pET22b (+) containing the myc-tag peptide at the C-terminus for ELISA detection.

Example 9

Isolation of VHH-aHER3 Mutant Monodomain Antibodies Specific Against HER3

To analyze the binding of the investigated mutant single-domain antibodies to human HER3, ELISA was used according to the procedure described in Example 5. The number of tested clones producing VHH-aHER3 mutant single-domain antibodies amounted to 1200 pieces. As a positive control, the original version of the VHH-aHER3 single domain antibody with the sequence of SEQ ID NO: 4 was used.

As a result of the analysis, 37 positive clones were selected that gave a signal higher or similar to the control initial VHH-aHER3 single domain antibody (values in the range of 0.7-1.0 rel. Units).

Example 10

Characterization of VHH-aHER3 mutant single domain antibodies specific for HER3

Thirty-seven ELISA screening candidates (Example 9) for positive clone candidates that gave a signal higher or similar to the control VHH-aHER3 single domain antibody were sequenced on an Applied Biosystems 3130 sequencer according to protocols recommended by the manufacturer. As a result, 26 clones unique in sequence were obtained. The obtained unique candidates were further analyzed by the quantitative characteristic of the kinetic dissociation constant on an OctetRED96 instrument according to the procedure described in Example 11.

The obtained quantitative kinetic data of the specific interaction of VHH-aHER3 mutant single domain antibodies and their sequences are shown in Table 2.

As a result, mutants of VHH-aHER3 single domain antibodies were obtained, having up to nine substitutions in all three CDRs and, at the same time, retaining or having enhanced affinity for the HER3 receptor. In addition, the amino acid composition of such substitutions is completely diverse. Thus, the possibility of creating high-affinity, specific mutant forms of the VHH-aHER3 polypeptide having up to 25% substitutions in the total CDR sequences was shown (Variants with a maximum number of substitutions — up to 9 substitutions were calculated, to the total number of CDRs positions — about 40).

Example 11

Comparative screening of VHH-aHER3 mutant single domain antibodies by koff

Koff screening was performed using a Pall Forte Bio Octet QK 96 instrument. Anti Streptavidin biosensors (SA) were rehydrated for 30 minutes in a working buffer containing 10 mM PBS, pH 7.2-7.4, 0.1% Tween-20, 0.1% BSA Next, biotinylated HER3HF antigen (20 μg / ml) was immobilized onto biosensors. Sensors with immobilized biotinylated antigen were transferred to wells with a working buffer, where the baseline was recorded (for 60 sec.). In the test samples of E. coli supernatants, 10 × working buffer was added to a final concentration of 1 ×. Then the biosensors were immersed in E. coli supernatants containing Fab fragments of antibody candidates for the association of the antigen-antibody complex (for 300 sec). Then the sensors were returned to the wells containing the working buffer for the subsequent stage of dissociation (within 600 sec). The analysis of the obtained curves was carried out using Octet Data Analysis software (version 7.0) according to the standard procedure using a 1: 1 interaction model. As a control was used Fab reproduced antibodies AV203 (WO 2011/136911 A2).

Example 12

Design and production of bispecific antibodies VHH-IgG1 against HER2 and HER3

Genes of the variable domains of the light and heavy chains of antibodies against human HER2 [WO / 1999/031140] were synthesized in the original codon composition and cloned into plasmids containing the genes of the constant domains of light and heavy chains, respectively, for transient expression in CHO-K1 cells, as described in Example 2. The resulting constructs were validated by sequencing (SEQ ID NO: 5 and SEQ ID NO: 6 amino acid sequences of the variable domains of the heavy and light chains, respectively).

In addition, based on the sequence of the detected high affinity VHH Fab against HER3 (Example 7), a gene was encoded that encodes the amino acid sequence of SEQ ID NO 4. In this case, E44G / R45T mutations were introduced at the intersubunit contact site of the VH and VL variable domains, which, as shown earlier, [application RU 2014138740], determine the stability of the VHH derivative in combination with the antibody light chain.

