Bispecific antibodies, including heterodimers based on MHC proteins

Abstract

The present invention relates to the field of biotechnology, in particular to bivalent, bispecific, chimeric antibodies comprising heterodimers based on the membrane proximal domain of MHC (major histocompatibility complex) or MHC-like molecules (CD1 (cluster of differentiation 1) or HFE (hemochromatosis protein)), as well as technologies for producing said bispecific antibodies. The present invention further relates to nucleic acids encoding said bispecific antibodies, expression vectors, host cells for producing said bivalent, chimeric, bispecific antibodies and methods for producing said cells.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, in particular to bivalent, bispecific, chimeric antibodies comprising heterodimers based on the membrane proximal domain of MHC (major histocompatibility complex) or MHC-like proteins (CD1 (cluster of differentiation 1) or HFE (hemochromatosis protein)), as well as technologies for producing said bispecific antibodies. The present invention further relates to nucleic acids encoding said bispecific antibodies, expression vectors, host cells for producing said bivalent, chimeric, bispecific antibodies and methods for producing said cells. [Background technology]

[0002] Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have proven useful as effective pharmaceutical agents for the treatment of several disorders and diseases. A naturally occurring human antibody molecule consists of two heavy chain homodimers, each pairing with two identical light chain molecules to form a heterodimer. Conventional monoclonal antibodies in the form of whole molecules consist of a bivalent ("two-arm") heterodimer of a heavy and a light chain.

[0003] Diseases are often caused as a result of several pathologies and are accompanied by many complications. Bispecific antibodies are able to bind and thereby neutralize two or more different antigens per antibody molecule. The possibility of significant improvements in the therapeutic properties (and value) of drugs compared to monoclonal antibodies has made bispecific antibodies an active research area. Over the past two decades, the literature has described many solutions regarding engineered versions of bispecific antibodies, such as those 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.

[0004] As mentioned above, there are many techniques for generating molecules with combined antigen-binding domains, i.e., with antigen-binding domains that are different from each other. However, each of these methods has its drawbacks.

[0005] Cross-linking by chemical methods is a time-consuming process, since homodimers and other undesirable by-products should be removed from the corresponding moieties. Moreover, the chemical modification step may alter the integrity of the protein, thus compromising its stability. Thus, the above methods are typically ineffective and may result in the loss of antibody activity.

[0006] Cell fusion-based methods (e.g., hybridoma production) are any assembly of two heavy chains and two light chains, resulting in 10 combinations of antibodies. Target heteromultimeric antibodies are only a portion of the antibodies produced in this manner. Isolation of target heteromultimeric proteins significantly reduces product yield and increases production costs.

[0007] Recombinant DNA technology has been used to generate a variety of heteromultimeric antibodies, e.g., single chain Fv fragments, diabodies, etc., that do not contain an Fc fragment. The main drawback of this type of antibody molecule is the absence of an Fc domain, resulting in an antibody that cannot trigger effector functions (e.g., complement activation, binding to Fc receptors, etc.). Thus, there is a need for bispecific antibodies that contain a functional Fc domain.

[0008] Recombinant DNA technology has further been used to engineer bispecific antibodies using the Knob-into-Holes technique. See International Applications WO9627011 and WO9850431, and Merchant AM ET ALL., An efficient route to human bispecific IgG, Nat Biotechnol. 1998 July;16(7):677-81. One factor limiting the use of the above method is the fact that the light chains of the two initial antibodies should be identical to prevent mispairing and the formation of undesirable and / or inactive molecules when expressed in a single cell.

[0009] The purity of the bispecific antibody product depends on two factors: a) heterodimeric assembly of two different heavy chains co-expressed in a cell, and b) The precise pairing of two different light chains with the corresponding heavy chains.

[0010] The "knobs-into-holes" technique for designing bispecific antibodies solves the problem of correct heterodimer assembly of two different heavy chains co-expressed in a cell. However, the use of the knobs-into-holes technique for designing bispecific antibodies allows the achievement of only about 25% yield of properly assembled bispecific products, since the problem of correct pairing of two different light chains with the corresponding heavy chains remains to be solved.

[0011] The problem of correct pairing of two different light chains with the corresponding heavy chains can be solved in various ways: 1. The use of (identical) light chains in the first and second antigen-binding portions 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).

[0012] The drawback of the above solution is its non-universality, because it may be problematic to select suitable light chains for both valencies.Furthermore, amino acid substitutions in the light chain to optimize the properties of the antigen-binding fragment may affect both valencies.Furthermore, the binding of the antibody to the second antigen may be destroyed.

[0013] 2. Use of a single chain format, ie, a format in which the light and heavy chains of the antigen-binding fragment specific for a first antigen are interconnected via a linker of several amino acids. This format has technical disadvantages because it fuses the antibody core (IgA, IgD, IgE, IgG or IgM) to an additional binding protein (e.g., scFv or scFab) or uses linkers that fuse, for example, the light and heavy chain variable domains (VH and VL) in an scFv or the light chain (VL-CK (or CL)) to VH-CH1 in an scFab. Linkers can cause problems in a therapeutic setting. In fact, these foreign peptides can provoke an immune response against the linker itself or against the binding region between the protein and the linker. Moreover, the flexibility of these peptides and their mobility make them more susceptible to proteolytic cleavage, potentially resulting in lower antibody stability, aggregation and increased immunogenicity.

[0014] 3. Modification of the CH1-CK domain in bispecific antibodies to alter the interaction interface in bispecific antibody expression technology to eliminate incorrect association of the light chain. For example, international application WO2017059551 provides various amino acid substitutions in CH1 and / or CK that facilitate preferred pairing between the desired heavy chain and the desired light chain.

[0015] Despite the various bispecific antibody expression technologies described above, there remains a need in the art for improved purity of the bispecific antibody product, as well as scalable production solutions for producing correctly assembled bispecific antibodies. Summary of the Invention

[0016] The new format of bispecific chimeric antibodies comprising heterodimers based on the membrane proximal domain of MHC or MHC-like proteins, such as CD1 (cluster of differentiation 1) or HFE (human homeostatic iron regulatory protein), developed by the authors of the present invention, as well as the technology for producing said bispecific chimeric antibodies, surprisingly allows the production of high yield products with the correct heterodimeric assembly of two different heavy chains co-expressed in a cell and the correct pairing between two different light chains and the corresponding heavy chains.

[0017] The new format of bispecific chimeric antibody comprising heterodimers based on the membrane proximal domains of MHC or MHC-like proteins, developed by the authors of the present invention, as well as the technology for producing said bispecific antibodies, surprisingly allows the production of a highly pure and correctly assembled bispecific chimeric antibody product.

[0018] As a result, the above results reduce the production costs for producing precisely assembled bispecific antibodies and provide a scalable production solution. In one aspect, the invention provides a bivalent, bispecific, chimeric antibody, comprising: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; wherein the first light chain comprises a light chain variable domain and a light chain constant domain; wherein said first heavy chain comprises a heavy chain variable domain and an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and a third (CH3) heavy chain constant domain; b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen; the second light chain comprises a light chain variable domain and a constant domain; The constant domain is selected from the group consisting of the first membrane proximal domain of the MHC (major histocompatibility complex) or the first membrane proximal domain of an MHC-like protein; wherein the second heavy chain is A heavy chain variable domain, a constant domain selected from the group consisting of the second membrane proximal domain of the MHC (major histocompatibility complex) or the second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains. Contains; wherein said first membrane proximal domain of an MHC or MHC-like protein and said second membrane proximal domain of an MHC or MHC-like protein form a heterodimer therebetween that is stabilized by a disulfide bond; wherein the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are modified to contact each other with their surface and form a bivalent, bispecific chimeric antibody, said modification in the heavy chain CH3 domain being a substitution that facilitates heterodimerization. The present invention relates to a bivalent, bispecific, chimeric antibody comprising:

[0019] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC or MHC-like protein and a second membrane proximal domain of an MHC or MHC-like protein that form a heterodimer therebetween that is stabilized by a disulfide bond due to a mutation or mutations in the first and / or second constant domain forming an S-S bond (disulfide cysteine ​​bridge) between the first and second membrane proximal domains of the MHC or MHC-like protein.

[0020] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC or MHC-like protein and a second membrane proximal domain of an MHC or MHC-like protein which form a heterodimer therebetween, stabilized by a disulfide bond by one or more (1-10) amino acids at the C-terminus which form an S-S bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and an extension of the first membrane proximal domain of the MHC or MHC-like protein by a terminal Cys at the C-terminus.

[0021] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an extension of the first membrane proximal domain of the MHC or MHC-like protein, which extension is the sequence of the three amino acids GSC.

[0022] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first light chain comprising a light chain variable domain (VL1) and a light chain constant domain; a first heavy chain comprising an antibody heavy chain constant domain comprising a heavy chain variable domain (VH1) and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain comprising a light chain variable domain (VL2) and the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; a second heavy chain comprising a heavy chain variable domain (VH2), a second membrane proximal domain of MHC or a second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Including, wherein a first membrane proximal domain of the MHC or MHC-like protein and a second membrane proximal domain of the MHC or MHC-like protein form a heterodimer therebetween that is stabilized by a disulfide bond due to a mutation or mutations in the first and / or second constant domain forming a S-S bond (disulfide cysteine ​​bridge) between the first and second membrane proximal domains of the MHC or MHC-like protein; Here, a knob-into-hole structure is formed between the first and second Fc variants in the CH3 domain.

[0023] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first light chain comprising a light chain variable domain (VL1) and a light chain constant domain; a first heavy chain comprising an antibody heavy chain constant domain comprising a heavy chain variable domain (VH1) and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain comprising a light chain variable domain (VL2) and the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; a second heavy chain comprising a heavy chain variable domain (VH2), a second membrane proximal domain of MHC or a second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Including, wherein a knob-into-hole structure is formed between the first Fc variant and the second Fc variant in the CH3 domain; wherein the first membrane proximal domain of the MHC or the first membrane proximal domain of the MHC-like protein further comprises an extension of the first membrane proximal domain of the MHC or MHC-like protein by one or more (1-10) amino acids at the C-terminus and a terminal Cys at the C-terminus which form an S-S bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and the hinge.

[0024] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first light chain comprising a light chain variable domain (VL1) and a light chain constant domain; a first heavy chain comprising an antibody heavy chain constant domain comprising a heavy chain variable domain (VH1) and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain comprising a light chain variable domain (VL2) and the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; a second heavy chain comprising a heavy chain variable domain (VH2), a second membrane proximal domain of MHC or a second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Including, wherein a knob-into-hole structure is formed between the first Fc variant and the second Fc variant in the CH3 domain; wherein the first membrane proximal domain of the MHC or the first membrane proximal domain of the MHC-like protein further comprises an extension of the first membrane proximal domain of the MHC or MHC-like protein by the amino acid sequence GSC, which forms an S-S bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and the hinge.

[0025] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane-proximal domain of MHC class I (major histocompatibility complex class I); the first membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the first membrane proximal domain of MHC class I, or Modified variants of the first membrane-proximal domain of MHC class II The first membrane proximal domain of MHC may be selected from the group comprising:

[0026] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the second membrane-proximal domain of MHC class I (major histocompatibility complex class I); the second membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the second membrane proximal domain of MHC class I, or Modified variants of the second membrane-proximal domain of MHC class II The second membrane proximal domain of MHC may be selected from the group comprising:

[0027] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises the membrane proximal domain of human MHC class I. In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises the membrane proximal domain of human MHC class II.

[0028] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a membrane proximal domain of MHC class II selected from the group of HLA-DM, HLA-DO, HLA-DP, HLA-DQ or HLA-DR.

[0029] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a membrane proximal domain of MHC class I selected from the group of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G.

[0030] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a membrane proximal domain of a human MHC-like protein. In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of CD1 (cluster of differentiation 1); the first membrane-proximal domain of HFE (hemochromatosis protein); a modified variant of the first membrane proximal domain of CD1, or Modified variants of the first membrane-proximal domain of HFE The first membrane proximal domain of an MHC-like protein may be selected from the group comprising:

[0031] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the second membrane-proximal domain of CD1 (cluster of differentiation 1); the second membrane-proximal domain of HFE (hemochromatosis protein); a modified variant of the second membrane proximal domain of CD1, or Modified variants of the second membrane-proximal domain of HFE The second membrane proximal domain of the MHC-like protein may be selected from the group comprising:

[0032] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a membrane proximal domain of CD1 selected from the group of CD1a, CD1b, CD1c, CD1d or CD1e. In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a variable fragment of a second light chain (VL) separated from a first membrane proximal domain of the MHC or MHC-like protein by a linker of 1-25 amino acids in length, and / or a variable fragment of a second heavy chain (VH) separated from a second membrane proximal domain of the MHC or MHC-like protein by a linker of 1-25 amino acids in length.

[0033] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a) a CH3 domain of one heavy chain, which has been modified in such a way that on the surface of the CH3 domain of one heavy chain that contacts the surface of the CH3 domain of the other heavy chain in the bivalent bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, resulting in the formation of a knob on the surface of the CH3 domain of one heavy chain that can fit into a hole on the surface of the CH3 domain of the other heavy chain, and b) a CH3 domain of the other heavy chain, which has been modified such that on the surface of the CH3 domain of the second heavy chain that contacts the surface of the CH3 domain of the first heavy chain in the bivalent bispecific antibody, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, resulting in the formation of a hole on the surface of the CH3 domain of the second heavy chain that can accept a knob on the interface of the CH3 domain of the first heavy chain; Includes; wherein the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W); And, wherein the amino acid residue having a smaller side chain volume is selected from the group including alanine (A), serine (S), threonine (T) and valine (V).

[0034] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a constant domain of the first light chain of the antibody selected from CK or CL. In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an antibody CH3 domain which is further modified by introduction of the amino acid cysteine ​​(C) into the corresponding position of each CH3 domain such that disulfide bridges can form between the CH3 domains.

[0035] In some embodiments of the invention, the bivalent, bispecific antibody comprises a CH3 domain of one heavy chain that is modified to form a knob and a CH3 domain of the other heavy chain that is modified to form a hole, or vice versa.

[0036] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a CH3 domain of one heavy chain with the amino acid substitutions S354C / T366W and a CH3 domain of the other heavy chain with the amino acid substitutions Y349C / T366S / L368A / Y407V.

[0037] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a CH3 domain of one heavy chain with the amino acid substitutions Y349C / T366S / L368A / Y407 and a CH3 domain of the other heavy chain with the amino acid substitutions S354C / T366W.

[0038] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, the α2 domain of MHC C1 and the β2 domain of MHC II, respectively, which form a heterodimer between them.

[0039] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, which are the β2 domain of MHC II and the α2 domain of MHC II, respectively, that form a heterodimer between them.

[0040] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an α2 domain of MHC II having an amino acid sequence selected from the group of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38, and a β2 domain of MHC II having an amino acid sequence selected from the group of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39.

[0041] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of the α2 domain of MHC II and the β2 domain of MHC II, respectively, and form a heterodimer therebetween.

[0042] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of the β2 domain of MHC II and the α2 domain of MHC II, respectively, and form a heterodimer therebetween.

[0043] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, which are the α3 domain of MHC I and β2 microglobulin (β2M), respectively, forming a heterodimer between them.

[0044] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, which are β2 microglobulin (β2M) and the α3 domain of MHC I, respectively, forming a heterodimer between them.

[0045] In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an α3 domain of MHC I having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29, and a β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO: 46.

[0046] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of the α3 domain of MHC I and β2 microglobulin (β2M), respectively, forming a heterodimer therebetween.

[0047] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of β2 microglobulin (β2M) and α3 domain of MHC II, respectively, forming a heterodimer therebetween.

[0048] In some aspects of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of β2 microglobulin (β2M) having an amino acid sequence selected from SEQ ID NO:47 or SEQ ID NO:48.

[0049] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are the α3 domain of CD1 and β2 microglobulin (β2M), respectively, forming a heterodimer between them.

[0050] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are β2 microglobulin (β2M) and the α3 domain of CD1, respectively, forming a heterodimer between them.

[0051] In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an α3 domain of CD1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44 and a β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO: 46.

[0052] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of the α3 domain of CD1 and a modified variant of β2 microglobulin (β2M), respectively, that form a heterodimer therebetween.

[0053] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of β2 microglobulin (β2M) and a modified variant of the α3 domain of CD1, respectively, forming a heterodimer therebetween.

