Bivalent bispecific antibody and method for its production

Abstract

FIELD: biotechnology.SUBSTANCE: bivalent bispecific antibody is presented, which includes a first light chain and a first heavy chain of an antibody, specifically binding to a first antigen, and a second light chain and a heavy chain of an antibody, specifically binding to a second antigen, in which the variable domains VL and VH are replaced by each other, wherein VL and VH have a modification at the C-terminus of FR4. A method for producing said antibody is also disclosed.EFFECT: invention makes it possible to efficiently produce bispecific antibodies.17 cl, 9 tbl, 9 dwg, 10 ex

Description

[0001] Field of technology to which the invention relates

[0002] The present invention relates to the field of biotechnology, namely to a bivalent bispecific antibody that includes a first light chain and a first heavy chain of an antibody that specifically bind to a first antigen, and a second light chain and a heavy chain of an antibody that specifically bind to a second antigen, in which the variable domains VL and VH are replaced by each other, wherein VL and VH have a modification at the C-terminus of FR4, as well as to a method for producing it.

[0003] Technology Level

[0004] Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have proven their value as effective drugs for the treatment of a number of disorders and diseases.

[0005] Natural human antibody molecules consist of two heavy chain homodimers, each of which forms a heterodimer partnered with two identical light chain molecules. Conventional monoclonal antibodies, as whole molecules, consist of bivalent ("two-armed") heterodimers of heavy and light chains.

[0006] Diseases often arise from multiple pathologies and are accompanied by numerous comorbidities. Bispecific antibodies have the ability to bind and thereby neutralize two distinct antigens per antibody molecule. The potential for significantly improving the therapeutic properties (and value) of drugs compared to monoclonal antibodies has made bispecific antibodies an active area of ​​research. Over the past two decades, numerous solutions for engineered versions of bispecific antibodies have been described in the scientific literature, as described in Brinkmann, U and RE Kontermann, 2017, The Making of Bispecific Antibodies, MAbs; 209 Feb / Mar; 9(2): 182–212, doi: 10.1080 / 19420862.2016.1268307.

[0007] There are many ways to create molecules with mixed antigen-binding domains, that is, with antigen-binding domains that are distinct from each other. However, each of these methods has its own drawbacks.

[0008] Chemical cross-linking is a labor-intensive process, as the relevant fragments must be purified of homodimers and other unwanted byproducts. Furthermore, chemical modification steps can alter the integrity of proteins, reducing their stability. Therefore, this method is often ineffective and can lead to loss of antibody activity.

[0009] A cell fusion method (e.g., hybridoma production) involves the random assembly of two heavy and two light chains, resulting in 10 antibody combinations. Target heteromultimeric antibodies constitute a small fraction of the antibodies produced in this manner. Isolation of the target heteromultimeric proteins significantly reduces product yield and increases production costs.

[0010] Recombinant DNA techniques are used to create various heteromultimeric antibodies, such as single-chain Fv fragments, diabodies, and others that lack an Fc fragment. The main drawback of this type of antibody molecule is the lack of an Fc domain and, consequently, the inability of such an antibody to initiate effector functions (such as complement activation, Fc receptor binding, etc.). Therefore, a bispecific antibody containing a functional Fc domain is required.

[0011] Recombinant DNA techniques are also used to create bispecific antibodies that link in a knob-into-hole fashion. See International Patent Applications WO 9627011 and WO 9850431, and Merchant AM ET ALL., An efficient route to human bispecific IgG, Nat Biotechnol. 1998 Jul; 16(7):677–81. One limiting factor in the use of this technique is that the light chains of the two parent antibodies must be identical to prevent mispairing and the formation of unwanted and / or inactive molecules when expressed in the same cell.

[0012] The purity of bispecific antibody product depends on two factors:

[0013] a) heterodimeric assembly of two different heavy chains coexpressed in a cell, and

[0014] b) correct pairing of two different light chains with the corresponding heavy chains.

[0015] Using Knob-into-Holes technology to create bispecific antibodies solves the problem of correctly assembling two different heavy chains coexpressed in a cell. However, using Knob-into-Holes alone to create bispecific antibodies only achieves a yield of approximately 25% of correctly assembled bispecifics, as the problem of correctly pairing two different light chains with their corresponding heavy chains remains unresolved.

[0016] The problem of correctly pairing two different light chains with their corresponding heavy chains is solved in various ways:

[0017] 1. Use of the same light chain in the first and second antigen-binding parts of the antibody (Van Blarcom T. et al., Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies, MAbs. 2018 Feb / Mar; 10(2):256-268. doi: 10.1080 / 19420862.2017.1406570).

[0018] A drawback of this solution is its limited universality, as it is not always possible to select a light chain suitable for both valences. Furthermore, when substituting amino acids in the light chain to optimize the properties of the antigen-binding fragment, the substitutions affect both valences. This may disrupt antibody binding to the second antigen.

[0019] 2. Use of single-chain formats, i.e. formats where the light and heavy chains of the antigen-binding fragment specific to the first antigen are connected to each other via a linker of several amino acids.

[0020] This format has technological drawbacks, as it uses linkers to either fuse the core antibody structure (IgA, IgD, IgE, IgG, or IgM) to an additional binding protein (e.g., scFv or scFab), or to fuse, for example, the light and heavy variable domains (VH and VL) of an scFv or the light chain (VL-CK (or CL)) to VH-CH1 of an scFab. Linkers can pose therapeutic challenges. In fact, these foreign peptides can elicit an immune response against the linker itself or the junction between the protein and the linker. Furthermore, the flexible nature of these peptides and their mobility makes them more susceptible to proteolytic cleavage, which can lead to poor stability, aggregation, and increased immunogenicity of the antibody.

[0021] 3. Modification of the CH1-CK domains in a bispecific antibody, which will allow for altering the interaction interface in bispecific antibody expression technologies to prevent inappropriate light chain association. For example, patent document WO 2017059551 describes various amino acid substitutions in CH1 and / or CK that promote preferential pairing of the desired heavy chain with the desired light chain.

[0022] Despite the above-mentioned various bispecific antibody expression technologies, there is still a need in the art for improved bispecific antibody product purity and a scalable manufacturing solution for producing properly assembled bispecific antibodies.

[0023] Disclosure of the essence of the invention

[0024] The new format of bivalent bispecific antibodies developed by the authors unexpectedly enables the production of a high-yield product with the correct assembly of two different heavy chains coexpressed in the cell and the correct pairing of two different light chains with the corresponding heavy chains. This format of bivalent bispecific antibodies comprises a first light and first heavy chain specifically binding to a first antigen, and a second light and second heavy chain specifically binding to a second antigen, in which the variable domains VL and VH are exchanged for each other, with VL and VH having a modification at the C-terminus of FR4. A technology for producing these bispecific antibodies was also developed.

[0025] The above results consequently reduce production costs and lead to a scalable production solution for producing properly assembled bispecific antibodies.

[0026] General Definitions and General Methods

[0027] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

[0028] Furthermore, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. The generally used classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and are widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as commonly practiced in the art, or as described herein.

[0029] The term “KD” in this specification refers to the affinity constant (or equilibrium constant) which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed as a molar concentration (M).

[0030] "Binding affinity" typically refers to the strength of the combined non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can typically be represented by the affinity constant (KD). It is desirable that the KD value is approximately 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less. The affinity can be measured by conventional methods known in the art, including the methods described herein.Low-affinity antibodies typically bind to antigen slowly and tend to dissociate easily, whereas high-affinity antibodies typically bind to antigen more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present invention.

[0031] The term "Kd," "Koff," or "kdis" refers to the dissociation rate constant of a specific interaction between a binding molecule and an antigen. The dissociation rate constant koff can be measured using biolayer interferometry, such as the Octet™ system.

[0032] The term "Ka", "kon" or "on-rate" refers to the association rate constant.

[0033] In the present description and in the following claims, unless the context otherwise requires, the words "include" and "comprise" or variations thereof such as "includes," "including," "comprises," or "containing" are to be understood as including the stated whole or group of wholes, but not excluding any other whole or group of wholes.

[0034] Bivalent bispecific antibody

[0035] The present invention relates to a bivalent bispecific antibody.

[0036] The antibody of the invention is a monoclonal antibody.

[0037] The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and secreted by a distinct clonal population of cells.

[0038] The antibody of the invention is a recombinant antibody.

[0039] The term "recombinant antibody" means an antibody that is expressed in a cell or cell line containing a nucleotide sequence(s) that encodes the antibody, wherein said nucleotide sequence(s) is(are) not associated with the cell in nature.

[0040] The divalent bispecific antibodies of the invention are isolated antibodies.

[0041] The term "isolated" as used to describe various antibodies described herein means an antibody that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Contaminants from the natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Typically, an isolated polypeptide is obtained through at least one purification step.

[0042] In one aspect, the present invention relates to a bivalent bispecific antibody that specifically binds to a first antigen and a second antigen, wherein said antibody comprises:

[0043] a) a first light chain and a first heavy chain of an antibody that specifically bind to a first antigen,

[0044] wherein the first light chain comprises a light chain variable domain VL and a light chain constant domain, and

[0045] wherein the first heavy chain comprises a heavy chain variable domain VH and heavy chain constant domains of an antibody comprising a first heavy chain constant domain CH1 and an Fc fragment monomer comprising a second CH2 and a third CH3 heavy chain constant domains; and

[0046] b) a second light chain and a second heavy chain of an antibody that specifically bind to a second antigen,

[0047] wherein the second light chain comprises:

[0048] 1) VH variable domain with a deletion of 4 amino acids at the C-terminus of FR4 of the VH variable domain (VH -4 ),

[0049] 2) amino acid sequence with EIK (Glu-Ile-Lys), and

[0050] 3) light chain constant domain; and

[0051] wherein the second heavy chain comprises:

[0052] 1) VL variable domain with a deletion of 3 amino acids at the C-terminus of FR4 of the VL variable domain (VL -3 ),

[0053] 2) the amino acid sequence with SEQ ID NO: 1 (TVSS), and

[0054] 3) constant domains of the heavy chain of the antibody, including the first constant domain CH1 of the heavy chain and the monomer of the Fc fragment containing the second CH2 and third CH3 constant domains of the heavy chain.

[0055] In some embodiments, the bivalent bispecific antibody comprises a second light chain having the following structure: VH -4 - EIK - CL.

[0056] In some embodiments, the divalent bispecific antibody comprises a second heavy chain having the following structure: VL -3 - SEQ ID NO: 1 - CH1-CH2-CH3.