Further, in a standard genetic engineering procedure, an anti-HER3 gene encoding the amino acid sequence of SEQ ID NO 4 separated from the light chain by a flexible amino acid linker was cloned into the light chain N-terminus of the above-described anti-HER2 antibody light chain construct.

Example 13

Enzyme-linked immunosorbent assay for the interaction of anti-HER3 antibodies with HER3 and other antigens

ELISA was used to measure the relative affinity of antibodies to HER3 and other antigens. For binding assays, plate wells (medium binding from Greiner bio one) were coated with 50 μl of HER3HF, 3DHER3HE, HER2-Fc, EGFR-Fc, IGFR-Fc, IL12-Fc, CD3ED-Fc, GCSF, cMET, IL17-Fc, IL6R- Fc (5 μg / ml in 1 × carbonate buffer), tightly closed and incubated overnight at 4 ° C. All subsequent steps were carried out according to the standard ELISA protocol described in Example 5. The tested antibodies were used at a concentration of 5 μg / ml in PBST.

The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The degree of antibody binding was proportional to the production of a color signal (FIG. 12). An anti-HER3 antibody, which is a full-sized IgG, where instead of the VH domain there is a VHH domain, specifically binds to full-size HER3 and its third domain and does not bind to other antigens studied.

Example 14

Enzyme-linked immunosorbent assay for interactions of bispecific anti-HER2 / antiHER3 (BCD090) antibodies with HER2, HER3 receptors of different organisms

ELISA was used to measure the comparative affinity of the bispecific antiHER2 / antiHER3 antibody (hereinafter BCD090) to the HER2, HER3 receptors of different organisms. For binding assays, plate wells (medium binding from Greiner bio one) were filled with 50 μl of human HER2-Fc, HER2His cynomolgus monkey, mouse, rat, HER2FE rabbit, guinea pig, human HER3-Fc, macaque, mouse, rat HER3His, guinea pig HER3FE (1 μg / ml in 1 × covering carbonate buffer), tightly closed and incubated overnight at 4 ° C. All subsequent steps were carried out according to the standard ELISA protocol described in Example 5. The BCD090 bispecific antibody affinity binds to human HER2, cynomolgus HER2, human HER3 and all HER3 orthologs antigens: macaque, mouse, rat, guinea pig. BCD090 is 2 times weaker bound to guinea pig HER2 and does not bind to other studied receptors (Fig. 13, Fig. 14).

Example 15

Analysis of interactions of antiHER2 / antiHER3 antibodies (BCD090) with human and macaque Rhesus HER3 and macaque cynomolgus HER2 receptors on an Octet RED 96 instrument

The affinity constants of binding of the antibody to the HER3 receptors of human and rhesus macaques and cynomolgus macaques HER2 were obtained using an OctetRed 96 instrument (from ForteBio). Antibodies were non-specifically immobilized to the surface of second-generation amine reactive sensors (ForteBio, Pall) according to the standard protocol according to the manufacturer's instructions for the preparation and immobilization of AR2G sensors. The analysis was performed at 30 ° C. using PBS containing 0.1% Tween-20 and 0.1% BSA as the working buffer. Receptor titration was performed using a working buffer of various concentrations depending on the intended affinity.

The binding curves with the deduction of the base signal were analyzed using the Octet Data Analysis software (version 7.0) according to the standard procedure using a 1: 1 interaction model. The BCD090 antibody specifically binds to human and Rhesus macaque HER3 and Macaque cynomolgus HER2 antigens (Fig. 15, 16, 17). The analysis showed that the BCD090 antibody with high affinity binds to human HER3, the affinity for HER3 rhesus macaques is 6 times lower. The BCD090 antibody also binds to macaque cynomolgus HER2, but the affinity constant for HER2 cynomolgus is inferior to the affinity constant for human HER2 by about 4 orders of magnitude. The resulting affinity constants are presented in Table 3.

