RU2780629C1 - Method for producing multispecific antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: proposed is a pharmaceutical composition for treating diseases, containing an effective amount of a multispecific antibody from the subclass of human IgG1, and a pharmaceutically acceptable carrier is presented, wherein said antibody includes: a first light chain, a first heavy chain containing a first heteromonomeric Fc-region polypeptide, a second light chain and a second heavy chain containing a second heteromonomeric Fc-region polypeptide; and is produced as a result of cultivation of a mammalian cell in a culture medium.

EFFECT: possibility of producing a pharmaceutical composition with an active product and a low content of impurities intended for treating diseases.

9 cl, 6 dwg, 11 tbl, 7 ex

Description

The field of technology to which the invention belongs

The present invention relates to the production of multispecific antibodies, especially those multispecific antibodies which contain a crossover of domains in one of their chains. In the method described herein, the level of expression of a recombinant mammalian cell secreting a multispecific antibody is increased by introducing an additional expression cassette for the domain-swapped chain into an already transfected or transduced cell.

Prerequisites for the creation of the invention

US No. 5,958,727 describes a method for producing a polypeptide, comprising culturing a mutant cell under conditions leading to the production of a polypeptide, in which the mutant cell is related to a parent cell containing a first DNA sequence that encodes a polypeptide, by introducing a nucleic acid construct into the genome of the parent cell in a locus that is not in the first DNA sequence is not in a second DNA sequence that codes for a protein that is a negative regulator of transcription, translation, or secretion of the polypeptide, and is not in a third DNA sequence that codes for a protease that hydrolyzes the polypeptide in data conditions; and the mutant cell produces more polypeptide than the parent cell when both cells are cultured under the given conditions; and isolation of the polypeptide.

Genzel Y. et al. have found that replacing glutamine with pyruvate reduces ammonia production and inhibits the growth of mammalian cells (Biotechnol. Prog. 21, 2005, pp. 58-69). De la Cruz Edmonds M. C. et al have developed transfection and high throughput screening protocols for the CHOH1 SV cell system (Mol. Biotechnol. 34, 2006, pp. 179-190). WO 2007/036291 describes an improved cell culture medium. EP 1482031 describes a serum-free mammalian cell culture medium and its use. Link T. et al. have developed a biological process for producing a recombinant MUC1 fusion protein expressed by CHO-K1 cells in a protein-free medium (J. Biotechnol. 110, 2004, pp. 51-62). EP 0481791 describes a culture medium for CHO cells and adapted cells. US 2007/161079 describes recombinant cell clones with improved stability and methods for their preparation and use. EP 0659880 describes a method for culturing animal cells or antibody-producing cells. Butler M. et al. describe the adaptation of mammalian cells to non-ammoniogenic media (Cytotechnol. 15, 1994, pp. 87-94). Altamirano C. et al. have described an improved composition of CHO cell culture medium: namely, the simultaneous replacement of glucose and glutamine (Biotechnol. Prog. 16, 2000, pp. 69-75).

EP 0 569 678 describes double transfectants for MHC genes as cell-based vaccines for the immunoprophylaxis of tumor metastases. WO 97/08342 describes an improved method for measuring the activity of a promoter sequence in a mammalian cell using a reporter gene. The use of anti-RhoA and anti-RhoC siRNAs to specifically inhibit RhoA or RhoC synthesis is described in WO 2005/113770. A method for the recombinant production or expression of a eukaryotic alkaline phosphatase mutant in yeast cells is described in US Pat. No. 7,202,072. WO 2001/038557 describes a method for screening multiply transformed cells using bicistronic expression of fluorescent proteins. A method for obtaining recombinant eukaryotic cell lines that express a variety of proteins or RNA of interest is described in WO 1999/47647. Systems, including methods, compositions and kits, for transfecting cells with transfection materials using encoded carriers are described in WO 2003/076588. US No. 5,089,397 describes an expression system for recombinant production of the desired protein, comprising CHO cells transformed with a DNA sequence that has the coding sequence of the desired protein under the control of the human metallothionein-P promoter. A method for obtaining recombinant proteins is described in US 2003/0040047. Lamango et al. (Lamango N.S. et al., Arch. Biochem. Biophys. 330, 1996, pp. 238-250) described the dependence of the production of the prohormone convertase 2 on the presence of the neuroendocrine polypeptide 7 B2. Transfection with a BPV-1 expression vector of cells carrying non-integrated BPV-1 replicating genomes is described in Waldenstroem, M. et al., Gene 120, 1992, pp. 175-181. US Pat. No. 4,912,038 describes methods and vectors for the production of canine and human alveolar surfactant protein 32K.

WO 89/10959 describes recombinant DNA technologies and the expression of mammalian polypeptides in genetically engineered eukaryotic cells. The repeated co-transfer of a human growth hormone expression vector and a selection marker gene expression vector is described in DD 287531. WO 93/01296 describes the production of antibodies in vaccinia infected cells. WO 95/17513 describes the re-transformation of filamentous fungi. WO 89/00999 describes the modular assembly of antibody genes, the antibodies produced therewith, and their use. US 2003/096341 describes the expression of alkaline phosphatase in yeast.

EP 1453966 describes a method for producing a recombinant polypeptide. WO 03/046187 describes a method for producing a recombinant polypeptide. US Pat. No. 5,550,036 describes a method for the co-amplification of human C protein genes in human cells. EP0921194 describes the TNF ligand gene family. EP 0319206 describes gene amplification. Lin F.K. et al. describe the cloning and expression of the human erythropoietin gene (Proc. Natl. Acad. Sci. USA 82, 1985, pp. 7580-7584). WO 00/28066 describes host cells expressing recombinant human erythropoietin. Chen S. et al. have described the production of recombinant proteins in mammalian cells (in: Curr. Prot. Prot. Sci., 1998, 5.10.1-5.10.41).

WO 89/00605 describes transfected cells containing vectors that have genes oriented in opposite directions and methods for their preparation. US Pat. No. 5,420,019 describes stable

bactericidal/penetrating protein products and pharmaceutical compositions containing them. US 5,639,275 describes biocompatible immune-isolated capsules containing genetically modified cells to deliver biologically active molecules.

Kemball-Cook G. et al have described the highly productive production of human blood coagulation factors VII and XI using a novel mammalian cell expression vector (Gene 139, 1994, pp. 275-279). EP 1010758 describes an expression system for the production of recombinant human erythropoietin, a method for purifying secreted human erythropoietin and its use.

Mulligan R.C. and Berg R. described the selection of animal cells that express the Escherichia coli gene encoding xanthine-

guanine phosphoribosyltransferase (Proc. Natl. Acad. Sci. USA 78, 1981, pp. 2072-2076). Colosimo A. et al. have described the transfer and expression of a foreign gene in mammalian cells (BioTechniques 29, 2000, pp. 314-331). Maruyama K. et al. have described the transfection of cultured mammalian cells with mammalian cell expression vectors (Meth. Nucleic Acids Res. 1991, pp. 283-305). Wang D.Z. et al described the treatment of acute stroke patients with intravenous tPA (Stroke 31, 2000, pp. 77-81). Sakamoto T. et al have described the prevention of arterial re-occlusion after thrombolysis using activated protein C (Circulation 90, 1994, pp. 427-432). Lee G.M. et al developed a serum-free medium for the production of erythropoietin using a suspension culture of recombinant Chinese hamster ovary cells using statistical design (J. Biotechnol. 69, 1999, pp. 85-93). Lusky M. and Botchan M.R. describes the characterization of the support sequences of the bovine papillomavirus vector (Cell 36, 1984, pp. 391-401).

US 2014/0242079 describes a vector ratio of 1:2:1:1 for vectors of a single expression cassette intended for transient expression in HEK cells.

WO 2015/052230 describes multispecific domain-swapped antibodies in a common variable light chain.

WO 2012/023053 describes methods for generating multispecific and multivalent antibodies.

WO 2005/072112 describes methods for the production and identification of multispecific antibodies.

WO 02/079255 describes recombinant antibodies co-expressed with GnTIII.

US 2002/06210 describes a method for producing multispecific antibodies having heteromultimeric and common components.

US 2013/045888 describes a multicopy strategy for producing high titers of highly purified multisubunit proteins such as antibodies in transformed microbes such as Pichia pastoris.

Frenzel et al. at Front. Immunol. 4, 2013, article 217 describes the expression of recombinant antibodies.

Wurm et al. have described the production of therapeutic agents as recombinant proteins in cultured mammalian cells (Nat. Biotechnol. 22, 2004, pp. 1393-1398).

Brief summary of the invention

It has been established that in order to create cell lines intended for the production of heterodimeric, i.e. multispecific antibodies, it is expedient to use for transfection an expression vector which contains, as the only expression cassette for the antibody chain, the expression cassette for the light chain. This vector can be used together with other expression vectors for co-transfection or separately in a second subsequent transfection step. Using this manufacturing approach, it is possible to obtain a cell line that produces a heterodimeric antibody with an improved product profile, i.e. with an increased amount of product and a reduced amount of product-related impurities.

One of the objects of the present invention is a method for obtaining a multispecific antibody that contains/consists of/includes at least three different polypeptides, which includes the following steps:

culturing the mammalian cell in a culture medium (under conditions suitable for expression of the multispecific antibody) wherein the mammalian cell is created by

a) transfection of a mammalian cell (not expressing the antibody) with the first expression vector and one, two or three additional expression vectors,

wherein the first expression vector contains exactly one nucleotide sequence that encodes a polypeptide of a multispecific antibody, and one, two or three additional expression vectors, each of which contains at least two nucleotide sequences, each of which encodes different polypeptide chains of a multispecific antibody,

where strictly one nucleotide sequence of the first expression vector is a nucleotide sequence that encodes a multispecific antibody light chain polypeptide, where transfection with the first expression vector is carried out either simultaneously, or before or after transfection with one, two or three additional expression vectors, and

b) selecting a cell (stably) transfected in step a) that grows under selective culture conditions,

isolating the multispecific antibody from the cell or culture medium,

and thereby obtaining a multispecific antibody.

One of the objects of the present invention is a method of creating/producing/obtaining a mammalian cell (having the ability to (stable) expression of a multispecific antibody that contains/consists of at least three different polypeptides, comprising the following step:

a) transfection of a mammalian cell (not expressing the antibody) with the first expression vector and one, two or three additional expression vectors,

wherein the first expression vector contains exactly one nucleotide sequence that encodes a polypeptide of a multispecific antibody, and one, two or three additional expression vectors, each of which contains at least two nucleotide sequences, each of which encodes different polypeptide chains of a multispecific antibody,

where strictly one nucleotide sequence of the first expression vector is a nucleotide sequence that encodes a multispecific antibody light chain polypeptide, where transfection with the first expression vector is carried out either simultaneously, or before or after transfection with one, two or three additional expression vectors, and

b) selecting the cell transfected in step a) which grows under selective culture conditions,

and thereby creating/producing/obtaining a mammalian cell that (stably) expresses the multispecific antibody.

In one embodiment of all aspects of the invention described herein, two of the multispecific antibody polypeptides contain/have an exchange of (cognate) domains.

In one embodiment of all aspects of the invention described herein, exactly one nucleic acid of the first expression vector encodes a multispecific antibody domain-exchange light chain polypeptide.

In one embodiment of all aspects of the invention described herein, step a) comprises: co-transfecting a mammalian cell (not expressing the antibody) with the first expression vector and one, two or three additional expression vectors.

In one of the embodiments of all the objects of the invention specified in the present description, stage a) includes the following stages: I) transfection (simultaneous or sequential) of a mammalian cell with one, two or three additional expression vectors, optionally II) selection of a (stably) transfected cell, III) transfection of the cell specified in subparagraph I) or II) with the first expression vector and optionally IV) selection of the (stably) transfected cell.

In one of the embodiments of all the objects of the invention specified in the present description, the selection is based on the level of expression and/or the amount of related by-products/impurities product.

In one of the embodiments of all the objects of the invention specified in the present description, selected (stably)

the transfected cell(s) that produces(s) the least amount (fraction) of product-related by-products/impurities.

In one of the embodiments of all objects of the invention specified in the present description, select (stably) transfection(s) cell(s), which(s) produces(s) the least amount (fraction) of related by-products/impurities and is characterized by ) with the highest yield.

In one of the embodiments of all the objects of the invention specified in the present description, the mammalian cell stably expresses a multispecific antibody.

In one of the embodiments of all the objects of the invention specified in the present description, the mammalian cell is a CHO cell.

In one embodiment of all aspects of the invention described herein, the domain exchange is a CH1-CL crossover or a VH-VL crossover.

In one of the embodiments of all the objects of the invention specified in the present description, the multispecific antibody is a bivalent bispecific antibody that contains

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the VL and VH variable domains of the second light chain and the second heavy chain are interchanged.

In one embodiment of all aspects of the invention described herein, the multispecific antibody is a bivalent bispecific antibody that comprises a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, in which the CL and CH1 constant domains of the second light chain and the second heavy chain are exchanged for each other.

In one of the embodiments of all the objects of the invention specified in the present description, the multispecific antibody is a trivalent bispecific antibody that contains

a) the first light chain and the first heavy chain of a full-length antibody that specifically binds to the first antigen,

b) a second full-length antibody heavy chain that, when paired with the first light chain, specifically binds to the first antigen, and

c) A Fab fragment that specifically binds to a second antigen, fused via a peptide linker to the C-terminus of one of the heavy chains specified in a) or b), in which the CL and CH1 constant domains of the second light chain and the second heavy chain are replaced Each other.

One object of the present invention is a (stably transfected) mammalian cell obtained using the method presented in the present description.

One of the objects of the present invention is a method for obtaining a multispecific antibody, which includes the following steps:

culturing a (stably transfected) cell as defined herein in a culture medium (under conditions suitable for expression of a multispecific antibody),

isolating the multispecific antibody from the cell or culture medium,

optional purification of the isolated antibody using one or more chromatography step(s),

and thereby obtaining a multispecific antibody.