[0057] The term "antibody" or "immunoglobulin" (Ig), as used herein, includes whole antibodies. The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Five types of mammalian antibody heavy chains are known, designated by the Greek letters α, δ, ε, γ, and μ. (Janeway C.A., Jr. et al., Immunobiology, 5th ed., Garland Publishing, 2001). The type of heavy chain present determines the class of the antibody; These chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades RA, Pflanzer RG, Human Physiology, 4th ed., Thomson Learning, 2002).The different heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, and μ and ε consist of approximately 550 amino acids. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α, and δ contain a constant region, which consists of three constant domains CHI, CH2, and CH3 (arranged in a row) and a hinge region that confers flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, pp. 89-99); The μ and ε heavy chains contain a constant region, which consists of four constant domains: CH1, CH2, CH3, and CH4 (Janeway, C.A., Jr., et al., Immunobiology, 5th ed., Garland Publishing, 2001). In mammals, only two types of light chains are known, designated lambda (λ) and kappa (κ). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.The approximate length of the light chain is 211-217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the CL constant domain is preferably a C-kappa (κ).

[0058] The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), located between more conserved regions, called framework regions (FRs). Framework regions are responsible for acting as a scaffold for the CDRs and help maintain the overall structure of the four variable domains of an antibody. FRs are highly conserved regions of the variable portion of an antibody, as they ensure proper folding of the antibody, which helps stabilize the CDRs. Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen.Constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0059] The term "antigen-binding portion" of an antibody or "antigen-binding fragment" (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. An example of a binding fragment included in the term "antigen-binding portion" of an antibody includes a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains.

[0060] “Kabat nomenclature” or “Kabat nomenclature” is used in this application to refer to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than the rest of the amino acid residues in the variable regions of the heavy and light chains of an antibody (Kabat et al. Arm. NY Acad. Sci., 190:382-93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0061] The antibody of the present invention that "binds" a target antigen is an antibody that binds the antigen with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent by targeting a protein or a cell or tissue expressing the antigen, and has little cross-reaction with other proteins. In such embodiments, the degree of binding of the antibody to a non-target protein (an "off-target protein") is less than 10% of the binding of the antibody to the specific target protein, as determined by fluorescence-activated cell sorting (FACS), radioimmunoprecipitation assay (RIA), or ELISA.With respect to the binding of an antibody to a target molecule, the term "specific binding" or the expressions "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target polypeptide means binding that is measurably different from non-specific interaction.

[0062] Specific binding can be determined quantitatively, for example, by determining the binding of a molecule relative to the binding of a control molecule. For example, specific binding can be determined by a competitive reaction with another molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target.As used herein, the term "specific binding" or the expressions "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target polypeptide may be characterized by an example of a molecule having a KD to the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or higher.In one embodiment of the invention, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide.

[0063] The term "bispecific antibody" means an antibody comprising antigen-binding domains that are capable of specifically binding to two different epitopes on a single biological molecule or are capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having "dual specificity" or as being an antibody with "dual specificity."

[0064] The crystallizable fragment of immunoglobulin (Fc region) is the terminal portion of the immunoglobulin molecule that interacts with the Fc receptor on the cell surface and with certain proteins of the complement system. This property allows antibodies to activate the immune system. The Fc region of IgG, IgA, and IgD isotypes consists of two identical protein fragments, the second and third constant domains of the two heavy chains, respectively. In the case of IgM and IgE isotypes, Fc contains three heavy chain constant domains (the CH2, CH3, and CH4 domains) in each polypeptide chain.

[0065] By "Fc fragment monomer" is meant the Fc region from the second and third constant domains of either of the two heavy chains.

[0066] In some embodiments of the invention, the bivalent bispecific antibody comprises a light chain constant domain of the antibody, which is selected from a light chain kappa constant domain (CK) or a light chain lambda constant domain (CL).

[0067] Only two types of light chains are known in mammals, designated lambda (λ) and kappa (κ). The constant domain of the lambda light chain is designated CL, and the constant domain of the kappa light chain is designated CK.

[0068] In some embodiments of the invention, the divalent bispecific antibody comprises a first antigen that is a cancer antigen.

[0069] In some embodiments of the invention, the divalent bispecific antibody comprises a first cancer antigen that is selected from the group consisting of BCMA or GD2.

[0070] B-cell maturation antigen (BCMA; TNFRSF17 and CD269) is a member of the tumor necrosis factor (TNF) receptor superfamily.

[0071] GD2 refers to ganglioside GD2.

[0072] In some embodiments of the invention, the divalent bispecific antibody comprises a second antigen that is a T cell receptor.

[0073] In some embodiments, the divalent bispecific antibody comprises a T cell receptor that is CD3.

[0074] CD3 (cluster of differentiation 3) is a molecule present on the surface of T lymphocytes.

[0075] In some embodiments of the invention, the divalent bispecific antibody comprises an Fc region that is related to human IgG.

[0076] In some embodiments of the invention, the divalent bispecific antibody comprises an Fc region isotype that is human IgG1.

[0077] In some embodiments of the invention, the bivalent bispecific antibody comprises the mutations L234A and L235A according to the amino acid numbering of EU antibodies in the CH2 region.

[0078] In some embodiments of the invention, the bivalent bispecific antibody comprises mutations M252Y, S254T, T256E according to the amino acid numbering of EU antibodies in the CH2 region.

[0079] In some embodiments, the bivalent bispecific antibody comprises a deletion of 446G and 447K according to the amino acid numbering of EU antibodies in the CH3 region.

[0080] In some embodiments of the invention, a bivalent bispecific antibody comprises a third CH3 constant domain of one heavy chain and a third constant domain of another heavy chain that are in contact with each other at surfaces that are altered to form the bivalent bispecific antibody, wherein these alterations in the third constant domains of the heavy chains are substitutions to allow for heterodimerization.

[0081] In some embodiments of the invention, the bivalent bispecific antibody comprises CH3 domains of the antibody that are further altered by introducing cysteine ​​as an amino acid at the corresponding positions of each CH3 domain such that a disulfide bridge can be formed between both CH3 domains.

[0082] In some embodiments, the bivalent bispecific antibody comprises a CH3 domain of one heavy chain that is altered to form a Knob, and a CH3 domain of the other heavy chain that is altered to form a Hole, or vice versa.

[0083] "Knobs-into-holes" (knob-hole interaction) is an approach that circumvents the problem of mismatched side products. This approach aims to enhance the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the interface at the contact site. On one chain, large amino acids were replaced with amino acids with short side chains to create a "hole." Conversely, amino acids with larger side chains were introduced into the other CH3 domain, creating a "knob." By co-expressing these two heavy chains, a high yield of the heterodimeric (knob-hole) formation was obtained relative to the homodimeric (hole-hole or knob-knob) formation (WO 9627011 and WO 9850431, and the article by Merchant AM ET ALL., An efficient route to human bispecific IgG, Nat Biotechnol. 1998 Jul; 16(7):677-81).

[0084] In some embodiments, the bivalent bispecific antibody comprises a CH3 domain of one heavy chain that has amino acid substitutions S354C / T366W, and a CH3 domain of the other heavy chain has amino acid substitutions Y349C / T366S / L368A / Y407V.

[0085] In some embodiments, the bivalent bispecific antibody comprises a CH3 domain of one heavy chain that has amino acid substitutions Y349C / T366S / L368A / Y407, and a CH3 domain of the other heavy chain has amino acid substitutions S354C / T366W.

[0086] In some embodiments of the invention, the divalent bispecific antibody comprises a first antigen that is BCMA and a second antigen that is CD3.

[0087] In some embodiments of the invention, the divalent bispecific antibody comprises a first antigen that is GD2 and a second antigen that is CD3.

[0088] The application materials provide the following antibodies: 12-001, 12-002, 12-003, 12-004, 12-005, 12-006, 13-001, 13-002, 13-003, 13-004, 13-005, 13-006, 02-002, 03-003, which are described in Table 1.

[0089] These antibodies are provided for illustrative purposes to demonstrate the viability of the bivalent bispecific antibody format of the invention, as well as its unexpected properties. These antibodies should not be construed as limiting the bivalent bispecific antibody of the invention in any way.

[0090] The yield parameters of the product with the correct assembly of two different heavy chains and the correct pairing of two different light chains with the corresponding heavy chains are independent of the variable regions of the heavy and light chains of the bispecific antibody and their specificities to antigens.

[0091] The divalent bispecific antibodies of the invention can be used for the treatment of various diseases, in particular oncological diseases.

[0092]

[0093]

[0094]

[0095] Method for producing antibodies

[0096] In one aspect, the present invention relates to a method for producing any of the above-mentioned bivalent bispecific antibodies, which comprises the following steps:

[0097] a) host cell transformation

[0098] - expression vectors that contain nucleic acid molecules encoding the first light chain and the first heavy chain of a bispecific antibody,

[0099] - expression vectors that contain nucleic acid molecules encoding the second light chain and the second heavy chain of the bispecific antibody,

[0100] b) culturing the host cell under conditions that allow the synthesis of said bivalent bispecific antibody; and

[0101] c) isolating the said divalent bispecific antibody from the cell culture.

[0102] The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. In some embodiments, vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors).In other embodiments, vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, thereby replicating along with the host gene. Furthermore, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors" or "expression vectors").

[0103] The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" refer not only to the specific cell claimed but also to the progeny of such a cell. Because modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein.

[0104] Transformation can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, transfection with a complex of a nucleic acid and a positively charged polymer, transfection with a nucleic acid-calcium phosphate precipitate, polybrene-mediated transfection, protoplast fusion, transfection with liposome-encapsulated polynucleotides, and direct microinjection of DNA into nuclei. Additionally, nucleic acid molecules can be introduced into mammalian cells via viral vectors.

[0105] Mammalian cell lines used as transformation hosts are well known in the art and include a variety of immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by determining which cell lines have high expression levels and provide the desired characteristics of the produced protein. Other cell lines that can be used include insect cell lines such as Sf9 or Sf21 cells.When recombinant expression vectors encoding the above-mentioned bispecific antibody or a portion thereof are introduced into mammalian host cells, the above-mentioned bispecific antibody is produced by culturing the host cells for a time sufficient to express the above-mentioned bispecific antibody of the invention in the host cells or, more preferably, by isolating the above-mentioned bispecific antibody into a nutrient medium in which the host cells are grown. The above-mentioned bispecific antibody can be isolated from the nutrient medium using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0106] Furthermore, the production level of the bispecific antibody of the present invention from a producing cell line can be enhanced using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is widely used to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with patents EP 0216846, 0256055, 0323997, and 0338841.

[0107] It is likely that the bispecific antibody of the invention will differ in its glycosylation profile in different cell lines or host cells. However, the bispecific antibody described herein is part of the present invention regardless of the glycosylation state of the binding molecules and, in general, regardless of the presence or absence of post-translational modifications.

[0108] The above host cell does not refer to the host cell obtained using human embryos.

[0109] The above host cell does not refer to a host cell obtained by modifying the genetic integrity of human germline cells.