Example 16

Analysis of interactions of antiHER2 / antiHER3 (BCD090) antibodies with human HER2 using a Biacore T200 instrument

Experiments on the binding affinity of BCD090 antibodies to human HER2 antigen were carried out on a Biacore T200 optical analyzer (Ge Healthcare Life Sciences), the principle of the method of which is based on the effect of surface plasmon resonance for detecting intermolecular interactions. We used standard CM5 optical chips with a carboxymethyldextran coating. The BCD090 antibody was nonspecifically (for NH2 groups) immobilized on the surface of the chip at a concentration of 20 μg / ml according to the protocol in accordance with the manufacturer's instructions. Then, the antigen was injected at concentrations of 1-15 μg / ml for 5 minutes at a flow rate of 10 μl / min in working buffer (HBS-EP, 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20). Complex dissociation was detected in real time at a temperature of 37 ° C.

Curves of specific interactions of antigens with antibodies immobilized on an optical chip were obtained as the signal difference between the experimental and control channels. The analysis of the data was performed using the BIA Evalution 3.1 program and the Heterogeneous Ligand model. The affinity constant was calculated as the average of the results obtained in the experiment.

The BCD090 bispecific antibody has high affinity for human HER2.

The affinity of BCD090 for HER2 was KD1 / KD2 -0.55 pMol. Association / dissociation curves are shown in FIG. eighteen.

Example 17

Analysis of the interaction of bispecific antibody BCD090 with both human HER2 and HER3 antigens on an OctetRED96 instrument

The analysis of the simultaneous interactions of BCD090 antibodies with human HER2 and HER3 antigens was performed using the classic “sandwich” analysis method on an OctetRed 96 instrument (Pall-ForteBio). The analysis was performed at 30 ° C using PBS containing 0.1% Tween-20 and 0.1% BSA as the working buffer. During the experiment, the first antigen (HER2) at a concentration of 5 μg / ml and a volume of 200 μl / well was immobilized on amine-reactive sensors. The sensors were then immersed in an antibody solution of 200 μl / well of antibodies with a concentration of 20 μg / ml. After that, the sensors were either immersed in a solution of the second HER3 antigen of 200 μl / well with a concentration of 10 μg / ml and in parallel in a physiological solution containing no protein.

The binding curves with the deduction of the base signal were analyzed using the Octet Data Analysis software (version 8.2) according to the standard procedure.

The sensogram shown in FIG. 19, indicates that the BCD090 antibody molecule is capable of simultaneously binding two antigens: human HER2 and HER3.

Example 18

Analysis of the interactions of BCD090 antibodies with FcRn and Fcγ receptors using Octet RED 96

To analyze the interaction of antibodies with FcgRIIIaV, FcgRIa, FcRn, a Forte bio Octet RED96 instrument and streptavidin coated biosensors (SA-Streptavidin) were used. The antibody Trastuzumab was used as a control.

In the course of the experiment, oriented immobilization of biotin-labeled receptors to the surface coated with streptavidin sensors was performed. Prepared a series of dilutions of antibodies from a concentration of 1000 (100 μg / ml in the case of FcgRIa) μg / ml in increments of 2 by 7 points and placed in the wells of a 96-well plate. Next, the association stage was carried out by immersing the sensors in antibody solutions with different concentrations, after which the dissociation stage was carried out by immersing the sensors in a working buffer.

For the analysis of the affinity of antibodies to FcgRIIIaV, FcgRIa, the working buffer PH7.4 (PBS, 0.1% Tween, 0.1% BSA) was used; for the analysis of the affinity for FcRn, the working buffer PH6.0 was used.

We performed a SteadyState analysis of the obtained curves with the deduction of the reference signal using a 2: 1 model (heterogeneous ligand) according to the standard procedure. The analysis showed that the bispecific antibody specifically binds to the receptors FcRn, FcgRIIIaV, FcgRIa.

An analysis of affinity for Fcg receptors suggests that the effector functions of BCD090 are expected to be comparable with the effector functions of Trastuzumab. An analysis of affinity for the FcRn receptor suggests that the pharmacokinetics of the bispecific antibody are similar to the pharmacokinetics of the trastuzumab antibody. The results are presented in Table 4.