[0054] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a modified variant of the α3 domain of CD1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 49-56 and / or SEQ ID NO: 109 and a modified variant of β2 microglobulin (β2M) having an amino acid sequence selected from SEQ ID NO: 47 or SEQ ID NO: 48.

[0055] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are the α3 domain of HFE and β2 microglobulin (β2M), respectively, forming a heterodimer between them.

[0056] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are β2 microglobulin (β2M) and the α3 domain of HFE, respectively, forming a heterodimer between them.

[0057] In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an α3 domain of HFE having the amino acid sequence of SEQ ID NO:45 and a β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO:46.

[0058] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of the α3 domain of HFE and a modified variant of β2 microglobulin (β2M), respectively, that form a heterodimer therebetween.

[0059] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of β2 microglobulin (β2M) and a modified variant of the α3 domain of HFE, respectively, that form a heterodimer therebetween.

[0060] In some embodiments of the present invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of β2 microglobulin (β2M) having an amino acid sequence selected from SEQ ID NO:47 or SEQ ID NO:48.

[0061] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that comprises a substitution of a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from the first and second membrane proximal domains of the MHC or MHC-like protein.

[0062] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that contains one or more substitutions at various positions in the membrane proximal domain of the MHC or MHC-like protein, respectively, resulting in an increase in thermodynamic stability Tm of more than 1° C. compared to the wild-type membrane proximal domain of the MHC or MHC-like protein.

[0063] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that contains one or more substitutions at various positions in the membrane proximal domain of the MHC or MHC-like protein, respectively, that results in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of the MHC or MHC-like protein.

[0064] In some aspects of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that comprises one or more substitutions at various positions in the membrane proximal domain of the MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of the MHC or MHC-like protein.

[0065] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first light chain variable domain and a second light chain variable domain that are identical. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment belonging to IgG.

[0066] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an Fc fragment selected from the group comprising human IgG1, IgG2, or IgG4. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment monomer into which further substitutions have been introduced, resulting in the absence of ADCC, CDC and / or ADCP properties in the bivalent, bispecific antibody.

[0067] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an Fc fragment monomer into which further LALA substitutions (L234A and L235A) have been introduced. In some embodiments of the invention, the bivalent, bispecific, chimeric antibodies comprise an Fc fragment monomer into which further substitutions have been introduced resulting in a prolongation of the effect of the antibody.

[0068] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment monomer into which YTE substitutions (M252Y, S254T and T256E) have been further introduced. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment monomer into which further substitutions have been introduced resulting in enhanced ADCC, CDC and / or ADCP properties in the bivalent, bispecific antibody.

[0069] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an Fc fragment monomer into which the substitution E345R has been further introduced. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to CD20 and CD3.

[0070] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to BCMA and CD3. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to PD-L1 and CD47.

[0071] In some embodiments of the invention, the bivalent, bispecific chimeric antibody specifically binds to coagulation factor 9 (FIX) and coagulation factor 10 (FX). In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to GD2 and CD3.

[0072] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to AXL and CD3. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to PD-L1 and TGF-beta.

[0073] In one aspect, the invention relates to an isolated nucleic acid encoding any of the above-described bivalent, bispecific, chimeric antibodies. In some aspects of the invention, the nucleic acid is DNA.

[0074] In one aspect, the invention relates to an expression vector comprising any of the above nucleic acids. In one aspect, the invention relates to a method for producing a host cell for producing any of the above-described bivalent, bispecific, chimeric antibodies, comprising transformation of the cell with an expression vector as described above.

[0075] In one aspect, the invention relates to a host cell for producing any of the above-described bivalent, bispecific, chimeric antibodies comprising any of the above-described nucleic acids. In one aspect, the invention provides a method for producing any of the bivalent, bispecific, chimeric antibodies described above, comprising the steps of: a) removing a host cell from - an expression vector comprising a nucleic acid molecule encoding a first light chain and a first heavy chain of the bispecific chimeric antibody, - an expression vector comprising a nucleic acid molecule encoding a second light chain and a second heavy chain of the bispecific chimeric antibody. Transforming with b) culturing the host cells under conditions suitable for the synthesis of said bivalent, bispecific, chimeric antibody; and c) isolating said bivalent, bispecific antibody from the cell culture. The present invention relates to a method comprising the steps of: [Brief description of the drawings]

[0076] [Figure 1] Figure 1 is a schematic diagram of the bivalent, bispecific chimeric antibody format. A is the "forward" orientation of the dimerization unit and B is the "reverse" orientation of the dimerization unit. VH1, VL1 are the heavy and light chain variable domains, respectively, responsible for binding to antigen 1; VH2, VL2 are the variable domains responsible for binding to antigen 2. CH1, CK are the heavy and light chain constant domains, respectively; b2M is β microglobulin and CD1b is the α3 domain of the CD1b protein. Knob, hole are mutations in the CH3 domain of the antibody that allow heterodimerization of the heavy chains. [Diagram 2] FIG. 2 is a schematic diagram of the heavy and light chain complex of a chimeric antibody in which the CH1 and CK domains are replaced by β2 microglobulin and the membrane proximal domain α3 of the CD1b protein. A is the "direct" orientation of the domains of the dimerization unit; B is the "reverse" orientation of the dimerization unit. VH, VL are the variable domains of the antibody; b2M is β microglobulin; CD1b is the membrane proximal domain α3 of the CD1b protein; hinge is the hinge region of the antibody, the first 5 amino acids are explicitly indicated; Fc is the Fc fragment of the antibody; b2M, amino acids from the C-terminus of the α3 domain of CD1b and the N-terminus of the hinge region are explicitly indicated; S-S bonds are shown as dotted lines. [Diagram 3]Figure 3 shows electropherograms of chimeric antibodies with and without additional disulfide bonds between the heavy and light chains on a 7.5% polyacrylamide gel, non-reducing conditions. Lanes: M is the standard marker Precision Plus Protein™ Dual Color Standards (BIO-RAD), the molecular weights of the corresponding lanes are shown in kDa in the left column on the gel. 1 - control prorugolimab-IgG1; 2 - monospecific chimeric antibody based on a domain of MHC-like protein replacing CH1-CK with the variable domain of prorugolimab and an additional disulfide bridge; 3 - monospecific chimeric antibody based on a domain of MHC-like protein replacing CH1-CK with the variable domain of prorugolimab and an additional disulfide bridge; 4 - monospecific chimeric antibody based on a domain of MHC-like protein replacing CH1-CK in the "reverse" orientation with the variable domain of prorugolimab and an additional disulfide bridge. [Figure 4]FIG. 4 is a diagram of an experiment to test the effect of MHC dimerization units on incorrect pairing between heavy and light chains. The ovals indicate the domains replacing CH1 and CK from MHC-like proteins. First group: Samples 01-001 to 01-003 are antibodies with a "correct" combination of VH and VL and an "incorrect" pair of constant domains. Second group: Samples 01-004 to 01-007 are antibodies with a "incorrect" combination of VH and VL and an "incorrect" pair of constant domains. Third group: Samples 01-008 to 01-010 are antibodies with a "incorrect" combination of VH and VL and an "incorrect" pair of constant domains. Fourth group: Samples 01-011 and 01-012 are control antibodies that are chimeric antibodies in which the constant domains are replaced by MHC dimerization units and classical antibodies of IgG1 format, respectively. An "exact" combination of VH and VL refers to a VH and VL pair that originates from a single antibody. An "incorrect" combination of VH and VL refers to a VH and VL that originate from different antibodies. An "exact" pair of constant domains refers to a CH1-CK pair or a pair of domains that originate from an MHC-like protein (in this case the α3 domains of β2 microglobulin and CD1b protein). An "incorrect" pair of constant domains refers to a CH1-CD1b or β2 microglobulin-CK pair. [Diagram 5]Figure 5 shows an electropherogram of a sample produced in an experiment containing strand mismatch. 7.5% polyacrylamide gel, non-reducing conditions. A-lane: M is a standard marker, Precision Plus Protein™ Dual Color Standards (BIO-RAD), and the molecular weight of the corresponding lane is indicated in kDa in the left column of the gel. 1-01-001:2-01-002;3-01-007;4-01-008;5-01-009;6-01-012;B-lane: standard markers of M-Precision Plus Protein™ Dual Color Standards (BIO-RAD);1-01-003;2-01-004;3-01-005;4-01-006;5-01-007;6-01-012;C-lane: standard markers of M-Precision Plus Protein™ Dual Color Standards (BIO-RAD);1-01-010;2-01-011. [Figure 6] Figure 6: Electropherograms of bispecific chimeric antibodies. A - 7.5% polyacrylamide gel, non-reducing conditions, B - 12.5% ​​polyacrylamide gel, reducing conditions. Lanes: M are standard markers from Precision Plus Protein™ Dual Color Standards (BIO-RAD), and the molecular weights of the corresponding lanes are indicated in kDa in the left column of the gel. 1-02-004, 2-02-005, 3-02-006, 4-02-007, 5-02-008, 6-02-009. [Figure 7] Figure 7 is a sensogram of an experiment involving the simultaneous interaction of antibodies 02-006 and 02-007 with two different antigens (hPD1ex-H6F and hCSF1R_His). The stages (steps of the experiment) are numbered at the top of the image and separated by vertical lines. Two sensograms are given for each antibody showing the interaction with hCSF1R_His at step 8 (an increase in signal level is observed) and the reference signal in 1xKB buffer without hCSF1R_His at step 8 (no increase in signal level). [Figure 8] Figure 8 is the deconvoluted mass spectrum of the total ion current chromatogram for sample 02-004. A-peak 4, B-peak 5, C-peak 6. [Figure 9-1]Figure 9 shows an electropherogram of SDS gel electrophoresis of bispecific chimeric antibodies after proteolysis with GingisKHAN protease. 7.5% polyacrylamide gel, non-reducing conditions. A-lane: M is a standard marker, Precision Plus Protein™ Dual Color Standards (BIO-RAD), the molecular weight of the corresponding lane is indicated in kDa in the left column on the gel.1 - 02-004 not processed by GingisKHAN, 2 - 02-009 not processed by GingisKHAN, 3 - ocrelizumab not processed by GingisKHAN, 4 - 01-011 not processed by GingisKHAN, 5 - prorugolimab not processed by GingisKHAN, 6 - 02-004 not processed by GingisKHAN in 100 mM ammonium bicarbonate buffer pH 7.2; B-lane: M-Precision Plus Protein™ Dual Color Standard markers from Standards (BIO-RAD); 1 - 02-004 after proteolysis with GingisKHAN, 2 - 02-009 after proteolysis with GingisKHAN, 3 - ocrelizumab after proteolysis with GingisKHAN, 4 - 01-011 after proteolysis with GingisKHAN, 5 - prorugolimab after proteolysis with GingisKHAN, 6 - 02-004 after proteolysis with GingisKHAN in 100 mM ammonium bicarbonate buffer pH 7.2, C-lane: M-Precision Plus Protein™ Dual Color Standard markers for Standards (BIO-RAD); 1 - 02-005 not processed by GingisKHAN, 2 - 02-005 after proteolysis by GingisKHAN, 3 - 02-006 not processed by GingisKHAN, 4 - 02-006 after proteolysis by GingisKHAN, 5 - 02-007 not processed by GingisKHAN, 6 - 02-007 after proteolysis by GingisKHAN, D-lane: Standard markers for M-Precision Plus Protein™ Dual Color Standards (BIO-RAD); 1 - 02-008 not processed by GingisKHAN, 2 - 02-008 after proteolysis by GingisKHAN, 3 - 02-004 not processed by GingisKHAN, 4 - 02-004 after proteolysis by GingisKHAN. [Figure 9-2]Figure 9 shows an electropherogram of SDS gel electrophoresis of bispecific chimeric antibodies after proteolysis with GingisKHAN protease. 7.5% polyacrylamide gel, non-reducing conditions. A-lane: M is a standard marker, Precision Plus Protein™ Dual Color Standards (BIO-RAD), the molecular weight of the corresponding lane is indicated in kDa in the left column on the gel.1 - 02-004 not processed by GingisKHAN, 2 - 02-009 not processed by GingisKHAN, 3 - ocrelizumab not processed by GingisKHAN, 4 - 01-011 not processed by GingisKHAN, 5 - prorugolimab not processed by GingisKHAN, 6 - 02-004 not processed by GingisKHAN in 100 mM ammonium bicarbonate buffer pH 7.2; B-lane: M-Precision Plus Protein™ Dual Color Standard markers from Standards (BIO-RAD); 1 - 02-004 after proteolysis with GingisKHAN, 2 - 02-009 after proteolysis with GingisKHAN, 3 - ocrelizumab after proteolysis with GingisKHAN, 4 - 01-011 after proteolysis with GingisKHAN, 5 - prorugolimab after proteolysis with GingisKHAN, 6 - 02-004 after proteolysis with GingisKHAN in 100 mM ammonium bicarbonate buffer pH 7.2, C-lane: M-Precision Plus Protein™ Dual Color Standard markers for Standards (BIO-RAD); 1 - 02-005 not processed by GingisKHAN, 2 - 02-005 after proteolysis by GingisKHAN, 3 - 02-006 not processed by GingisKHAN, 4 - 02-006 after proteolysis by GingisKHAN, 5 - 02-007 not processed by GingisKHAN, 6 - 02-007 after proteolysis by GingisKHAN, D-lane: Standard markers for M-Precision Plus Protein™ Dual Color Standards (BIO-RAD); 1 - 02-008 not processed by GingisKHAN, 2 - 02-008 after proteolysis by GingisKHAN, 3 - 02-004 not processed by GingisKHAN, 4 - 02-004 after proteolysis by GingisKHAN. [Figure 10] Figure 10: Electropherogram of samples with modifications introduced into the dimerization unit based on the membrane proximal domain of MHC-like proteins. 7.5% polyacrylamide gel, non-reducing conditions. A-lane: M is a standard marker of Precision Plus Protein™ Dual Color Standards (BIO-RAD), the molecular weight of the corresponding lane is indicated in kDa in the left column on the gel. 1-prorugolimab; 2-01-011; 3-03-003; 4-03-004; 5-03-005; 6-03-006; 7-03-007; 8-03-008. B-lane: M-standard marker of Precision Plus Protein™ Dual Color Standards (BIO-RAD); 1-prorugolimab; 2-01-011; 3-03-001; 4-03-002. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] General definitions and general methods Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0078] Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Typically, the classification of the present invention and the methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal chemistry and pharmaceutical chemistry, as well as protein and nucleic acid hybridization and chemistry described herein are well known to those of skill in the art and are widely used in the art. Enzymatic reactions and purification methods are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein.

[0079] As used herein, the term "KD" refers to the affinity constant (or equilibrium constant) calculated from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). "Binding affinity" generally refers to the strength of the sum of 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 indicated, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be expressed by an affinity constant (KD). Preferred Kd values ​​are about 200nM, 150nM, 100nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 8nM, 6nM, 4nM, 2nM, 1nM, or less. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0080] The terms "Kd", "koff" or "kdis" refer to the dissociation rate constant of a particular interaction between a binding molecule and an antigen. The dissociation rate constant koff can be measured using biolayer interferometry, for example, using the Octet™ system.

[0081] The terms "Ka," "kon" or "on-rate" refer to the binding rate constant. The term “R 2 " refers to the coefficient of determination.

[0082] The term "response" refers to an antibody-antigen binding signal. As used in this specification and the claims that follow, unless the context indicates otherwise, the words "include" and "comprise" or variations thereof such as "includes," "including," "comprises," or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0083] Detailed Description of the Invention Bivalent, bispecific chimeric antibodies The present invention relates to bivalent, bispecific, chimeric antibodies.

[0084] The antibody according to the invention is a monoclonal antibody. The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and isolated by a separate clonal cell population.

[0085] The antibody of the present invention is a recombinant antibody. The term "recombinant antibody" refers to an antibody expressed in a cell or cell line that contains a nucleotide sequence encoding an antibody that is not naturally associated with the cell.

[0086] A bivalent, bispecific, chimeric antibody according to the invention is an isolated antibody. The term "isolated" as used to describe the various antibodies described herein refers to an antibody that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Impurities (contaminating components) from the natural environment are typically materials that would interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. An isolated polypeptide is typically prepared by at least one purification step.