[0110] The present invention relates to methods for producing bivalent bispecific antibodies according to the present invention. One embodiment of the invention relates to a method for producing bivalent bispecific antibodies as defined herein, comprising obtaining a recombinant host cell capable of expressing a bivalent bispecific antibody, culturing said host cell under conditions suitable for expressing bivalent bispecific antibodies, and isolating the resulting bivalent bispecific antibodies. The bivalent bispecific antibody obtained by such expression in such recombinant host cells is referred to herein as a "bivalent bispecific antibody."

[0111] Brief description of drawings

[0112] Figure 1 is a schematic representation of the structure of the anti-GD2 / anti-CD3 bispecific antibody molecule in the classical knob-into-hole antibody format, where the following are indicated:

[0113] VH - variable domain of the heavy chain;

[0114] VL - variable domain of light chain;

[0115] CH1 - the first constant domain of the heavy chain;

[0116] CH2 - the second constant domain of the heavy chain;

[0117] CH3 - the third constant domain of the heavy chain;

[0118] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain.

[0119] Figure 2 is a schematic representation of the structure of the molecule of the bispecific anti-BCMA / anti-CD3 antibody, in the anti-CD3 part of which the variable domains VL and VH are replaced by each other, while VL and VH have a modification at the C-terminus of FR4, where the following are indicated:

[0120] VH -4- variable domain of the heavy chain with a deletion of 4 amino acids at the C-terminus of FR4 of the variable domain;

[0121] VL -3 - variable domain of the light chain with a deletion of 3 amino acids at the C-terminus of FR4 of the variable domain;

[0122] CH1 - the first constant domain of the heavy chain;

[0123] CH2 - the second constant domain of the heavy chain;

[0124] CH3 - the third constant domain of the heavy chain;

[0125] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain;

[0126] 2 - additional 4 terminal amino acids (TVSS) at the C-terminus of FR4 of the VL variable domain -3 ;

[0127] 3 - additional 3 terminal amino acids (EIK) at the C-terminus of FR4 of the VH variable domain -4 .

[0128] Figure 3 is a schematic representation of the structure of the molecule of the bispecific anti-GD2 / anti-CD3 antibody, in the anti-CD3 part of which the variable domains VL and VH are replaced by each other, while the replacement of VL and VH occurs incompletely, where the following are indicated:

[0129] VH - variable domain of the heavy chain;

[0130] VL - variable domain of light chain;

[0131] CH1 - the first constant domain of the heavy chain;

[0132] CH2 - the second constant domain of the heavy chain;

[0133] CH3 - the third constant domain of the heavy chain;

[0134] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain;

[0135] 2 - additional 4 terminal amino acids (TVSS) at the C-terminus of FR4 of the VL variable domain -3 ;

[0136] 3 - additional 3 terminal amino acids (EIK) at the C-terminus of FR4 of the VH variable domain -4 .

[0137] Figure 4 is a schematic representation of the structure of the molecule of the bispecific anti-BCMA / anti-CD3 antibody, in the anti-BCMA part of which the variable domains VL and VH are replaced by each other, where the following are indicated:

[0138] VH - variable domain of the heavy chain;

[0139] VL - variable domain of light chain;

[0140] CH1 - the first constant domain of the heavy chain;

[0141] CH2 - the second constant domain of the heavy chain;

[0142] CH3 - the third constant domain of the heavy chain;

[0143] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain.

[0144] Figure 5 is a schematic representation of the structure of the molecule of the bispecific anti-BCMA / anti-CD3 antibody, in the anti-CD3 portion of which the Fab domains of VL-CK and VH-CH1 are replaced by each other, where the following are indicated:

[0145] VH - variable domain of the heavy chain;

[0146] VL - variable domain of light chain;

[0147] CH1 - the first constant domain of the heavy chain;

[0148] CH2 - the second constant domain of the heavy chain;

[0149] CH3 - the third constant domain of the heavy chain;

[0150] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain.

[0151] Figure 6 is a schematic representation of the structure of the molecule of the bispecific anti-BCMA / anti-CD3 antibody, in the anti-BCMA part of which the Fab domains VL-CK and VH-CH1 are replaced by each other, where the following are indicated:

[0152] VH - variable domain of the heavy chain;

[0153] VL - variable domain of light chain;

[0154] CH1 - the first constant domain of the heavy chain;

[0155] CH2 - the second constant domain of the heavy chain;

[0156] CH3 - the third constant domain of the heavy chain;

[0157] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain.

[0158] Figure 7 is a schematic representation of the structure of the molecule of the bispecific anti-BCMA / anti-CD3 antibody, in the anti-CD3 part of which the constant domains CK and CH1 are replaced by each other, where the following are indicated:

[0159] VH - variable domain of the heavy chain;

[0160] VL - variable domain of light chain;

[0161] CH1 - the first constant domain of the heavy chain;

[0162] CH2 - the second constant domain of the heavy chain;

[0163] CH3 - the third constant domain of the heavy chain;

[0164] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain.

[0165] Figure 8 is a schematic representation of the structure of the molecule of the bispecific anti-BCMA / anti-CD3 antibody, in the anti-BCMA part of which the constant domains CK and CH1 are replaced by each other, where the following are indicated:

[0166] VH - variable domain of the heavy chain;

[0167] VL - variable domain of light chain;

[0168] CH1 - the first constant domain of the heavy chain;

[0169] CH2 - the second constant domain of the heavy chain;

[0170] CH3 - the third constant domain of the heavy chain;

[0171] 1 - the position of the knob-into-hole mutations S354C and T366W, as well as Y349C, T366S, L368A and Y407V - in the third constant domain of the heavy chain.

[0172] Figure 9 is a gradient polyacrylamide gel electropherogram of the bispecific antibody that specifically binds to CD3 and GD2, where:

[0173] 1) Protein molecular weight marker;

[0174] 2) 02-002 2 mcg under reducing conditions;

[0175] 3) 02-002 2 mcg under non-reducing conditions.

[0176] Examples

[0177] For a better understanding of the invention, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0178] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example to avoid ambiguity, those skilled in the art, based on the teachings disclosed herein, will readily understand that certain changes and modifications can be made without departing from the spirit and scope of the accompanying embodiments of the invention.

[0179] Materials and General Methods

[0180] General information concerning the nucleotide sequences of the light and heavy chains of human immunoglobulin is given in: Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The amino acids of the antibody chains are numbered according to the EU numbering (Edelman GM et al., Proc. Natl. Acad. Sci. USA 63, 1969, ee. 78-85; Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0181] Recombinant DNA methods

[0182] DNA manipulations were performed using standard methods 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 manufacturers' instructions.

[0183] Gene synthesis

[0184] The desired gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments ranging from 300 to 1400 bp in length, flanked by unique restriction sites, were assembled by annealing and ligation of the oligonucleotides, including PCR amplification and subsequent cloning through the restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0185] DNA Sequencing

[0186] DNA sequences were determined by Sanger sequencing.

[0187] DNA and protein sequence analysis and sequence data processing

[0188] Unipro UGENE version 1.29 and SnapGene Viewer software were used to create, map, analyze, annotate, and illustrate sequences.

[0189] Expression vectors

[0190] For expression of the antibodies described in the application materials, variants of expression plasmids designed for antibody expression in prokaryotic cells (E. coli) and short-term expression in eukaryotic cells (e.g., CHO cells) were used. In addition to the antibody expression cassette, the vectors contained a replication origin site, ensuring replication of the plasmid in E. coli, and genes conferring resistance to various antibiotics (e.g., ampicillin and kanamycin) in E. coli.

[0191] Fusions of genes containing the described antibody chains, as described below, were accomplished by PCR and / or gene synthesis and assembly using known recombination methods and procedures by joining corresponding nucleic acid segments, for example, by using unique restriction sites in the corresponding vectors. Subcloned nucleotide sequences were confirmed by DNA sequencing. For short-term transfections, large quantities of plasmids were generated by obtaining plasmids from transformed E. coli cultures.

[0192] Example 1. Design and production of genetic constructs of bispecific antibodies that specifically bind to CD3 and the BCMA tumor antigen

[0193] To obtain bispecific antibodies, various variants of constructs were developed in which regions were replaced.

[0194] Options: 12-001, 12-002, 12-003, 12-004, 12-005, 12-006, 13-001, 13-002, 13-003, 13-004,13-005, 13-006.

[0195] For candidates 12-001, 12-003, 13-001, 13-004, the format of replacing Fab chains VH-CH1 with VL-CK is used, while for candidates 12-001 and 13-001, the replacement occurs for anti-CD3 chains (Figure 5), and for candidates 12-003 and 13-004, for anti-BCMA chains (Figure 6).

[0196] For candidates 12-002, 12-004, 13-002, 13-005, the CH1 to CK substitution format is used, while for candidates 12-002 and 13-002 (Figure 7), the substitution occurs for anti-CD3 chains, and for candidates 12-004 and 13-005 (Figure 8), for anti-BCMA chains.

[0197] For candidates 12-005 and 13-003 (Figure 2), a VL to VH substitution format for anti-CD3 chains is used, namely, a VL variable domain with a deletion of 3 amino acids at the C-terminus of FR4 of the VL variable domain (VL -3 ) and additional amino acids of SEQ ID NO: 1 (TVSS) at the C-terminus are replaced with the VH variable domain with a deletion of 4 amino acids at the C-terminus of FR4 of the VH variable domain (VH -4) and additional amino acids EIK (Glu-Ile-Lys) at the C-terminus; and where the substitution of VL and VH occurs to obtain the following heavy and light chains:

[0198] VL -3 - SEQ ID NO: 1 - CH1-CH2-CH3, where VL -3 This is a VL variable domain with a deletion of 3 amino acids at the C-terminus of FR4 of the VL variable domain, and

[0199] VH -4 - EIK - CL, where VH -4 VH variable domain with a deletion of 4 amino acids at the C-terminus of FR4 of the VH variable domain.

[0200] For candidates 12-006 and 13-006, the VL and VH substitution format for anti-BCMA chains is used (Figure 4).

[0201] For all candidates, the heavy chain constant domains are human IgG1 HC, with the CH3 domain of one heavy chain having the amino acid substitutions S354C / T366W, and the CH3 domain of the other heavy chain having the amino acid substitutions Y349C / T366S / L368A / Y407V, for proper heterodimerization of the heavy chains of the bispecific antibody.

[0202] The variable domain of the light chain VL is fused to the constant domain of the light chain CK of human IgG1.

[0203] The heavy and light chain genes of antibodies to BCMA and CD3, as well as the dimerization domain genes, were synthesized de novo. Fusion of the heavy chain variable domain and the human IgG1 Fc fragment, the light chain variable domain and CK, or the variable domains and dimerization domains was achieved by PCR and / or gene synthesis and assembly using established restriction and ligation methods.

[0204] The heavy and light chain genes of the antibody were cloned into pEE-Hc and pEE-Lc plasmids to produce IgG1 protein in mammalian cells.