Example 19

Analysis of the inhibition of tumor cell proliferation by bispecific antibody BCD090

To assess the ability of the BCD090 antibody to inhibit choregulin-dependent proliferation of tumor cells, the MCF7, BT474, A431NS, SKBR-3 cell lines were used.

The composition of the medium for analysis on the MCF7 cell line: DMEM, 2 mM Glutamine, 0.3% FBS, 10 μg / ml, 25 ng / ml HRG (rhHRG1-β1).

The composition of the medium for analysis on the BT474 cell line: DMEM, 2 mM Glutamine, 5% FBS, 10 μg / ml, 2.5 ng / ml HRG.

Solutions of the antibodies to be analyzed were prepared in the assay medium (RPMI, 2 mM Glutamine, 0.3% FBS, 10 μg / ml insulin in the case of MCF7 and BT474, 5 ng / ml HRG in the case of SKBR-3) in concentrations: 400 μg / ml, 80 μg / ml, 16 μg / ml, 3.2 μg / ml, 640 ng / ml, 128 ng / ml, 25.6 ng / ml, 5.1 ng / ml, 1.02 ng / ml. The resulting solutions were transferred to the wells of the 96-well in triplicate.

4000 cell / well cells were added to the antibody solutions in assay medium.

The plates were incubated at 37 ° C, 5% CO ₂ for 72 hours. Then, the number of living proliferating cells was measured by adding the vital dye AlamarBlue and analyzing the level of fluorescence on a Fluoroskan Ascent instrument.

As shown in FIG. 20, 21, 22, 23 bispecific monoclonal antibody BCD090 significantly inhibits the proliferation of cell lines MCF7, BT474, A431NS, SKBR-3 than the control antibodies AV203 and Trastuzumab.

Table 5 shows EC50 values for various antibodies for all cell lines.

Example 20

Determination of thermal stability by the point of protein aggregation by dynamic light scattering (DLS).

The aggregation point of the studied samples was determined on a Zetasizer Nano ZSP instrument. For this, the solution was placed in a quartz dedusted cuvette, which was gradually heated in the apparatus from 50 to 85 ° C. A heating step of 1.5 ° C with constant measurement of the intensity of the scattered light. The scattered light intensity was detected at an angle θ = 173 °. The data are presented in FIG. 24 and in Table 6.

According to the results of this test, it can be concluded that the BCD090 molecule has good thermocolloidal stability and may be suitable for technological and pharmaceutical use.

Example 21

Determination of thermal stability during prolonged exposure with thermostat method 50 ° C.

The studied samples in protein concentration were divided into 3 parts, 1 tube for each composition was stored in a refrigerator at + 4 ° C, the rest were installed in a tube thermostat and thermostated at 50 ° C for 24 and 72 hours. After warming up, it was kept at room temperature for about 15 minutes, centrifuged at 8000 rpm for 5 minutes. Supernatants were transferred for analysis by size exclusion HPLC (SEC HPLC) with a UV detector. Chromatography was performed on an Agilent 1100 HPLC system on a Tosoh TSK-Gel G3000SWXL column, 7.8 mm × 30 cm, cat. No. 08541 with Tosoh TSKgel Guard SWXL pre-column, 6.0 mm × 4.0 cm, with a particle diameter of 7 μm, cat. No. 08543. Detection was carried out at wavelengths of 220 and 280 nm.

The resulting stability data of BCD090 in various buffers during prolonged heating at 50 ° C. in FIG. 25. In FIG. 26 shows combined chromatograms: red — intact, blue — 72 hours of incubation at 50 ° C.

According to the results of this test, we can conclude that the BCD090 molecule has good thermal stability (the difference between the content of aggregates in the solution before and after stress thermostress was not more than 5%) and may be suitable for technological and pharmaceutical use.

Example 22

The study of the stability of monoclonal bespecifically BCD090 antibodies in human serum

To assess the stability of BCD090, antibodies were added to human serum obtained from 7 healthy donors and stabilized with 0.01% merthiolate to concentrations of 4, 20, and 100 μg / ml. The resulting mixture was incubated for 7 days at 37 ° C. After 7 days, the concentration of BCD090 was determined by two methods (Fig. 27).