One of the objects of the present invention is a method for obtaining a preparation of a multispecific antibody with a low/reduced amount of product-related impurities, which includes the following steps:

obtaining / producing / creating a (stably transfected) mammalian cell that (stably) expresses a multispecific antibody using the method presented in the present description,

culturing the resulting/produced/generated mammalian cell in a culture medium,

isolating the antibody preparation from the cell or culture medium,

optional purification of the isolated antibody using one or more chromatography step(s),

and thereby obtaining a multispecific antibody preparation with a low/reduced amount of product-related impurities.

One aspect of the present invention is the use of the method described herein to reduce the amount of product-related impurities in a multispecific antibody preparation.

The present description provides a method for producing a multispecific antibody that contains at least one domain crossover chain in a recombinant mammalian cell. The method provides an improved process, where the improvement is, among other things, a reduction in the amount of product-related by-products and an increase in the amount of correctly folded/correctly assembled multispecific antibody.

One object of the present invention is a method for producing a multispecific antibody (comprising at least one domain crossover polypeptide chain), which includes the following steps:

a) obtaining a (stably transfected) mammalian cell that (stably) expresses a multispecific antibody,

b) transfection of the mammalian cell indicated in step a) with an expression cassette encoding a polypeptide chain of a multispecific antibody that has a domain crossover,

c) culturing the cell indicated in step b) and isolating the antibody from the cell or culture medium and thereby obtaining a multispecific antibody,

d) optionally purifying the recovered antibody by one or more chromatography step(s).

In one embodiment of all aspects of the invention, a mammalian cell expressing the multispecific antibody stably expresses the multispecific antibody.

In one embodiment, the expression cassette indicated in step b) is an expression vector.

In one embodiment of all aspects of the invention, the polypeptide chain of the multispecific antibody that has the domain crossover is an antibody light chain.

In one embodiment of all aspects of the invention, the domain crossover is a CH1-CL crossover or a VH-VL crossover.

In one embodiment of all aspects of the invention, the multispecific antibody is a bivalent bispecific antibody, or a trivalent bispecific antibody, or a tetravalent bispecific antibody.

In one embodiment of all aspects of the invention, a mammalian cell expressing a multispecific antibody is obtained by transfecting a mammalian cell with one or more nucleic acid(s) molecule(s) that encode(s) a multispecific antibody, and stably selected transfected cell.

In one embodiment of all aspects of the invention, the multispecific antibody is a bivalent bispecific antibody that contains

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the VL and VH variable domains of the second light chain and the second heavy chain are interchanged.

In one embodiment of all aspects of the invention, the multispecific antibody is a bivalent bispecific antibody that contains

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, in which the CL and CH1 constant domains of the second light chain and the second heavy chain are exchanged for each other.

In one embodiment of all aspects of the invention, the multispecific antibody is a trispecific or tetraspecific antibody that contains

a) the first light chain and the first heavy chain of a full-length antibody that specifically binds to the first antigen, and

b) a second (modified) light chain and a second (modified) heavy chain of a full length antibody that specifically binds to a second antigen in which the VL and VH variable domains are swapped and/or the CL and CH1 constant domains are swapped, and

c) wherein one or two antigen-binding peptides that specifically bind to one or two additional antigens (i.e. a third and/or fourth antigen) are fused via a peptide linker to the C- or N-terminus of the light chains or heavy chains, referred to in subparagraphs a) and / or b).

In one embodiment of all aspects of the invention, the multispecific antibody is a bispecific tetravalent antibody that contains

a) two light chains and two heavy chains of an antibody that specifically binds to the first antigen (and contains two Fab fragments),

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, in which said additional Fab fragments are both fused via a peptide linker to either the C- or N-terminus of the heavy chains specified in subparagraph a),

and

where the following modifications have been made in the Fab fragments: I) in both Fab fragments referred to in subparagraph a), or both Fab fragments referred to in subparagraph b), the variable domains VL and VH are exchanged for each other and/or the constant domains CL and CH1 are replaced by each other,

or

ii) in both Fab fragments referred to in subparagraph a), the variable domains VL and VH are exchanged for each other and the constant domains CL and CH1 are exchanged for each other, and

in both Fab fragments referred to in b), the VL and VH variable domains are exchanged, or the CL and CH1 constant domains are exchanged, or

iii) in both Fab fragments referred to in subparagraph a), the variable domains VL and VH are exchanged for each other or the constant domains CL and CH1 are exchanged for each other, and

in both Fab fragments referred to in b), the variable domains VL and VH are exchanged for each other and the constant domains CL and CH1 are exchanged for each other, or

iv) in both Fab fragments referred to in b), the VL and VH variable domains are exchanged for each other and in both Fab fragments referred to in b), the CL and CH1 constant domains are exchanged for each other, or

V) in both Fab fragments referred to in subparagraph a), the constant domains CL and CH1 are exchanged for each other and in both Fab fragments indicated in subparagraph b), the variable domains VL and VH are exchanged for each other.

In one of the embodiments of the invention, the following modifications are made to the Fab fragments

I) in both Fab fragments referred to in subparagraph a), or in both Fab fragments referred to in subparagraph b), the variable domains VL and VH are exchanged for each other and/or the constant domains CL and CH1 are exchanged for each other.

In one embodiment of all aspects of the invention, the multispecific antibody is a bispecific tetravalent antibody that contains:

a) a (modified) heavy chain of a first antibody that specifically binds to a first antigen and contains a first VH-CH1 domain pair, wherein the C-terminus of said heavy chain is fused to the N-terminus of a second VH-CH1 domain pair of said first antibody via a peptide linker,

b) two light chains of the specified first antibody specified in subparagraph a),

c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and contains a first VH-CL domain pair, where the C-terminus of said heavy chain is fused to the N-terminus of the second VH-CL domain pair of said second antibody via a peptide linker, and

d) two (modified) light chains of said second antibody of c), each containing a CL-CH1 domain pair.

In all aspects of the invention presented herein, the first light chain contains a VL domain and a CL domain, and the first heavy chain contains a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

In one of the embodiments of all aspects of the invention, the antibody obtained by the method described in the present description is a multispecific antibody that requires heterodimerization of at least two heavy chain polypeptides.

In one embodiment, the full length antibody is

a) a full-length antibody of the human IgG1 subclass,

b) a full-length antibody of the human IgG4 subclass,

c) a full-length antibody of the human IgG1 subclass with mutations L234A, L235A and P329G,

d) a full-length antibody of the human IgG4 subclass with mutations S228P, L235E and P329G,

e) a full length antibody of the human IgG1 subclass with L234A, L235A and P329G mutations in both heavy chains and T366W and S354C or Y349C mutations in one heavy chain and T366S, L368A, Y407V and Y349C or S354C mutations in the corresponding other heavy chain,

f) a full-length human IgG4 subclass antibody with S228P and P329G mutations in both heavy chains and T366W and S354C mutations in one heavy chain and T366S, L368A, Y407V and Y349C mutations in the corresponding other heavy chain,

g) a full-length human IgG1 subclass antibody with mutations L234A, L235A, P329G, 1253A, H310A and H435A in both heavy chains and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the corresponding other heavy chain, or

h) a full-length antibody of the human IgG1 subclass with mutations L234A, L235A, P329G, M252Y, S254T and T256E in both heavy chains and mutations T366W and S354C in one heavy chain and mutations T366S, L368A, Y407V and Y349C in the corresponding other heavy chain.

One of the objects of the invention referred to in the present description is a cell that contains a nucleic acid encoding a bispecific antibody, which is obtained using the method presented in the present description.

One of the objects of the invention referred to in the present description is a method for obtaining a multispecific antibody presented in the present description, which includes the following steps:

a) culturing a cell as defined herein that produces/expresses a multispecific antibody, and

b) isolating the multispecific antibody from the cell or culture medium,

and thereby obtaining a multispecific antibody presented in the present description.

One of the objects of the invention specified in the present description is an antibody, which is obtained by the method presented in the present description.

One of the objects of the invention referred to in the present description is a pharmaceutical composition that contains an antibody obtained by the method presented in the present description, and a pharmaceutically acceptable carrier.

One of the objects of the invention referred to in the present description is an antibody obtained by the method, which is presented in the present description, for use as a drug.

One of the objects of the invention referred to in the present description is the use of a bispecific antibody obtained by the method presented in the present description, for the preparation of a medicinal product.

In one embodiment of all aspects of the invention, the bispecific antibody is selected from the group of bispecific antibodies that consists of an anti-Abeta protein/transferrin receptor antibody, an anti-CO20/transferrin receptor antibody, an anti-PD1/Tim3 antibody, and an anti-FAP/DR5 antibody.

In one embodiment of all aspects of the invention, the multispecific antibody is a bispecific tetravalent antibody that contains

a) two light chains and two heavy chains of an antibody that specifically binds to the first antigen (and contains two Fab fragments),

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, in which each of said additional Fab fragments is fused via a peptide linker to an individual C-terminus of one of the heavy chains specified in subparagraph a), and

in which the following modifications are made in the additional Fab fragments: in both additional Fab fragments referred to in subparagraph b), the variable domains VL and VH are replaced with each other and/or the constant domains CL and CH1 are replaced with each other,

wherein I) the first antigen is DR5 and the second antigen is FAP, or ii) the first antigen is FAP and the second antigen is DR5,

where the two heavy chains of the antibody that specifically binds to the first antigen are of the human IgG1 subclass with mutations L234A, L235A and P329G.

In one embodiment of all aspects of the invention, the multispecific antibody is a bivalent bispecific antibody that contains

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and

b) the second light chain and the second heavy chain of an antibody that specifically binds to the second antigen, where the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other,

wherein I) the first antigen is PD1 and the second antigen is Tim3 or ii) the first antigen is Tim3 and the second antigen is PD1,

where the first heavy chain and the second heavy chain are both of the human IgG1 subclass with mutations L234A, L235A and P329G and with mutation T366W and optionally S354C or Y349C in one heavy chain, and with mutations T366S, L368A, Y407V and optionally Y349C or S354C in the corresponding other heavy chain, while the terminal glycine or glycine-lysine dipeptide may be absent,

wherein the first light chain contains in the light chain constant domain (CL) at position 123 an arginine amino acid residue (instead of the wild-type glutamic acid residue present in the molecule; E123R mutation) and at position 124 a lysine amino acid residue (instead of the wild-type glutamine residue present in the molecule ; mutation Q124K) (numbering according to Cabot),

in which the first heavy chain contains in the first heavy chain constant domain (CH1) at position 147 a glutamic acid residue (instead of the wild-type lysine residue present in the molecule; mutation K147E) and at position 213 a glutamic acid residue (instead of the amino acid residue present in the wild-type molecule lysine; mutation K213E) (numbering according to Cabot's EU index).

In one embodiment of all aspects of the invention, the multispecific antibody is a trivalent bispecific antibody that contains

a) two light chains and two heavy chains of an antibody that specifically binds to the first antigen (and contains two Fab fragments),

b) one additional Fab fragment of an antibody that specifically binds to a second antigen, in which said additional Fab fragment is fused via a peptide linker to the C-terminus of one of the heavy chains specified in subparagraph a),

and

in which the following modifications are made in the additional Fab fragment: the variable domains VL and VH are replaced with each other and / or the constant domains CL and CH1 are replaced with each other, while I) the first antigen is A-beta, and the second antigen is a transferrin receptor; or II) the first antigen is CD20 and the second antigen is a transferrin receptor.

In one embodiment of all aspects of the invention, the multispecific antibody is a bispecific antibody that contains

a) one full-length antibody containing two pairs of chains, each of which consists of a light chain of a full-length antibody and a heavy chain of a full-length antibody, where the binding sites formed by each of the pairs, consisting of a full-length heavy chain and a full-length light chain, specifically bind to the first antigen , and

b) one additional Fab fragment, where the additional Fab fragment is fused to the C-terminus of one of the heavy chains of the full-length antibody, while the binding site of the additional Fab fragment specifically binds to the second antigen,

in which each of the light chains of the full-length antibody contains in the light chain constant domain (CL) at position 123 the amino acid residue of arginine (instead of the glutamic acid residue present in the wild-type molecule; mutation E123R) and at position 124 the amino acid residue of lysine (instead of type of glutamine residue; mutation Q124K) (numbering according to Cabot),

in which each of the heavy chains of the full-length antibody contains in the first constant domain of the heavy chain (CH1) at position 147 a glutamic acid residue (instead of a lysine residue present in the wild-type molecule; mutation K147E) and at position 213 a glutamic acid residue (instead of present in the wild-type molecule type of lysine amino acid residue; mutation K213E) (numbering according to Cabot's EU index),

wherein the additional Fab fragment specifically binding to the second antigen comprises a crossover of domains, such as an exchange of a light chain constant domain (CL) and a heavy chain constant domain 1 (CH1), and

where the first antigen is the human A-beta protein and the second antigen is the human transferrin receptor.

In one embodiment of all aspects of the invention, the multispecific antibody is a bispecific antibody that contains

a) one full-length antibody containing two pairs of chains, each of which consists of a light chain of a full-length antibody and a heavy chain of a full-length antibody, where the bindings formed by each of the pairs, consisting of a full-length heavy chain and a full-length light chain, specifically bind to the first antigen, and

b) one additional Fab fragment, where the additional Fab fragment is fused to the C-terminus of one of the heavy chains of the full-length antibody, while the binding site of the additional Fab fragment specifically binds to the second antigen,

in which each of the light chains of the full-length antibody contains in the light chain constant domain (CL) at position 123 the amino acid residue of arginine (instead of the glutamic acid residue present in the wild-type molecule; mutation E123R) and at position 124 the amino acid residue of lysine (instead of type of glutamine residue; mutation Q124K) (numbering according to Cabot),

in which each of the heavy chains of the full-length antibody contains in the first constant domain of the heavy chain (CH1) at position 147 a glutamic acid residue (instead of a lysine residue present in the wild-type molecule; mutation K147E) and at position 213 a glutamic acid residue (instead of present in the wild-type molecule type of lysine amino acid residue; mutation K213E) (numbering according to Cabot's EU index),

wherein the additional Fab fragment specifically binding to the second antigen comprises a crossover of domains, such as an exchange of a light chain constant domain (CL) and a heavy chain constant domain 1 (CH1), and

where the first antigen is human CD20 and the second antigen is the human transferrin receptor.