[0205] The cloned nucleotide sequences were confirmed by DNA sequencing. The resulting plasmids were produced in the required quantities in E. coli cells and purified using a plasmid DNA isolation kit.

[0206] The obtained genetic constructs were transferred to transient protein production in the CHO cell line.

[0207] Example 2. Production of full-length bispecific antibodies that specifically bind to CD3 and the BCMA tumor antigen

[0208] Full-length antibodies 12-005, 12-006, 13-001, 13-002, 13-003, 13-004, 13-005, 13-006 were produced in cells of a permanent cell line derived from Chinese hamster ovary cells (CHO-T line) using transient transfection in duplicate. Suspension cultivation was carried out in bioreactors on an orbital shaker, using serum-free media. For transient expression, cells at a concentration of 2-2.2×10 6 Cells / ml were transfected using linear polyethyleneimine. The DNA / PEI ratio was 1:7. On day 10 of culture, the cell suspension was centrifuged at 2000 g for 15 minutes and filtered through a 0.22 μm filter.

[0209] Antibodies were purified by affinity chromatography on columns using a robotic station. The column was equilibrated with 50 mM NaPB (sodium phosphate buffer), 150 mM NaCl (pH 7.5), filtered culture liquid with antibodies was applied, then the column was washed with 8 column volumes of 50 mM NaPB, 150 mM NaCl, and 4 column volumes of 50 mM NaPB (pH 7.5). Protein elution was performed with a solution of 50 mM NaPB, 100 mM NaCl (pH 3) in 6 column volumes. The resulting samples were neutralized with 25 μl of 1 M phosphate buffer (pH 8).

[0210] PAGE was used to control the purity of the bispecific antibodies. Electrophoresis was performed on a 7.5% polyacrylamide gel under denaturing, non-reducing conditions. Protein purity was determined based on band staining intensity at a protein loading of 10 μg per lane.

[0211]

[0212] The bispecific antibodies in the new format of the invention (12-005 and 13-003) unexpectedly demonstrated the best productivity parameters (≥248), purification yield (≥3.4), and high monomer yield (≥64.3%) among bispecific antibodies in other formats studied. Therefore, these anti-CD3 chains were used to produce other antibodies.

[0213] Example 3. Design and production of genetic constructs of bispecific antibodies that specifically bind to CD3 and tumor antigen GD2.

[0214] The chains of the second antigen-binding fragment, which specifically binds to GD2, of any bispecific antibody selected from 02-002 or 03-003, comprise the variable domains of the heavy and light chains fused to the constant domains of the heavy and light chains, respectively.

[0215] The heavy chain constant domains are represented by human IgG1 HC, with the CH3 domain of one heavy chain having the amino acid substitutions S354C / T366W, and the CH3 domain of the other heavy chain having the amino acid substitutions Y349C / T366S / L368A / Y407V, to ensure proper heterodimerization of the heavy chains of the bispecific antibody. The variable domain of the light chain VL is fused to the constant domain of the human IgG1 CK light chain.

[0216] The chains of the first antigen-binding fragment that specifically binds to CD3 of any bispecific antibody selected from 02-002 or 03-003 comprise heavy and light chain variable domains, and candidate 03-003 also contains deletions 446G, 447K at the C-terminus of Fc to reduce antibody heterogeneity.

[0217] Also for candidates 02-002, 03-003 (Figure 3), the VL to VH substitution format for anti-CD3 chains is used, namely the VL variable domain with a deletion of 3 amino acids at the C-terminus of FR4 of the VL variable domain (VL -3) and additional amino acids of SEQ ID NO: 1 (TVSS) at the C-terminus are replaced with the VH variable domain with a deletion of 4 amino acids at the C-terminus of FR4 of the VH variable domain (VH -4 ) and additional amino acids EIK (Glu-Ile-Lys) at the C-terminus; and where the substitution of VL and VH occurs to obtain the following heavy and light chains:

[0218] VL -3 - SEQ ID NO: 1 - CH1-CH2-CH3, where VL -3 This is a VL variable domain with a deletion of 3 amino acids at the C-terminus of FR4 of the VL variable domain, and

[0219] VH -4 - EIK- CL, where VH -4 VH variable domain with a deletion of 4 amino acids at the C-terminus of FR4 of the VH variable domain.

[0220] The heavy and light chain genes of antibodies to GD2 and CD3, as well as the dimerization domain genes, were synthesized de novo. Fusion of the heavy chain variable domain and the Fc fragment of human IgG1, the light chain variable domain and CK, or the variable domains and dimerization domains was achieved by PCR and / or gene synthesis and assembly using established restriction and ligation methods.

[0221] The heavy and light chain genes of the antibody were cloned into pEE-Hc and pEE-Lc plasmids to produce IgG1 protein in mammalian cells.

[0222] The cloned nucleotide sequences were confirmed by DNA sequencing. The resulting plasmids were produced in the required quantities in E. coli cells and purified using a plasmid DNA isolation kit.

[0223] The obtained genetic constructs - pEE-anti-GD2-Hc, pEE-anti-GD2-Lc, pEE-anti-CD3-Hc and pEE-anti-CD3-Lc - were transferred to transient protein production in the CHO cell line.

[0224] Example 4. Production, isolation and purification of bispecific antibodies aCD3 / aGD2 from suspension culture of mammalian cells.

[0225] Full-length bispecific antibodies, selected from 02-002 or 03-003, were produced in CHO cell growth medium. After transfection of the cells with expression vectors, orbital feed-batch cultivation was performed in serum-free medium for 7 days. Secretion of the studied antibodies was monitored using a Pall ForteBio Octet RED96 molecular interaction analysis system with protein A biosensors.

[0226] Target proteins were isolated from the culture fluid using affinity chromatography on a chromatographic system. The culture fluid was applied to a Protein A column, then washed with PBS and the protein was eluted with a 0.1 M glycine buffer solution, pH 3.0, after which the protein solutions were neutralized. The proteins were then transferred to PBS, pH 7.4, by dialysis, and the resulting solutions were filtered (0.22 μm). The preparations were stored at -70°C. The purity of the resulting protein solutions was assessed by SDS gel electrophoresis in a 4-15% gradient polyacrylamide gel. Figure 9 shows data for bispecific antibody 02-002 for illustrative purposes only. Bispecific antibody 03-003 showed similar results.

[0227] Example 5. Affinity of interaction of bispecific antibodies to human CD3 antigen (epsilon and delta subunits)

[0228] A study of the interaction of bispecific antibodies with the human CD3 antigen (epsilon and delta subunits) was conducted on a ForteBio Octet RED96 instrument using AR2G biosensors (ForteBio). Recombinant human CD3 antigen (epsilon and delta subunits) was used for the study. The experiment consisted of the following steps: sensor activation, protein loading onto the sensors, quenching of unreacted activated groups, baseline recording, analyte association recording, and dissociation recording. Measurements were conducted at 30°C. Sensor activation was carried out in an aqueous solution containing 20 mM EDC and 10 mM sNHS for 300 s. Antibody 02-002 was loaded onto the biosensor surface in a 10 mM sodium acetate buffer solution with a pH of 5.0 for 1000 s. The protein concentration for loading was 20 μg / ml.Quenching of unreacted active sites on the sensor surface was carried out for 300 s in a 1 M aqueous ethanolamine solution with a pH of 8.5 (the pH was adjusted by adding hydrochloric acid). The baseline and all subsequent steps of the experiment were conducted in a kinetic buffer solution. During the association step (duration of the step was 300 s), the sensors with the loaded antibody were immersed in wells containing antigen solutions at a concentration of 6.25 μg / mL, prepared in a kinetic buffer solution. During the dissociation step (600 s), the sensors were immersed in wells containing the kinetic buffer, in which the baseline was recorded.

[0229] Binding curves were processed using Octet Data Analysis (version 8.2) with a 1:1 interaction model. The results are presented in Table 3. Bispecific antibodies 02-002 and 13-003 were chosen for illustrative purposes only. Bispecific antibody 02-002 has the same complete set of 6 CDRs (LCDR1+LCDR2+LCDR3+HCDR1+HCDR2+HCDR3) of the antigen-binding fragment to CD3 as bispecific antibodies 03-003, 13-003, and 12-005.

[0230]

[0231] Thus, it can be concluded that all bispecific antibodies specifically bind to the human CD3 antigen.

[0232] Example 6. Affinity study of a bispecific antibody that specifically binds to CD3 and BCMA tumor antigen

[0233] Antibody 13-003 at a concentration of 10 μg / mL was immobilized on the surface of Protein A biosensors (ForteBio) for 300 s. The analysis was performed at 30°C using a kinetic buffer solution. After a baseline of 120 s was recorded in the kinetic buffer solution, the sensors with immobilized antibody were immersed in wells containing the analyte solution (BCMA), where the complex association occurred for 300 seconds. For each antibody tested, a sensorgram was recorded for an antigen solution (Avi-His-hBCMA-HSA) with concentrations of 5 μg / mL (65.2 nM), 2.5 μg / mL (32.6 nM), and 1.25 μg / mL (16.3 nM), along with a reference signal (reference sensorgram) of a kinetic buffer solution without antigen. Dissociation of the complex in the buffer solution was then detected for 600 seconds. Sensors not loaded with antibodies were used to test for nonspecific interactions between the analyte and the sensors (negative control).During the loading step, negative control sensors were immersed in a kinetic buffer solution that did not contain antibodies, all other steps were similar to antibody-loaded sensors.

[0234] The reference signal subtracted binding curves were analyzed by Octet Data Analysis software (version 9.0) using the 1:1 interaction model from three sensorgrams, the sensorgrams are presented in Table 4.

[0235]

[0236] Thus, it can be concluded that the studied bispecific antibody specifically binds to the human BCMA antigen.

[0237] Example 7. Thermal stability analysis of bispecific antibodies

[0238] Antibodies were heated in PBS pH 7.4 using a thermocycler in plastic tubes at 50°C for 48 hours, followed by a transition to +4°C. After completion of the program, samples before and after heating were analyzed using analytical gel chromatography on a TSK Gel G3000 SWxl column. The target peak areas of the samples before and after heating were compared, and the delta change in monomer peak area was calculated. The results are presented in Table 5.

[0239]

[0240]

[0241] Thus, all studied bispecific antibodies that specifically bind to CD3 and the GD2 antigen have satisfactory aggregation stability.

[0242] Example 8. Aggregation stability of a bispecific antibody that specifically binds to CD3 and the BCMA tumor antigen

[0243] Study of aggregation stability of monoclonal bispecific antibodies using size-exclusion (S-EC) HPLC and dynamic light scattering (DLS) methods.

[0244] The study consisted of the following stages: determination of aggregation temperature (DLS), thermal stress of samples at 40°C for 144 hours, analysis of stressed samples and control intact samples by (E) HPLC, analysis of particle sizes of stressed samples and control samples by DLS.