Using the first method, recombinant human HER2 was immobilized on a plate, detection of the level of the bound antiHER2 / antiHER3 antibody was carried out by 3DHER3HE human labeled with biotin and a streptavidin conjugate with horseradish peroxidase. Using the second method, recombinant human HER2 was also immobilized on a plate, and the detection of bound antiHER2 / antiHER3 was carried out using a conjugate of goat polyclonal antibodies against the Fc fragment of human IgG with horseradish peroxidase. As a control of 100% stability, human serum images were used to which antiHER2 / antiHER3 were added in similar concentrations immediately before analysis.

The concentration of antiHER2 / antiHER3 was measured as follows. Control and stored for 7 days at 37 ° C blood serum samples with added antiHER2 / antiHER3 were diluted to a concentration of 100 ng / ml, respectively, 40, 200 and 1000 times in PBST. The concentration was determined by the calibration curve (values of calibration points from 6.25 to 200 ng / ml), calibration standards were also diluted PBST. Further, the analysis was carried out according to a standard method. 100 μl of pre-diluted test samples and calibration standards were added to the wells of a pre-immobilized recombinant human HER2 plate. Immobilization was carried out at a HER2 concentration of 2 μg / ml from 20 mM carbonate buffer, pH 9.5, free binding sites were blocked with 200 μl of 0.5% cattle milk powder in PBS. The plates were incubated with constant shaking of 300 rpm at room temperature. After washing the wells 3 times with 250 μl of PBST, 100 μl of biotinylated 3DHER3HE, 3 μg / ml (first method) or 100 μl of polyclonal goat anti-human IgG Fc fragment in working dilution 1: 25000 in PBST (second method) were added to the wells. The plates were incubated with constant shaking (300 rpm) at room temperature. After washing the wells 3 times with 250 μl of PBST, 100 μl of streptavidin peroxidase conjugate at a working dilution of 1: 20,000 were added to the wells to which biotinylated 3DHER3HE was added. After incubation and washing, peroxidase activity was manifested with 100 μl of TMB substrate for 10-15 minutes, the reaction was stopped by adding 50 μl of 10% sulfuric acid. The optical density in the wells was determined using a plate spectrophotometer Tecan Sunrise (Tecan) at 450 nm. The amount of bound antiHER2 / antiHER3 antibody was directly proportional to the optical density. The concentration of antiHER2 / antiHER3 calculated by the calibration curve was multiplied by the dilution factor of the sample and expressed as% of the concentration theoretically added to the serum (Openability,%).

According to the results of both experiments (Fig. 28), no significant differences were found between the concentration of antiHER2 / antiHER3 determined in human serum after incubation for 7 days at 37 ° C and the concentration determined in serum without incubation. Thus, we can conclude that the monoclonal bispecific antiHER2 / antiHER3 BCD090 antibody is stable in human serum.

Example 23

Determination of antibody dependent cytotoxicity (ADCC)

For the analysis of ADCC, target cells of the BT474 line (ATCC, NTV-20) and mononuclear cells of peripheral blood (PBMC) of a healthy donor were used.

The cells of the line were cultured in DMEM medium with 10% fetal bovine serum (FBS) at 37 ° C and 5% CO ₂ . Cells were fluorescently labeled with calcein AM (Santa Cruz Biotechnology, SC-203865) and resuspended in RPMI1640 medium to a density of 2E + 5 cells / ml.

Peripheral blood mononuclear cells (PBMCs) were isolated from the venous blood of a healthy donor using cell separation in a density gradient. After this, the cells were washed twice in PBS and resuspended in RPMI1640 with 10% FBS to a density of 5E + 6 cells / ml.