In one embodiment of all aspects of the invention described herein, each polypeptide is in an expression cassette, each of which contains in the 5'->3' direction a promoter, a structural gene encoding the polypeptide, a polyadenylation sequence, and optionally a termination sequence. In one embodiment, all expression cassettes have the same promoter, the same polyadenylation site, and optionally the same termination sequence. In one embodiment, the promoter is a human CMV (cytomegalovirus) promoter. In one embodiment, the CMV promoter contains intron A. In one embodiment, the polyadenylation site is a BGH (bovine growth hormone) polyadenylation site. In one embodiment, the terminator is present and is HGT (human growth hormone terminator). In one embodiment, the promoter is a CMV promoter, optionally containing intron A, and the polyadenylation site is a BGH polyadenylation site. In one embodiment, the promoter is a CMV promoter optionally containing intron A, the polyadenylation site is a BGH polyadenylation site, and the terminator is HGT.

In one of the embodiments of the invention, the additional expression vector contains or each of the additional expression vectors contains at least two nucleotide sequences, each of which encodes different polypeptide chains of the multispecific antibody, with each nucleic acid present/contained exactly once in the corresponding vector.

Detailed description of the invention

Modules for dimerization type "knobs-into-holes" ("protrusions in the "hollows"") and their use for the creation of antibodies are described in Carter R.; Ridgway J.W.B.; Presta L.G.: Immunotechnology, Vol. 2, No. 1, February 1996, pp. 73-73(1).

General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulins is provided by: Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

For the purposes of this specification, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, and is referred to herein as "numbering according to Cabot". In particular, the Cabot numbering system (see pp. 647-660) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, is used for the kappa and lambda isotype CL light chain constant domain, and the Cabot EU numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3 , which in this case is additionally specified using the designation "numbering according to the Cabot EU-index).

Methods and technologies suitable for the implementation of the present invention are described, for example, in: Current Protocols in Molecular Biology, vol. I-III, ed. Ausubel F.M., 1997; DNA Cloning: A Practical Approach, vol. I and II, ed. Glover N.D. and Hames B.D., Oxford University Press, 1985; Animal Cell Culture - a practical approach, ed. Freshney R.I., IRL Press Limited, 1986; Watson J.D. et al., Recombinant DNA, 2nd ed., CHSL Press, 1992; Winnacker E.L., From Genes to Clones; N.Y., VCH Publishers, 1987; Cell Biology, ed. Celis J., 20th ed., Academic Press, 1998; Freshney R.I., Culture of Animal Cells: A Manual of Basic Technique, 20th ed., Alan R. Liss, Inc., N.Y., 1987.

The use of recombinant DNA technology makes it possible to create nucleic acid derivatives. These derivatives, for example, may be modified at one or more nucleotide positions by substitution, alteration, exchange, deletion or insertion. Modification or derivatization can, for example, be carried out using site-directed mutagenesis techniques. These modifications can easily be made by one skilled in the art (see, for example, Sambrook J. et al., Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, New York, USA, 1999; Hames B.D. and Higgins S.G. , Nucleic acid hybridization a practical approach, IRL Press, Oxford, England, 1985).

Definitions

"Multispecific antibody" means an antibody having binding specificities for at least two different epitopes on the same antigen or on two different antigens. Multispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F(ab') ₂ type bispecific antibodies) or combinations thereof (eg full length antibody plus additional scFv or Fab fragments). Engineered antibodies with two, three, or more (eg, four) functional antigen-binding sites have been described (see, for example, US 2002/0004587 A1).

The concept of "rule laid / correctly assembled" in the context of the present description means that the antibody has the correct stoichiometry, i.e. contains compatible amounts and copies of individual/relevant light and heavy chains. For example, a "native human IgG-type antibody" is "rule folded/correctly assembled" when the isolated molecule contains two light chain polypeptides and two heavy chain polypeptides. For example, if a multispecific antibody is a bivalent bispecific native human IgG-type antibody, then it is rule folded/correctly assembled when the isolated molecule consists of a first pair of a cognate first light chain and a cognate first heavy chain binding to the first antigen, and a second pair a cognate second light chain; and a cognate second heavy chain that binds to a second antigen, i. e. consists of four different polypeptides. All antibodies that are not normally folded/correctly assembled, ie. which contain fewer or more than the required number of strands and/or contain incorrectly associated strands, ie. those that do not form a heavy and light chain cognate pair are referred to as "product-related by-products".

The concept of "domain crossover" in the context of the present description means that in a pair consisting of a VH-CHl fragment of the heavy chain of the antibody and the corresponding cognate light chain of the antibody, i. in the binding arm of an antibody (i.e., in a Fab fragment), the sequence of domains differs from the naturally occurring sequence in that at least one heavy chain domain is replaced with a corresponding light chain domain and vice versa. There are three general types of domain crossover (I ) CH1 and CL domain crossover, which results in a light chain domain crossover that contains the VL-CH domain sequence and a heavy chain fragment domain crossover that contains the VH-CL domain sequence (or full-length antibody heavy chain that contains the sequence of the VH-CH-Hinge-CH2-CH3 domains), (II) a crossover of the VH and VL domains, which results in a crossover of the light chain domains, which contains the sequence of VH-CL domains, and a domain crossover of the heavy chain fragment, which contains a sequence of VL-CH1 domains, and (III) a crossover of the full light chain domains (VL-CL) and a full heavy chain fragment of VH-CH1 (“crossover, Cross-Fab” ), which results in a light chain domain crossover that contains a VH-CH1 domain sequence and a heavy chain fragment domain crossover that contains a VL-CL domain sequence (all of the above domain sequences are in N-terminal to C-terminal direction) .

In the context of the present description, the term "replaced for each other" in relation to the respective domains of heavy and light chains refers to the above-described domain crossovers. Thus, when the CH1 and CL domains are "replaced with each other", this refers to the domain crossover referred to in subparagraph (I) and the heavy and light chain domain sequences thus formed. Accordingly, when VH and VL are "replaced with each other", then this refers to the domain crossover referred to in subparagraph (II); and when CH1 and CL domains are "replaced with each other" and VH1 and VL domains are "replaced with each other", then this refers to the domain crossover referred to in subparagraph (III). Bispecific antibodies comprising domain crossovers are described, for example, in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254 and Schaefer W. et al., Proc. Natl. Acad. Sci USA 108, 2011, p. 11187-11192.

The multispecific antibody produced by the method described herein mainly contains Fab fragments comprising the CH1 and CL domain crossover as described in subparagraph (I) above or the VH and VL domain crossover as described in subparagraph above. (II). Fab fragments that specifically bind to the same(them) and the same(and) antigen(s) are designed so that they have the same sequence of domains. Thus, when more than one Fab fragment with a domain crossover is contained in a multispecific antibody, said Pab fragment(s) specifically bind(s) to the same antigen.

In the context of the present description, the term "antibody" is used in its broadest sense, and it refers to various structures of antibodies, including (but not limited to) monoclonal antibodies, polyclonal antibodies and multispecific antibodies (for example, bispecific antibodies), provided, that they have the desired antigen-binding activity.

The term "antibody fragment" refers to a molecule, other than an intact antibody, which contains a portion of the intact antibody that has the ability to bind to the antigen to which the intact antibody binds. Examples of antibody fragments are (but not limited to) Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , dimeric antibodies (diabodies); linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments.

The term "chimeric" antibody refers to an antibody in which part of the heavy and/or light chain is from a particular source or species, and the remainder of the heavy and/or light chain is from a different source or species.

The term "class" of an antibody refers to the type of constant domain or constant region included/included in the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

The term "Fc receptor" in the context of the present description refers to activating receptors, characterized by the presence of a cytoplasmic ITAM sequence associated with the receptor (see, for example, Ravetch J.V. and Bolland S., Annu. Rev. Immunol. 19, 2001, ss 275-290). These receptors are FcγRI, FcγRIIA and FcγRIIIA. The term "not binding to FcγR" means that the antibody obtained by the method presented in the present description, at a concentration of 10 μg/ml, binding to NK cells is 10% or less of the binding of the antibody to OX40L LC.001, which is described in WO 2006/029879.

While IgG4 has reduced FcR binding, antibodies from other IgG subclasses have strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc-fused carbohydrate), Pro329 and 234, 235, 236 and 237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are residues that, when altered, also reduce the ability bind to FcR (Shields R. L. et al., J. Biol. Chem. 276, 2001, pp. 6591-6604; Lund J. et al., FASEB J. 9, 1995, pp. 115-119; Morgan A. and et al., Immunology 86, 1995, pp. 319-324 and EP 0307434).

In one of the embodiments of the invention, the antibody obtained by the method presented in the present description belongs to the IgG1 or IgG2 subclass and contains a PVA236, GLPSS331 and/or L234A/L235A mutation. In one of the embodiments of the invention, the antibody obtained by the method presented in the present description belongs to the IgG4 subclass and contains the L235E mutation. In one embodiment, the antibody further comprises the S228P mutation.

The term "Fc region" in the context of the present description refers to the C-terminal region of the heavy chain of an immunoglobulin, which contains at least part of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may either be present or absent. Unless otherwise noted, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. NIH Publication 91-3242.

Antibodies obtained by the method presented in the present description, contain as the Fc region in one of the embodiments of the invention the Fc region of human origin. In one embodiment, the Fc region contains all parts of the human constant region. The Fc region of an antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. While the effect of the antibody on the complement system depends on certain conditions, binding to C1q depends on certain binding sites in the Fc region. These binding sites are known in the art and are described by Lukas T.J. et al., J. Immunol. 127, 1981, pp. 2555-2560; Brunhouse R. and Cebra J.J., Mol. Immunol. 16, 1979, pp. 907-917; Burton D.R. et al., Nature 288, 1980, pp. 338-344; Thommesen J.E. and others, Mol. Immunol. 37, 2000, pp. 995-1004; Idusogie E.E. et al., J. Immunol. 164, 2000, pp. 4178-4184; Hezareh M. et al., J. Virol. 75, 2001, pp. 12161-12168; Morgan A. et al., Immunology 86, 1995, pp. 319-324; and EP 0307434. These binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to Cabot's EU index; unless specifically indicated otherwise, the numbering of amino acid residues in Fc- region or constant region is carried out according to the EU numbering system, which is also referred to as the EU index, described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. NIH Publication 91-3242). Antibodies of the IgG1, IgG2, and IgG3 subclasses generally have the ability to activate complement, bind to C1q, and activate C3, while IgG4 does not activate the complement system, does not bind to C1q, and does not activate C3. "Antibody Fc region" is a concept well known to one skilled in the art and is defined based on papain digestion of antibodies. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is a human IgG4 subclass Fc region containing S228P and/or L235E mutations (numbered according to Cabot's EU index). In one embodiment, the Fc region is a human IgG1 subclass Fc region containing mutations L234A and L235A (numbered according to Cabot's EU index).

The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody, or having heavy chains that contain the Fc region specified herein. A "full-length antibody" is an antibody that contains an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domains may have a native sequence of constant domains (eg, constant domains having a human native sequence) or amino acid sequence variants thereof. More specifically, a full-length antibody contains two antibody light chains (each containing a light chain variable domain and a light chain constant domain) and two antibody heavy chains (each containing a heavy chain variable domain, a hinge region, and heavy chain CH1, CH2, and CH3 constant domains). chains). C-terminal amino acid residues K or GK may or may not be present independently of each other in the two heavy chains of a full-length antibody.

As used herein, "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including progeny of said cells. Cells include "transformants" and "transformed cells", which include primary transformed cells, as well as progeny derived from them, regardless of the number of passages. The offspring may not be strictly identical to the parent cell in terms of nucleic acid composition, but may contain mutations. This concept includes mutant progeny that have the same function or biological activity as the original transformed cell selected by screening or selection.

The term "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to regions of the non-human antibody and all or substantially all of the FRs correspond to regions human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The term "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic assays (such as, for example, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic assays (such as, for example, as ion exchange chromatography or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman S. et al., J. Chrom. In 848, 2007, pp. 79-87.

The term "monoclonal antibody" in the context of the present description refers to an antibody obtained from a population of substantially homogeneous antibodies, i. individual antibodies in a population are identical and/or bind to the same epitope, with the exception of possible variants of antibodies, for example, containing naturally occurring mutations or arising during the production of a preparation of a monoclonal antibody, these variants are usually present in minor quantities. Unlike polyclonal antibody preparations, which typically include different antibodies to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single antigen determinant. Thus, the adjective "monoclonal" defines the characteristics of an antibody, characterizing it as derived from a substantially homogeneous population of antibodies, rather than being engineered to meet the requirements for producing an antibody by any particular method. For example, monoclonal antibodies that can be used according to the present invention can be created using various technologies, including (but not limited to) the hybridoma method, recombinant DNA methods, phage display methods, and transgenic animal methods that contain all or part immunoglobulin loci, these methods and other exemplary methods for obtaining monoclonal antibodies are presented in the present description.

The term "native antibodies" refers to naturally occurring immunoglobulin molecules with different structures. For example, native IgG-type antibodies are heterotetrameric glycoproteins of approximately 150,000 Da, consisting of two identical light chains and two identical heavy chains linked by disulfide bridges. Each heavy chain in the direction from the N- to the C-terminus contains a variable region (VH), which is also called the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also called the constant region heavy chain. Similarly, each light chain in the direction from the N - to the C-terminus contains a variable region (VL), which is also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. A light chain can be one of two types, designated kappa (k) or lambda (X), based on the amino acid sequence of its constant domain. A "native-like" antibody has the same structure as a "native antibody" but differs in binding specificity.