[0087] In one aspect, the invention provides a bivalent, bispecific, chimeric antibody, comprising: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; wherein the first light chain comprises a light chain variable domain and a light chain constant domain; wherein said first heavy chain comprises a heavy chain variable domain and an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and a third (CH3) heavy chain constant domain; b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen; wherein the second light chain comprises a light chain variable domain and a constant domain; The constant domain is selected from the group consisting of the first membrane proximal domain of the MHC (major histocompatibility complex) or the first membrane proximal domain of an MHC-like protein; wherein the second heavy chain comprises: A heavy chain variable domain, a constant domain selected from the group consisting of the second membrane proximal domain of the MHC (major histocompatibility complex) or the second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains. Contains; wherein said first membrane proximal domain of an MHC or MHC-like protein and said second membrane proximal domain of an MHC or MHC-like protein form a heterodimer therebetween that is stabilized by a disulfide bond; wherein the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are modified to contact each other with their surface and form a bivalent, bispecific chimeric antibody, said modification in the heavy chain CH3 domain being a substitution that facilitates heterodimerization. The present invention relates to a bivalent, bispecific, chimeric antibody comprising:

[0088] A heterodimer formed by a first membrane proximal domain of an MHC or MHC-like protein and a second membrane proximal domain of an MHC or MHC-like protein and stabilized by a disulfide bond is 1) a heterodimer comprising a mutation or a plurality of mutations in the first and / or second membrane proximal domain of an MHC or MHC-like protein to form an S-S bond (disulfide bond, cysteine ​​bridge) between the first and second membrane proximal domains of the MHC or MHC-like protein; or, 2) extension of the first membrane proximal domain of the MHC or MHC-like protein by one or more (1-10) amino acids at the C-terminus and a terminal Cys at the C-terminus to form an SS bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and the hinge; means.

[0089] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC or MHC-like protein and a second membrane proximal domain of an MHC or MHC-like protein which form a heterodimer therebetween that is stabilized by a disulfide bond due to a mutation or mutations in the first and / or second membrane proximal domain of the MHC or MHC-like protein that form an S-S bond (disulfide cysteine ​​bridge) between the first and second membrane proximal domains of the MHC or MHC-like protein.

[0090] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC or MHC-like protein and a second membrane proximal domain of an MHC or MHC-like protein which form a heterodimer therebetween stabilized by a disulfide bond by one or more (1-10) amino acids at the C-terminus forming an S-S bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and the hinge and an extension of the first membrane proximal domain of the MHC or MHC-like protein with a terminal Cys at the C-terminus.

[0091] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an extension of the first membrane proximal domain of the MHC or MHC-like protein, which extension is the sequence of the three amino acids GSC.

[0092] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first light chain comprising a light chain variable domain (VL1) and a light chain constant domain; a first heavy chain comprising an antibody heavy chain constant domain comprising a heavy chain variable domain (VH1) and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain comprising a light chain variable domain (VL2) and the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; a second heavy chain comprising a heavy chain variable domain (VH2), a second membrane proximal domain of MHC or a second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Including, a first membrane proximal domain of the MHC or MHC-like protein and a second membrane proximal domain of the MHC or MHC-like protein form a heterodimer therebetween that is stabilized by a disulfide bond due to a mutation or mutations in the first and / or second membrane proximal domain of the MHC or MHC-like protein forming an S-S bond (disulfide cysteine ​​bridge) between the first and second membrane proximal domains of the MHC or MHC-like protein; A knobs-into-hole structure is formed between the first and second Fc variants in the CH3 domain.

[0093] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first light chain comprising a light chain variable domain (VL1) and a light chain constant domain; a first heavy chain comprising an antibody heavy chain constant domain comprising a heavy chain variable domain (VH1) and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain comprising a light chain variable domain (VL2) and the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; a second heavy chain comprising a heavy chain variable domain (VH2), a second membrane proximal domain of MHC or a second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Including, a knob-into-hole structure is formed between the first and second Fc variants in the CH3 domain; The first membrane proximal domain of the MHC or the first membrane proximal domain of the MHC-like protein further comprises an extension of the first membrane proximal domain of the MHC or MHC-like protein by one or more (1-10) amino acids at the C-terminus and a terminal Cys at the C-terminus which form an S-S bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and the hinge.

[0094] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first light chain comprising a light chain variable domain (VL1) and a light chain constant domain; a first heavy chain comprising an antibody heavy chain constant domain comprising a heavy chain variable domain (VH1) and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain comprising a light chain variable domain (VL2) and the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; a second heavy chain comprising a heavy chain variable domain (VH2), a second membrane proximal domain of MHC or a second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Including, a knob-into-hole structure is formed between the first and second Fc variants in the CH3 domain; The first membrane proximal domain of the MHC or the first membrane proximal domain of the MHC-like protein further comprises an extension of the first membrane proximal domain of the MHC or MHC-like protein by a GSC that forms an S-S bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and the hinge.

[0095] In the above bivalent, bispecific chimeric antibodies, the variable and constant domains are arranged in the heavy and light chains in the following order: First light chain: 1) a light chain variable domain, 2) light chain constant domain; First heavy chain of the antibody: 1) a heavy chain variable domain, 2) the first (CH1) heavy chain constant domain; 3) the second (CH2) heavy chain constant domain; and 4) the third (CH3) heavy chain constant domain; Second light chain: 1) a light chain variable domain, 2) the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; Second heavy chain of the antibody: 1) a heavy chain variable domain, 2) the second membrane proximal domain of MHC or the second membrane proximal domain of an MHC-like protein; 3) the second (CH2) heavy chain constant domain; and 4) The third (CH3) heavy chain constant domain.

[0096] FIG. 1 shows several variants of the bivalent, bispecific, chimeric antibody formats described above. As used herein, the term "antibody" or "immunoglobulin" (Ig) includes whole antibodies. The term "antibody" refers to a glycoprotein that contains at least two heavy (H) chains and two light (L) chains, or antigen-binding moieties, interconnected by disulfide bonds. 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 CA, Jr. et al., Immunobiology, 5th Edition, published by Garland Publishing, 2001). The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades RA, Pflanzer RG, Human Physiology, 4th Edition, published by Thomson Learning, 2002). Different heavy chains differ in size and composition; α and γ contain about 450 amino acids, while μ and ε have about 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 have a constant region composed of three constant domains CH1, CH2 and CH3 (in line), as well as a hinge region for added flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); heavy chains μ and ε have a constant region composed of four constant domains CH1, CH2, CH3 and CH4 (Janeway CA, Jr. et al., Immunolobiology, 5th ed., published by Garland Publishing, 2001). In mammals, only two types of light chains are known, designated lambda (λ) and kappa (κ). Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain has an approximate length of 211-217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably C kappa (κ).

[0097] An "antibody" according to the present invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, preferably IgG1).

[0098] The VL and VH regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are located between more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the 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.

[0099] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL and CH1 domains or a Fab-like fragment, i.e., a monovalent fragment consisting of the VL, VH domains, a first membrane proximal domain of an MHC or MHC-like protein and a second membrane proximal domain of an MHC or MHC-like protein.

[0100] Preferably, the CDRs of the antigen-binding portion, or the entire antigen-binding portion of an antibody of the invention, are substantially of human origin, derived from a mouse, llama or human donor library, or in which certain amino acid residues have been altered, e.g., replaced with different amino acid residues to optimize certain properties of the antibody, e.g., KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are of human origin or are substantially of human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human origin).

[0101] In other embodiments, the antigen-binding portions of the invention may be derived from other non-human species, including, but not limited to, mouse, llama, rabbit, rat or hamster, or the antigen-binding region may be derived from a human species.

[0102] The term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies. The V domains mediate antigen binding and determine the specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of invariant segments called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" or CDRs. Naturally occurring heavy and light chain variable domains each contain four FRs, often adopting a beta-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs, and the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0103] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions typically contain amino acid residues from the "complementarity determining regions" or "CDRs" and / or residues from the "hypervariable loops".

[0104] As used in this application, "Kabat numbering scheme" or "Kabat numbering" refers to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of antibody heavy and light chains (Kabat et al., Ann. NY Acad. Sci., 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0105] An antibody of the present invention that "binds" to a target antigen refers to an antibody that binds to the antigen with sufficient affinity and cross-reacts to a small extent with other proteins such that the antibody can be used as a diagnostic and / or therapeutic agent targeting cells or tissues expressing the protein or antigen. In such embodiments, the extent of antibody binding to non-target proteins is less than 10% of the antibody binding to the specific target protein by analytical methods: fluorescence activated cell sorting (FACS), radioimmunoassay (RIA) or ELISA. With respect to the binding of an antibody to a target molecule, the terms "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular target polypeptide refer to binding that is significantly (measurably) different from non-specific interactions.

[0106] Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule.For example, specific binding can be determined by competition with another molecule similar to the target, for example, an excess of unlabeled target.In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.As used herein, the term "specific binding" or phrase "specifically binds to" a specific polypeptide or epitope on a specific target polypeptide can be described by the example of a molecule having a Kd for target of at least about 200nM, or at least about 150nM, or at least about 100nM, or at least about 60nM, or at least about 50nM, or at least about 40nM, or at least about 30nM, or at least about 20nM, or at least about 10nM, or at least about 8nM, or at least about 6nM, or at least about 4nM, or at least about 2nM, or at least about 1nM, or higher. In one aspect, the term "specific binding" refers to binding of a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or epitopes on polypeptides.

[0107] The term "bispecific antibody" refers to an antibody that has antigen-binding domains that can specifically bind to two different epitopes on a single biomolecule or that can specifically bind to epitopes on two different biomolecules. Bispecific antibodies are also referred to herein as having "dual specificity" or as being "dual specificity" antibodies.

[0108] The fragment crystallizable region of an immunoglobulin ("Fc region, Fc") is the "tail" region of an immunoglobulin molecule that interacts with cell surface Fc receptors as well as with some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of two heavy chains, respectively; in IgM and IgE isotypes, Fc contains three heavy chain constant domains (CH2, CH3 and CH4 domains) in each polypeptide chain.

[0109] "Fc fragment monomer" is understood to mean an Fc region derived from the second and third constant domains of one of the two heavy chains (for IgG, IgA and IgD isotypes); for IgM and IgE isotypes, the Fc monomer comprises the three constant domains (CH2, CH3 and CH4 domains) of one of the two heavy chains.

[0110] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first light chain comprising a light chain variable domain (VL1) and a light chain constant domain; a first heavy chain comprising an antibody heavy chain constant domain comprising a heavy chain variable domain (VH1) and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain comprising a light chain variable domain (VL2) and the first membrane proximal domain of MHC or the first membrane proximal domain of an MHC-like protein; a second heavy chain comprising a heavy chain variable domain (VH2), a second membrane proximal domain of MHC or a second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Including, a knob-into-hole structure is formed between the first and second Fc variants in the CH3 domain; An additional peptide linker is inserted between the second membrane proximal domain of the MHC and / or the second membrane proximal domain of the MHC-like protein and the hinge.

[0111] As used herein, the term "peptide linker" is intended to mean any peptide capable of combining domains with a length depending on the domains to be linked together and containing any amino acid sequence. Preferably, the peptide linker has a length of 4 amino acids or more and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

[0112] MHC (major histocompatibility complex) refers to major histocompatibility complex molecules that are central components of the vertebrate immune system present on the surface of all nucleated cells. There are two main forms of MHC, specifically MHC class I and II.

[0113] MHC-like proteins refer to proteins that have structural similarity to the extracellular portion of MHC class I or MHC class II molecules. In particular, MHC-like proteins refer to, for example, CD1 (cluster of differentiation 1) proteins or HFE proteins (hereditary hemochromatosis proteins).

[0114] General structural similarities between the MHC I, II, CD1 and HFE molecules are evident, including uniformity in the spatial organization of the entire molecule, the number of domains (four), structural similarity of the membrane proximal domains of MHC and MHC-like proteins as well as antigen binding sites, and similar molecular weights.

[0115] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane-proximal domain of MHC class I (major histocompatibility complex class I); the first membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the first membrane proximal domain of MHC class I, or Modified variants of the first membrane-proximal domain of MHC class II The first membrane proximal domain of MHC may be selected from the group comprising:

[0116] In humans, classical MHC I genes are called HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G. Class I molecules consist of two polypeptide chains: a polymorphic α chain (sometimes called the heavy chain) and a smaller chain called β2 microglobulin (also known as the light chain), which is not generally polymorphic. These two chains form a non-covalent heterodimer on the cell surface. The α chain contains three domains (α1, α2, and α3). Exon 1 of the α chain gene codes for the leader sequence, exons 2 and 3 code for the α1 and α2 domains, exon 4 codes for the α3 domain, exon 5 codes for the transmembrane domain, and exons 6 and 7 code for the cytoplasmic tail. The α chain forms a peptide-binding region that includes the α1 and α2 domains.

[0117] The α2 domain is followed by the α3 domain, which is located at the C-terminus of the extracellular portion of the α chain of MHC I and forms a heterodimeric non-covalent complex with β2 microglobulin. Said heterodimeric non-covalent complex composed of the α3 domain of MHC I and β2 microglobulin is referred to herein as a heterodimer based on the membrane proximal domain of MHC I.

[0118] MHC class I molecules are expressed on all nucleated cells, including tumor cells. They are specifically expressed on T and B lymphocytes, macrophages, dendritic cells, and neutrophils, and function to present peptide fragments (typically 8-10 amino acids long) on ​​the surface of CD8+ cytotoxic T lymphocytes (CTLs).

[0119] In humans, classical MHC II genes are called HLA-DM, HLA-DO, HLA-DP, HLA-DQ or HLA-DR. MHC class II molecules are heterodimers composed of non-covalently linked alpha and beta chains. The extracellular portion of each chain consists of two domains (α1 (alpha 1), α2 (alpha 2) and β1 (beta 1), β2 (beta 2), respectively), connected by a short peptide (about 30 amino acid residues long) with a transmembrane segment. Following the transmembrane segment is a cytoplasmic domain containing about 10-15 residues.

[0120] The antigen-binding region of MHC class II molecules is formed by alpha helical sections of interacting chains similar to those of class I molecules, except that the antigen-binding groove of MHC class II molecules is formed not by two domains of a single alpha chain, but by two domains from different chains, the α1 and β1 domains.

[0121] The α1 domain is followed by an α2 domain which forms a heterodimeric non-covalent complex with the β2 domain. Said heterodimeric non-covalent complex composed of the α2 domain of MHC II and the β2 domain of MHC II is referred to in the present specification as a heterodimer based on the membrane proximal domain of MHC II.

[0122] In the structure of MHC class II molecules, the antigen-binding groove is more open than that of MHC class I molecules, thus accommodating longer peptides. In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the second membrane-proximal domain of MHC class I (major histocompatibility complex class I); the second membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the second membrane proximal domain of MHC class I, or Modified variants of the second membrane-proximal domain of MHC class II The second membrane proximal domain of MHC may be selected from the group comprising:

[0123] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises the membrane proximal domain of human MHC class I. In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises the membrane proximal domain of human MHC class II.

[0124] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane-proximal domain of MHC class I (major histocompatibility complex class I); the first membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the first membrane proximal domain of MHC class I, or Modified variants of the first membrane-proximal domain of MHC class II The first membrane proximal domain of MHC, which may be selected from the group comprising: And, the second membrane-proximal domain of MHC class I (major histocompatibility complex class I); the second membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the second membrane proximal domain of MHC class I, or Modified variants of the second membrane-proximal domain of MHC class II The second membrane proximal domain of MHC may be selected from the group comprising:

[0125] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of MHC, which is the first membrane proximal domain of MHC class I (major histocompatibility complex class I); and The second membrane-proximal domain of MHC, which is the second membrane-proximal domain of MHC class I (major histocompatibility complex class I) Includes.

[0126] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of MHC, which is the first membrane proximal domain of MHC class II (major histocompatibility complex class II); and The second membrane-proximal domain of MHC, which is the second membrane-proximal domain of MHC class II (major histocompatibility complex class II) Includes.

[0127] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of MHC which is a modified variant of the first membrane proximal domain of MHC class I; and A second membrane proximal domain of MHC that is a modified variant of the second membrane proximal domain of MHC class I Includes.