[0245] The results of the study of the average particle size, hydrodynamic radius of the target fraction, polydispersity of the target fraction and scattering intensity of the target fraction obtained by the DLS method for the intact sample (not subjected to stress) and the stressed sample are presented in Table 6. The results of the HPLC study of the intact sample (not subjected to stress) and the stressed sample are presented in Table 7.

[0246]

[0247]

[0248]

[0249] The analyzed bispecific antibody, which specifically binds to CD3 and the BCMA tumor antigen, has satisfactory aggregation stability.

[0250] Example 9. Test for determining the specific activity of aCD3 / aGD2 antibodies on the Jurkat-NFAT-Luc reporter cell line

[0251] This assay is carried out using the Jurkat-NFAT-luc reporter cell line, which is based on the Jurkat cell line containing the firefly luciferase gene under the control of the NFAT promoter; SK-N-BE(2) cells were used as the target cells. Jurkat-NFAT-Luc cells were cultured at 37°C with 5% CO2 in RPMI-1640 medium (10% FBS, 10 μg / ml gentamicin, 2 mM L-glutamine, and 200 μg / ml hygromycin), and SK-N-BE(2) cells were cultured in DMEM / F12 medium (10% FBS, 10 μg / ml gentamicin, and 2 mM L-glutamine).

[0252] The assay was performed in a white 96-well luminescence culture plate. Dilutions of the analyzed antibodies were prepared at 20 μg / mL. Each well was inoculated with 25,000 Jurkat-NFAT-Luc effector cells, 25,000 SK-N-BE(2) target cells, and the analyzed antibodies. The final volume of cell and antibody suspension per well was 100 μL. All suspension components were prepared in RPMI-1640 medium (10%) (FBS), 10 μg / mL gentamicin, and 2 mM L-glutamine).

[0253] The plate was incubated for 4 hours at 37°C, 5% CO2. Luciferase substrate (BIOKAD) was then added to all wells, and the luminescence intensity in the wells was measured. Luminescence was measured using a Spark plate reader (Tecan). Data processing and graphing were performed using Magellan software. The obtained experimental data on the dependence of luminescence on the concentration of the studied drugs were approximated using a four-parameter logistic function. The key parameters for evaluation were the maximum luminescence / reporter activation value and EC50.

[0254] Thus, it was demonstrated that antibodies 02-002 and 03-003 activate the reporter with comparable efficacy in the Jurkat-NFAT-Luc cell line in the presence of GD2-expressing cells - Sk-N-BE(2) (neuroblastoma). EC50 values ​​are presented in Table 8.

[0255]

[0256] It was also shown that when using a line that does not express GD2 - A431 (human lung carcinoma) in the test, no reporter activation was observed, which indicates the specificity of the action of these antibodies.

[0257] Example 10. Analysis of the ability of a bispecific antibody that specifically binds to CD3 and the BCMA tumor antigen to activate the Jurkat NFAT-luc C1.1 reporter T-lymphocyte line in the presence of RPMI8226 multiple myeloma cells.

[0258] The ability of anti-BCMA / anti-CD3 antibodies to activate T cell signaling through binding to the CD3 subunit of the T cell receptor (TCR) in the presence of the targeted multiple myeloma cell line RPMI8226 was assessed by detecting the luminescent signal after incubation of antibodies with Jurkat NFAT-luc Cl.1 human T lymphocyte reporter T cells in the presence of RPMI8226 multiple myeloma cells.

[0259] 20,000 RPMI8226 cells and 20,000 Jurkat NFAT-luc Cl.1 cells were incubated in culture medium for 24 hours with serial dilutions of the antibodies to be analyzed. After the indicated time, the cells were incubated in the dark for 8 minutes, and the luminescent signal was then recorded using a plate reader.

[0260] Half of the effective concentration of EC 50 was calculated by constructing a four-parameter logistic regression model, the data are presented in Table 9.

[0261]

[0262] Thus, the analyzed bispecific antibody, which specifically binds to CD3 and the BCMA tumor antigen, has the ability to activate the Jurkat NFAT-luc Cl.1 reporter cell line in the presence of multiple myeloma.

[0263] --->

[0264] <?xml version="1.0" encoding="UTF-8"?>

[0265] <!DOCTYPE ST26SequenceListing PUBLIC "- / / WIPO / / DTD Sequence Listing

[0266] 1.3 / / EN" "ST26SequenceListing_V1_3.dtd">

[0267] <st26sequencelisting dtdversion="V1_3" filename="Crossmab VH-4-VL-3

[0268] Перечень последовательностей.xml" softwarename="WIPO Sequence"

[0269] softwareversion="2.3.0" productiondate="2024-08-28">

[0270] <applicantfilereference>2-Crossmab

[0271] VH-4-VL-3< / applicantfilereference>

[0272] <applicantname languagecode="ru">JOINT-STOCK COMPANY

[0273] "BIOCAD"< / applicantname>

[0274] <applicantnamelatin>JOINT STOCK COMPANY BIOCAD< / applicantnamelatin>

[0275] <inventiontitle languagecode="ru">DIVALENT BISPECIFIC

[0276] ANTIBODY AND METHOD FOR ITS PRODUCTION< / inventiontitle>

[0277] <sequencetotalquantity> 26< / sequencetotalquantity>

[0278] <sequencedata sequenceidnumber="1">

[0279] <insdseq>

[0280] <INSDSeq_length> 4< / INSDSeq_length>

[0281] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0282] <INSDSeq_division> PAT< / INSDSeq_division>

[0283] <INSDSeq_feature-table>

[0284] <insdfeature>

[0285] <INSDFeature_key>source< / INSDFeature_key>

[0286] <INSDFeature_location>1..4< / INSDFeature_location>

[0287] <INSDFeature_quals>

[0288] <insdqualifier>

[0289] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0290] <INSDQualifier_value>protein< / INSDQualifier_value>

[0291] < / insdqualifier>

[0292] <insdqualifier id="q4">

[0293] <INSDQualifier_name>organism< / INSDQualifier_name>

[0294] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0295] < / insdqualifier>

[0296] < / INSDFeature_quals>

[0297] < / insdfeature>

[0298] < / INSDSeq_feature-table>

[0299] <INSDSeq_sequence> TVSS< / INSDSeq_sequence>

[0300] < / insdseq>

[0301] < / sequencedata>

[0302] <sequencedata sequenceidnumber="2">

[0303] <insdseq>

[0304] <INSDSeq_length>226< / INSDSeq_length>

[0305] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0306] <INSDSeq_division>PAT< / INSDSeq_division>

[0307] <INSDSeq_feature-table>

[0308] <insdfeature>

[0309] <INSDFeature_key>source< / INSDFeature_key>

[0310] <INSDFeature_location>1..226< / INSDFeature_location>

[0311] <INSDFeature_quals>

[0312] <insdqualifier>

[0313] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0314] <INSDQualifier_value>protein< / INSDQualifier_value>

[0315] < / insdqualifier>

[0316] <insdqualifier id="q6">

[0317] <INSDQualifier_name>organism< / INSDQualifier_name>

[0318] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0319] < / insdqualifier>

[0320] < / INSDFeature_quals>

[0321] < / insdfeature>

[0322] < / INSDSeq_feature-table>

[0323] <INSDSeq_sequence>EVQLVQSGGGVVQPGRSLRLSCAASGFTFNDYTMHWIRQAPGKGLEWVS

[0324] GISWNGGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDIWGQGTTV

[0325] TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

[0326] SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC< / INSDSeq_sequence>

[0327] < / insdseq>

[0328] < / sequencedata>

[0329] <sequencedata sequenceidnumber="3">

[0330] <insdseq>

[0331] <INSDSeq_length>441< / INSDSeq_length>

[0332] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0333] <INSDSeq_division>PAT< / INSDSeq_division>

[0334] <INSDSeq_feature-table>

[0335] <insdfeature>

[0336] <INSDFeature_key>source< / INSDFeature_key>

[0337] <INSDFeature_location>1..441< / INSDFeature_location>

[0338] <INSDFeature_quals>

[0339] <insdqualifier>

[0340] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0341] <INSDQualifier_value>protein< / INSDQualifier_value>

[0342] < / insdqualifier>

[0343] <insdqualifier id="q8">

[0344] <INSDQualifier_name>organism< / INSDQualifier_name>

[0345] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0346] < / insdqualifier>

[0347] < / INSDFeature_quals>

[0348] < / insdfeature>

[0349] < / INSDSeq_feature-table>

[0350] <INSDSeq_sequence>EIVMTQSPGTLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIY

[0351] GSSTRATGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQHYNNWPLTFGGGTKVEIKRTVAAPSVFIFP

[0352] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

[0353] KVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVD

[0354] VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT

[0355] ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0356] LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[0357] < / insdseq>

[0358] < / sequencedata>

[0359] <sequencedata sequenceidnumber="4">

[0360] <insdseq>

[0361] <INSDSeq_length>212< / INSDSeq_length>

[0362] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0363] <INSDSeq_division>PAT< / INSDSeq_division>

[0364] <INSDSeq_feature-table>

[0365] <insdfeature>

[0366] <INSDFeature_key>source< / INSDFeature_key>

[0367] <INSDFeature_location>1..212< / INSDFeature_location>

[0368] <INSDFeature_quals>

[0369] <insdqualifier>

[0370] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0371] <INSDQualifier_value>protein< / INSDQualifier_value>

[0372] < / insdqualifier>

[0373] <insdqualifier id="q10">

[0374] <INSDQualifier_name>organism< / INSDQualifier_name>

[0375] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0376] < / insdqualifier>

[0377] < / INSDFeature_quals>

[0378] < / insdfeature>

[0379] < / INSDSeq_feature-table>

[0380] <INSDSeq_sequence>EIVMTQSPGTLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIY

[0381] GSSTRATGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQHYNNWPLTFGGGTKVEIKSSASTKGPSVFP

[0382] LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

[0383] YICNVNHKPSNTKVDKRVEPKSC< / INSDSeq_sequence>

[0384] < / insdseq>

[0385] < / sequencedata>

[0386] <sequencedata sequenceidnumber="5">

[0387] <insdseq>

[0388] <INSDSeq_length>457< / INSDSeq_length>

[0389] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0390] <INSDSeq_division>PAT< / INSDSeq_division>

[0391] <INSDSeq_feature-table>

[0392] <insdfeature>

[0393] <INSDFeature_key>source< / INSDFeature_key>

[0394] <INSDFeature_location>1..457< / INSDFeature_location>

[0395] <INSDFeature_quals>

[0396] <insdqualifier>

[0397] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0398] <INSDQualifier_value>protein< / INSDQualifier_value>

[0399] < / insdqualifier>

[0400] <insdqualifier id="q12">

[0401] <INSDQualifier_name>organism< / INSDQualifier_name>

[0402] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0403] < / insdqualifier>