In the context of the present description, the term "vector" refers to a nucleic acid molecule that has the ability to amplify another nucleic acid with which it is associated. The term refers to a vector as a self-replicating structure containing a nucleic acid, as well as to a vector inserted into the genome of the host cell into which the vector is introduced. Some vectors have the ability to control the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "expression cassette" refers to a construct that contains the necessary regulatory elements, such as a promoter and a polyadenylation site, for the expression of at least the nucleic acid contained therein in a cell.

The term "expression vector" means a nucleic acid containing all the necessary elements for the expression of the structural gene(s) it contains(s) in a cell. Typically, the expression vector contains a plasmid unit for propagating a prokaryotic organism, for example in E. coli, containing an origin of replication, a selection marker, a eukaryotic selection marker, and one or more expression cassettes for expression of the structural gene(s) of interest. (s), each of which contains a promoter nucleic acid, a structural gene and a transcription terminator, including a polyadenylation signal. Gene expression is typically under the control of a promoter nucleic acid, and such a structural gene is referred to as "operably linked" to the promoter nucleic acid. Similarly, a regulatory element and a core promoter nucleic acid are operably linked if the regulatory element modulates the activity of the core promoter nucleic acid.

The concept of "functionally related" means the mutual arrangement of two or more components, in which these components are in a relationship that provides them with the desired function. For example, a promoter and/or enhancer is operably linked to a coding sequence if it acts(s) in the cis direction to control or modulate the transcription of the linked sequence. Typically, but not necessarily, DNA sequences that are "operably linked" are contiguous and, if two protein-coding regions such as a secretory leader and a polypeptide are to be joined, contiguous and in-frame (readouts). However, although an operably linked promoter is typically located upstream of a coding sequence, it is not necessary that it be adjacent to it. An enhancer is operably linked to a coding sequence if the enhancer enhances transcription of the coding sequence. Functionally linked enhancers can be located upstream, within coding sequences, or upstream of coding sequences and at a significant distance from the promoter. A polyadenylation site is operably linked to a coding sequence if it is located at the forward end of the coding sequence such that transcription occurs through the coding sequence in the polyadenylation sequence. A translation stop codon is operably linked to an exonic nucleic acid sequence if it is located at the forward end (3' end) of the coding sequence such that translation proceeds through the coding sequence to and terminates at the stop codon. Linking is carried out using recombination methods known in the art, for example using PCR methodology and/or by ligation at suitable restriction sites. If suitable restriction sites do not exist, then synthetic adapters or linkers are used in accordance with accepted practice.

The term "polypeptide" means a polymer consisting of amino acids linked by peptide bonds, obtained either naturally or by synthesis. Polypeptides of less than about 20 amino acid residues may be referred to as "peptides", while molecules consisting of two or more polypeptides or containing a single polypeptide of more than 100 amino acid residues may be referred to as "proteins" . The polypeptide may also contain non-amino acid components such as carbohydrate groups, metal ions, or carboxylic acid esters. Non-amino acid components may be supplied by the cell in which the polypeptide is expressed and may vary depending on the cell type. In the context of the present description, polypeptides are defined depending on the structure of their amino acid backbone or nucleic acid encoding them. Additives such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term "obtaining" means the expression of a structural gene of interest inserted into an expression cassette in a cell. The concept includes the processes of transcription and translation of a nucleic acid. The production is carried out in the appropriate prokaryotic or eukaryotic cells, and expressed, i. the resulting polypeptide can be isolated from the cells after lysis or from the culture supernatant.

The term "nucleic acid promoter, nucleotide promoter" means a polynucleotide sequence that controls the transcription of a gene/structural gene or nucleotide sequence to which it is operably linked. The nucleotide promoter includes signals for binding RNA polymerase and initiating transcription. The nucleotide promoter used must be functional in the cell in which the chosen structural gene is to be expressed. A large number of nucleotide promoters are well known in the art, including constitutive, inducible, and repressible promoters from a wide variety of different sources (and can be identified in databases such as GenBank), and are available as or as part of cloned polynucleotides (e.g., found depositories such as ATCC, as well as other commercial or individual sources).

As a rule, the nucleotide promoter is located in the 5' non-coding or non-translated region of the gene, proximal to the transcription start site of the structural gene. Sequence elements in nucleotide promoters whose function is to initiate transcription are often characterized by consensus nucleotide sequences. These elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs), cyclic AMP response elements (CREs), serum response elements (SREs), glucocorticosteroid response elements (GREs), and binding sites for other transcription factors. such as CRE/ATF, AP2, SP1, cAMP responder element binding protein (CREB), and octamer factors. If the nucleotide promoter is an inducible nucleotide promoter, then the rate of transcription increases in response to the inducing agent, as in the case of the CMV nucleotide promoter followed by two tet operator sites, metallothionein nucleotide promoters and heat shock proteins. The rate of transcription is not regulated by the inducing agent if the nucleotide promoter is a constitutively active nucleotide promoter. Eukaryotic nucleotide promoters that have been identified as strong nucleotide promoters for expression are the SV40 nucleotide early promoter, adenovirus nucleotide major late promoter, mouse metallothionein-I nucleotide promoter, Rousa sarcoma virus long terminal repeat, Chinese hamster elongation factor 1 alpha (CHEF-1 ), human EF-1 alpha, ubiquitin, and the human cytomegalovirus major immediate-early nucleotide promoter (hCMV MIE).

The term "marker for selection (selection marker)" means a nucleic acid that provides a specific selection of cells that carry it, in relation to or against the presence of the corresponding selection agent (culturing under selective culturing conditions). Typically, a selectable marker can confer drug resistance or compensate for a metabolic or catabolic defect in the cell into which it is introduced. The selection marker may be positive, negative, or bifunctional. A valuable positive selection marker is an antibiotic resistance gene that selects cells transformed by it in the presence of an appropriate selection agent, such as an antibiotic. An untransformed cell cannot grow or survive under selective conditions, ie. in the presence of a selective agent. Negative selection markers provide selective elimination of cells carrying the marker. Selection markers useful for eukaryotic cells include, for example, structural genes encoding aminoglycoside phosphotransferase (APH), such as markers for selection for resistance to hygromycin (hyg), neomycin (neo) and G418, encoding dihydrofolate reductase (DHFR), thymidine kinase (tk), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (selection agent indole), histidinol dehydrogenase (selection agent histidinol D), and nucleic acids conferring resistance to puromycin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid.

Ways

The present invention is based at least in part on the discovery that, in order to establish cell lines for the production of heterodimeric antibodies, it is expedient to use for transfection an expression vector that contains, as the only nucleic acid encoding (antibody) polypeptide, a nucleic acid that encodes a light chain polypeptide, i.e. e. a vector containing, as a single expression cassette of the antibody polypeptide, a light chain expression cassette. The specified vector is used in combination with additional expression vectors for co-transfection or transfection separately in the second subsequent stage. Using this approach, it is possible to obtain a producer cell line that produces a heterodimeric antibody with an improved product profile, i.e. with an increased amount of product and a reduced amount of product-related impurities.

One approach to the creation of multispecific antibodies is known as "CrossMab technology". This approach is based on crossover of domains between heavy and light chains, thereby leading to the creation of different arrangements of domains in heavy chains and light chains of different specificity (see, for example, WO 2009/080251, WO 2009/080252, WO 2009/080253 , WO 2009/080254, in Schaefer, W. et al., Proc. Natl, Acad. Sci. USA 108, 2011, pp. 11187-11192, for data concerning bivalent IgG-type bispecific antibodies with domain crossover, in WO 2010/145792 and WO 2010/145792 data concerning quadrivalent antigen-binding proteins with domain crossover).

Multispecific antibodies with VH/VL substitution/exchange at one binding site to prevent light chain mismatch (CrossMabVH-VL), which are described in WO 2009/080252 (see also Schaefer W. et al., Proc. Natl. Acad. Sci USA, 108, 2011, pp. 11187-11191, significantly reduced the by-product formation due to the mismatch of the light chain that binds to the first antigen with the "wrong" heavy chain that binds to the second antigen (compared to approaches in However, their preparation is not completely free from the formation of by-products. The main by-product is formed as a result of the interaction of the Bence-Jones type of the "wrong" light chain with the heavy chain with exchanged domains (see also Schaefer W. et al. ., Proc. Natl, Acad. Sci. USA, 108, 2011, pp. 11187-11191; Fig. S11 in the appendix).

WO 2015/101588 A1 describes shuttle modules for the blood-brain barrier. WO2015/101588 A1 mentions bivalent bispecific antibodies with a VH/VL crossover in one of the binding arms with mutations at the CH1/CL interface. WO 2015/101588 A1 does not describe the technical effect of these mutations.

There are various methods for creating cell lines for obtaining four-chain homodimeric divalent antibodies, i.e. antibodies such as native antibodies. To improve the performance (productivity) of these cell lines, some of these methods are based on the so-called "supertransfection" approach. Using this approach, the cells are transfected at least twice with the selection of the intermediate cell line. Each of the vectors used in this supertransfection approach typically contains complete information for encoding the antibody to be expressed, i.e. for the light chain and for the heavy chain. Some special supertransfection methods use very similar or even identical vectors, differing only in a selection marker, in order to achieve tight integration into the genome in a known productive region. Like DHFR gene amplification methods, supertransfection methods serve to increase the level (yield) of expression by increasing the number of functional expression cassettes in cells.

For novel complex trivalent bispecific antibody formats containing a heterodimeric Fc region and a so-called domain swap, which are both introduced to limit or even eliminate mismatches and thereby increase the yield of a correctly folded and assembled resulting multispecific antibody, a complex co-transfection procedure has been described 3-4 vectors, each containing one expression cassette, using different ratios of vectors (see, for example, WO 2013/026833).

The present invention is based at least in part on the discovery that the level of expression of a multispecific antibody in a recombinant cell can be increased if the cell is re-transfected with an expression cassette to express the light chain of said multispecific antibody. This is especially valuable if the multispecific antibody contains a crossover heavy and light chain variant.

One of the objects of the invention presented in the present description is a method for producing a multispecific antibody (containing at least one domain crossover polypeptide), including the following steps:

a) obtaining a mammalian cell that expresses the antibody,

b) transfection of a mammalian cell referred to in subparagraph a) with an expression vector that contains an expression cassette encoding an antibody polypeptide that has a domain crossover,

c) culturing the cell referred to in b) and isolating the antibody from the cell or culture medium, thereby obtaining a multispecific antibody.

The modified cells produced by the method presented herein "secrete" more of the multispecific antibody in a properly folded and assembled form and are referred to herein as cells in which the amount of correctly folded and correctly assembled multispecific antibody released into the extracellular environment, is increased compared to the parent cell. Western blot analysis, biological activity assays, and physicochemical separation methods can be used to determine the absolute amounts of correctly folded and assembled multispecific antibody released from the modified cell compared to the parent cell.

One of the objects of the invention presented in the present description is a method for obtaining a multispecific antibody, which includes the following steps:

a) cultivation of the modified cell under conditions suitable / favorable for obtaining a multispecific antibody, while

I) the modified cell is related to the parent cell, where the parent cell contains the first DNA sequences encoding the multispecific antibody by introducing a nucleic acid into the genome of the parent cell at a locus that is not in the first DNA sequences; and

ii) the modified cell produces more multispecific antibody than the parent cell when both cells are cultured under the same conditions; and

b) isolation of the polypeptide.

Domain Crossover Antibody Formats

The method presented herein is generally suitable for the production of any multispecific antibody that contains a separately encoded heavy and light chain.

In one embodiment, the multispecific antibody is a bivalent bispecific antibody that comprises a) a first light chain and a first heavy chain of an antibody that specifically binds to the first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the VL and VH variable domains of the second light chain and the second heavy chain are interchanged.

The antibody in a) does not contain the modification in b) and the heavy chain and light chain in a) are isolated chains.

In the antibody referred to in subparagraph b),

in the light chain

the VL light chain variable domain is replaced by the VH heavy chain variable domain of said antibody, and

in the heavy chain

the VH heavy chain variable domain is replaced by the VL light chain variable domain of said antibody.

In one embodiment, the multispecific antibody is a bivalent bispecific antibody that contains

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, in which the VL and VH variable domains of the second light chain and the second heavy chain are interchanged, and in which the CL and CH1 constant domains of the second light chain and the second heavy chain replaced with each other.

The antibody in a) does not contain the modification in b) and the heavy chain and light chain in a) are isolated chains.

In the antibody referred to in subparagraph b),

in the light chain

the light chain variable domain VL is replaced by the heavy chain variable domain VH of said antibody, and the light chain constant domain CL is replaced by the heavy chain constant domain CH1 of said antibody; and

in the heavy chain

the VH heavy chain variable domain is replaced by the VL light chain variable domain of said antibody, and the CH1 heavy chain constant domain is replaced by the CL light chain constant domain of said antibody.

In one embodiment, the multispecific antibody is a bivalent bispecific antibody that contains

a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and

b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, in which the CL and CH1 constant domains of the second light chain and the second heavy chain are exchanged for each other.

The antibody in a) does not contain the modification in b) and the heavy chain and light chain in a) are isolated chains.

In the antibody referred to in subparagraph b),

in the light chain

the constant domain of the light chain CL is replaced by the constant domain of the heavy chain CH1 of the indicated antibody; and

in the heavy chain

the CH1 heavy chain constant domain is replaced by the CL light chain constant domain of said antibody.

In one embodiment of all aspects of the invention, the multispecific antibody is a trispecific or tetraspecific antibody that contains

a) the first light chain and the first heavy chain of a full-length antibody that specifically binds to the first antigen, and

b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to a second antigen, in which the VL and VH variable domains are interchanged and/or in which the CL and CH1 constant domains are interchanged , and

c) wherein 1-4 antigen-binding peptides that specifically bind to one or two additional antigens (i.e. a third and/or fourth antigen) are fused via a peptide linker to the C- or N-terminus of the light chains or heavy chains, referred to in subparagraphs a) and / or b).

The antibody in a) does not contain the modification in b) and the heavy chain and light chain in a) are isolated chains.