[0128] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of MHC which is a modified variant of the first membrane proximal domain of MHC class II; and A second membrane proximal domain of MHC that is a modified variant of the second membrane proximal domain of MHC class II Includes.

[0129] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of MHC, which is the first membrane proximal domain of MHC class I (major histocompatibility complex class I); and A second membrane proximal domain of MHC that is a modified variant of the second membrane proximal domain of MHC class I Includes.

[0130] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of MHC which is a modified variant of the first membrane proximal domain of MHC class I; and The second membrane-proximal domain of MHC, which is the second membrane-proximal domain of MHC class I (major histocompatibility complex class I) Includes.

[0131] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of MHC, which is the first membrane proximal domain of MHC class II (major histocompatibility complex class II); and A second membrane proximal domain of MHC that is a modified variant of the second membrane proximal domain of MHC class II Includes.

[0132] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of MHC which is a modified variant of the first membrane proximal domain of MHC class II; and The second membrane-proximal domain of MHC, which is the second membrane-proximal domain of MHC class II (major histocompatibility complex class II) Includes.

[0133] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a membrane proximal domain of MHC class II selected from the group of HLA-DM, HLA-DO, HLA-DP, HLA-DQ or HLA-DR.

[0134] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a membrane proximal domain of MHC class I selected from the group of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G.

[0135] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a membrane proximal domain of a human MHC-like protein. In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of CD1 (cluster of differentiation 1); the first membrane-proximal domain of HFE (hemochromatosis protein); a modified variant of the first membrane proximal domain of CD1, or Modified variants of the first membrane-proximal domain of HFE The first membrane proximal domain of an MHC-like protein may be selected from the group comprising:

[0136] CD1 (cluster of differentiation 1) refers to the cluster of differentiation 1 molecule, a component of the immune system that is located on the surface of various antigen-presenting cells, such as dendritic cells, macrophages, and other cells. In the same manner as MHC classes I and II, CD1 presents antigens for recognition by T cells through interaction with the T cell receptor. Unlike MHC classes I and II, CD1 proteins present lipids and their derivatives rather than peptides.

[0137] The multiple CD1 variants found in humans are divided into five groups: CD1a, CD1b, CD1c, CD1d, and CD1e, which differ in the structure of the antigen-binding fragment and, as a consequence, in their specificity for lipids of different structures. The proteins of the CD1e group, unlike the proteins of the other groups, are not expressed on the cell surface, but are soluble and are responsible for lipid transport (Kaczmarek, R., Pasciak, M., Szymczak-Kulus, K., & Czerwinski, M. (2017), CD1: A Singed Cat of the Three Antigen Presentation Systems. Archivum Immunologiae et Therapiae Experimentalis, 65(3), 201-214).

[0138] CD1 molecules are similar in structure to MHC class I. In a similar manner, one CD1 molecule is a non-covalent complex consisting of two polypeptide chains: a polymorphic α-chain (sometimes referred to as the heavy chain) and a smaller chain called β2-microglobulin (also known as the light chain), which is generally not polymorphic.

[0139] The α chain forms an antigen-binding region comprising the α1 and α2 domains. Following the α2 domain, the α3 domain is located at the C-terminus of the extracellular portion of the α chain of CD1, and forms a heterodimeric non-covalent complex together with β2 microglobulin. Said heterodimeric non-covalent complex composed of the α3 domain of CD1 and β2 microglobulin is referred to in the present specification as a heterodimer based on the membrane proximal domain of MHC-like proteins.

[0140] HFE (hereditary hemochromatosis protein) refers to the hemochromatosis protein molecule. The human HFE locus is located on chromosome 6 and consists of 5 exons and 4 introns. Hemochromatosis protein is a membrane protein associated with β2 microglobulin and is responsible for regulating iron absorption by regulating transferrin-transferrin receptor interaction. Mutations in the HFE gene result in hemochromatosis, an iron metabolism-related latent pathology associated with iron accumulation in various organs. The HFE molecule is identical in structure to MHC I. One HFE molecule is a non-covalent complex consisting of two polypeptide chains: a polymorphic α chain (sometimes referred to as the heavy chain) and a smaller chain called β2 microglobulin (also known as the light chain), which is not generally polymorphic.

[0141] The α chain comprises α1, α2 and α3 domains. The α3 domain is followed by the α2 domain, which is located at the C-terminus of the extracellular portion of the α chain of HFE and forms a heterodimeric non-covalent complex with β2 microglobulin. The heterodimeric non-covalent complex composed of the α3 domain of HFE and β2 microglobulin is referred to in the present specification as a heterodimer based on the membrane proximal domain of MHC-like protein.

[0142] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the second membrane-proximal domain of CD1 (cluster of differentiation 1); the second membrane-proximal domain of HFE (hemochromatosis protein); a modified variant of the second membrane proximal domain of CD1, or Modified variants of the second membrane-proximal domain of HFE The second membrane proximal domain of the MHC-like protein may be selected from the group comprising:

[0143] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of CD1 (cluster of differentiation 1); the first membrane-proximal domain of HFE (hemochromatosis protein); a modified variant of the first membrane proximal domain of CD1, or Modified variants of the first membrane-proximal domain of HFE a first membrane proximal domain of an MHC-like protein, which may be selected from the group comprising: And the second membrane-proximal domain of CD1 (cluster of differentiation 1); the second membrane-proximal domain of HFE (hemochromatosis protein); a modified variant of the second membrane proximal domain of CD1, or Modified variants of the second membrane-proximal domain of HFE The second membrane proximal domain of the MHC-like protein may be selected from the group comprising:

[0144] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of an MHC-like protein, which is the first membrane proximal domain of CD1; and The second membrane proximal domain of the MHC-like protein, which is the second membrane proximal domain of CD1 Includes.

[0145] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of an MHC-like protein, which is the first membrane proximal domain of HFE; and The second membrane proximal domain of the MHC-like protein, which is the second membrane proximal domain of HFE Includes.

[0146] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of an MHC-like protein that is a modified variant of the first membrane proximal domain of CD1; and A second membrane proximal domain of an MHC-like protein that is a modified variant of the second membrane proximal domain of CD1. Includes.

[0147] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of an MHC-like protein that is a modified variant of the first membrane proximal domain of HFE; and A second membrane proximal domain of an MHC-like protein that is a modified variant of the second membrane proximal domain of HFE. Includes.

[0148] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of an MHC-like protein, which is the first membrane proximal domain of CD1; and A second membrane proximal domain of an MHC-like protein that is a modified variant of the second membrane proximal domain of CD1. Includes.

[0149] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of an MHC-like protein that is a modified variant of the first membrane proximal domain of CD1; and The second membrane proximal domain of the MHC-like protein, which is the second membrane proximal domain of CD1 Includes.

[0150] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: the first membrane proximal domain of an MHC-like protein, which is the first membrane proximal domain of HFE; and A second membrane proximal domain of an MHC-like protein that is a modified variant of the second membrane proximal domain of HFE. Includes.

[0151] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a first membrane proximal domain of an MHC-like protein that is a modified variant of the first membrane proximal domain of HFE; and The second membrane proximal domain of HFE. The second membrane proximal domain of MHC-like proteins. Includes.

[0152] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a membrane proximal domain of CD1 selected from the group of CD1a, CD1b, CD1c, CD1d or CD1e. In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a variable fragment of a second light chain (VL) separated from a first membrane proximal domain of the MHC or MHC-like protein by a linker of 1-25 amino acids in length, and / or a variable fragment of a second heavy chain (VH) separated from a second membrane proximal domain of the MHC or MHC-like protein by a linker of 1-25 amino acids in length.

[0153] In some aspects of the invention, a bivalent, bispecific chimeric antibody comprises: a) a CH3 domain of one heavy chain, which has been modified in such a way that on the surface of the CH3 domain of one heavy chain that contacts the surface of the CH3 domain of the other heavy chain in the bivalent bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, resulting in the formation of a knob on the surface of the CH3 domain of one heavy chain that can fit into a hole on the surface of the CH3 domain of the other heavy chain, and b) a CH3 domain of the other heavy chain, which has been modified such that on the surface of the CH3 domain of the second heavy chain that contacts the surface of the CH3 domain of the first heavy chain in the bivalent bispecific antibody, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, resulting in the formation of a hole on the surface of the CH3 domain of the second heavy chain that can accept a knob on the interface of the CH3 domain of the first heavy chain; Includes; wherein the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W); And, wherein the amino acid residue having a smaller side chain volume is selected from the group including alanine (A), serine (S), threonine (T) and valine (V).

[0154] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a constant domain of the first light chain of the antibody selected from CK or CL. In mammals, only two types of light chains are known, designated lambda (λ) and kappa (κ). The constant domain of the lambda light chain is designated CL and that of the kappa light chain is designated CK.

[0155] In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an antibody CH3 domain which is further modified by introduction of cysteine ​​(C) as an amino acid into the corresponding position of each CH3 domain such that disulfide bridges can form between both CH3 domains.

[0156] In some embodiments of the invention, the bivalent, bispecific antibody comprises a CH3 domain of one heavy chain that is modified to form a knob and a CH3 domain of the other heavy chain that is modified to form a hole, or vice versa.

[0157] "Knobs-into-holes" ("knobs-into-holes" type of interaction) is a technique that can avoid problems associated with mispairing by-products. This technique aims to force pairing of two different antibody heavy chains by introducing mutations in the CH3 domain to modify the contact interface. On one chain, bulky amino acids were replaced with amino acids with short side chains to create "holes". Conversely, amino acids with large side chains were introduced into the CH3 domain of the other to create "knobs". Co-expression of these two heavy chains resulted in a high yield of heterodimer formation ("knobs-holes") compared to homodimer formation ("hole-holes" or "knobs-knobs") (WO9627011 and WO9850431, and Merchant AM ET ALL.,An efficient route to human bispecific IgG, Nat Biotechnol.1998 Jul;16(7):677-81).

[0158] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a CH3 domain of one heavy chain with the amino acid substitutions S354C / T366W and a CH3 domain of the other heavy chain with the amino acid substitutions Y349C / T366S / L368A / Y407V.

[0159] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a CH3 domain of one heavy chain with the amino acid substitutions Y349C / T366S / L368A / Y407 and a CH3 domain of the other heavy chain with the amino acid substitutions S354C / T366W.

[0160] In some embodiments of the invention, a bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, which are the α2 domain of MHC I and the β2 domain of MHC II, respectively, forming a heterodimer between them.

[0161] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, which are the β2 domain of MHC II and the α2 domain of MHC II, respectively, that form a heterodimer between them.

[0162] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an α2 domain of MHC II having an amino acid sequence selected from the group of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38, and a β2 domain of MHC II having an amino acid sequence selected from the group of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39.

[0163] SEQ ID NO:30 is the α2 membrane proximal domain of MHC class II HLA-DM (HLA-DMA * 01:01:01:01) amino acid sequence. SEQ ID NO: 31 is the β2 membrane proximal domain of MHC class II HLA-DM (HLA-DMB * 01:01:01:01) amino acid sequence.

[0164] SEQ ID NO: 32 is the α2 membrane proximal domain of MHC class II HLA-DO (HLA-DOA * 01:01:01) amino acid sequence. SEQ ID NO: 33 is the β2 membrane proximal domain of MHC class II HLA-DO (HLA-DOB * 01:01:01:01) amino acid sequence.

[0165] SEQ ID NO: 34 is the α2 membrane proximal domain of MHC class II HLA-DP (HLA-DP A1 * 01:03:01:01) amino acid sequence. SEQ ID NO: 35 is the β2 membrane proximal domain of MHC class II HLA-DP (HLA-DPB1 * 01:01:01:01) amino acid sequence.

[0166] SEQ ID NO: 36 is the α2 membrane proximal domain of MHC class II HLA-DQ (HLA-DQA1 * 01:01:01:01) amino acid sequence. SEQ ID NO: 37 is the β2 membrane proximal domain of MHC class II HLA-DQ (HLA-DQB1 * 05:01:01:01) amino acid sequence.

[0167] SEQ ID NO: 38 is the α2 membrane proximal domain of MHC class II HLA-DR (HLA-DR * 01:01:01:01) amino acid sequence. SEQ ID NO: 39 is the β2 membrane proximal domain of the MHC class HLA-DR (HLA-DRB1 * 01:01:01:01) amino acid sequence.

[0168] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of the α2 domain of MHC II and the β2 domain of MHC II, respectively, and form a heterodimer therebetween.

[0169] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of the β2 domain of MHC II and the α2 domain of MHC II, respectively, and form a heterodimer therebetween.

[0170] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, which are the α3 domain of MHC I and β2 microglobulin (β2M), respectively, forming a heterodimer between them.

[0171] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC, which are β2 microglobulin (β2M) and the α3 domain of MHC I, respectively, forming a heterodimer between them.

[0172] β2 microglobulin is a 12 kDa non-glycosylated protein; one of its functions is to stabilize the α chain of MHC class I, as well as the CD1 and HFE proteins. Human β2 microglobulin within the protein has a substantially identical structure, and therefore, in the present specification, human β2 microglobulin is described without indicating that the protein is a part of it.

[0173] Unlike the α chain, β2 microglobulin does not cross the membrane. The human β2 microglobulin locus is located on chromosome 15. The β2 microglobulin gene consists of 4 exons and 3 introns.

[0174] In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an α3 domain of MHC I having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29, and a β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO: 46.

[0175] SEQ ID NO: 1 represents the α3 membrane proximal domain of MHC class I HLA-A (allele A * 01:01:01:01) amino acid sequence. SEQ ID NO:2 is the α3 membrane proximal domain of MHC class I HLA-A (A * 02:01:01:01) amino acid sequence.

[0176] SEQ ID NO:3 represents the α3 membrane proximal domain of MHC class I HLA-A (A * 03:01:01:01) amino acid sequence. SEQ ID NO: 4 represents the α3 membrane proximal domain of MHC class I HLA-A (A * 11:01:01:01) is the amino acid sequence.

[0177] SEQ ID NO:5 is the α3 membrane proximal domain of MHC class I HLA-A (A * 23:01:01:01) is the amino acid sequence. SEQ ID NO:6 is the α3 membrane proximal domain of MHC class I HLA-B (B * 07:02:01:01) amino acid sequence.

[0178] SEQ ID NO: 7 is the α3 membrane proximal domain of MHC class I HLA-B (B * 08:01:01:01) amino acid sequence. SEQ ID NO:8 is the α3 membrane proximal domain of MHC class I HLA-B (B * 13:01:01:01) is the amino acid sequence.

[0179] SEQ ID NO: 9 is the α3 membrane proximal domain of MHC class I HLA-B (B * 14:01:01:01) amino acid sequence. SEQ ID NO: 10 is the α3 membrane proximal domain of MHC class I HLA-B (B * 15:01:01:01) is the amino acid sequence.

[0180] SEQ ID NO: 11 is the α3 membrane proximal domain of MHC class I HLA-C (C * 01:02:01:01) amino acid sequence. SEQ ID NO: 12 is the α3 membrane proximal domain of MHC class I HLA-C (C * 03:02:01) amino acid sequence.

[0181] SEQ ID NO: 13 is the α3 membrane proximal domain of MHC class I HLA-C (C * 04:01:01:01) amino acid sequence. SEQ ID NO: 14 represents the α3 membrane proximal domain of MHC class I HLA-C (C * 05:01:01:01) amino acid sequence.

[0182] SEQ ID NO: 15 is the α3 membrane proximal domain of MHC class I HLA-C (C * 06:02:01:01) amino acid sequence. SEQ ID NO: 16 is the α3 membrane proximal domain of MHC class I HLA-E (E * 01:01:01:01) amino acid sequence.

[0183] SEQ ID NO: 17 is the α3 membrane proximal domain of MHC class I HLA-E (E * 01:03:01:01) amino acid sequence. SEQ ID NO: 18 is the α3 membrane proximal domain of MHC class I HLA-E (E * 01:05) amino acid sequence.

[0184] SEQ ID NO: 19 is the α3 membrane proximal domain of MHC class I HLA-E (E * 01:06) amino acid sequence. SEQ ID NO: 20 represents the α3 membrane proximal domain of MHC class I HLA-E (E * 01:09) amino acid sequence.

[0185] SEQ ID NO: 21 represents the α3 membrane proximal domain of MHC class I HLA-F (F * 01:01:01:01) amino acid sequence. SEQ ID NO: 22 is the α3 membrane proximal domain of MHC class I HLA-F (F * 01:02) amino acid sequence.