For ADCC analysis, 100 μl of antibodies with concentrations from 20 pg / ml to 20 μg / ml were added to the wells of a 96-well plate. To them were added 50 μl of target cells and 50 μl of effector cells. The plate was incubated for 4 hours at 37 ° C and 5% CO ₂ . After the tablet was centrifuged, then 100 ml of the supernatant was transferred to a new tablet. The fluorescence was determined at an excitation / emission wavelength of 485/538 nm. Fluorescence of spontaneously released calcein was determined in samples containing 50 μl of target cells and 150 μl of RPMI1640 with 10% FBS. Calcein fluorescence during complete lysis of the target cells was determined in samples containing 50 μl of target cells and 150 μl of RPMI1640 with 10% FBS and 1.4% Triton X100. The effectiveness of ADCC was determined by the formula:

The results are shown in FIG. 29.

Example 24

Evaluation of the antitumor activity of BCD090 in a subcutaneous xenograft model using human tumor lines MCF-7, BT474, SK-BR-3 in immunodeficient mice

A suspension of tumor cells was injected subcutaneously into the right flank of immunodeficient mice. 3 days before and weekly after the introduction of tumor lines MCF-7 and BT474 (estrogen-dependent tumor lines), the hormone estradiol was administered to mice. The volume of the introduced suspension of tumor cells was 0.5 ml.

The concentration of MCF-7 cells was 10E + 6 cells / ml in serum-free medium RPMI1640. The concentration of BT474 cells was 10E + 6 cells / ml in serum-free medium RPMI1640. The concentration of SK-BR-3 cells was 4E + 6 cells / ml in serum-free medium RPMI1640. Together with the tumor cells, Matrigel (reduced growth factor) was introduced in a ratio of 1: 1.

The administration of antibody preparations began when the tumor volume reached 100 mm3. The study drug and placebo solution was administered intraperitoneally to animals twice a week. During the experiment, the size of the tumor node was measured twice a week and its volume was calculated by the formula:

V = π / 6 * L * W * H, where L, W, H are the linear sizes of the tumor.

The criterion for the effectiveness of the drug was the tumor growth inhibition index (SRW), which was calculated according to the formula:

where Vk and V0 are the average tumor volume (mm3) in the control and experimental groups, respectively.

The tumor growth index was also calculated, which characterizes the increase in tumor volume at each time point, compared with the size at the time of treatment initiation. The calculation of the indicator was carried out according to the formula:

Ii = Vi / V0,

where, I is the tumor growth index, i is the day of the experiment, V0 is the tumor volume on the day treatment is started.

Observation of the animals was carried out for 35 days, from the start of treatment.

The drug BCD090 showed a more pronounced antitumor activity compared with monospecific antibodies against HER2 and HER3 in all tumor lines according to the TPO index. The data are presented in FIG. 30, 31, 32, 33, 34, 35.

Example 25

Creating a stable cell line for BCD090

The stable cell line producing the monoclonal antibody BCD090 was obtained by electroporation using the Neon Transfection System (Life Technologies) instrument of the parent suspension cell line CHO-S with vector constructs containing the light and heavy chains of the antibody in an optimized ratio. Clonal lines with a high level of productivity (more than 1000 mg / l) were obtained using the ClonePix robotic platform (Molecular Devices) and preliminary stages of mini-pool selection using antibiotics in different cultivation formats. Productivity analysis was performed using the Octet RED96 analytical system (Pall Life Sciences). The design of the experiment on the selection of the basic medium and the cultivation scheme was carried out on the basis of the automated system Biomek FX robotics (Beckman Coulter). For the cultivation of the producer used serum-free media and feeds that do not contain proteins of animal origin. The preparation of BCD090 for preclinical studies was performed in a HyClone single-use bioreactor (Thermo Fisher Scientific) fermenter with a working volume of 50 l.

Example 26

Purification of BCD090 from production medium of a stable cell line.

Clarification of the culture fluid containing the BCD090 bispecific antibody was performed on a Millistak C0HC depth filter (Merck-Millipore). The primary purification of the antibody was carried out on an affinity sorbent with protein A using specific elution of the target protein at pH 3.3-3.8 in glycine buffer. The resulting eluate was incubated at acidic pH for 30-60 minutes for viral inactivation, and then neutralized with a 1M Tris-OH solution. Final chromatographic purification was performed on a CHT ™ Ceramic Hydroxyapatite (Bio-Rad) sorbent to remove residual DNA, producer cell proteins, cleaved affinity sorbent ligand, aggregates and antibody fragments. For this, a protein solution was applied to the prepared sorbent at pH 7.5, followed by specific elution of the bispecific antibody in a gradient of sodium chloride. The purified protein was subjected to antiviral filtration using a Viresolve PRO filter kit (Merck Millipore). Concentration was performed in a tangential flow on cassettes with a cutoff limit of 50 kDa followed by diafiltration against histidine buffer (pH 6.0). The protein concentration in the final solution was at least 20 mg / ml.