In one of the embodiments of the invention trispecific or tetraspecific antibody contains one or two antigennegative peptides specified in subparagraph c), which specifically bind to one or two additional antigens.

In one embodiment, the antigen binding peptides are selected from the group consisting of an scFv fragment and a scFab fragment.

In one embodiment, the antigen-binding peptides are scFv fragments.

In one embodiment, the antigen-binding peptides are scFab fragments.

In one embodiment, the antigen-binding peptides are fused to the C-terminus of the heavy chains of a) and/or b).

In one embodiment, the trispecific or tetraspecific antibody comprises one or two of the antigen-binding peptides listed in c), which specifically bind to one additional antigen.

In one of the embodiments of the invention trispecific or tetraspecific antibody contains two identical antigen-binding peptides specified in subparagraph c), which specifically bind to the third antigen. In one preferred embodiment of the invention, two identical antigen-binding peptides are both fused via the same peptide linker to the C-terminus of the heavy chains indicated in subparagraphs a) and b). In one preferred embodiment of the invention, said two identical antigen-binding peptides are either an scFv fragment or a scFab fragment.

In one of the embodiments of the invention trispecific or tetraspecific antibody contains two antigen-binding peptides specified in subparagraph c), which specifically bind to the third and fourth antigen.

In one of the embodiments of the invention, these two antigen-binding peptides are both fused through the same peptide connector to the C-terminus of the heavy chains indicated in subparagraphs a) and b). In one preferred embodiment of the invention, said two antigen-binding peptides are either an scFv fragment or a scFab fragment.

In one embodiment, the multispecific antibody is a bispecific tetravalent antibody that contains

a) two light chains and two heavy chains of an antibody that specifically binds to the first antigen (and contains two Fab fragments),

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, in which said additional Fab fragments are both fused via a peptide linker to either the C- or N-terminus of the heavy chains specified in subparagraph a),

and

where the following modifications have been made in Fab fragments

i) in both Fab fragments referred to in subparagraph a), or in both Fab fragments referred to in subparagraph b), the variable domains VL and VH are exchanged for each other and/or the constant domains CL and CH1 are exchanged for each other,

or

ii) in both Fab fragments referred to in subparagraph a), the variable domains VL and VH are exchanged for each other and the constant domains CL and CH1 are exchanged for each other, and

in both Fab fragments referred to in b), the VL and VH variable domains are exchanged, or the CL and CH1 constant domains are exchanged, or

iii) in both Fab fragments referred to in subparagraph a), the variable domains VL and VH are exchanged for each other or the constant domains CL and CH1 are exchanged for each other, and

in both Fab fragments referred to in b), the variable domains VL and VH are exchanged for each other and the constant domains CL and CH1 are exchanged for each other, or

iv) in both Fab fragments referred to in subparagraph a), the variable domains VL and VH are exchanged for each other and in both Fab fragments indicated in subparagraph b), the constant domains CL and CH1 are exchanged for each other,

or

V) in both Fab fragments referred to in subparagraph a), the constant domains CL and CH1 are exchanged for each other and in both Fab fragments indicated in subparagraph b), the variable domains VL and VH are exchanged for each other.

In one embodiment, said additional Fab fragments are both fused via a peptide linker to either the C-terminus of the heavy chains of subparagraph a) or the N-terminus of the heavy chains of subparagraph a).

In one of the embodiments of the invention, these additional Fab fragments are both fused via a peptide linker to the C-terminus of the heavy chains specified in subparagraph a).

In one of the embodiments of the invention, these additional Fab fragments are both fused through a peptide connector to the N-terminus of the heavy chains specified in subparagraph a).

In one of the embodiments of the invention, the following modifications are made to the Fab fragments:

i) in both Fab fragments referred to in subparagraph a), or in both Fab fragments referred to in subparagraph b), the variable domains VL and VH are replaced with each other,

and/or

constant domains CL and CH1 are replaced with each other.

In one of the embodiments of the invention, the following modifications are made to the Fab fragments:

i) in both Fab fragments referred to in subparagraph a), the variable domains VL and VH are substituted for each other and/or

constant domains CL and CH1 are replaced with each other.

In one of the embodiments of the invention, the following modifications are made to the Fab fragments:

I) in both Fab-fragments specified in subparagraph a), the constant domains CL and CH1 are replaced with each other.

In one of the embodiments of the invention, the following modifications are made to the Fab fragments:

i) in both Fab fragments referred to in subparagraph b), the variable domains VL and VH are substituted for each other and/or

constant domains CL and CH1 are replaced with each other. In one of the embodiments of the invention, the following modifications are made to the Fab fragments:

I) in both Fab-fragments specified in subparagraph b), the constant domains CL and CH1 are replaced with each other.

In one embodiment, the multispecific antibody is a bispecific tetravalent antibody that contains:

a) a (modified) heavy chain of a first antibody that specifically binds to a first antigen and contains a first VH-CH1 domain pair, in which the N-terminus of the second VH-CH1 domain pair of said first antibody is fused to the C-terminus of said heavy chain via a peptide linker ,

b) two light chains of the specified first antibody specified in subparagraph a),

c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and contains a first VH-CL domain pair, in which the N-terminus of the second VH-CL domain pair of said second antibody is fused to the C-terminus of said heavy chain via a peptide linker , and

d) two (modified) light chains of the second antibody in c), each containing a CL-CH1 domain pair.

In one embodiment, the multispecific antibody is a bispecific antibody that contains:

a) the heavy chain and light chain of the first full-length antibody that specifically binds to the first antigen, and

c) a heavy chain and a light chain of a second full-length antibody that specifically binds to a second antigen, in which the N-terminus of the heavy chain is connected to the C-terminus of the light chain via a peptide linker.

The antibody in a) does not contain the modification in b) and the heavy chain and light chain in a) are isolated chains.

In all aspects of the invention presented herein, the first light chain contains a VL domain and a CL domain, and the first heavy chain contains a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

In one of the embodiments of the invention, the antibody obtained by the method presented in the present description is a multispecific antibody that needs heterodimerization of at least two heavy chain polypeptides.

Several approaches to CH3 modifications promoting heterodimerization are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO2010/129304, WO 2011/ 90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291, which are incorporated herein by reference. Typically, in approaches known in the art, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are both designed in a complementary fashion, whereby a heavy chain containing one of the designed CH3 domains can no longer form a homodimer with the other. a heavy chain of the same structure (e.g., a first CH3 engineered heavy chain can no longer homodimer with another first CH3 engineered heavy chain; and a second CH3 engineered heavy chain can no longer homodimer with another second CH3 engineered heavy chain). Thus, the first heavy chain, which contains a single designed CH3 domain, is enhanced in its ability to heterodimerize with another heavy chain, which contains a complementary designed CH3 domain. According to this embodiment, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are created in a complementary manner with amino acid substitutions that enhance the heterodimerization ability of the first heavy chain and the second heavy chain, while the first heavy chain and the second heavy chain can no longer form a homodimer (eg due to steric hindrance).

Various approaches are known in the art to promote heavy chain heterodimerization as cited and described above, and are considered as various alternatives for use in a multispecific antibody of the invention that contains an "uncrossed Fab region" from a first antibody that specifically binds with the first antigen, and a "crossed Fab region" from a second antibody that specifically binds to the second antigen, in combination with the specific amino acid substitutions described above in the invention.

The CH3 domains of a multispecific antibody produced by the method described herein can be altered using the "knob-into-holes" technique, which is described in detail with several examples, e.g. , in WO 96/027011, Ridgway J.B. et al., Protein Eng 9, 1996, pp. 617-621 and Merchant A.M. et al., Nat Biotechnol 16, 1998, pp. 677-681. In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing the two said CH3 domains. Each of the two CH3 domains (two heavy chains) may contain a "ridge" and the other may contain a "trough". The introduction of a disulfide bridge additionally stabilizes heterodimers (Merchant A.M. et al., Nature Biotech 16, 1998, pp. 677-681; Atwell S. et al., J. Mol. Biol. 270, 1997, pp. 26-35) and increases exit.

In one preferred embodiment of the invention, the multispecific antibody obtained using the method presented in the present description contains the T366W mutation in the "knob chain" CH3 domain and the T366S, L368A, Y407V mutations in the "gap chain" CH3 domain (numbering according to Cabot's EU index). An additional interchain disulfide bridge between CH3 domains can also be used (Merchant A.M. et al., Nature Biotech 16, 1998, pp. 677-681), for example, by introducing a Y349C mutation in the "knock chain" CH3 domain and an E356C mutation or an S354C mutation in the "gap chain" CH3 domain. Thus, according to another preferred embodiment of the invention, the multispecific antibody obtained using the method presented in the present description contains mutations Y349C and T366W in one of the two CH3 domains and mutations E356C, T366S, L368A and Y407V in the second of the two CH3 domains , or a multispecific antibody obtained using the method presented in the present description, contains mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A and Y407V in the second of the two CH3 domains (an additional Y349C mutation in one of CH3 domains and an additional E356C or S354C mutation in another CH3 domain leads to the formation of an interchain disulfide bridge) (numbering according to Cabot's EU index).

However, in an alternative or additional embodiment, other variants of the "knobs-in-holes" technology presented in EP 1870459A1 can also be used. In one of the embodiments of the invention, the multispecific antibody obtained using the method presented in the present description, contains mutations R409D and K370E in the CH3 domain of the chain with "lumps" and mutations D399K and E357K in the CH3 domain of the "chain with a trough" (numbering according to Cabot's EU index).

In one of the embodiments of the invention, the multispecific antibody obtained using the method presented in the present description, contains the T366W mutation in the CH3-domain of the chain with "lumps" and mutations T366S, L368A and Y407V in the CH3-domain of the "trough chain" (numbering according to Cabot's EU index).

In one of the embodiments of the invention, a multispecific antibody obtained using the method presented in the present description contains mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A and Y407V in the second of the two CH3 domains, or multispecific the antibody obtained using the method presented in the present description contains mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A and Y407V in the second of the two CH3 domains and additionally contains mutations R409D and K370E in CH3- the “knob chain” domain and the D399K and E357K mutations in the “gap chain” CH3 domain (numbering according to Cabot's EU index).

In addition to the "knob-into-hole" technology, other technologies are known in the art to modify the CH3 domains of the heavy chains of a multispecific antibody to enhance heterodimerization. These technologies are primarily described in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754/143,545 WO 2012/058768, WO 2013/157954 and WO 2013/096291 are considered herein as an alternative to the "knob-into-hole" technique for a multispecific antibody produced by the method described herein.

In one embodiment of all objects and embodiments of the invention that are presented in the present description, multispecific antibody is a bispecific antibody or trispecific antibody. In one of the preferred embodiments of the invention, the multispecific antibody is a bispecific antibody.

In one of the embodiments of all objects of the invention, which are presented in the present description, the antibody is a divalent or trivalent antibody. In one embodiment, the antibody is a divalent antibody.

In one of the embodiments of all objects of the invention, which are presented in the present description, the multispecific antibody has a constant domain structure corresponding to an IgG-type antibody. In another embodiment of all aspects of the invention as provided herein, the multispecific antibody is characterized in that said multispecific antibody is of the human IgG1 subclass or human IgG1 subclass with the L234A and L235A mutations. In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgC2 subclass. In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgG3 subclass. In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgG4 subclass or human IgG4 subclass with an additional S228P mutation. In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgG1 subclass or human IgG4 subclass. In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgG1 subclass with mutations L234A and L235A (numbering according to Cabot's EU index). In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgG1 subclass with mutations L234A, L235A and P329G (numbering according to Cabot's EU index). In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgG4 subclass with mutations S228P and L235E (numbering according to Cabot's EU index). In one of the additional embodiments of all the objects of the invention that are presented in the present description, the multispecific antibody is characterized in that the specified multispecific antibody belongs to the human IgG4 subclass with mutations S228P, L235E and P329G (numbering according to Cabot's EU index).

In one of the embodiments of all the objects of the invention, which are presented in the present description, an antibody that contains a heavy chain that includes the CH3 domain specified in the present description contains an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU- Cabot index). In one embodiment of all aspects of the invention as provided herein, an antibody that contains a heavy chain comprising the CH3 domain as defined herein contains an additional C-terminal glycine residue (G446, numbered according to Cabot's EU index).

Bispecific trivalent antibody to human A-beta/human transferrin receptor

Said antibody is a bispecific antibody consisting of a full length base antibody and fused to a Fab fragment in which certain domains are cross-exchanged. Thus, the resulting bispecific antibody is asymmetric. For this purpose, a bispecific antibody was prepared using a heterodimerization technique referred to as “knobs-into-holes”, using a first heavy chain with mutations resulting in the formation of so-called “knobs” (HC “knob”) and a second heavy chain with mutations , leading to the formation of so-called depressions (HC "depression").

In this example, co-transfection was used.

Antibody 0012, Antibody 0015, Antibody 0020 and Antibody 0024 are described in WO 2017/055540 A1 (SEQ ID NO: 06-09, SEQ ID NO: 01-03 and SEQ ID NO: 10, SEQ ID NO: 11-13 and SEQ ID NO: 14-17 in WO 2017/055540 A1 respectively). "ledge", fused via a linker VL-domain, where in the Fab VH- and VL-domains are exchanged (crossover of VH-VL domains). Both Fabs without full-length antibody domain crossover were modified to contain charges to aid in proper assembly.

Antibody 0015 is a full-length antibody containing one heavy chain with mutations leading to the formation of a "trough", and one heavy chain with mutations leading to the formation of a "ledge", in which the C-terminus of the heavy chain with mutations leading to the formation of " overhang" is fused via a Fab VH-domain linker where the CH1 and CL domains are exchanged in the Fab (CH-CL domain crossover). Both Fabs without full-length antibody domain crossover were modified to contain charges to aid in proper assembly.