[0186] SEQ ID NO: 23 is the α3 membrane proximal domain of MHC class I HLA-F (F * 01:03:01:01) amino acid sequence. SEQ ID NO:24 represents the α3 membrane proximal domain of MHC class I HLA-F (F * 01:04:01:01) amino acid sequence.

[0187] SEQ ID NO:25 is the α3 membrane proximal domain of MHC class I HLA-F (F * 01:05) amino acid sequence. SEQ ID NO:26 is the α3 membrane proximal domain of MHC class I HLA-G (G * 01:01:01:01) amino acid sequence.

[0188] SEQ ID NO:27 is the α3 membrane proximal domain of MHC class I HLA-G (G * 01:03:01:01) amino acid sequence. SEQ ID NO:28 is the α3 membrane proximal domain of MHC class I HLA-G (G * 01:04:01:01) amino acid sequence.

[0189] SEQ ID NO:29 is the α3 membrane proximal domain of MHC class I HLA-G (G * 01:06) amino acid sequence. SEQ ID NO:46 is the amino acid sequence of universal β2 microglobulin (β2M), an MHC class I or MHC-like protein (CD1, HFE).

[0190] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of the α3 domain of MHC I and β2 microglobulin (β2M), respectively, forming a heterodimer therebetween.

[0191] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of MHC and a second membrane proximal domain of MHC that are modified variants of β2 microglobulin (β2M) and α3 domain of MHC I, respectively, forming a heterodimer therebetween.

[0192] In some aspects of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of β2 microglobulin (β2M) having an amino acid sequence selected from SEQ ID NO:47 or SEQ ID NO:48.

[0193] SEQ ID NO: 47 is the amino acid sequence of universal β2 microglobulin (β2M) (within the unit S-S bridge) of MHC class I or MHC-like protein (CD1, HFE) having the mutation R12C.

[0194] SEQ ID NO: 48 is the amino acid sequence of universal β2 microglobulin (β2M) of MHC class I or MHC-like protein (CD1, HFE) with mutation R12C (SS bridge is within the unit) + hinge with mutation C220A (SS bridge has been removed from the C-terminus of the unit and moved within the unit).

[0195] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are the α3 domain of CD1 and β2 microglobulin (β2M), respectively, forming a heterodimer between them.

[0196] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are β2 microglobulin (β2M) and the α3 domain of CD1, respectively, forming a heterodimer between them.

[0197] In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an α3 domain of CD1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44 and a β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO: 46.

[0198] SEQ ID NO: 40 is the amino acid sequence of the α3 membrane proximal domain of CD1a. SEQ ID NO: 41 is the amino acid sequence of the α3 membrane proximal domain of CD1b. SEQ ID NO: 42 is the amino acid sequence of the α3 membrane proximal domain of CD1c.

[0199] SEQ ID NO: 43 is the amino acid sequence of the α3 membrane proximal domain of CD1d. SEQ ID NO: 44 is the amino acid sequence of the α3 membrane proximal domain of CD1e. In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of the α3 domain of CD1 and a modified variant of β2 microglobulin (β2M), respectively, that form a heterodimer therebetween.

[0200] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of β2 microglobulin (β2M) and a modified variant of the α3 domain of CD1, respectively, forming a heterodimer therebetween.

[0201] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a modified variant of the α3 domain of CD1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 49-56 or SEQ ID NO: 109, and a modified variant of β2 microglobulin (β2M) having an amino acid sequence selected from SEQ ID NO: 47 or SEQ ID NO: 48.

[0202] SEQ ID NO: 49 is the amino acid sequence of the α3 membrane proximal domain of CD1b with the mutation N57C (with an additional SS bridge within the unit) and a C-terminal 3 amino acid GSC extension.

[0203] SEQ ID NO:50 is the amino acid sequence of the α3 membrane proximal domain of CD1b with the mutation N57C (the SS bridge is within the unit) and a two amino acid GS extension at the C-terminus (the SS bridge has been removed from the C-terminus of the unit and moved within the unit).

[0204] SEQ ID NO:51 is the amino acid sequence of the α3 membrane proximal domain of CD1b with the mutation N59A (a substitution at a predicted N-glycosylation site) and a C-terminal 3 amino acid GSC extension.

[0205] SEQ ID NO: 52 is the amino acid sequence of the α3 membrane proximal domain of CD1b with the mutation N59D (a substitution at a predicted N-glycosylation site) and a C-terminal 3 amino acid GSC extension.

[0206] SEQ ID NO: 53 is the amino acid sequence of the α3 membrane proximal domain of CD1b with the mutations N57C, N59A and a C-terminal 3 amino acid GSC extension (with an additional S-S bridge within the unit + predicted N-glycosylation site removed).

[0207] SEQ ID NO: 54 is the amino acid sequence of the α3 membrane proximal domain of CD1b with the mutations N57C, N59D and a C-terminal 3 amino acid GSC extension (with an additional S-S bridge within the unit + predicted N-glycosylation site removed).

[0208] SEQ ID NO: 55 is the amino acid sequence of the α3 membrane proximal domain of CD1b with mutations N57C, N59A and a two amino acid GS extension at the C-terminus (SS bridge moved intraunit + predicted N-glycosylation site removed).

[0209] SEQ ID NO: 56 is the amino acid sequence of the α3 membrane proximal domain of CD1b with mutations N57C, N59D and a two amino acid GS extension at the C-terminus (SS bridge moved intraunit + predicted N-glycosylation site removed).

[0210] SEQ ID NO: 109 is the amino acid sequence of the α3 membrane proximal domain of CD1b with a C-terminal 3 amino acid GSC extension. In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are the α3 domain of HFE and β2 microglobulin (β2M), respectively, forming a heterodimer between them.

[0211] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein, which are β2 microglobulin (β2M) and the α3 domain of HFE, respectively, forming a heterodimer between them.

[0212] In some embodiments of the present invention, the bivalent, bispecific chimeric antibody comprises an α3 domain of HFE having the amino acid sequence of SEQ ID NO:45 and a β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO:46.

[0213] SEQ ID NO: 45 is the amino acid sequence of the α3 membrane proximal domain of HFE. In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of the α3 domain of HFE and a modified variant of β2 microglobulin (β2M), respectively, that form a heterodimer therebetween.

[0214] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first membrane proximal domain of an MHC-like protein and a second membrane proximal domain of an MHC-like protein that are a modified variant of β2 microglobulin (β2M) and a modified variant of the α3 domain of HFE, respectively, that form a heterodimer therebetween.

[0215] In some embodiments of the present invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of β2 microglobulin (β2M) having an amino acid sequence selected from SEQ ID NO:47 or SEQ ID NO:48.

[0216] Over 1,000 characterized structures of MHC, CD1 and HFE are known. Any of the above structures can be used in the bispecific antibodies according to the invention. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that comprises a substitution of a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from the first and second membrane proximal domains of the MHC or MHC-like protein.

[0217] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that contains one or more substitutions at various positions in the membrane proximal domain of the MHC or MHC-like protein, respectively, resulting in an increase in thermodynamic stability Tm of more than 1° C. compared to the wild-type membrane proximal domain of the MHC or MHC-like protein.

[0218] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that contains one or more substitutions at various positions in the membrane proximal domain of the MHC or MHC-like protein, respectively, that results in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of the MHC or MHC-like protein.

[0219] In some aspects of the invention, the bivalent, bispecific, chimeric antibody comprises a modified variant of an MHC or MHC-like protein, where modified variant refers to a variant that comprises one or more substitutions at various positions in the membrane proximal domain of the MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of the MHC or MHC-like protein.

[0220] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises a first light chain variable domain and a second light chain variable domain that are identical. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment belonging to IgG.

[0221] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an Fc fragment selected from the group comprising human IgG1, IgG2, or IgG4. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment monomer into which further substitutions have been introduced, resulting in the absence of ADCC, CDC and / or ADCP properties in the bivalent, bispecific antibody.

[0222] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an Fc fragment monomer into which further LALA substitutions (L234A and L235A) have been introduced. In some embodiments of the invention, the bivalent, bispecific, chimeric antibodies comprise an Fc fragment monomer into which further substitutions have been introduced resulting in a prolongation of the effect of the antibody.

[0223] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment monomer into which YTE substitutions (M252Y, S254T and T256E) have been further introduced. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody comprises an Fc fragment monomer into which further substitutions have been introduced resulting in enhanced ADCC, CDC and / or ADCP properties in the bivalent, bispecific antibody.

[0224] In some embodiments of the invention, the bivalent, bispecific chimeric antibody comprises an Fc fragment monomer into which the substitution E345R has been further introduced. The above mutations in the Fc fragment are numbered according to the EU numbering system for the amino acid chains of antibodies (Edelman, GM et al., Proc. Natl. Acad. Sci. USA 63 (1969), pp. 78-85; Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0225] Mutation in Fc fragment is understood to mean modification of the amino acid sequence of the antibody described in this application. The amino acid sequence variant of the antibody is prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution is made to arrive at the final construct, provided that the final construct has the desired characteristics.

[0226] Variants of antibody amino acid sequence modifications using amino acid substitutions are those that replace at least one amino acid residue in the antibody molecule with another residue. Conservative substitutions are shown in Table A under "preferred substitutions."

[0227] [Table 1]

[0228] The term antibody "effector function" refers to a biological activity attributable to the Fc region of an antibody (either a native Fc region sequence or an Fc region amino acid variant) and varies with the antibody isotype. Examples of antibody effector functions include C1 q These include binding and complement dependent cytotoxicity; Fc receptor binding; antibody dependent cellular cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor, BCR); and B cell activation.

[0229] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and then cause lysis or phagocytosis of the target cells. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRIII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS (USA), 95:652-656 (1998).

[0230] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from their natural sources, e.g., blood or PBMCs, as described herein.

[0231] "Complement-dependent cytotoxicity" and "CDC" refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J.Immunol.Methods, 202:163 (1996).

[0232] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to CD20 and CD3. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to BCMA and CD3.

[0233] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to PD-L1 and CD47. In some embodiments of the invention, the bivalent, bispecific chimeric antibody specifically binds to coagulation factor 9 (FIX) and coagulation factor 10 (FX).

[0234] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to GD2 and CD3. In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to AXL and CD3.

[0235] In some embodiments of the invention, the bivalent, bispecific, chimeric antibody specifically binds to PD-L1 and TGF-beta. The material of the present application provides the following antibodies and antibody-like molecules: 01-001, 01-002, 01-003, 01-004, 01-005, 01-006, 01-007, 01-008, 01-009, 01-010, 01-011, 01-012, 02-004, 02-005, 02-006, 02-007, 02-008, 02-009, 03-001, 03-002, 03-003, 03-004, 03-005, 03-006, 03-007, 03-008.

[0236] 01-001 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1, and a light chain based on VL and CD1b, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, VL is the amino acid sequence of the light chain variable domain of the antibody prorugolimab (prorugolimab_VL) having SEQ ID NO: 104, and CD1b is the α3 domain of CD1b having the amino acid sequence of SEQ ID NO: 109.

[0237] 01-002 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and b2M and a light chain based on VL and CK, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, b2M is β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and VL is the amino acid sequence of the light chain variable domain of the antibody prorugolimab (prorugolimab_VL) having SEQ ID NO: 104.

[0238] 01-003 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and CD1b, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having SEQ ID NO: 105, VL is the amino acid sequence of the light chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having SEQ ID NO: 106, and CD1b is the α3 domain of CD1b having the amino acid sequence of SEQ ID NO: 109.

[0239] 01-004 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and CK, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab having SEQ ID NO: 103 (prorugolimab_VH) and VL is the amino acid sequence of the light chain variable domain of the antibody ocrelizumab having SEQ ID NO: 106 (ocrelizumab_VL).

[0240] 01-005 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and b2M, where VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having the amino acid sequence of SEQ ID NO: 103, b2M is β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, VL is the amino acid sequence of the light chain variable domain of the antibody ocrelizumab (ocrelizumab_VL) having the amino acid sequence of SEQ ID NO: 106, and CD1b is the α3 domain of CD1b having the amino acid sequence of SEQ ID NO: 109.

[0241] 01-006 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and CK, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 105 (ocrelizumab_VH) and VL is the amino acid sequence of the light chain variable domain of the antibody prorugolimab having SEQ ID NO: 104 (prorugolimab_VL).

[0242] 01-007 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and b2M, where VH is the amino acid sequence of the heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having the amino acid sequence of SEQ ID NO: 105, b2M is β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, VL is the amino acid sequence of the light chain variable domain of the antibody prorugolimab (prorugolimab_VL) having the amino acid sequence of SEQ ID NO: 104, and CD1b is the α3 domain of CD1b having the amino acid sequence of SEQ ID NO: 109.

[0243] 01-008 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and CD1b, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, VL is the amino acid sequence of the light chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having SEQ ID NO: 106, and CD1b is the α3 domain of CD1b having the amino acid sequence of SEQ ID NO: 109.

[0244] 01-009 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and b2M and a light chain based on VL and CK, where VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, b2M is β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and VL is the amino acid sequence of the light chain variable domain of the antibody ocrelizumab (ocrelizumab_VL) having SEQ ID NO: 106.

[0245] 01-010 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and CD1b, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having SEQ ID NO: 105, VL is the amino acid sequence of the light chain variable domain of the antibody prorugolimab (prorugolimab_VL) having SEQ ID NO: 104, and CD1b is the α3 domain of CD1b having the amino acid sequence of SEQ ID NO: 109.

[0246] 01-011 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and b2M, where VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having the amino acid sequence of SEQ ID NO: 103, b2M is β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, VL is the amino acid sequence of the light chain variable domain of the antibody prorugolimab (prorugolimab_VL) having the amino acid sequence of SEQ ID NO: 104, and CD1b is the α3 domain of CD1b having the amino acid sequence of SEQ ID NO: 109.

[0247] 01-012 is a model monospecific antibody-like molecule comprising a heavy chain based on VH and CH1 and a light chain based on VL and CK, wherein VH is the amino acid sequence of the heavy chain variable domain of the antibody prorugolimab having SEQ ID NO: 103 (prorugolimab_VH) and VL is the amino acid sequence of the light chain variable domain of the antibody prorugolimab having SEQ ID NO: 104 (prorugolimab_VL).