Example 27

Homologous modeling of the HER3 and antiHER3 VHH complex

The structure of the interaction complex was obtained using the Schrodinger Suite 2016-1 software. The crystal structure of PDB 4LEO [Cancer Res. 2013 73: 5183-5194]. To model the structure of the antibody, a homologous folding protocol of the antibody was used using the template structure PDB 2X10 [Cell.Mol.Life Sci. 2010 67: 1519-1535] for constructing framework regions. The HCDR3 loop was modeled de novo using the Prime algorithm [Proteins 2014 82 (2): 1646-1655]. The obtained ten conformations of this loop with the best characteristics (energy, similarity of the loop structure to those available in the database, etc.) were ranked by the free energy value, after which the best structure was selected for this parameter and docking was performed with the target structure using the Piper algorithm [ Proteins 2006 65 (2): 392-406]. As a result, a repertoire of poses was obtained, from which a pose with the best binding energy was chosen. The final structure was subjected to 5ns molecular dynamics using the Desmond package [Bowers KJ, et al (2006) Scalable Algorithms for Molecular Dynamics Simulations on Commodity Clusters, Proceedings of the ACM / IEEE Conference on Supercomputing (SC06), Tampa, Florida, November 11- 17, 2006] and the energy field OPLS_2005 [Banks, JL, et al (2005) Integrated Modeling Program, Applied Chemical Theory (IMPACT), J Comput Chem. 26 (16): 1752-1780]. From the simulation trajectory, a period of time was obtained at which the complex is inactive, and the final structure was taken from it.

, LLC].The image (Fig. 36) was built using the program for viewing structures PyMol [The PyMOL Molecular Graphics System, Version 1.8

, LLC].

Claims (33)