Antibody 0020 is a full-length antibody containing one heavy chain with mutations leading to the formation of a "trough", and one heavy chain with mutations leading to the formation of a "ledge", in which the C-terminus of the heavy chain with mutations leading to the formation of " overhang" fused via a linker VL domain of a single stranded Fab (no domain crossover). Both Fabs without full-length antibody domain crossover were modified to contain charges to aid in proper assembly.

Antibody 0024 is a full-length antibody containing one heavy chain with mutations leading to the formation of a "trough" and one heavy chain with mutations leading to the formation of a "ledge", in which the C-terminus of the heavy chain with mutations leading to the formation of " overhang" is fused via a Fab VH-domain linker where the CH1 and CL domains are exchanged in the Fab (CH-CL domain crossover).

For recombinant production of bispecific antibodies, different distributions/different combinations of the respective polypeptides in different expression vectors, different ratios of generated vectors and different transfection sequences have been used.

EC+HC pit: an expression vector that contains one expression cassette for a heavy chain with a pit mutation and a light chain.

LCcross+ HC"BbicTyn": an expression vector that contains a single expression cassette for a heavy chain with a bud mutation and a light chain with a domain crossover.

LC: Expression vector that contains one light chain expression cassette.

LCcross: An expression vector that contains a single crossover light chain expression cassette.

HC Overhang: An expression vector that contains a single heavy chain expression cassette with a overhang mutation fused to a scFab.

The table below shows the results obtained using CHO-K1 cells.

Bispecific antibodies were generated on a small scale in CHO-S cells and by-product distribution was analyzed after a first purification step using protein A affinity chromatography and after a second purification step using preparative gel filtration. The results are presented in the table below.

Using this approach, it is possible to obtain a producer cell line that produces a heterodimeric antibody with an improved product profile, i.e. with an increased amount of product and a reduced amount of product-related impurities.

To create stable producer cell lines, co-transfection with an expression plasmid containing an expression cassette for the antibody light chain only was used.

CHO-K1 cells were transfected using the ratio of plasmids 1(LC): 1(LC+HC"trough"):3(LCcross+HC"trough"). Cells with stable foreign DNA integrated into their genome were selected using methotrexate. Stable cell lines were isolated and evaluated using a four-day fed-batch culture for product quality. The product was isolated by protein A affinity chromatography and analyzed by SDS-CE.

Bispecific trivalent antibody to human CP20/human transferrin receptor

Said antibody is a bispecific antibody consisting of a full length base antibody and fused to a Fab fragment in which certain domains are cross-exchanged. Thus, the resulting bispecific antibody is asymmetric. For this purpose, a bispecific antibody was prepared using a heterodimerization technique referred to as “knobs-into-holes”, using a first heavy chain with mutations resulting in the formation of so-called “knobs” (HC “knob”) and a second heavy chain with mutations , leading to the formation of so-called depressions (HC "depression").

In this example, co-transfection was used.

Antibody 0039, Antibody 0041, Antibody 0040 and Antibody 0042 are described in WO 2017/055542 Al (SEQ ID NO: 06-09, SEQ ID NO: 01-03 and SEQ ID NO: 10, SEQ ID NO: 11-13 and SEQ ID NO: 22 and SEQ ID NO: 14-17 in WO 2017/055542 A1, respectively).

Antibody 0038 is a full-length antibody containing one heavy chain with mutations that lead to the formation of a "trough", and one heavy chain with mutations that lead to the formation of a "ledge", in which the C-terminus of the heavy chain with mutations leading to the formation of "ledge", merged through the scFab linker. Both arms of the canonical Fab have been modified to contain charges to aid in proper assembly.

Antibody 0039 is a full-length antibody containing one heavy chain with mutations leading to the formation of a "trough" and one heavy chain with mutations leading to the formation of a "ledge", in which the C-terminus of the heavy chain with mutations leading to the formation of "ledge", merged through the Fab VL-domain linker, where in the Fab the VH- and VL-domains are exchanged (VH-VL domain crossover). Both Fabs with unchanged domains have been modified to contain charges to aid in proper assembly.

Antibody 0041 is a full-length antibody containing one heavy chain with mutations leading to the formation of a "trough", and one heavy chain with mutations leading to the formation of a "ledge", in which the C-terminus of the heavy chain with mutations leading to the formation of "ledge", fused through a linker VH-domain, where in the Fab CH1- and CL-domains are exchanged (crossover of CH-CL domains). Both heavy and light chain pairs of the full length antibody, as well as Fab, were modified to contain charges to aid in proper assembly. Both Fabs with unchanged domains have been modified to contain charges to aid in proper assembly.

Antibody 0040 is a full length antibody containing one heavy chain with mutations leading to the formation of a "trough" and one heavy chain with mutations leading to the formation of a "ledge", in which the C-terminus of the heavy chain with mutations leading to the formation of overhang, fused via a VH Fab linker where CH1 and CL domains are exchanged in the Fab (CH-CL domain crossover).

Antibody 0042 is a full-length antibody containing a single heavy chain with trough mutations and

one heavy chain with mutations leading to the formation of a "lip", in which the N-terminus of the heavy chain with mutations leading to the formation of a "lip" is fused with a CH1 Fab, where in the Fab the VH and VL domains of the fused heavy chain are exchanged ( crossover of VH-VL domains). Both Fabs with unchanged domains have been modified to contain charges to aid in proper assembly.

For recombinant production of bispecific antibodies, different distributions/different combinations of the respective polypeptides in different expression vectors, different ratios of generated vectors and different transfection sequences have been used.

EC+HC pit: an expression vector that contains one expression cassette for a heavy chain with a pit mutation and a light chain.

LCcross+HC"BbicTyn": an expression vector that contains one expression cassette for a heavy chain with a bud mutation and a light chain with a domain crossover.

LC: Expression vector that contains one light chain expression cassette.

LCcross: An expression vector that contains a single crossover light chain expression cassette.

HC Overhang: An expression vector that contains a single heavy chain expression cassette with a overhang mutation fused to a scFab.

For recombinant production of bispecific antibodies in HEK cells, different distributions/different combinations of the respective polypeptides in different expression vectors, different ratios of generated vectors and different sequences for transfection were used. The results are presented in the table below.

For recombinant production of bispecific antibodies in CHO-K1 cells, a different distribution/different combination of the corresponding polypeptides in different expression vectors, different ratios of the resulting vectors and different sequences for transfection were used. The results are presented in the table below.

Bispecific antibodies were generated on a small scale in CHO-S cells and by-product distribution was analyzed after a first purification step using protein A affinity chromatography and after a second purification step using preparative gel filtration. The results are presented in the table below.

Bispecific antibodies were obtained in various cell lines. The results are presented in the table below.

Using this approach, it is possible to obtain a producer cell line that produces a heterodimeric antibody with an improved product profile, i.e. with an increased amount of product and a reduced amount of product-related impurities.

Bispecific bivalent antibody to human PD1/human Tim3

The specified antibody is a bispecific antibody consisting of a full-length antibody with “knob-into-hole” mutations in the Fc region and an artificial disulfide bridge between the CH3 domains, in which in the pair of heavy and light chains that form the binding site for PD1, VH- and VL domains are replaced with each other. Thus, the resulting bispecific antibody is asymmetric. For this purpose, a bispecific antibody was prepared using a heterodimerization technique referred to as “knobs-into-holes”, using a first heavy chain with mutations leading to the formation of so-called “knobs” (HC “knob”) and a second heavy chain with mutations , leading to the formation of so-called depressions (HC "depression"). The sequences are described in WO 2017/055404 A1

In this example, co-transfection was used.

To do this, several versions of the expression plasmids were combined to obtain a cell line expressing the above antibody. These approaches differed in the combination of plasmids, but did not differ in the resulting antibodies.

To obtain antibody 0516, co-transfection was carried out in a 1:1 ratio with vector 1 used for transfection, which contains expression cassettes for the first light chain (LC-1) and the first heavy chain with mutations leading to the formation of a "hollow" (HC-1-" trough"), an IgG1 subclass, and vector 2, which contains expression cassettes for a second domain-exchange light chain (CrossLC-2) and a second domain-exchange heavy chain with bump-producing mutations (CrossHC-2- "protrusion"), IgG1 subclass.

To obtain antibody 0517, a 1:1:1 co-transfection was carried out with vector 1 used for transfection, which contains expression cassettes for the first light chain (LC-1) and the first heavy chain with mutations leading to the formation of a "hollow" (HC-1 - "trough"), IgG1 subclass, vector 2, which contains expression cassettes for the second light chain with domain exchange (CrossLC-2), and the second heavy chain with domain exchange with mutations leading to the formation of a "ledge" (CrossHC-2 - "protrusion"), IgG1 subclass, and vector 3, which contains the expression cassette for the second light chain with domain exchange (CrossLC-2).

To obtain antibody 0518, a 1:1 co-transfection was carried out with the vector 1 used for transfection, which contains expression cassettes for the second light chain with domain exchange (CrossLC-2) and the first heavy chain with mutations leading to the formation of a "trough" (HC- 1-"trough"), IgG1-subclass, and vector 2, which contains expression cassettes for the first light chain (LC-1) and the second heavy chain with the exchange of domains with mutations leading to the formation of a "protrusion" (CrossHC-2-" protrusion"), IgG1 subclass.

To obtain antibody 0519, a 1:1:1 co-transfection was carried out with vector 1 used for transfection, which contains expression cassettes for the second light chain with domain exchange (CrossLC-2) and the first heavy chain with mutations leading to the formation of a "hollow" ( HC-1-"hollow"), IgG1-subclass, vector 2, which contains expression cassettes for the first light chain (LC-1) and the second heavy chain with the exchange of domains with mutations leading to the formation of a "protrusion" (CrossHC-2- "BbicTyn"), an IgG1 subclass, and vector 3, which contains an expression cassette for a domain exchange second light chain (CrossLC-2). The results are presented in the table below.

A correctly assembled antibody has a stoichiometry corresponding to ABCD, where A denotes the second heavy chain with mutations that lead to the formation of a "ledge" (CrossHC-2-"BbicTyn"), IgG1 subclass, B denotes the first heavy chain with mutations that lead to the formation of " cavities" (HC-1-"cavity"), IgG1 subclass, C denotes the second light chain with an exchange of domains (CrossLC-2), and D denotes the first light chain (LC-1).

Most of the related compound by-products formed were misassembled antibodies. The two main by-products were both four chain antibodies. The first was a hetero-"hollow"-"protrusion"-HC-dimer, in which the crossed light chain is replaced by an uncrossed light chain (ABD2). The second was a homo-"cavity"-"cavity"-dimer semi-antibody (B2D2).

It has been found that when an additional plasmid containing only the domain-swapped light chain expression cassette is used for transfections, improved results can be obtained, ie. reducing the presence of product-related by-products (see FIGS. 1A-1D).

As shown in FIG. 1, it is possible to reduce the amount of product-related by-products, especially the by-product ABD2. This simultaneously reduces product loss during subsequent purification steps. For example, either the number of purification steps needed can be reduced, or the product loss associated with peak overlap and fractionation can be reduced (the peaks are more separated and can be "cut out" with reduced product loss), or both. Thus, the output obtained can be increased.

Using this approach, it is possible to obtain a producer cell line that produces a heterodimeric antibody with an improved product profile, i.e. with an increased amount of product and a reduced amount of product-related impurities.

Bispecific quadrivalent antibody to human FAP/DR5

Bispecific antibodies to FAP-DR5 were generated by fusion of the FAP-binding domain to the heavy chain of anti-DRS IgG at the C-terminus via a (G4S)4 linker. The corresponding DR5 antibody portion consisted of the variable light chain (VL) and variable heavy chain (VH) of drositumab (see US 2007/003141401) or novel anti-DR5 antibodies generated by phage display. CrossMab domain crossover technology was used to minimize mismatched light chain by-products. The FAP binding module was designed as a crossed Fab in which the VH domain is fused to the constant light chain (CL) and the VL domain is fused to the CH1 domain (constant heavy chain 1). The sequences are described in WO 2016/055432.

In this example, co-transfection was used.

Expression cassettes corresponding to the polypeptides were distributed into various expression vectors.

EC+HC pit: an expression vector that contains one expression cassette for a heavy chain with a pit mutation and a light chain.

LCcross+HC"BbicTyn": an expression vector that contains one expression cassette for a heavy chain with a bud mutation and a light chain with a domain crossover.

LC: Expression vector that contains one light chain expression cassette.

LCcross: An expression vector that contains a single crossover light chain expression cassette.

Clone 131 was generated by standard transfection with two plasmids each containing two expression cassettes for the expression of a bispecific antibody (full length antibody with each one of the CH1/CL cross-Fabs attached to each of the heavy chain C-termini).

This clone produced products of the following composition.

This clone was used as the master clone for the second transfection using a plasmid that only contained a crossed FAP binding site light chain.

The table below shows, by way of example, the characteristics of some clones obtained.

The results obtained by SDS-CE (231=5/6 antibody; 242=monomer) are shown in the table below and in FIG. 2.

Using this approach, it is possible to obtain a producer cell line that produces a heterodimeric antibody with an improved product profile, i.e. with an increased amount of product and a reduced amount of product-related impurities.

General methods of recombination and compositions used to obtain antibodies

Antibodies can be obtained using recombination and composition techniques such as those described in US No. 4,816,567. When implementing these methods, one or more isolated(s) nucleic acid(s) encoding(s) the antibody is obtained.

In the case of a native antibody or native antibody fragment, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Said nucleic acid(s) encode(s) an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of an antibody (for example, an antibody light(s) and/or heavy chain(s) ). These nucleic acids may be in the same expression vector or in different expression vectors.