[0248] 02-004 specifically binds to PD1 and CD20, a) a first light chain and a first heavy chain of an antibody that specifically binds to PD1; wherein the first light chain comprises a light chain variable domain (prorugolimab_VL) and a light chain constant domain of the antibody prorugolimab having SEQ ID NO: 104; wherein the first heavy chain comprises an antibody heavy chain constant domain comprising the heavy chain variable domain of the antibody prorugolimab having SEQ ID NO: 103 (prorugolimab_VH), and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a hole; b) a second light chain and a second heavy chain of an antibody that specifically binds to CD20; wherein the second light chain comprises a light chain variable domain of the antibody ocrelizumab (ocrelizumab_VL) having SEQ ID NO: 106 and an α3 membrane proximal domain of CD1b having the amino acid sequence of SEQ ID NO: 109; wherein the second heavy chain comprises a heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having SEQ ID NO: 105, a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a knob; and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0249] 02-004 specifically binds to PD1 and CD20, a) a first light chain and a first heavy chain of an antibody that specifically binds to PD1; wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 58 (Prorugolimab_VL_CK); wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO:57 (Prorugolimab_VH_HC_hole); b) a second light chain and a second heavy chain of an antibody that specifically binds to CD20; wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 60 (ocrelizumab_VL_CD1b); wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 59 (ocrelizumab_VH_b2m_Fc_knob); and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0250] 02-005 specifically binds to PD1 and CD20, a) a first light chain and a first heavy chain of an antibody that specifically binds to CD20; wherein the first light chain comprises a light chain variable domain (ocrelizumab_VL) and a light chain constant domain of the antibody ocrelizumab having SEQ ID NO: 106; wherein the first heavy chain comprises a heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 105 (ocrelizumab_VH), and an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution that forms a hole; b) a second light chain and a second heavy chain of an antibody that specifically binds to PD1; wherein the second light chain comprises a light chain variable domain of the antibody prorugolimab (prorugolimab_VL) having SEQ ID NO: 104 and an α3 membrane proximal domain of CD1b having the amino acid sequence of SEQ ID NO: 109; wherein the second heavy chain comprises a heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a knob; and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0251] 02-005 specifically binds to PD1 and CD20, a) a first light chain and a first heavy chain of an antibody that specifically binds to CD20; wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 62 (ocrelizumab_VL_CK); wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 61 (Ocrelizumab_VH_HC_hole); b) a second light chain and a second heavy chain of an antibody that specifically binds to PD1; wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 64 (Prorugolimab_VL_CD1b); wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 63 (Prorugolimab_VH_b2m_Fc_knob); and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0252] 02-006 specifically binds to PD1 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to PD1; wherein the first light chain comprises a light chain variable domain (prorugolimab_VL) and a light chain constant domain of the antibody prorugolimab having SEQ ID NO: 104; wherein the first heavy chain comprises an antibody heavy chain constant domain comprising the heavy chain variable domain of the antibody prorugolimab having SEQ ID NO: 103 (prorugolimab_VH), and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a hole; b) a second light chain and a second heavy chain of an antibody that specifically binds to CSF1R; wherein the second light chain comprises a light chain variable domain of an antibody against CSF1R having the amino acid sequence of SEQ ID NO: 108 and an α3 membrane proximal domain of CD1b having the amino acid sequence of SEQ ID NO: 109; wherein the second heavy chain comprises an Fc fragment monomer comprising a heavy chain variable domain of an antibody against CSF1R having SEQ ID NO: 107, a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a knob; and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0253] 02-006 specifically binds to PD1 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to PD1; wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 58 (Prorugolimab_VL_CK); the first heavy chain comprises the amino acid sequence of SEQ ID NO:57 (Prorugolimab_VH_HC_hole); b) a second light chain and a second heavy chain of an antibody that specifically binds to CSF1R; wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 66 (anti-CSF1R_VL_CD1b); wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 65 (anti-CSF1R_VH_b2m_Fc_knob); and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0254] 02-007 specifically binds to PD1 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to CSF1R; wherein the first light chain comprises a light chain variable domain and a light chain constant domain of an antibody against CSF1R having SEQ ID NO: 108; wherein the first heavy chain comprises a heavy chain variable domain of an antibody against CSF1R having SEQ ID NO: 107, and an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution that forms a hole; b) a second light chain and a second heavy chain of an antibody that specifically binds to PD1; wherein the second light chain comprises a light chain variable domain of the antibody prorugolimab (prorugolimab_VL) having SEQ ID NO: 104 and an α3 membrane proximal domain of CD1b having the amino acid sequence of SEQ ID NO: 109; wherein the second heavy chain comprises a heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a knob; and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0255] 02-007 specifically binds to PD1 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to CSF1R; wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 68 (anti-CSF1R_VL_CK); wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 67 (Anti-CSF1R_VH_HC_Hole); b) a second light chain and a second heavy chain of an antibody that specifically binds to PD1; wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 64 (Prorugolimab_VL_CD1b); wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 63 (Prorugolimab_VH_b2m_Fc_knob); and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0256] 02-008 specifically binds to CD20 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to CD20; wherein the first light chain comprises a light chain variable domain (ocrelizumab_VL) and a light chain constant domain of the antibody ocrelizumab having SEQ ID NO: 106; wherein the first heavy chain comprises a heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 105 (ocrelizumab_VH), and a heavy chain constant domain of an antibody comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution that forms a hole; b) a second light chain and a second heavy chain of an antibody that specifically binds to CSF1R; wherein the second light chain comprises a light chain variable domain of an antibody against CSF1R having the amino acid sequence of SEQ ID NO: 108 and an α3 membrane proximal domain of CD1b having the amino acid sequence of SEQ ID NO: 109; wherein the second heavy chain comprises an Fc fragment monomer comprising a heavy chain variable domain of an antibody against CSF1R having SEQ ID NO: 107, a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a knob; and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0257] 02-008 specifically binds to CD20 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to CD20; wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 62 (ocrelizumab_VL_CK); wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 61 (Ocrelizumab_VH_HC_hole); b) a second light chain and a second heavy chain of an antibody that specifically binds to CSF1R; wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 66 (anti-CSF1R_VL_CD1b); wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 65 (anti-CSF1R_VH_b2m_Fc_knob); and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0258] 02-009 specifically binds to CD20 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to CSF1R; wherein the first light chain comprises a light chain variable domain and a light chain constant domain of an antibody against CSF1R having SEQ ID NO: 108; wherein the first heavy chain comprises a heavy chain variable domain of an antibody against CSF1R having SEQ ID NO: 107, and a heavy chain constant domain of an antibody comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution that forms a hole; b) a second light chain and a second heavy chain of an antibody that specifically binds to CD20; wherein the second light chain comprises a light chain variable domain of the antibody ocrelizumab (ocrelizumab_VL) having SEQ ID NO: 106 and an α3 membrane proximal domain of CD1b having the amino acid sequence of SEQ ID NO: 109; wherein the second heavy chain comprises an Fc fragment monomer comprising a heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 105 (ocrelizumab_VH), a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and a second (CH2) and a third (CH3) heavy chain constant domain, wherein CH3 comprises an amino acid substitution forming a knob; and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0259] 02-009 specifically binds to CD20 and CSF1R, a) a first light chain and a first heavy chain of an antibody that specifically binds to CSF1R; wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 68 (anti-CSF1R_VL_CK); wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 67 (Anti-CSF1R_VH_HC_Hole); b) a second light chain and a second heavy chain of an antibody that specifically binds to CD20; wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 60 (ocrelizumab_VL_CD1b); wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 59 (ocrelizumab_VH_b2m_Fc_knob). and wherein the chimeric antibody is a bivalent, bispecific chimeric antibody comprising:

[0260] 03-001 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutation N57C (with an additional S-S bridge in the unit) and an extension of the C-terminal three amino acids GSC, having the amino acid sequence of SEQ ID NO: 49; A heavy chain comprising the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, the membrane proximal domain of β2 microglobulin having the mutation R12C, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains, having the amino acid sequence of SEQ ID NO: 47. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0261] 03-001 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 72; and Heavy chain comprising the amino acid sequence of SEQ ID NO:69 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0262] 03-002 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutation N57C (an additional S-S bridge is within the unit) and a C-terminal two amino acid GS extension (the S-S bridge has been removed from the C-terminus of the unit and moved within the unit) having the amino acid sequence of SEQ ID NO: 50; A heavy chain comprising the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, the membrane proximal domain of β2 microglobulin having the mutation R12C and an Fc fragment monomer comprising a hinge having the mutation C220A and the second (CH2) and third (CH3) heavy chain constant domains having the amino acid sequence of SEQ ID NO: 48. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0263] 03-002 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 73; and Heavy chain comprising the amino acid sequence of SEQ ID NO:71 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0264] 03-003 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutation N59A (a substitution at a predicted N-glycosylation site on the light chain) and a C-terminal 3 amino acid GSC extension having the amino acid sequence of SEQ ID NO: 51; A heavy chain comprising a heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0265] 03-003 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 74; and A heavy chain comprising the amino acid sequence of SEQ ID NO: 70 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0266] 03-004 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutation N59D (a substitution at a predicted N-glycosylation site on the light chain) and a C-terminal 3 amino acid GSC extension having the amino acid sequence of SEQ ID NO: 52; A heavy chain comprising a heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, a membrane proximal domain of β2 microglobulin having the amino acid sequence of SEQ ID NO: 46, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0267] 03-004 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 75; and A heavy chain comprising the amino acid sequence of SEQ ID NO: 70 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0268] 03-005 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutations N57C, N59A and a C-terminal 3 amino acid GSC extension (with an additional S-S bridge in the unit + predicted N-glycosylation site removed) having the amino acid sequence of SEQ ID NO: 53; A heavy chain comprising the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, the membrane proximal domain of β2 microglobulin having the mutation R12C, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains, having the amino acid sequence of SEQ ID NO: 47. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0269] 03-005 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 76; and Heavy chain comprising the amino acid sequence of SEQ ID NO:69 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0270] 03-006 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutations N57C, N59D and a C-terminal 3 amino acid GSC extension (with an additional S-S bridge in the unit + predicted N-glycosylation site removed) having the amino acid sequence of SEQ ID NO: 54; A heavy chain comprising the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, the membrane proximal domain of β2 microglobulin having the mutation R12C, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains, having the amino acid sequence of SEQ ID NO: 47. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0271] 03-006 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 77; and Heavy chain comprising the amino acid sequence of SEQ ID NO:69 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0272] 03-007 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutations N57C, N59A and a C-terminal two amino acid GS extension (SS bridge moved intraunit + predicted N-glycosylation site removed) having the amino acid sequence of SEQ ID NO: 55; A heavy chain comprising the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, the membrane proximal domain of β2 microglobulin having the mutation R12C and an Fc fragment monomer comprising a hinge having the mutation C220A and the second (CH2) and third (CH3) heavy chain constant domains having the amino acid sequence of SEQ ID NO: 48. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0273] 03-007 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 78; and Heavy chain comprising the amino acid sequence of SEQ ID NO:71 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0274] 03-008 specifically binds to PD1, a light chain comprising the light chain variable domain of the antibody prorugolimab (Prorugolimab_VL) having SEQ ID NO: 104 and the α3 membrane proximal domain of CD1b with the mutations N57C, N59D and a C-terminal two amino acid GS extension (SS bridge moved intraunit + predicted N-glycosylation site removed) having the amino acid sequence of SEQ ID NO: 56; A heavy chain comprising the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 103, the membrane proximal domain of β2 microglobulin having the mutation R12C and an Fc fragment monomer comprising a hinge having the mutation C220A and the second (CH2) and third (CH3) heavy chain constant domains having the amino acid sequence of SEQ ID NO: 48. and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0275] 03-008 specifically binds to PD1, a light chain comprising the amino acid sequence of SEQ ID NO: 79; and Heavy chain comprising the amino acid sequence of SEQ ID NO:71 and wherein the chimeric antibody is a bivalent, monospecific chimeric antibody comprising:

[0276] These antibodies are provided for illustrative purposes to confirm the operability of the bivalent, bispecific, chimeric antibodies according to the invention as well as to confirm their surprising properties, and should not be construed as limiting in any way the bivalent, bispecific, chimeric antibodies according to the invention.

[0277] The product yield parameters, which show the correct heterodimeric assembly of two different heavy chains and the correct pairing between two different light chains and the corresponding heavy chains, are independent of the heavy and light chain variable fragments of the bispecific chimeric antibody and its specificity for the antigen.

[0278] The bivalent, bispecific chimeric antibodies according to the invention can be used to treat various diseases, in particular tumor diseases, autoimmune diseases or diseases associated with blood coagulation (clotting) disorders. nucleic acid In one aspect, the invention relates to an isolated nucleic acid encoding any of the above-described bivalent, bispecific, chimeric antibodies or fragments thereof.

[0279] The terms "nucleic acid," "nucleic sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence," used interchangeably herein, refer to a precise sequence of nucleotides, modified or unmodified, that defines a fragment or region of a nucleic acid, either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA, that may or may not contain non-naturally occurring nucleotides.

[0280] It should also be included herein that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in the natural state. The sequences of the present invention are isolated and / or purified, i.e. they have been sampled directly or indirectly, for example by copying their environment, which has been at least partially modified. Thus, isolated nucleic acids obtained, for example, by recombinant genetics using a host cell or by chemical synthesis, should also be described herein.

[0281] A reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

[0282] In any of the above aspects, the nucleic acid molecule may be isolated. An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule-contaminant, the former being associated with the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different from the form or set in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions.

[0283] In one aspect, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 1-79 or SEQ ID NO: 109. The nucleic acid molecule may also comprise any combination of said nucleotide sequences.

[0284] In some embodiments, the nucleic acid is DNA. In one variant, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding the light or heavy chain amino acid sequence of said bispecific antibody according to the invention, said amino acid sequence being a first light chain amino acid sequence comprising a light chain variable domain and a light chain constant domain; a first heavy chain amino acid sequence comprising an antibody heavy chain constant domain comprising a heavy chain variable domain and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain amino acid sequence comprising a light chain variable domain and a constant domain selected from the group of a first membrane proximal domain of an MHC (major histocompatibility complex) or a first membrane proximal domain of an MHC-like protein; a heavy chain variable domain and a second membrane proximal domain of an MHC (major histocompatibility complex) or a second membrane proximal domain of an MHC-like protein, and a second heavy chain amino acid sequence comprising a constant domain selected from the group of Fc fragment monomers comprising a second (CH2) and a third (CH3) heavy chain constant domain; is selected from.

[0285] The nucleic acid molecule may also comprise any combination of the above nucleotide sequences. Those skilled in the art will appreciate that a first light chain amino acid sequence comprising a light chain variable domain and a light chain constant domain; a first heavy chain amino acid sequence comprising an antibody heavy chain constant domain comprising a heavy chain variable domain and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain amino acid sequence comprising a light chain variable domain and a constant domain selected from the group of a first membrane proximal domain of an MHC (major histocompatibility complex) or a first membrane proximal domain of an MHC-like protein; a heavy chain variable domain and a second membrane proximal domain of an MHC (major histocompatibility complex) or a second membrane proximal domain of an MHC-like protein, and a second heavy chain amino acid sequence comprising a constant domain selected from the group of Fc fragment monomers comprising a second (CH2) and a third (CH3) heavy chain constant domain; It will be understood that peptides comprising the amino acid sequence of the light or heavy chain of the bispecific antibody according to the present invention selected from the following may be encoded by a variety of different DNA sequences due to the degeneracy of the genetic code. It is within the skill of a person trained in the art to create these alternative DNA sequences that code for the same amino acid sequence. Such variant DNA sequences are within the scope of the present invention.

[0286] The nucleic acid molecules according to the invention may be isolated from any source that produces a bispecific antibody according to the invention. In certain aspects of the invention, the nucleic acid molecules of the invention may be synthesized rather than isolated.

[0287] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the first heavy chain (Prorugolimab_VH_HC_hole) of candidates 02-004 and 02-006 and comprises a nucleotide sequence having SEQ ID NO:80.

[0288] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the first light chain (Prorugolimab_VL_CK) of candidates 02-004 and 02-006 and comprises a nucleotide sequence having SEQ ID NO:81.

[0289] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the second heavy chain (ocrelizumab_VH_b2m_Fc_knob) of candidates 02-004 and 02-009 and comprises a nucleotide sequence having SEQ ID NO:82.

[0290] In some aspects of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the second light chain (ocrelizumab_VL_CD1b) of candidates 02-004 and 02-009 and comprises a nucleotide sequence having SEQ ID NO:83.

[0291] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the first heavy chain (ocrelizumab_VH_HC_hole) of candidates 02-005 and 02-008 and comprises a nucleotide sequence having SEQ ID NO:84.

[0292] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the first light chain (ocrelizumab_VL_CK) of candidates 02-005 and 02-008 and comprises a nucleotide sequence having SEQ ID NO:85.

[0293] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the second heavy chain (prorugolimab_VH_b2m_Fc_knob) of candidates 02-005 and 02-007 and comprises a nucleotide sequence having SEQ ID NO:86.

[0294] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the second light chain (prorugolimab_VL_CD1b) of candidates 02-005 and 02-007 and comprises a nucleotide sequence having SEQ ID NO:87.

[0295] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the second heavy chain (anti-CSF1R_VH_b2m_Fc_knob) of candidates 02-006 and 02-008 and comprises a nucleotide sequence having SEQ ID NO:88.

[0296] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the second light chain (anti-CSF1R_VL_CD1b) of candidates 02-006 and 02-008 and comprises a nucleotide sequence having SEQ ID NO:89.

[0297] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the first heavy chain (Anti-CSF1R_VH_HC_Hole) of candidates 02-007 and 02-009 and comprises a nucleotide sequence having SEQ ID NO:90.

[0298] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the first light chain (anti-CSF1R_VL_CK) of candidates 02-007 and 02-009 and comprises a nucleotide sequence having SEQ ID NO:91.

[0299] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of candidates 03-001 and 03-005, including the universal β2 microglobulin (β2M) having the mutation R12C, and comprises a nucleotide sequence having SEQ ID NO:92.

[0300] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of candidates 03-003 and 03-004, including universal β2 microglobulin (β2M), and comprises a nucleotide sequence having SEQ ID NO:93.