[0404] < / INSDFeature_quals>

[0405] < / insdfeature>

[0406] < / INSDSeq_feature-table>

[0407] <INSDSeq_sequence>EVQLVQSGGGVVQPGRSLRLSCAASGFTFNDYTMHWIRQAPGKGLEWVS

[0408] GISWNGGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDIWGQGTTV

[0409] TVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY

[0410] SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKD

[0411] TLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0412] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN

[0413] NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_se

[0414] quence>

[0415] < / insdseq>

[0416] < / sequencedata>

[0417] <sequencedata sequenceidnumber="6">

[0418] <insdseq>

[0419] <INSDSeq_length>214< / INSDSeq_length>

[0420] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0421] <INSDSeq_division>PAT< / INSDSeq_division>

[0422] <INSDSeq_feature-table>

[0423] <insdfeature>

[0424] <INSDFeature_key>source< / INSDFeature_key>

[0425] <INSDFeature_location>1..214< / INSDFeature_location>

[0426] <INSDFeature_quals>

[0427] <insdqualifier>

[0428] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0429] <INSDQualifier_value>protein< / INSDQualifier_value>

[0430] < / insdqualifier>

[0431] <insdqualifier id="q14">

[0432] <INSDQualifier_name>organism< / INSDQualifier_name>

[0433] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0434] < / insdqualifier>

[0435] < / INSDFeature_quals>

[0436] < / insdfeature>

[0437] < / INSDSeq_feature-table>

[0438] <INSDSeq_sequence>EIVMTQSPGTLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIY

[0439] GSSTRATGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQHYNNWPLTFGGGTKVEIKRTVAAPSVFIFP

[0440] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

[0441] KVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[0442] < / insdseq>

[0443] < / sequencedata>

[0444] <sequencedata sequenceidnumber="7">

[0445] <insdseq>

[0446] <INSDSeq_length>453< / INSDSeq_length>

[0447] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0448] <INSDSeq_division>PAT< / INSDSeq_division>

[0449] <INSDSeq_feature-table>

[0450] <insdfeature>

[0451] <INSDFeature_key>source< / INSDFeature_key>

[0452] <INSDFeature_location>1..453< / INSDFeature_location>

[0453] <INSDFeature_quals>

[0454] <insdqualifier>

[0455] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0456] <INSDQualifier_value>protein< / INSDQualifier_value>

[0457] < / insdqualifier>

[0458] <insdqualifier id="q16">

[0459] <INSDQualifier_name>organism< / INSDQualifier_name>

[0460] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0461] < / insdqualifier>

[0462] < / INSDFeature_quals>

[0463] < / insdfeature>

[0464] < / INSDSeq_feature-table>

[0465] <INSDSeq_sequence>EVQLVQSGGGVVQPGRSLRLSCAASGFTFNDYTMHWIRQAPGKGLEWVS

[0466] GISWNGGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDIWGQGTTV

[0467] TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

[0468] SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYI

[0469] TREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV

[0470] SNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT

[0471] TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequen

[0472] ce>

[0473] < / insdseq>

[0474] < / sequencedata>

[0475] <sequencedata sequenceidnumber="8">

[0476] <insdseq>

[0477] <INSDSeq_length>229< / INSDSeq_length>

[0478] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0479] <INSDSeq_division>PAT< / INSDSeq_division>

[0480] <INSDSeq_feature-table>

[0481] <insdfeature>

[0482] <INSDFeature_key>source< / INSDFeature_key>

[0483] <INSDFeature_location>1..229< / INSDFeature_location>

[0484] <INSDFeature_quals>

[0485] <insdqualifier>

[0486] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0487] <INSDQualifier_value>protein< / INSDQualifier_value>

[0488] < / insdqualifier>

[0489] <insdqualifier id="q18">

[0490] <INSDQualifier_name>organism< / INSDQualifier_name>

[0491] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0492] < / insdqualifier>

[0493] < / INSDFeature_quals>

[0494] < / insdfeature>

[0495] < / INSDSeq_feature-table>

[0496] <INSDSeq_sequence>EVQLVQSGGGVVQPGRSLRLSCAASGFTFNDYTMHWIRQAPGKGLEWVS

[0497] GISWNGGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDIWGQGTTV

[0498] EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

[0499] LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[0500] < / insdseq>

[0501] < / sequencedata>

[0502] <sequencedata sequenceidnumber="9">

[0503] <insdseq>

[0504] <INSDSeq_length> 438< / INSDSeq_length>

[0505] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0506] <INSDSeq_division> PAT< / INSDSeq_division>

[0507] <INSDSeq_feature-table>

[0508] <insdfeature>

[0509] <INSDFeature_key>source< / INSDFeature_key>

[0510] <INSDFeature_location>1..438< / INSDFeature_location>

[0511] <INSDFeature_quals>

[0512] <insdqualifier>

[0513] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0514] <INSDQualifier_value>protein< / INSDQualifier_value>

[0515] < / insdqualifier>

[0516] <insdqualifier id="q20">

[0517] <INSDQualifier_name>organism< / INSDQualifier_name>

[0518] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0519] < / insdqualifier>

[0520] < / INSDFeature_quals>

[0521] < / insdfeature>

[0522] < / INSDSeq_feature-table>

[0523] <INSDSeq_sequence> EIVMTQSPGTLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIY

[0524] GSSTRATGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQHYNNWPLTFGGGTKVTVSSASTKGPSVFPL

[0525] APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

[0526] ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKPDTLYITREPEVTCVVVDVSH

[0527] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIC

[0528] AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0529] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[0530] < / insdseq>

[0531] < / sequencedata>

[0532] <sequencedata sequenceidnumber="10">

[0533] <insdseq>

[0534] <INSDSeq_length>436< / INSDSeq_length>

[0535] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0536] <INSDSeq_division>PAT< / INSDSeq_division>

[0537] <INSDSeq_feature-table>

[0538] <insdfeature>

[0539] <INSDFeature_key>source< / INSDFeature_key>

[0540] <INSDFeature_location>1..436< / INSDFeature_location>

[0541] <INSDFeature_quals>

[0542] <insdqualifier>

[0543] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0544] <INSDQualifier_value>protein< / INSDQualifier_value>

[0545] < / insdqualifier>

[0546] <insdqualifier id="q22">

[0547] <INSDQualifier_name>organism< / INSDQualifier_name>

[0548] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0549] < / insdqualifier>

[0550] < / INSDFeature_quals>

[0551] < / insdfeature>

[0552] < / INSDSeq_feature-table>

[0553] <INSDSeq_sequence>EIVMTQSPGTLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIY

[0554] GSSTRATGVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQHYNNWPLTFGGGTKVTVSSASTKGPSVFPL

[0555] APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

[0556] ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSH

[0557] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0558] AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0559] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP< / INSDSeq_sequence>

[0560] < / insdseq>

[0561] < / sequencedata>

[0562] <sequencedata sequenceidnumber="11">

[0563] <insdseq>

[0564] <INSDSeq_length>217< / INSDSeq_length>

[0565] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0566] <INSDSeq_division>PAT< / INSDSeq_division>

[0567] <INSDSeq_feature-table>

[0568] <insdfeature>

[0569] <INSDFeature_key>source< / INSDFeature_key>

[0570] <INSDFeature_location>1..217< / INSDFeature_location>

[0571] <INSDFeature_quals>

[0572] <insdqualifier>

[0573] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0574] <INSDQualifier_value>protein< / INSDQualifier_value>

[0575] < / insdqualifier>

[0576] <insdqualifier id="q24">

[0577] <INSDQualifier_name>organism< / INSDQualifier_name>

[0578] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0579] < / insdqualifier>

[0580] < / INSDFeature_quals>

[0581] < / insdfeature>

[0582] < / INSDSeq_feature-table>

[0583] <INSDSeq_sequence>QPVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLI

[0584] FKNNQRPSGVPDRFSGSKSGTSASLVISGLQSEDEADYYCAAWDGSLNAWMFGGGTKLTVLRTVAAPSVF

[0585] IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0586] EKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[0587] < / insdseq>

[0588] < / sequencedata>

[0589] <sequencedata sequenceidnumber="12">

[0590] <insdseq>

[0591] <INSDSeq_length>451< / INSDSeq_length>

[0592] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0593] <INSDSeq_division>PAT< / INSDSeq_division>

[0594] <INSDSeq_feature-table>

[0595] <insdfeature>

[0596] <INSDFeature_key>source< / INSDFeature_key>

[0597] <INSDFeature_location>1..451< / INSDFeature_location>

[0598] <INSDFeature_quals>

[0599] <insdqualifier>

[0600] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0601] <INSDQualifier_value>protein< / INSDQualifier_value>

[0602] < / insdqualifier>

[0603] <insdqualifier id="q26">

[0604] <INSDQualifier_name>organism< / INSDQualifier_name>

[0605] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0606] < / insdqualifier>

[0607] < / INSDFeature_quals>

[0608] < / insdfeature>

[0609] < / INSDSeq_feature-table>

[0610] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGLEWVS

[0611] GYNGGSTRAGYADSVEGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLVTV

[0612] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0613] VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITR

[0614] EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0615] KALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP

[0616] PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence

[0617] >

[0618] < / insdseq>

[0619] < / sequencedata>

[0620] <sequencedata sequenceidnumber="13">

[0621] <insdseq>

[0622] <INSDSeq_length>451< / INSDSeq_length>

[0623] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0624] <INSDSeq_division>PAT< / INSDSeq_division>

[0625] <INSDSeq_feature-table>

[0626] <insdfeature>

[0627] <INSDFeature_key>source< / INSDFeature_key>

[0628] <INSDFeature_location>1..451< / INSDFeature_location>

[0629] <INSDFeature_quals>

[0630] <insdqualifier>

[0631] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0632] <INSDQualifier_value>protein< / INSDQualifier_value>

[0633] < / insdqualifier>

[0634] <insdqualifier id="q28">

[0635] <INSDQualifier_name>organism< / INSDQualifier_name>

[0636] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0637] < / insdqualifier>

[0638] < / INSDFeature_quals>

[0639] < / insdfeature>

[0640] < / INSDSeq_feature-table>

[0641] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGFEWVS

[0642] GYNGGSDRAGFADSVEGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLVTV

[0643] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0644] VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITR

[0645] EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0646] KALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP

[0647] PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence

[0648] >

[0649] < / insdseq>

[0650] < / sequencedata>

[0651] <sequencedata sequenceidnumber="14">

[0652] <insdseq>

[0653] <INSDSeq_length>224< / INSDSeq_length>

[0654] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0655] <INSDSeq_division>PAT< / INSDSeq_division>

[0656] <INSDSeq_feature-table>

[0657] <insdfeature>

[0658] <INSDFeature_key>source< / INSDFeature_key>

[0659] <INSDFeature_location>1..224< / INSDFeature_location>

[0660] <INSDFeature_quals>

[0661] <insdqualifier>

[0662] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0663] <INSDQualifier_value>protein< / INSDQualifier_value>