In the case of a heterodimeric heavy chain bispecific antibody, four nucleic acids are required, one for the first light chain, one for the second light chain, which contains the first heteromonomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain, which contains the second heteromonomeric Fc region polypeptide. For example, one heterodimeric heavy chain contains so-called "knob mutations" (T366W and optionally one of S354C or Y349C) and another contains so-called "trough mutations" (T366S, L368A and Y407V and optionally Y349C or S354C) (see, for example, Carter P. et al., Immunotechnol. 2, 1996, p. 73). Said nucleic acid(s) encode(s) an amino acid sequence that contains a first VL and/or an amino acid sequence that contains a first VH that includes a first heteromonomeric Fc region and/or an amino acid sequence that contains second VL, and/or amino acid sequence,

which contains a second VH, including a second heteromonomeric Fc region of the antibody (for example, the first and/or second light and/or the first and/or second heavy chains of the antibody). These nucleic acids can be located in one expression vector or in different expression vectors, as a rule, these nucleic acids are localized in two or three expression vectors, i. one vector may contain more than one of these nucleic acids. Examples of such bispecific antibodies are CrossMab and T-cell bispecific antibodies.

One of the embodiments of the invention are selected nucleic acids encoding the antibody, which is used in the methods presented in the present description.

The following embodiment of the invention is(are) one or more vectors (eg, expression vectors), which(s) contains(at) the specified(s) nucleic acid(s).

The next embodiment of the invention is a host cell containing the specified(s) nucleic(s) acid(s).

In one of these embodiments of the invention, the host cell contains (for example, transformed):

- in the case of a native antibody or native antibody fragment:

(1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or

(2) a first vector containing a nucleic acid that encodes an amino acid sequence containing an antibody VL and a second vector containing a nucleic acid that encodes an amino acid sequence containing an antibody VH;

- in the case of a bispecific antibody with heterodimeric heavy chains:

(1) a first vector containing a first pair of nucleic acids that encode the amino acid sequences of one of them containing the first VL and the other containing the first VH of the antibody, and a second vector containing a second pair of nucleic acids that encode the amino acid sequences of one of them, containing a second VL and another containing a second VH antibody, or

(2) a first vector containing a first nucleic acid that encodes an amino acid sequence containing one of the variable domains (preferably a light chain variable domain), a second vector containing a pair of nucleic acids that encodes the amino acid sequences of one of them containing a light chain variable domain , and the other containing the variable domain of the first heavy chain, and the third vector containing a pair of nucleic acids that encode the amino acid sequences of one of them containing the corresponding light chain variable domain different from that present in the second vector, and the other of them containing the variable domain second heavy chain, or

(3) a first vector containing a nucleic acid that encodes an amino acid sequence containing the first VL of an antibody, a second vector containing a nucleic acid that encodes an amino acid sequence containing the first VH of an antibody, a third vector containing a nucleic acid that encodes an amino acid sequence containing the second VL of the antibody; and the fourth vector containing the nucleic acid that encodes the amino acid sequence containing the second VH of the antibody.

In one embodiment, the host cell is eukaryotic, eg, a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, Y0-, NS0-, Sp20 cell). One embodiment of the invention is a method for producing an anti-[PRO]] antibody, wherein the method comprises culturing a host cell that contains nucleic acids encoding the antibody described above under conditions suitable for expressing the antibody, and optionally isolating the antibody from the cell. host (or medium for culturing the host cell).

For recombinant production of anti-[[PRO]] antibodies, nucleic acids encoding an antibody, such as those described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. These nucleic acids can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that have the ability to specifically bind to genes encoding antibody heavy and light chains), or obtained by recombination methods, or obtained by chemical synthesis.

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc-related effector function are not required. For information on the expression of antibody fragments and polypeptides in bacteria, see, for example, US No. 5648237, US No. 5789199 and US No. 5840523 (see also Charlton K.A. in: Methods in Molecular Biology, edited by Lo B.K. C, Humana Press, Totowa, NJ, vol.248, 2003, pp. 245-254 for a description of the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified .

In addition to prokaryotic organisms, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been "humanized" can be used as hosts suitable for cloning or expression of vectors that encode antibodies, which makes it possible to obtain an antibody with partially or a fully human glycosylation scheme (see Gerngross T.U., Nat. Biotech. 22, 2004, pp. 1409-1414 and Li H. et al., Nat. Biotech. 24, 2006, pp. 210-215).

Host cells that can be used to express the glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells are insect cells, and plant cells can also be used. Numerous strains of baculoviruses and corresponding suitable insect cells as hosts have been identified, primarily for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts (see, for example, US No. 5959177, US No. 6040498, US No. 6420548, US No. 7125978 and US No. 6417429 (a description of the PLANTIBODIES™ technology for producing antibodies in transgenic plants)).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to growth in suspension can be used. Other examples of suitable mammalian host cell lines are the CV1 monkey kidney cell line transformed with OB40 (COS-7); a human embryonic kidney cell line (HEK 293 cells or 293 cell line, described, for example, in Graham, F. L. et al., J. Gen. Virol., 36, 1977, pp. 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells described, for example, in Mather J.P., Biol. Reprod., 23, 1980, pp. 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; dog kidney cells (MDCK); bovine rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells described, for example, in Mather J.P. et al., Annals N.Y. Acad. Sci., 383, 1982, pp. 44-68); MRC 5 cells and FS4 cells. Other valuable mammalian host cell lines are Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub G. et al., Proc. Natl. Acad. Sci. USA, 77, 1980, pp. 4216-4220) ; and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of specific mammalian host cell lines that can be used to produce antibodies, see, for example, Yazaki P. and Wu A.M., in: Methods in Molecular Biology, ed. V.K.S. Lo, Humana Press, Totowa, NJ, vol. 248, 2003, pp. 255-268.

Brief description of the drawings

The drawings show:

in fig. 1 total ionic ESI-MS results after antibody deglycosylation for the following options:

(A) transfection 0516;

(B) transfection 0517;

(B) transfection 0518;

(D) transfection 0519;

A: second heavy chain with mutations leading to the formation

"protrusion" (CrossHC-2-"BbicTyn"), IgG1 subclass; B: the first heavy chain with mutations leading to the formation of a "hollow" (HC-1 - "hollow"), IgG1 subclass; B: second light chain with domain exchange (CrossLC-2), D: first light chain (LC-1);

in fig. 2 relative content of monomer (A) and by-product in the form of 5/6 antibodies (B) when using the reference clone 0131 and clones obtained using the method presented in the present description, according to data obtained using CE-SDS.

Examples

The following are examples of the methods and compositions of the invention. As should be obvious, various other embodiments of the invention may be practiced in view of the above description of the invention as a whole.

Materials and General Methods

For general information regarding the nucleotide sequences of human immunoglobulin light and heavy chains, see: Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Antibody chain amino acids were numbered and labeled according to the Kabat numbering system (Kabat E A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991)

Recombinant DNA methods

For DNA manipulation, standard methods were used as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

Gene synthesis

The desired gene segments were created from oligonucleotides obtained by chemical synthesis. Long gene segments flanked by single sites recognized by restriction endonucleases were assembled by annealing and oligonucleotide ligation, including PCR amplification and subsequent cloning through the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Synthesis of gene fragments was ordered in accordance with these specifications from Geneart (Regensburg, Germany).

DNA sequencing

DNA sequences were determined by double strand sequencing, which was carried out by MediGenomix GmbH (Martinsried, Germany) or by SequiServe GmbH (Vaterstetten, Germany).

Analysis of DNA and protein sequences and processing of sequence data

GCG (Genetics Computer Group, Madison, WI), version 10.2 and Infomax's Vector NT1 Advance suite, version 8.0 were used to create, map, analyze, annotate, and illustrate sequences.

Expression vectors

Expression vectors for transient expression (eg, in HEK293 cells) based on a cDNA structure with a CMV promoter with or without intron A, or a genomic structure with a CMV promoter can be used to express the described bispecific antibodies.

In addition to the expression cassette for the antibody, the vectors contain:

an origin of replication that allows the vector to replicate in E. coli, and

p-lactamase gene, which confers ampicillin resistance in E. coli.

The transcription unit of an antibody gene consists of the following elements:

unique restriction site(s) at the 5' end, immediate early enhancer and promoter from human cytomegalovirus,

intron A sequence in the case of a cDNA-based structure, 5'-untranslated region from a human antibody gene, immunoglobulin heavy chain signal sequence, nucleic acid encoding the corresponding antibody chain either as a cDNA structure or genomic exon-intron structure, 3'-untranslated a region with a polyadenylation signal sequence and a unique restriction site(s) at the 3' end.

Fusion genes encoding antibody chains were generated by PCR and/or gene synthesis and assembled using known recombination techniques and techniques by joining nucleic acid segments, for example using unique restriction sites in appropriate vectors. The subcloned nucleic acid sequences were confirmed by DNA sequencing. For short term transfections, large numbers of vectors were prepared using vector preparations from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

All constructs used a trough-to-trough heterodimerization technique with a typical trough-forming substitution (T366W) in the first CH3 domain and corresponding trough-forming substitutions (T366S, L368A, and Y407V) at second CH3 domain (as well as two introduced cysteine residues S354C/Y349'C) (which were in the respective heavy chain (HC) sequences above).

Cell culture techniques

Standard cell culture techniques were used as described in Current Protocols in Cell Biology, ed. Bonifacino J.S., Dasso M., Harford J.B., Lippincott-Schwartz J. and Yamada K.M., John Wiley & Sons, Inc., 2000.

Short-term transfections in the HEK293-F system

Bispecific antibodies were obtained by transient expression. For this purpose, transfection with the appropriate vectors was carried out using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. In summary, the method was as follows: HEK293-F cells (Invitrogen) grown in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) were transfected with a mixture of the appropriate expression vectors and 293fectin ™ or Fectin (Invitrogen). Using a 2-liter shake flask (Corning), HEK293-F cells were seeded at a density of 1.0x10 ⁶ cells/ml in 600 ml and incubated at 120 rpm in an atmosphere with 8% CO ₂ . The next day, cells were transfected at a cell density of about 1.5×10 ⁶ cells/ml using about 42 ml of mixture A) 20 ml of Opti-MEM (Invitrogen) containing 600 μg of total vector DNA (1 μg/ml), and B) 20 ml Opti-MEM supplemented with 1.2 ml 293fectin™ or Fectin (2 μl/ml). Due to the uptake of glucose, a glucose solution was added during fermentation. The supernatant containing the secreted antibody was collected after 5-10 days and the antibodies were either purified directly from the supernatant or the supernatant was frozen and stored. Protein definition

The protein concentration of the purified antibodies and derivatives was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated from the amino acid sequence according to Race et al., Protein Science 4, 1995, ss. 2411-1423. Determination of antibody concentration in supernatants The concentration of antibodies and their derivatives in cell culture supernatants was determined by immunoprecipitation using protein A-agarose beads (Roche Diagnostics GmbH, Mannheim, Germany). For this, 60 μl of protein A-agarose beads were washed three times in TBS-NP40 (50 mM Tris buffer, pH 7.5, supplemented with 150 mM NaCl and 1% Nonidet-P40). Then 1-15 ml of the cell culture supernatant was applied to protein A-agarose beads pre-equilibrated in TBS-NP40. After incubation for 1 hour at room temperature, the beads were washed on an Ultrafree-MC filter column (Amicon), once with 0.5 ml of TBS-NP40, twice with 0.5 ml of 2x phosphate buffered saline (2xPBS, Roche Diagnostics GmbH, Mannheim, Germany) and briefly 4 times with 0.5 ml of 100 M Na-citrate buffer (pH 5.0). Bound antibody was eluted by adding 35 μl of NuPAGE® LDS sample buffer (Invitrogen). Half of the sample was combined with NuPAGE® sample reductant or left unreduced, respectively, and incubated for 10 min at 70°C. Then 5-30 µl was loaded onto 4-12% NuPAGE® Bis-Tris SDS-PAGE gel (Invitrogen) (with MOPS buffer for SDS-PAGE without reducing agent and MES buffer as running buffer for NuPAGE® with antioxidant additive (Invitrogen) for SDS-PAGE using a reducing agent) and stained with Coomassie blue.

The concentration of antibodies in cell culture supernatants was quantified by HPLC affinity chromatography. In general, the method was as follows: cell culture supernatants containing antibodies that bind to protein A were applied to an Applied Biosystems Poros A/20 column in a buffer containing 200 mM KH ₂ PO ₄ , 100 mM sodium citrate, pH 7.4 , and eluted with a buffer containing 200 mM NaCl, 100 mM citric acid, pH 2.5, on an Agilent HPLC 1100 system. The eluted antibody concentration was quantified using UV absorption and peak area integration. A purified IgG1 subclass standard antibody served as a standard.

Alternatively, the concentration of antibodies and their derivatives in cell culture supernatants was determined using a sandwich IgG-ELISA. In general, the method was as follows: 96-well microtiter plates with high binding ability to streptavidin (StreptaWell High Bind Streptavidin A) (Roche Diagnostics GmbH, Mannheim, Germany) were sensitized using 100 μl/well of a biotinylated capture antibody molecule to human IgG F(ab') ₂ <h-Fcy>BI (Dianova) at a concentration of 0.1 μg/ml, for 1 h at room temperature or alternatively overnight at 4°C and then washed three times, using 200 μl/well PBS, 0.05% Tween (PBS-T, Sigma). Then, serial dilutions in PBS (Sigma) containing the appropriate antibody cell culture supernatants were added to the wells at a rate of 100 μl/well and incubated for 1-2 hours on a shaker at room temperature. The wells were washed three times with 200 μl/well PBS-T, and the bound antibody was detected using 100 μl F(ab') ₂ <hFcγ>POD (Dianova) at a concentration of 0.1 μg/ml as identifying antibody, by incubation for 1-2 h on a shaker at room temperature. Unbound identifying antibody was removed by washing three times with 200 μl/well PBS-T. Bound identifying antibody was detected by adding 100 μl ABTS/well followed by incubation. The absorbance determination was carried out with a Tecan Fluor spectrometer, measuring at a wavelength of 405 nm (reference wavelength 492 nm).