[0301] In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of candidates 03-002, 03-007 and 03-008, which comprises a universal β2 microglobulin (β2M) having the mutation R12C and a hinge having the mutation C220A, and comprises a nucleotide sequence having SEQ ID NO: 94.

[0302] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-001, comprising the α3 membrane proximal domain of CD1b with the mutation N57C and a C-terminal three amino acid GSC extension, and comprising a nucleotide sequence having SEQ ID NO: 95.

[0303] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-002, comprising the α3 membrane proximal domain of CD1b having the mutation N57C and a two amino acid GS extension at the C-terminus, and comprising a nucleotide sequence having SEQ ID NO: 96.

[0304] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-003, comprising the α3 membrane proximal domain of CD1b with the mutation N59A and a C-terminal three amino acid GSC extension, and comprising a nucleotide sequence having SEQ ID NO: 97.

[0305] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-004, comprising the α3 membrane proximal domain of CD1b with the mutation N59D and a C-terminal three amino acid GSC extension, and comprising a nucleotide sequence having SEQ ID NO: 98.

[0306] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-005, comprising the α3 membrane proximal domain of CD1b with the mutations N57C, N59A and a C-terminal three amino acid GSC extension, and comprising a nucleotide sequence having SEQ ID NO: 99.

[0307] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-006, comprising the α3 membrane proximal domain of CD1b with the mutations N57C, N59D and a C-terminal three amino acid GSC extension, and comprising a nucleotide sequence having SEQ ID NO: 100.

[0308] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-007, comprising the α3 membrane proximal domain of CD1b with the mutations N57C, N59A and a two amino acid GS extension at the C-terminus, and comprising a nucleotide sequence having SEQ ID NO: 101.

[0309] In some embodiments of the invention, the nucleic acid is a nucleic acid encoding the amino acid sequence of the light chain of candidate 03-008, comprising the α3 membrane proximal domain of CD1b having the mutations N57C, N59D and a two amino acid GS extension at the C-terminus, and comprising a nucleotide sequence having SEQ ID NO: 102.

[0310] Nucleic acid molecules can be used to express a bivalent, bispecific, chimeric antibody according to the invention. Expression vector In one aspect, the invention relates to an expression vector comprising the isolated nucleic acid described above.The invention relates to vectors suitable for the expression of any of the nucleotide sequences described herein.

[0311] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some aspects of the invention, the vector is a plasmid, i.e., a circular double-stranded piece of DNA to which additional DNA segments may be ligated. In some aspects of the invention, the vector is a viral vector, to which additional DNA segments may be ligated into the viral genome. In some aspects of the invention, the vector is capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having an origin of replication). In further aspects of the invention, the vector (e.g., non-episomal mammalian vectors) is integrated into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genes. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0312] The present invention relates to a method for producing a method for treating a cancer cell comprising the steps of: a first light chain amino acid sequence comprising a light chain variable domain and a light chain constant domain; a first heavy chain amino acid sequence comprising an antibody heavy chain constant domain comprising a heavy chain variable domain and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain amino acid sequence comprising a light chain variable domain and a constant domain selected from the group of a first membrane proximal domain of an MHC (major histocompatibility complex) or a first membrane proximal domain of an MHC-like protein; a heavy chain variable domain and a second membrane proximal domain of an MHC (major histocompatibility complex) or a second membrane proximal domain of an MHC-like protein, and a second heavy chain amino acid sequence comprising a constant domain selected from the group of Fc fragment monomers comprising a second (CH2) and a third (CH3) heavy chain constant domain; The present invention relates to a vector comprising the above-mentioned nucleic acid molecule encoding any of the above-mentioned bivalent, bispecific antibodies or structural parts thereof, selected from the group consisting of:

[0313] Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. DNA molecules can be ligated into vectors such that the transcriptional and translational control sequences in the vectors perform their intended function of regulating DNA transcription and translation. Expression vectors and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules partially or completely encoding the sequences of the first and second binding domains (e.g., heavy and light chain sequences in the case where the binding domain comprises heavy and light chain sequences) can be introduced into individual vectors. In one embodiment, any combination of said DNA molecules is introduced into the same expression vector. DNA molecules can be inserted into expression vectors by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0314] In some aspects of the invention, suitable vectors include restriction sites that allow easy insertion and expression of any VH or VL sequence, as described above. Polyadenylation and transcription termination may occur at native chromosomal sites downstream of the coding region. The recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0315] In some embodiments of the invention, in addition to the antibody chain genes, the recombinant vector expression of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. One skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the desired level of expression of the protein, and the like. Preferred regulatory sequences for mammalian expression host cells include, for example, viral elements that ensure high levels of protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma virus derived promoters and / or enhancers, as well as strong mammalian promoters such as the native immunoglobulin promoter and actin promoter. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0316] In some aspects of the invention, in addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes that facilitate selection of host cells into which the vector has been introduced.

[0317] In some aspects of the present invention, vectors may contain expression control sequences. As used herein, the term "expression control sequences" refers to polynucleotide sequences that are necessary for the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if necessary. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" includes at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0318] Host cells and methods for their production In one aspect, the invention relates to a method for producing a host cell for producing any of the above-described bivalent, bispecific, chimeric antibodies, comprising transformation of the cell with an expression vector as described above.

[0319] In one aspect, the invention relates to a host cell for producing any of the above-described bivalent, bispecific, chimeric antibodies comprising any of the above-described nucleic acids. As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may, for example, comprise a vector according to the invention as described above.

[0320] The present invention further relates to a host cell comprising, for example, a nucleotide sequence encoding the first heavy chain of a bivalent, bispecific, chimeric antibody according to the invention, a nucleotide sequence encoding the first light chain of a bivalent, bispecific, chimeric antibody according to the invention, a nucleotide sequence encoding the second heavy chain of a bivalent, bispecific, chimeric antibody according to the invention, or a nucleotide sequence encoding the second light chain of a bivalent, bispecific, chimeric antibody according to the invention, or all four of the above sequences. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not in fact be identical to the parent cell, since modifications may occur in subsequent generations due to mutations or environmental influences, but such cells are still included within the scope of the term "host cell" as used herein.

[0321] a first light chain amino acid sequence comprising a light chain variable domain and a light chain constant domain; a first heavy chain amino acid sequence comprising an antibody heavy chain constant domain comprising a heavy chain variable domain and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; a second light chain amino acid sequence comprising a light chain variable domain and a constant domain selected from the group of a first membrane proximal domain of an MHC (major histocompatibility complex) or a first membrane proximal domain of an MHC-like protein; a heavy chain variable domain and a second membrane proximal domain of an MHC (major histocompatibility complex) or a second membrane proximal domain of an MHC-like protein, and a second heavy chain amino acid sequence comprising a constant domain selected from the group of Fc fragment monomers comprising a second (CH2) and a third (CH3) heavy chain constant domain; Nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence of the light or heavy chain of the above bispecific antibody selected from, as well as vectors comprising these nucleic acid molecules, can be used to transfect suitable mammalian or cells thereof, plant or cells thereof, bacterial or yeast host cells. Transformation can be carried out by any known technique for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors.

[0322] Mammalian cell lines used as hosts for transformation are well known in the art and include several immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and several other cell lines. Cell lines are selected by determining that the cell line has high expression levels and provides the required characteristics of the protein produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When a recombinant expression vector encoding the bispecific antibody or a portion thereof is introduced into a mammalian host cell, the bispecific antibody is produced by culturing the host cell for a period of time sufficient to express the bispecific antibody or a portion thereof according to the invention in the host cell, or more preferably to secrete the bispecific antibody into the culture medium in which the host cell is cultured. The bispecific antibody can be isolated from the culture medium using standard protein purification techniques. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia species and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0323] Furthermore, the production level of the bispecific antibodies of the present invention from the production cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, 0323997 and 0338841.

[0324] Bispecific chimeric antibodies of the invention in different cell lines or host cells are likely to have different glycosylation patterns from each other. However, the bispecific antibodies disclosed herein are part of the present invention regardless of the glycosylation state of the binding molecules and generally regardless of the presence or absence of post-translational modifications.

[0325] The above host cells do not refer to host cells produced using human embryos. The above host cells do not refer to host cells produced by modifying the genetic integrity of human germline cells.

[0326] Methods for Producing Antibodies In one aspect, the invention provides a method for producing any of the bivalent, bispecific, chimeric antibodies described above, comprising the steps of: a) removing a host cell from - an expression vector comprising a nucleic acid molecule encoding the first light chain and the first heavy chain of a bivalent, bispecific, chimeric antibody according to the invention, - an expression vector comprising a nucleic acid molecule encoding the second light chain and the second heavy chain of a bivalent, bispecific, chimeric antibody according to the invention; Transforming with b) culturing the host cells under conditions suitable for the synthesis of said bivalent, bispecific antibody; and c) isolating said bivalent, bispecific antibody from the cell culture. The present invention relates to a method comprising the steps of:

[0327] The present invention relates to a method for producing a bivalent, bispecific, chimeric antibody according to the present invention. One aspect of the present invention relates to a method for producing a bivalent, bispecific, chimeric antibody as defined herein, comprising the steps of producing a recombinant host cell capable of expressing the bivalent, bispecific, chimeric antibody, culturing said host cell under conditions suitable for expression of the bivalent, bispecific, chimeric antibody, and isolating the bivalent, bispecific, chimeric antibody obtained. The bivalent, bispecific, chimeric antibody produced by such expression in such a recombinant host cell is referred to herein as a "bivalent, bispecific, chimeric antibody". EXAMPLES

[0328] The following examples are provided for a better understanding of the present invention. These examples are illustrative only and should not be construed as limiting the scope of the present invention in any manner. All publications, patents, and patent applications cited herein are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the accompanying embodiments.

[0329] Materials and General Methods Recombinant DNA Technology DNA was manipulated using standard methods as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to manufacturer's protocols.

[0330] Gene synthesis The desired gene segments were prepared from oligonucleotides made by chemical synthesis. Gene segments of 300-4,000 bp in length, flanked by single restriction sites, were assembled by annealing and ligation of oligonucleotides containing PCR amplification, and then cloned via unique restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0331] DNA sequencing The DNA sequence was determined by Sanger sequencing. DNA and protein sequence analysis and sequence data management The Ylab2 (Biocad) software package was used for sequence generation, mapping, analysis, annotation and illustration.

[0332] Expression vector The expression plasmid variants were applied for the transient expression of antibodies, antibody-like proteins and antigens of interest in eukaryotic cells (e.g., CHO cells). In addition to the expression cassette for the target protein, the (plasmid) vector contained an origin of replication allowing the plasmid to replicate in E. coli, genes conferring resistance in E. coli to various antibiotics (e.g., ampicillin and kanamycin).

[0333] Fusion genes containing the above antibody chains were generated by PCR and / or gene synthesis and assembled using known methods and techniques, for example, by joining corresponding nucleic acid segments using unique restriction sites in the corresponding vectors. Subcloned nucleic acid sequences were verified by DNA sequencing. The required amount of plasmid for transient transfection was generated in E. coli cell culture and isolated using known techniques.

[0334] Production and purification of recombinant antigens in mammalian cell suspension cultures Recombinant proteins were produced in cells of an established cell line derived from Chinese Hamster Ovary cells (CHO line). Suspension cultures were performed in flasks on an orbital incubator shaker using serum-free medium supplemented with 8 mM L-glutamine and 1 g / l Pluronic® 68. For transient expression, cells (2–2.2x10 6 Cells / ml) were transfected using linear polyethyleneimine. Nine days after transfection, the culture medium was separated from the cells by filtration through 0.5 / 0.22 μm depth filters.

[0335] The histidine-tagged proteins were purified by metal chelate chromatography. The purified proteins were filtered through 0.22 μm and stored at −70° C. The purity of the resulting protein solutions was assessed using SDS gel electrophoresis, which was performed in denaturing 12% polyacrylamide gels supplemented with mercaptoethanol and in native 8% polyacrylamide gels.

[0336] Production of antibodies and antibody-like proteins in mammalian cell suspension cultures The control antibody, the bispecific chimeric antibody according to the invention and the antibody-like proteins were produced in cells of a permanent cell line derived from Chinese Hamster Ovary cells (CHO line). Suspension cultures were carried out in flasks on an orbital incubator shaker using serum-free medium supplemented with 8 mM L-glutamine and 1 g / l Pluronic® 68. For transient expression, cells (2-2.2x10 6Cells / ml) were transfected using linear polyethyleneimine. The DNA / PEI ratio was 1:3 / 1:10. Nine days after transfection, the culture medium was separated from the cells by filtration through 0.5 / 0.22 μm depth filters, after which protein titers were measured on a ForteBio using standard methods. The clarified culture medium was passed through a column of protein A affinity adsorbent at 10–20 mg per ml of adsorbent, and the column was equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to remove nonspecifically bound components. Bound proteins were eluted using 0.1 M glycine buffer (pH 3). The main protein elution peak was collected and brought to pH 6.8–7.0 using 1 M Tris-HCl buffer (pH 8). All steps were performed at a flow rate of 110 cm / h. The protein was then dialyzed into PBS (pH 7.4), filtered (0.22 μm), transferred to tubes and stored at -70°C.

[0337] The purity of the resulting protein solution was assessed using SDS gel electrophoresis under reducing and non-reducing conditions, as well as using size-exclusion high performance liquid chromatography (SE HPLC).

[0338] SE HPLC was carried out on a TSK-Gel G3000SWXL column, 7.8x300mm, particle size: 5μm, pore size: 250A, and a TSK-Gel Guard SWxl precolumn.

[0339] Example 1 Selection of CH1 / CK domain replacements in bispecific antibodies For correct heterodimerization of heavy and light chains in bispecific antibodies, one of the two pairs of CH1 / CK domains was replaced with a structurally identical domain from another protein (Figure 1). The suitable replacement domain was selected in three steps: 1) Preliminary generation of a three-dimensional model of the CH1 / CK domain; 2) structural alignment of the three-dimensional model of the CH1 / CK domain with all objects in the Protein Data Bank ( www.rcsb.org ), except for antibodies and T-cell receptors; 3) Selection of suitable substitutions according to the results of structural alignment based on the RMSD metric and the number of amino acids that form identical secondary structure elements.

[0340] The first step, the preliminary generation of a three-dimensional model of the CH1 / CK domain, was the addition of missing atoms in the CH1 / CK domain from the PDBid 4nyl structure using the Prepwizard utility from the Schrodinger Suite. In the second step, the obtained model was aligned against all structures from the Protein Data Bank (except for antibodies and T-cell receptors) using the Protein Structure Alignment utility from the Schrodinger Suite. The degree of similarity between any structure and the CH1 / CK domain model was evaluated using two metrics: 1) RMSD, reflecting the root-mean-square deviation with respect to atoms between one structure and the other (polypeptide chain backbone atoms were used for the calculation), and 2) the number of amino acids that form identical secondary protein structure elements according to the alignment results. Table 1 shows the best results of the alignment using the RMSD metric.

[0341] [Table 2-1]

[0342] [Table 2-2]

[0343] The structures with the highest structural similarity with CH1 / CK domain by RMSD metric belong to major histocompatibility complex class I and II (MHC) proteins, and their structural analogues, CD1 and HFE. Based on the comparable values ​​of structural similarity degree (RMSD and number of amino acids from the same secondary structure), any of the above proteins can be selected for replacement. For further study and illustrative purposes involving CH1 / CK domain replacement, we have selected CD1b, especially its membrane proximal domain.

[0344] Example 2 Selection of the location of the disulfide bond between the heavy and light chains in antibody molecules with CH1 / CK substitutions to the membrane proximal domain of MHC or MHC-like proteins A human IgG isotype antibody molecule consists of two light chains and two heavy chains. Each of the two light chains of an antibody is connected to a heavy chain via an S-S bridge, which is a covalent bond between sulfur atoms in cysteine ​​residues. This bond is formed by the C-terminal cysteine ​​residue of the light chain and the N-terminal proximal cysteine ​​residue of the hinge region of the heavy chain (in the case of the IgG1 isotype).

[0345] The membrane proximal domains of MHC and MHC-like proteins are only connected by non-covalent bonds and do not form S-S bridges between them; therefore, the substitution of the CH1 / CK domain with the membrane proximal domain of MHC could potentially reduce the thermostability of the antibody molecule and the yield of the target product during production. To avoid this, a single cysteine ​​substitution was required in the light chain of the chimeric antibody molecule at a position suitable for the formation of S-S bridges with a cysteine ​​residue close to the N-terminus of the hinge region.