1. A bispecific antibody that specifically binds to human or cynomolgus HER2 and HER3, including: 1) the VHH heavy chain variable domain that specifically binds to HER3, where the VHH heavy chain variable domain contains at least 75% amino acid sequences homologous to SEQ ID NO: 1-3; 2) an antibody or antigen binding domain thereof that specifically binds to human or cynomolgus monkey HER2. 2. The bispecific antibody of claim 1, wherein the VHH heavy chain variable domain that specifically binds to HER3 and the antibody or antigen binding domain thereof that specifically binds to human or cynomolgus macaques HER2 are linked by a peptide linker. 3. Bispecific antibody according to claims. 1, 2, where the variable domain of the VHH heavy chain contains amino acid sequences that are at least 90% homologous to the sequences of SEQ ID NO: 1-3. 4. The bispecific antibody of claim 3, wherein the VHH heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 1-3. 5. The bispecific antibody according to any one of paragraphs. 1, 2, where the variable domain of the VHH heavy chain contains an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 4. 6. The bispecific antibody of claim 5, wherein the VHH heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4. 7. The bispecific antibody according to any one of paragraphs. 1, 2, where the antibody specific for HER2 is a full-length IgG antibody. 8. The bispecific antibody of claim 7, wherein the full-length IgG antibody is of the human IgG1, IgG2, IgG3, IgG4 isotype. 9. The bispecific antibody according to any one of paragraphs. 1, 2, comprising an antigen binding domain that specifically binds to HER2. 10. The bispecific antibody of claim 8, wherein the full length antibody or antigen binding domain specific for human or cynomolgus HER2 contains heavy and light chains that include amino acid sequences that are at least 80% homologous to SEQ ID NO: 5 and SEQ ID NO: 6, respectively. 11. The bispecific antibody of claim 10, wherein the full length antibody or antigen binding domain specific for human or cynomolgus HER2 contains heavy and light chains that include the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively. 12. The bispecific antibody of claim 2, wherein the peptide linker is more than 5 amino acids long and consists of any set of amino acids selected from G, A, S, P, E, T, D, K. 13. The bispecific antibody according to claims. 2 and 12, where a portion of VHH is attached to the N-terminus or C-terminus of the light or heavy chain of an antibody or antigen binding domain specific for HER2 of a human or cynomolgus monkey. 14. The bispecific antibody according to any one of paragraphs. 1, 2, having a dissociation constant of Kd binding to human HER2 not more than 10 ^-10 M and monkeys not more than 10 ^-7 M, K _D binding to human HER3 not more than 10 ^-9 M and monkeys not more than 10 ^-8 M. 15. A solution for inhibiting the biological activity of human HER2 or cynomolgus monkey and HER3 in a subject, comprising the bispecific antibody according to any one of paragraphs. 1-14 and one or more pharmaceutically acceptable excipients. 16. The solution according to claim 15, which has such aggregation stability that, at concentrations of more than 10 mg / ml and storage at t = 4 ° C for more than 6 months, the content of aggregates does not increase by more than 5% of the initial content in the solution . 17. The solution according to claim 15, which has such aggregation stability that at concentrations of more than 10 mg / ml and when the temperature rises to 37 ° C for more than 2 weeks, the content of aggregates does not increase by more than 5% of the initial content in the solution . 18. The solution according to claim 15, which has such aggregation stability that at concentrations of more than 10 mg / ml and a temperature increase of up to 50 ° C for more than 6 hours, the content of aggregates does not increase by more than 5% of the initial content in the solution. 19. Isolated, a nucleic acid molecule that encodes an antibody according to any one of paragraphs. 1-14. 20. The selected nucleic acid molecule according to claim 19, where the nucleic acid is DNA. 21. The expression vector containing the nucleic acid according to any one of paragraphs. 19, 20. 22. A method of obtaining a host cell to obtain an antibody according to any one of paragraphs. 1-14, including the transformation of the cell by the vector according to p. 21. 23. A host cell for producing an antibody according to any one of claims. 1-14, containing the nucleic acid according to any one of paragraphs. 19, 20. 24. The method of producing antibodies according to any one of paragraphs. 1-14, which consists in cultivating the host cell according to claim 23 in a culture medium under conditions sufficient to obtain the specified antibodies, if necessary, followed by isolation and purification of the obtained antibody. 25. A pharmaceutical composition for the prophylaxis or treatment of a disease or disorder mediated by human HER2 or cynomolgus monkey and HER3, comprising the antibody according to any one of claims. 1-14, in combination with one or more pharmaceutically acceptable excipients. 26. The pharmaceutical composition according to p. 25, intended for the prevention or treatment of a disease or disorder mediated by human HER2 or cynomolgus monkey and HER3 selected from the group: breast cancer (BC), gastric malignancies, non-small cell lung cancer, head malignancies and / or neck. 27. A method of inhibiting the biological activity of human HER2 or cynomolgus monkey and HER3 in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody according to any one of claims. 1-14. 28. A method of treating a disease or disorder mediated by human HER2 or cynomolgus monkey and HER3, comprising administering to the subject an antibody according to any one of claims. 1-14, or a solution according to claim 15, or a pharmaceutical composition according to claims. 25, 26, in need of such treatment, in a therapeutically effective amount. 29. A method of treating a disease or disorder according to claim 28, wherein the disease or disorder is selected from the group: breast cancer (BC), gastric malignancies, non-small cell lung cancer, head and / or neck malignancies. 30. The use of antibodies according to any one of paragraphs. 1-14, a solution according to claim 15 or a pharmaceutical composition according to claims 25, 26 for treating a subject in need of such treatment with a disease or disorder mediated by human HER2 or cynomolgus monkey and HER3. 31. The use of claim 30, wherein the disease or disorder is selected from the group: breast cancer (BC), gastric malignancies, non-small cell lung cancer, head and / or neck malignancies.

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