Preparative purification of antibodies

Antibodies were purified from filtered cell culture supernatants according to a standard protocol. In general, the method was as follows: antibodies were applied to a protein A-Sepharose column (GE Healthcare) and washed with PBS. Proteins were eluted at pH 2.8, after which the sample was immediately neutralized. Aggregated proteins were separated from monomeric antibodies by gel filtration (Superdex 200, GE Healthcare) in PBS or 20 mM histidine buffer containing 150 mM NaCl (pH 6.0). Monomeric antibody fractions were pooled, concentrated (if necessary) using, for example, a MILLIPORE Amicon Ultra centrifuge concentrator (30 MWCO), frozen and stored at -20°C or -80°C. Some of the samples were used for subsequent analytical analysis of proteins and analytical characterization, for example, using SDS-PAGE, gel filtration (SEC) or mass spectrometry.

SDS-PAGE

NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions. Specifically, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and NuPAGE® MES running buffer (reconstituted gels with an antioxidant supplement for NuPAGE® running buffer) or MOPS (unreduced gels).

EC-DSN

The purity and integrity of the antibodies were analyzed by SDS-CE using Labchip microfluidic technology (PerkinElmer, USA). For this, 5 μl of the antibody solution was prepared for SDS-CE analysis using the HT protein expression reagent kit (Protein Express Reagent Kit) according to the manufacturer's instructions and analyzed using the LabChip GXII system using the HT Protein Express chip. Data was analyzed using LabChip GX software.

Analytical Gel Filtration

Gel filtration (SEC) to determine the aggregated and oligomeric state of the antibodies was performed using HPLC chromatography. In general, the method was as follows: protein-A-purified antibodies were applied to a Tosoh TSKgel G3000SW column in buffer containing 300 M NaCl, 50 mM KH2PO4/K2HPO4 (pH 7.5), in a Dionex Ultimate® system (Thermo Fischer Scientific) or into a Superdex 200 column (GE Healthcare) in 2 x PBS in a Dionex HPLC system. Eluted proteins were quantified based on UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151 1901 served as the standard.

Mass spectrometer

This section describes the characterization of bispecific antibodies with respect to their correct assembly. Expected primary structures were analyzed by mass spectrometry using electrospray ionization (ESI-MS) of the deglycosylated intact antibody and, in special variants, the deglycosylated/limitedly cleaved LysC antibody.

Antibodies were deglycosylated with N-glycosidase F in phosphate or Tris buffer at 37° C. for up to 17 hours using 1 mg/ml protein. Limited digests of LysC (Roche Diagnostics GmbH, Mannheim, Germany) were performed using 100 μg of deglycosylated antibody in Tris buffer (pH 8) at room temperature for 120 h or at 37° C. for 40 min, respectively. Prior to mass spectrometry, the samples were desalted by HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined by ESI-MS using a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

Example 1

Expression and purification

Bispecific antibodies were obtained according to the method described above in the section "Materials and General Methods".

Bispecific antibodies were purified from the supernatant by a combination of protein A affinity chromatography and gel filtration. The resulting products were characterized for their identity by mass spectrometry and for analytical properties such as SDS-CE purity, monomer content and stability.

Expected primary structures were analyzed by mass spectrometry using electrospray ionization (ESI-MS) of a deglycosylated intact antibody and a deglycosylated/plasmin-cleaved or alternatively deglycosylated/limitedly-cleaved LysC antibody according to the method described in the General Methods section.

Additional analytical methods (eg, thermostability methods, mass spectrometry, and functional evaluation) were used only after purification with protein A and SEC.

Example 2

Determination of Apl-40 fibril binding in vitro by ELISA Binding of bispecific antibodies to fibrillar Ap was assessed by ELISA assay. In general, the method was as follows: Maxisorb plates were sensitized using Ap(1-40) at a concentration of 7 μg/ml in PBS for 3 days at 37°C to obtain Abeta fibrillar protein, and then dried for 3 hours at RT . The plate was blocked with 1% CroteinC and 0.1% RSA (rat serum albumin) in PBS (blocking buffer) for 1 hour at RT, then washed once with wash buffer. Bispecific antibodies or controls were added at concentrations up to 100 nM in blocking buffer and incubated at 4° C. overnight. After four washings, constructs were detected by adding anti-human-IgG-HRP (Jackson Immunoresearch) at a 1:10,000 dilution in blocking buffer (1 h, RT), followed by 6 washes and incubation in TMB (Sigma). The absorbance was determined at 450 N after the color reaction was stopped with 1N. HCl.

Example 3

In Vitro Transferrin Receptor Binding Determination Binding of bispecific antibodies to the mouse transferrin receptor was assessed by FACS analysis using mouse myeloma cell line X63.AG8-563. If anti-A(3) antibodies show a definite tendency towards non-specific binding to AgS cells, then specific binding can be quantified by co-incubation with a 20-fold excess of anti-mouse TfR antibody. Cells were harvested by centrifugation, washed once with PBS and ⁵ x 104 cells. incubated with serial dilutions of 1.5 pM to 10 nM polypeptide fusions with or without addition of 200 nM anti-mouse TfR antibody in 100 µl RPMI/10% FCS for 1.5 h on ice After two washes with RPMI/10% FCS, cells were incubated with phycoerythrin-linked goat anti-human IgG (Jackson Immunoresearch) at a 1:600 dilution in RPMI/19% FCS for 1.5 h on ice. using a FACS-Array (Becton-Dickinson).

Example 4

Surface Plasmon Resonance Binding Assay to Evaluate Human TfR-Antibody Interaction

The binding experiment was performed using a BIAcore B 4000 device (GE Healthcare) equipped with a C1 sensor chip (GE Healthcare, cat. no. BR1005-35) pretreated with an anti-human Fab antibody (GE Healthcare, cat. no. 28-9583- 25) using standard amine coupling chemistry according to the supplier's manual.

For kinetic measurements, the antibody sample was immobilized by contacting for 60 s using a flow rate of 10 µl/min in phosphate buffered saline (pH 7.4) supplemented with 0.05% Tween 20 at 25°C. Recombinant labeled His6 human transferrin receptor (R&D systems cat. no. 2474-TR-050) was applied at increasing concentrations and the signal was monitored over time. It was found that at a flow rate of 30 μl/min, the average duration of the association time was 150 s, and the dissociation time was 600 s. Data were fitted using a 1:1 binding model (Langmuir isotherm).

Example 5

Staining of Native Human P-Amyloid Plaques in Alzheimer's Patient Brain Sections by Indirect Immunofluorescence Using a Bispecific Antibody Produced by the Method Presented in this Description

It is possible to test the ability of bispecific antibodies to stain native human (3-amyloid) plaques by immunohistochemical analysis using indirect immunofluorescence. It was possible to demonstrate specific and sensitive staining of genuine human (3-amyloid) plaques. Cryostat sections of unfixed tissue from the temporal cortex obtained after the death of patients diagnosed with Alzheimer's disease were labeled for indirect immunofluorescence A two step incubation was used to detect bound bispecific antibody which was detected with affinity purified goat anti-human (GAH555) IgG (H+L) conjugated with Alexa 555 dye (Molecular Probes). included unrelated human antibodies of the IgG1 subclass (Sigma) and an individually secondary antibody, all of which were negative.

Example 6

In vivo detection of p-amyloid plaques with a bispecific antibody obtained by the method presented in the present description in a mouse model of Alzheimer's disease

The bispecific antibody can be tested in the dual transgenic mouse model APP/PS2, the mouse model of related AD amyloidosis (Richards J. Neuroscience, 23, 2003, pp. 8989-9003), for the ability to immune-decorate (detect) amyloid-beta plaques in vivo. . This allows assessing the degree of penetration into the brain and binding to plaques of amyloid-r. The fusion polypeptide can be administered at various doses compared to naked anti-Ap monoclonal antibody and, after 6 days, perfuse animals with phosphate-bored saline, freeze brain samples on dry ice, and prepare for cryosectioning.

The presence of antibodies associated with amyloid-β plaques can be assessed using non-fixed cryostat sections using a single-label indirect immunofluorescence method using goat anti-human IgG (H+L) conjugated with Alexa555 (GAH555) dye (Molecular Probes), in concentrations of 15 µg/ml for 1 h at room temperature. Amyloid plaques can be counterstained by incubation with BAP-2, a mouse anti-Ap monoclonal antibody conjugated to Alexa 488, at a concentration of 0.5 μg/ml for 1 hour at room temperature. Microscopic preparations were embedded in fluorescent maintenance medium (S3023, Dako) and visualized using a confocal laser microscope.

Although some details of the invention have been described above, given for the purpose of illustration and example for ease of understanding, the description and examples should not be construed as limiting the scope of the invention. The description of all cited patent and scientific literature is expressly incorporated herein by reference in its entirety.

Example 7

Transfection of a stable cell line expressing a bispecific aHTH-DR5/FAP antibody with an expression vector containing a domain-swapped light chain expression cassette

A 0131 cell clone was transfected with a CrossLC expression vector containing a single crossover light chain expression cassette. Transfections were carried out using linearized DNA in a chemically defined medium using nucleofection (Ataha) and 0.6/1.2/2.4 pM (total) plasmid, obtaining 2×3 cell pools, which were subjected to differential selection.

Pools of transfected clones were selected in a chemically defined medium supplemented with glutamine at a concentration of 10 mmol/l and 250 nM MTX (for DHFR) plus 500 nM and 700 nM hygromycin B. After 3 weeks, the pools were analyzed by SDS-CE and immunohistochemistry (IHC) in in relation to the reduction of side peaks and the increase in the main peak.

Based on the results obtained, three pools were selected for serial dilution (LD), which were selected using 250nM MTX and 700nM HygB (0314, 0316, 0318), and each of them was sown in 3x384-well plates with a medium of a certain chemical composition, supplemented with glutamine at a concentration of 10 mmol/l and MTX at a concentration of 250 nmol/l and 700nM HygB.

After 1 week, supernatants from 3×384 well plates were tested for binding to DR5 and FAP using ELISA and (double) DR5-EAP binding ELISA methods. 158 clones with high titers and high reactivity for both antigens were selected and propagated starting from 24-well plates up to 6 wells, in which they were evaluated in the form of a four-day batch obtained in the experiment (“estimated titers of sequentially propagated culture”) with respect to target binding by ELISA and double-binding ELISA, growth, productivity and by-product profile were assessed by SDS-CE. Of these, 46 clones with titers up to 830 μg/mL and acceptable product quality were further characterized in 14-day fed-batch cultures using the Ambr15 system and analyzed for target binding, growth characteristics, and by-product profile by CE- SDS and IHC. Of these, 20 clones were selected and further evaluated by mass spectrometry (MS). Of these, 10 selected clones were cultured in shake flasks in a defined chemical medium and stored as PSB (broth containing peptone, sorbitol and bile salts).

Claims (30)

1. Pharmaceutical composition for the treatment of diseases, containing a multispecific antibody in an effective amount that belongs to the subclass of human IgG1, and a pharmaceutically acceptable carrier, and the specified antibody includes: - first light chain - the first heavy chain containing the first heteromonomeric Fc region polypeptide, - a second light chain and - a second heavy chain containing a second heteromonomeric Fc region polypeptide, and received as a result - culturing the mammalian cell in a culture medium where the mammalian cell is created by a) transfecting a mammalian cell with a first expression vector and one, two or three additional expression vectors, wherein the first expression vector contains exactly one nucleotide sequence that encodes a polypeptide of the multispecific antibody, and one, two or three additional expression vectors each contains at least two nucleotide sequences, each of which encodes a different polypeptide chain of the multispecific antibody, thus, that one, two or three additional expression vectors together contain all the nucleotide sequences necessary to encode the polypeptide chains of a multispecific antibody, where strictly one nucleotide sequence of the first expression vector is a nucleotide sequence that encodes a multispecific antibody light chain polypeptide, - isolating the multispecific antibody from the cell or culture medium, where the two polypeptide chains of the multispecific antibody contain a domain exchange, where the domain exchange is a CH1-CL crossover or a VH-VL crossover, where exactly one nucleic acid of the first expression vector encodes a light chain polypeptide with an exchange of multispecific antibody domains, where the multispecific antibody is a bivalent bispecific antibody, or a trivalent bispecific antibody, or a tetravalent bispecific antibody, where the bispecific antibody contains the T366W mutation in the CH3 domain of one heavy chain (“lump chain”) and the T366S, L368A, Y407V mutations in the CH3 domain of another heavy chain (“gap chain”) (numbering according to Kabat index EU). 2. A pharmaceutical composition according to one of claim 1, wherein step a) comprises co-transfection with a first expression vector and one, two or three additional expression vectors. 3. Pharmaceutical composition according to one of paragraphs. 1 and 2, wherein the mammalian cell is first transfected with one, two or three additional expression vectors and then transfected with the first expression vector. 4. Pharmaceutical composition according to one of paragraphs. 1-3, in which the mammalian cell stably expresses the multispecific antibody. 5. Pharmaceutical composition according to one of paragraphs. 1-4, wherein the mammalian cell is a CHO cell. 6. Pharmaceutical composition according to one of paragraphs. 1-5, in which the multispecific antibody is a bivalent bispecific antibody that contains a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the VL and VH variable domains of the second light chain and the second heavy chain are interchanged. 7. Pharmaceutical composition according to one of paragraphs. 1-6, in which the multispecific antibody is a bivalent bispecific antibody that contains a) the first light chain and the first heavy chain of an antibody that specifically binds to the first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, in which the CL and CH1 constant domains of the second light chain and the second heavy chain are exchanged for each other. 8. Pharmaceutical composition according to one of paragraphs. 1-7, in which the multispecific antibody is a trivalent bispecific antibody that contains a) the first light chain and the first heavy chain of a full-length antibody that specifically binds to the first antigen, b) a second full-length antibody heavy chain that, when paired with the first light chain, specifically binds to the first antigen, and c) A Fab fragment that specifically binds to a second antigen, fused via a peptide linker to the C-terminus of one of the heavy chains specified in a) or b), in which the CL and CH1 constant domains of the second light chain and the second heavy chain are replaced Each other. 9. Pharmaceutical composition according to one of paragraphs. 1-8, wherein the multispecific antibody is an antibody with a low/reduced amount of product-related impurities.

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