[0664] < / insdqualifier>

[0665] <insdqualifier id="q30">

[0666] <INSDQualifier_name>organism< / INSDQualifier_name>

[0667] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0668] < / insdqualifier>

[0669] < / INSDFeature_quals>

[0670] < / insdfeature>

[0671] < / INSDSeq_feature-table>

[0672] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGLEWVS

[0673] GYNGGSTRAGYADSVEGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLVTV

[0674] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0675] VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC< / INSDSeq_sequence>

[0676] < / insdseq>

[0677] < / sequencedata>

[0678] <sequencedata sequenceidnumber="15">

[0679] <insdseq>

[0680] <INSDSeq_length>224< / INSDSeq_length>

[0681] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0682] <INSDSeq_division>PAT< / INSDSeq_division>

[0683] <INSDSeq_feature-table>

[0684] <insdfeature>

[0685] <INSDFeature_key>source< / INSDFeature_key>

[0686] <INSDFeature_location>1..224< / INSDFeature_location>

[0687] <INSDFeature_quals>

[0688] <insdqualifier>

[0689] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0690] <INSDQualifier_value>protein< / INSDQualifier_value>

[0691] < / insdqualifier>

[0692] <insdqualifier id="q32">

[0693] <INSDQualifier_name>organism< / INSDQualifier_name>

[0694] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0695] < / insdqualifier>

[0696] < / INSDFeature_quals>

[0697] < / insdfeature>

[0698] < / INSDSeq_feature-table>

[0699] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGFEWVS

[0700] GYNGGSDRAGFADSVEGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLVTV

[0701] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0702] VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC< / INSDSeq_sequence>

[0703] < / insdseq>

[0704] < / sequencedata>

[0705] <sequencedata sequenceidnumber="16">

[0706] <insdseq>

[0707] <INSDSeq_length>444< / INSDSeq_length>

[0708] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0709] <INSDSeq_division>PAT< / INSDSeq_division>

[0710] <INSDSeq_feature-table>

[0711] <insdfeature>

[0712] <INSDFeature_key>source< / INSDFeature_key>

[0713] <INSDFeature_location>1..444< / INSDFeature_location>

[0714] <INSDFeature_quals>

[0715] <insdqualifier>

[0716] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0717] <INSDQualifier_value>protein< / INSDQualifier_value>

[0718] < / insdqualifier>

[0719] <insdqualifier id="q34">

[0720] <INSDQualifier_name>organism< / INSDQualifier_name>

[0721] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0722] < / insdqualifier>

[0723] < / INSDFeature_quals>

[0724] < / insdfeature>

[0725] < / INSDSeq_feature-table>

[0726] <INSDSeq_sequence>QPVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLI

[0727] FKNNQRPSGVPDRFSGSKSGTSASLVISGLQSEDEADYYCAAWDGSLNAWMFGGGTKLTVLRTVAAPSVF

[0728] IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0729] EKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCV

[0730] VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI

[0731] EKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

[0732] SFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[0733] < / insdseq>

[0734] < / sequencedata>

[0735] <sequencedata sequenceidnumber="17">

[0736] <insdseq>

[0737] <INSDSeq_length>215< / INSDSeq_length>

[0738] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0739] <INSDSeq_division>PAT< / INSDSeq_division>

[0740] <INSDSeq_feature-table>

[0741] <insdfeature>

[0742] <INSDFeature_key>source< / INSDFeature_key>

[0743] <INSDFeature_location>1..215< / INSDFeature_location>

[0744] <INSDFeature_quals>

[0745] <insdqualifier>

[0746] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0747] <INSDQualifier_value>protein< / INSDQualifier_value>

[0748] < / insdqualifier>

[0749] <insdqualifier id="q36">

[0750] <INSDQualifier_name>organism< / INSDQualifier_name>

[0751] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0752] < / insdqualifier>

[0753] < / INSDFeature_quals>

[0754] < / insdfeature>

[0755] < / INSDSeq_feature-table>

[0756] <INSDSeq_sequence>QPVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLI

[0757] FKNNQRPSGVPDRFSGSKSGTSASLVISGLQSEDEADYYCAAWDGSLNAWMFGGGTKLTVLSSASTKGPS

[0758] VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0759] TQTYICNVNHKPSNTKVDKRVEPKSC< / INSDSeq_sequence>

[0760] < / insdseq>

[0761] < / sequencedata>

[0762] <sequencedata sequenceidnumber="18">

[0763] <insdseq>

[0764] <INSDSeq_length>455< / INSDSeq_length>

[0765] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0766] <INSDSeq_division>PAT< / INSDSeq_division>

[0767] <INSDSeq_feature-table>

[0768] <insdfeature>

[0769] <INSDFeature_key>source< / INSDFeature_key>

[0770] <INSDFeature_location>1..455< / INSDFeature_location>

[0771] <INSDFeature_quals>

[0772] <insdqualifier>

[0773] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0774] <INSDQualifier_value>protein< / INSDQualifier_value>

[0775] < / insdqualifier>

[0776] <insdqualifier id="q38">

[0777] <INSDQualifier_name>organism< / INSDQualifier_name>

[0778] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0779] < / insdqualifier>

[0780] < / INSDFeature_quals>

[0781] < / insdfeature>

[0782] < / INSDSeq_feature-table>

[0783] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGLEWVS

[0784] GYNGGSTRAGYADSVEGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLVTV

[0785] SSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0786] SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL

[0787] YITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

[0788] KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY

[0789] KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequ

[0790] ence>

[0791] < / insdseq>

[0792] < / sequencedata>

[0793] <sequencedata sequenceidnumber="19">

[0794] <insdseq>

[0795] <INSDSeq_length>455< / INSDSeq_length>

[0796] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0797] <INSDSeq_division>PAT< / INSDSeq_division>

[0798] <INSDSeq_feature-table>

[0799] <insdfeature>

[0800] <INSDFeature_key>source< / INSDFeature_key>

[0801] <INSDFeature_location>1..455< / INSDFeature_location>

[0802] <INSDFeature_quals>

[0803] <insdqualifier>

[0804] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0805] <INSDQualifier_value>protein< / INSDQualifier_value>

[0806] < / insdqualifier>

[0807] <insdqualifier id="q40">

[0808] <INSDQualifier_name>organism< / INSDQualifier_name>

[0809] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0810] < / insdqualifier>

[0811] < / INSDFeature_quals>

[0812] < / insdfeature>

[0813] < / INSDSeq_feature-table>

[0814] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGFEWVS

[0815] GYNGGSDRAGFADSVEGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLVTV

[0816] SSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0817] SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL

[0818] YITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

[0819] KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY

[0820] KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequ

[0821] ence>

[0822] < / insdseq>

[0823] < / sequencedata>

[0824] <sequencedata sequenceidnumber="20">

[0825] <insdseq>

[0826] <INSDSeq_length>227< / INSDSeq_length>

[0827] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0828] <INSDSeq_division>PAT< / INSDSeq_division>

[0829] <INSDSeq_feature-table>

[0830] <insdfeature>

[0831] <INSDFeature_key>source< / INSDFeature_key>

[0832] <INSDFeature_location>1..227< / INSDFeature_location>

[0833] <INSDFeature_quals>

[0834] <insdqualifier>

[0835] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0836] <INSDQualifier_value>protein< / INSDQualifier_value>

[0837] < / insdqualifier>

[0838] <insdqualifier id="q42">

[0839] <INSDQualifier_name>organism< / INSDQualifier_name>

[0840] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0841] < / insdqualifier>

[0842] < / INSDFeature_quals>

[0843] < / insdfeature>

[0844] < / INSDSeq_feature-table>

[0845] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGLEWVS

[0846] GYNGGSTRAGYADSVEGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLLTV

[0847] LRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0848] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[0849] < / insdseq>

[0850] < / sequencedata>

[0851] <sequencedata sequenceidnumber="21">

[0852] <insdseq>

[0853] <INSDSeq_length>441< / INSDSeq_length>

[0854] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0855] <INSDSeq_division>PAT< / INSDSeq_division>

[0856] <INSDSeq_feature-table>

[0857] <insdfeature>

[0858] <INSDFeature_key>source< / INSDFeature_key>

[0859] <INSDFeature_location>1..441< / INSDFeature_location>

[0860] <INSDFeature_quals>

[0861] <insdqualifier>

[0862] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0863] <INSDQualifier_value>protein< / INSDQualifier_value>

[0864] < / insdqualifier>

[0865] <insdqualifier id="q44">

[0866] <INSDQualifier_name>organism< / INSDQualifier_name>

[0867] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0868] < / insdqualifier>

[0869] < / INSDFeature_quals>

[0870] < / insdfeature>

[0871] < / INSDSeq_feature-table>

[0872] <INSDSeq_sequence>QPVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLI

[0873] FKNNQRPSGVPDRFSGSKSGTSASLVISGLQSEDEADYYCAAWDGSLNAWMFGGGTKVTVSSASTKGPSV

[0874] FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

[0875] QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVD

[0876] VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT

[0877] ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0878] LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[0879] < / insdseq>

[0880] < / sequencedata>

[0881] <sequencedata sequenceidnumber="22">

[0882] <insdseq>

[0883] <INSDSeq_length>232< / INSDSeq_length>

[0884] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0885] <INSDSeq_division>PAT< / INSDSeq_division>

[0886] <INSDSeq_feature-table>

[0887] <insdfeature>

[0888] <INSDFeature_key>source< / INSDFeature_key>

[0889] <INSDFeature_location>1..232< / INSDFeature_location>

[0890] <INSDFeature_quals>

[0891] <insdqualifier>

[0892] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0893] <INSDQualifier_value>protein< / INSDQualifier_value>

[0894] < / insdqualifier>

[0895] <insdqualifier id="q46">

[0896] <INSDQualifier_name>organism< / INSDQualifier_name>

[0897] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0898] < / insdqualifier>

[0899] < / INSDFeature_quals>

[0900] < / insdfeature>

[0901] < / INSDSeq_feature-table>

[0902] <INSDSeq_sequence>EVQLVQSGGGLVQPGGSLRLSCAVSGFTFSSSVMSWVRQAPGKGFEWVS

[0903] GYNGGSDRAGFADSVEGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARYIANSGTYYWDYWGQGTLVTV

[0904] SSLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

[0905] TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[0906] < / insdseq>

[0907] < / sequencedata>

[0908] <sequencedata sequenceidnumber="23">

[0909] <insdseq>

[0910] <INSDSeq_length>441< / INSDSeq_length>

[0911] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0912] <INSDSeq_division>PAT< / INSDSeq_division>

[0913] <INSDSeq_feature-table>

[0914] <insdfeature>

[0915] <INSDFeature_key>source< / INSDFeature_key>

[0916] <INSDFeature_location>1..441< / INSDFeature_location>

[0917] <INSDFeature_quals>

[0918] <insdqualifier>

[0919] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0920] <INSDQualifier_value>protein< / INSDQualifier_value>

[0921] < / insdqualifier>

[0922] <insdqualifier id="q48">

[0923] <INSDQualifier_name>organism< / INSDQualifier_name>

[0924] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0925] < / insdqualifier>

[0926] < / INSDFeature_quals>

[0927] < / insdfeature>

[0928] < / INSDSeq_feature-table>

[0929] <INSDSeq_sequence>QPVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVNWYQQLPGKAPKLLI

[0930] FKNNQRPSGVPDRFSGSKSGTSASLVISGLQSEDEADYYCAAWDGSLNAWMFGGGTKVTVSSASTKGPSV

[0931] FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

[0932] QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVD

[0933] VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT

[0934] ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0935] LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[0936] < / insdseq>

[0937] < / sequencedata>

[0938] <sequencedata sequenceidnumber="24">

[0939] <insdseq>

[0940] <INSDSeq_length>220< / INSDSeq_length>

[0941] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0942] <INSDSeq_division>PAT< / INSDSeq_division>

[0943] <INSDSeq_feature-table>

[0944] <insdfeature>

[0945] <INSDFeature_key>source< / INSDFeature_key>

[0946] <INSDFeature_location>1..220< / INSDFeature_location>

[0947] <INSDFeature_quals>

[0948] <insdqualifier>

[0949] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0950] <INSDQualifier_value>protein< / INSDQualifier_value>

[0951] < / insdqualifier>

[0952] <insdqualifier id="q50">

[0953] <INSDQualifier_name>organism< / INSDQualifier_name>

[0954] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0955] < / insdqualifier>

[0956] < / INSDFeature_quals>

[0957] < / insdfeature>

[0958] < / INSDSeq_feature-table>

[0959] <INSDSeq_sequence>DIVMTQTPLSLSVTPGERASLSCRSSQNLVHRNGNTYLHWYLQKPGQSP

[0960] KLLIHKVNNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCGQSTHVPPLTFGQGTKLELKRTVAAP

[0961] SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

[0962] ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[0963] < / insdseq>

[0964] < / sequencedata>

[0965] <sequencedata sequenceidnumber="25">

[0966] <insdseq>

[0967] <INSDSeq_length>443< / INSDSeq_length>

[0968] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0969] <INSDSeq_division>PAT< / INSDSeq_division>

[0970] <INSDSeq_feature-table>

[0971] <insdfeature>

[0972] <INSDFeature_key>source< / INSDFeature_key>

[0973] <INSDFeature_location>1..443< / INSDFeature_location>

[0974] <INSDFeature_quals>

[0975] <insdqualifier>

[0976] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0977] <INSDQualifier_value>protein< / INSDQualifier_value>

[0978] < / insdqualifier>

[0979] <insdqualifier id="q52">

[0980] <INSDQualifier_name>organism< / INSDQualifier_name>

[0981] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0982] < / insdqualifier>

[0983] < / INSDFeature_quals>

[0984] < / insdfeature>

[0985] < / INSDSeq_feature-table>

[0986] <INSDSeq_sequence>QVQLVQSGAEVKKPGASVKVSCKASGSSFTGKNMNWVRQNIGQGLEWMG

[0987] AIDPFYGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCVSGMFYWGQGTLVTVSSASTKGP

[0988] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0989] GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVV

[0990] VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0991] KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[0992] FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[0993] < / insdseq>

[0994] < / sequencedata>

[0995] <sequencedata sequenceidnumber="26">

[0996] <insdseq>

[0997] <INSDSeq_length>441< / INSDSeq_length>

[0998] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0999] <INSDSeq_division>PAT< / INSDSeq_division>

[1000] <INSDSeq_feature-table>

[1001] <insdfeature>

[1002] <INSDFeature_key>source< / INSDFeature_key>

[1003] <INSDFeature_location>1..441< / INSDFeature_location>

[1004] <INSDFeature_quals>

[1005] <insdqualifier>

[1006] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1007] <INSDQualifier_value>protein< / INSDQualifier_value>

[1008] < / insdqualifier>

[1009] <insdqualifier id="q54">

[1010] <INSDQualifier_name>organism< / INSDQualifier_name>

[1011] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[1012] < / insdqualifier>

[1013] < / INSDFeature_quals>

[1014] < / insdfeature>

[1015] < / INSDSeq_feature-table>

[1016] <INSDSeq_sequence>QVQLVQSGAEVKKPGASVKVSCKASGSSFTGKNMNWVRQNIGQGLEWMG

[1017] AIDPFYGGTSYNQKFKGRVTLTVDKSISTAYMELSRLRSDDTAVYYCVSGMFYWGQGTLVTVSSASTKGP

[1018] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[1019] GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVV

[1020] VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[1021] KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[1022] FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP< / INSDSeq_sequence>

[1023] < / insdseq>

[1024] < / sequencedata>

[1025] < / st26sequencelisting>

[1026] <---

Claims

1. A bivalent bispecific antibody that specifically binds to a first cancer antigen and a second CD3 antigen, wherein said antibody comprises: a) a first light chain and a first heavy chain of an antibody that specifically bind to a first cancer antigen, wherein the first light chain comprises a light chain variable domain VL and a light chain constant domain, and wherein the first heavy chain comprises a heavy chain variable domain VH and heavy chain constant domains of an antibody comprising a first heavy chain constant domain CH1 and an Fc fragment monomer comprising a second CH2 and a third CH3 heavy chain constant domains; and b) a second light chain and a second heavy chain of an antibody that specifically bind to a second CD3 antigen, where the second light chain contains: 1) VH variable domain with a deletion of 4 amino acids at the C-terminus of FR4 of the VH variable domain (VH -4 ), 2) amino acid sequence with EIK (Glu-Ile-Lys), and 3) the light chain constant domain; and where the second heavy chain contains: 1) variable domain VL with a deletion of 3 amino acids at the C-terminus of FR4 of the variable domain VL (VL -3 ), 2) the amino acid sequence with SEQ ID NO: 1 (TVSS), and 3) constant domains of the heavy chain of the antibody, including the first constant domain CH1 of the heavy chain and the monomer of the Fc fragment containing the second CH2 and third CH3 constant domains of the heavy chain, where the Fc fragment refers to human IgG.

2. A bivalent bispecific antibody according to claim 1, wherein the second light chain has the following structure: VH -4 - EIK - CL.

3. The bivalent bispecific antibody of claim 1, wherein the second heavy chain has the following structure: VL -3 - SEQ ID NO: 1 - CH1-CH2-CH3.

4. The bivalent bispecific antibody of claim 1, wherein the constant domain of the light chain of the antibody is selected from the constant domain of the kappa light chain (CK) or the constant domain of the lambda light chain (CL).

5. The divalent bispecific antibody of claim 1, wherein the first cancer antigen is selected from the group consisting of BCMA or GD2.

6. The divalent bispecific antibody of claim 1, wherein the isotype of the Fc fragment is human IgG1.

7. A bivalent bispecific antibody according to claim 6, wherein the antibody contains the mutations L234A and L235A according to the amino acid numbering of EU antibodies in the CH2 region.

8. A bivalent bispecific antibody according to claim 6, wherein the antibody contains the mutations M252Y, S254T, T256E according to the amino acid numbering of EU antibodies in the CH2 region.

9. The bivalent bispecific antibody of claim 6, wherein the antibody comprises a deletion of 446G and 447K according to the amino acid numbering of EU antibodies in the CH3 region.

10. The bivalent bispecific antibody of claim 1, wherein the third constant domain CH3 of one heavy chain and the third constant domain of the other heavy chain are in contact with each other at surfaces that are altered to form the bivalent bispecific antibody, wherein these alterations in the third constant domains of the heavy chains are substitutions to ensure heterodimerization.

11. The bivalent bispecific antibody of claim 10, wherein the CH3 domains of the antibody are further altered by introducing cysteine ​​as an amino acid into the corresponding positions of each CH3 domain such that a disulfide bridge can be formed between both CH3 domains.

12. The bivalent bispecific antibody of claim 11, wherein the CH3 domain of one heavy chain is altered to form a Knob and the CH3 domain of the other heavy chain is altered to form a Hole or vice versa.

13. The bivalent bispecific antibody of claim 12, wherein the CH3 domain of one heavy chain has the amino acid substitutions S354C / T366W, and the CH3 domain of the other heavy chain has the amino acid substitutions Y349C / T366S / L368A / Y407V.

14. The bivalent bispecific antibody of claim 13, wherein the CH3 domain of one heavy chain has the amino acid substitutions Y349C / T366S / L368A / Y407, and the CH3 domain of the other heavy chain has the amino acid substitutions S354C / T366W.

15. The bivalent bispecific antibody of claim 1, wherein the first antigen is BCMA and the second antigen is CD3.

16. The divalent bispecific antibody of claim 1, wherein the first antigen is GD2 and the second antigen is CD3.

17. A method for producing a bivalent bispecific antibody, wherein the method comprises the following steps: a) synthesizing a nucleic acid that encodes the first light chain and the first heavy chain of a bispecific antibody that specifically binds to a first cancer antigen, wherein the first light chain comprises a light chain variable domain VL and a light chain constant domain, and wherein the first heavy chain comprises a heavy chain variable domain VH and heavy chain constant domains of an antibody comprising a first heavy chain constant domain CH1 and an Fc fragment monomer comprising a second CH2 and a third CH3 heavy chain constant domains; and b) synthesizing a nucleic acid that encodes the second light chain and the second heavy chain of a bispecific antibody that specifically binds to the second CD3 antigen, where the second light chain contains: 1) VH variable domain with a deletion of 4 amino acids at the C-terminus of FR4 of the VH variable domain (VH -4 ), 2) amino acid sequence with EIK (Glu-Ile-Lys), and 3) the light chain constant domain; and where the second heavy chain contains: 1) variable domain VL with a deletion of 3 amino acids at the C-terminus of FR4 of the variable domain VL (VL -3 ), 2) the amino acid sequence with SEQ ID NO: 1 (TVSS), and 3) constant domains of the heavy chain of the antibody, including the first constant domain CH1 of the heavy chain and the monomer of the Fc fragment containing the second CH2 and third CH3 constant domains of the heavy chain, where the Fc fragment refers to human IgG; c) transformation of the host cell - expression vectors that contain nucleic acid molecules encoding the first light chain and the first heavy chain of a bispecific antibody, - expression vectors that contain nucleic acid molecules encoding the second light chain and the second heavy chain of the bispecific antibody, d) culturing the host cell under conditions that allow the synthesis of said bivalent bispecific antibody; and d) isolating the said divalent bispecific antibody from the cell culture.

Images