[0346] The location for the introduction of a cysteine ​​residue into the membrane proximal domain of the MHC or MHC-like protein was selected based on a structural alignment of the MHC or MHC-like protein with the full-length antibody molecule. As a result of the alignment, a cysteine ​​residue was introduced into the MHC or MHC-like protein membrane proximal domain located at the site of the initial CL(CK) domain of the antibody, close to the hinge region cysteine ​​residue that forms an SS bridge with the light chain in a natural IgG1 isotype antibody molecule. By analogy with the light chain of a natural antibody, the introduced cysteine ​​residue is terminal. The light chain was extended by two amino acids GS to ensure that the distance between the cysteine ​​residues was sufficient to establish a disulfide bond. Figure 2 illustrates the location of the SS bridge between the heavy and light chains in a chimeric antibody with the CH1-CK domain replaced by the membrane proximal domain of the MHC-like protein CD1b.

[0347] Example 3 Production of genetic constructs for generating bivalent, bispecific chimeric antibodies To produce a construct encoding the sequences of the first light and first heavy chains of an antibody that specifically binds to a first antigen, a PCR product containing genes of the heavy and light chain variable domains of the antibody was generated using primers containing restriction sites. The heavy chain variable domain was cloned into the vector pSXn-HChole-NR_VH1 using the SalI / XbaI restriction sites. The light chain variable domain was cloned into the vector pSXn-CL-BR_VL1 using the SalI / XbaI restriction sites.

[0348] To produce constructs encoding the second light and second heavy chain sequences of an antibody that specifically binds to a second antigen, we synthesized constructs containing genes for the heavy and light chain variable domains of the antibody and the membrane proximal domain of the human CD1 sequence (http: / / www.rcsb.org / structure / 5wl1) with or without various modifications.

[0349] The sequences were synthesized from oligonucleotides by PCR using primers containing restriction sites. The heavy chain variable domain with the first membrane proximal domain of the human CD1 sequence with or without modification was cloned into the vector pSX-FCknob-PR using the SalI / XbaI restriction sites. The light chain variable domain with the second membrane proximal domain of the human CD1 sequence with or without modification was cloned into the vector pSX-HR using the SalI / XbaI restriction sites.

[0350] The above four vectors were combined in a transfection step to produce the bivalent, bispecific, chimeric antibody according to the invention. Required quantities of all the above plasmids were produced in E. coli cells and purified using the Maxiprep Qiagen kit.

[0351] Example 4 Effect of introduced disulfide bonds on the assembly of full-length chimeric antibodies To test the operability of the introduced disulfide bonds, we generated a model monospecific chimeric antibody, a format consisting of two heavy and two light chains in which the heavy chain CH1 and light chain CK domains were replaced by the α3 domains of β2 microglobulin and CD1b proteins, respectively.

[0352] The sequences of the VH (SEQ ID NO: 103) and VL (SEQ ID NO: 104) variable domains were obtained from the antibody prorugolimab (CJSC Biocad). There was no disulfide bond between the heavy and light chains in the first variant of the sample. In the second variant, a cysteine ​​was added to the light chain C-terminus (as described in Example 2); the cysteine ​​is believed to form a disulfide bond with the cysteine ​​in the upper hinge region, thereby stabilizing the heavy and light chain heterodimer.

[0353] Figure 3 shows the results of non-reducing SDS gel electrophoresis of the generated samples. A prorugolimab sample (classical IgG1) was applied in lane 3 as a control. The results show that the full-length molecule assembled only in the presence of an additional disulfide bond (lane 2), whereas in its absence we observed only fragments whose mobility was consistent with the heavy chain dimer (lane 3).

[0354] We also generated chimeric antibodies with the dimerization units in the opposite orientation compared to the heavy and light chains (the heavy chain CH1 domain and the light chain CK domain were replaced by the α3 domain of the CD1b protein and β2 microglobulin, respectively; see FIG. 1B). FIG. 3 (lane 4) shows the results of SDS gel electrophoresis of the samples under non-reducing conditions; the results confirm that this orientation is also capable of assembling chimeric antibodies.

[0355] Example 5 Examining the effect of dimerization units based on MHC or MHC-like proteins on the correct pairing of heavy and light chains To prove the functionality of the dimerization units based on MHC or MHC-like proteins, in particular to prevent incorrect pairing of light chains with inappropriate heavy chains, the inventors carried out the following experiments involving monospecific molecules. The inventors generated four groups of molecules, as follows: 1. Molecules with the "exact" combination of VH and VL and the "inexact" pairing of constant domains. 2. Molecules with an "incorrect" combination of VH and VL and a "correct" pairing of constant domains. 3. Molecules with "incorrect" combinations of VH and VL and "incorrect" pairs of constant domains. 4. Control molecules including classical IgG1 format antibodies and chimeric antibodies in which the constant domains are replaced by the membrane proximal domains of MHC or MHC-like proteins (in this case the α3 domains of β2 microglobulin and CD1b protein).

[0356] An "exact" combination of VH and VL means a VH and VL pair obtained from a single antibody. An "incorrect" combination of VH and VL means a VH and VL obtained from different antibodies.

[0357] An "exact" pair of constant domains refers to a CH1-CK pair or a pair of interaction domains derived from an MHC-like protein (in this case the α3 domains of the β2 microglobulin and CD1b proteins). An "incorrect" pair of constant domains refers to a CH1-CD1b or β2 microglobulin-CK pair.

[0358] Figure 4 shows the general scheme of the experiment: the variable domain sequences of the antibody prorugolimab were used as the variable domains VH1 and VL1, and the corresponding variable domain sequences of the antibody ocrelizumab (Genentech) were used as the VH2 and VL2 variable domains.

[0359] Table 2 shows the protein productivity data. The results show that candidates with the CH1 constant domain in their heavy chain and the membrane proximal domain of the CD1b protein in their light chain showed low productivity (antibodies 01-001, 01-003, 01-008, 01-010). This is in agreement with the known fact that correct folding and transport from the endoplasmic reticulum (ER) requires the interaction of the CH1 domain with the CL domain (Feige MJ, Groscurth S, Marcinowski M, Shimizu Y, Kessler H, Hendershot LM, Buchner J. An unfolded CH1 domain controls the assembly and secretion of IgG antibodies. Mol Cell. 2009 Jun 12:34(5):569-79. doi:10.1016 / j.molcel.2009.04.028. PMID:19524537; PMCID:PMC2908990). Antibodies 01-002 and 01-009 did not pass such quality controls in the ER and demonstrated productivity of over 200 mg / l; however, despite the high productivity levels, their mobility in PAGE did not match that of the full-length molecule (Figure 5A, lanes 2, 5).

[0360] [Table 3]

[0361] Figure 5 shows the results of SDS gel electrophoresis under non-reducing conditions. The results show that the electrophoretic mobility of antibodies from group 1 (lanes 1 and 2 in Figure 5A, lane 1 in Figure 5B) and group 3 (lanes 4 and 5 in Figure 5A, lane 1 in Figure 5B) is higher than that of the full-length molecule (lane 2 in Figure 5B), indicating that the molecules were unable to assemble. The electrophoretic mobility of antibodies from group 2 (lanes 2, 3, 4 and 5 in Figure 5B (same as lane 3 in Figure 5A)) coincides with that of the full-length molecule (lane 2 in Figure 5B), even though the VH and VL sequences were obtained from different antibodies. As expected, antibodies from the control group assembled (lane 2 in Figure 5B, lane 6 in Figure 5A (same as lane 6 in Figure 5B)).

[0362] The data obtained show that dimerization units based on the membrane proximal domains of MHC or MHC-like proteins prevent the formation of incorrect pairings between heavy and light chains when the chains contain constant domains of different nature.

[0363] Example 6 Generation of bivalent, bispecific, chimeric antibodies containing MHC-like dimerization units To verify the generality of the subject approach as a platform solution for assembling bispecific molecules using any pair of antigen-binding fragments (hereinafter light and heavy chain variable fragments), we selected three random pairs of light and heavy chain variable fragments (see Table 3) derived from known antibodies and used them to generate six bispecific chimeric antibodies. Table 3 shows the results of the productivity of the produced proteins. Figure 6 shows the results of SDS gel electrophoresis of the produced samples under non-reducing and reducing conditions. For all six samples, there is a major band at approximately 150 kDa, corresponding to the full-length molecule. Table 3 also shows the results of sample purity by SE HPLC.

[0364] [Table 4]

[0365] Example 7 Determination of affinity of full-length bispecific chimeric antibodies on Forte Bio Octert RED 384 To verify that the generated bispecific chimeric antibodies had not lost their antigen-binding capacity, we performed affinity analysis on a Forte Bio Octert RED 384. For antibodies 02-004 and 02-005, we measured the affinity for the extracellular domain of human PD-1 protein (hPD1ex-H6F) and the biotinylated peptide [NH2]CEPANPSEKNSPSTQYCYSIQS[CH2CH2]biotin, which contains a fragment of the human CD20 sequence in its amino acid sequence; for antibodies 02-006 and 02-007, we measured the affinity for the extracellular domain of human PD-1 protein (hPD1ex-H6F) and the extracellular domain of human CSF1R protein (hCSF1R_His); for antibodies 02-008 and 02-009, we measured the affinity for the extracellular domain of human CSF1R protein (hCSF1R_His) and the CD20 peptide.

[0366] The sequence of amino acids 20-512 of human CSF1R protein with C-terminal His and FLAG tags, molecular weight 57.4 kDa, was used as the hCSF1R antigen. The sequence of amino acids 21-170 of human PD-1 protein with C-terminal His and FLAG tags, molecular weight 20.6 kDa, was used as the hPD-1ex-H6F antigen (see Table 6).

[0367] As described in the general examples, gene sequences encoding the antigens were synthesized de novo, cloned into expression vectors, produced in CHO cells and purified using affinity chromatography.

[0368] The experiment was carried out using a dynamic buffer solution (hereinafter referred to as 1xKB) with the following formulation: 4.3mM Na2HPO4; 136.9mM NaCl, 1.5mM KH2PO4; 2.7mM KCl; the volume fraction of added Tween 20 was 0.1%; the mass fraction of added BSA (bovine serum albumin) was 0.1%; pH 7.4. Prior to the measurement, the ProA sensor was regenerated with a solution of 50mM glycine and hydrochloric acid, pH 1.8 (5 seconds in regeneration solution, 5 seconds in 1xKB, repeated three times).

[0369] For hPD-1ex-H6F and hCSF1R_His antigens, a Protein A (ProA) biosensor (ForteBio) was immersed in a solution containing the antibody at a concentration of 10 μg / ml for 60 seconds to immobilize it. A baseline was recorded for 60 seconds in 1xKB. The antibody-loaded sensor was then immersed in a well containing a solution of the target antigen (analyte) in dynamic buffer for 90 seconds. Measurements were performed on solutions containing the hPD1ex-H6F analyte at concentrations of 2.50 μg / ml (121.4 nM), 1.25 μg / ml (60.7 nM), and 0.625 μg / ml (30.35 nM). Measurements were carried out on solutions containing the hCSF1R_His analyte at concentrations of 2.50 μg / ml (43.6 nM), 1.25 μg / ml (21.8 nM) and 0.625 μg / ml (10.9 nM). We then detected the dissociation of the complex in 1xKB over 210 s for hPD1ex-H6F and 300 s for hCSF1R_His.

[0370] The reference sensor was passed through all steps as the sensor used to record the analyte sensorgrams, except for the association step, in which the sensor was immersed in 1xKB solution without analyte (the signal of the reference sensor was measured simultaneously with the recording of the main sensorgrams). The reference signal was subtracted from the signal received on the sensor interacting with the analyte during the processing of the sensorgrams.

[0371] To check for non-specific interactions between the analyte and the sensor, we used the sensor without antibody (in the loading step, the sensor was immersed in 1xKB solution; all other steps were identical to those used for the antibody-loaded sensor).

[0372] For biotinylated CD20 peptides, peptides at a concentration of 5 μg / ml were immobilized on the surface of a SAX sensor (High Precision Streptavidin (SAX) biosensor, ForteBio) for 120 seconds. A baseline was recorded for 120 seconds in 1xKB solution. The sensor containing the loaded peptide was immersed in a well containing an antibody solution (analyte) in 1xKB for 15 seconds. Measurements were performed on solutions containing analyte (antibody in case of CD20 peptide) at concentrations of 150 μg / ml, 75 μg / ml, 37.5 μg / ml. Dissociation of the complex was recorded for 120 seconds in 1xKB.

[0373] All measurements were performed at 30° C. and the orbital mixing speed was 1,000 revolutions per minute. Rate constant (k on is the on / binding rate constant and k dis The obtained sensorgrams were processed according to a 1:1 interaction model (selection of one set of k, kdis, and KD constants to analyze several sensorgrams of different concentrations) using Global Fit in the ForteBio Octet Data Analysis 9.0 software to obtain values ​​of k, kd, and KD (k is the dissociation rate constant and K is the equilibrium dissociation constant or affinity constant). The results are shown in Table 4.

[0374] [Table 5]

[0375] Table 5 shows the validation results for non-specific interactions between the analyte and the unloaded sensor. Table 5 shows that there was no non-specific interaction between the analyte and the unloaded sensor.

[0376] [Table 6]

[0377] The results show that all bispecific chimeric antibodies 02-004 to 02-009 show specific binding to the target antigens with KD values ​​of the same order of magnitude regardless of the location of the corresponding antigen-binding fragment (within the Fab fragment or within the Fab-like fragment containing the α3 domains of β2 microglobulin and CD1b protein replacing the CK and CH1 domains).

[0378] Example 8 Analysis of non-specific binding of bispecific chimeric antibodies to non-target antigens Experimental studies of non-specific binding of bispecific chimeric antibodies to non-target antigens were carried out on a Forte Bio Octert RED 384.

[0379] Protein A (ProA) biosensor (ForteBio) was immersed in a solution containing an antibody at a concentration of 10 μg / ml for 150 seconds to immobilize it. A baseline was recorded for 30 seconds in 1xKB solution. The antibody-loaded sensor was then immersed in a well containing a non-target antigen at a concentration of 30 μg / ml for 150 seconds. The dissociation of the complex was then recorded for 30 seconds.

[0380] The reference sensor was passed through all steps as was the sensor used to record the analyte sensorgram, except for the association step, in which the sensor was immersed in a dynamic buffer solution without analyte (the reference sensor signal was measured simultaneously with the recording of the main sensorgram). The reference signal was subtracted from the signal received on the sensor interacting with the analyte during processing of the sensorgram.

[0381] The antigen panel included hAng2_H6F, hPD1ex-H6F, hCSF1R_His, IL5R-His, hIL4R-His, hCD38-Avi, C-Avi-His-TEVs-HSA_hBCMA, Her3-H6F, hisOX40, Macaca CSF1R_C-His (see Table 6). Each tested antibody in the antigen panel has a specific (target) antigen, and the results of the interaction with the target antigen are given as a positive control.

[0382] The sensorgrams were processed using ForteBio Octet Data Analysisn 9.0 software. The recorded signal levels were checked at the end of the association step to report the absence of non-specific interactions (response parameters). Table 7 shows the results of the validation of the binding of the bispecific chimeric antibodies. The experiment showed that there was no interaction between the antibodies 02-004 to 02-009 and non-target antigens.

[0383] [Table 7]

[0384] [Table 8-1]

[0385] [Table 8-2]

[0386] [Table 8-3]

[0387] Example 9 Analysis of the simultaneous binding of bispecific chimeric antibodies according to the invention to two antigens Antibodies 02-006 and 02-007 were analyzed for simultaneous binding to two different target antigens (hPD1ex-H6F and hCSF1R_His) on a Forte Bio Octet RED 384. The experiment was carried out on an AR2G sensor (Amine Reactive Second-Generation (AR2G) biosensor, ForteBio). The experimental steps are shown in Table 8.

[0388] [Table 9]

[0389] The sensor was activated for 300 s in an aqueous solution containing 20 mM EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 10 mM sNHS (N-hydroxysulfosuccinimide). The antigen (hPD1ex-H6F) was loaded onto the surface of the biosensor in 10 mM sodium acetate buffer at pH 5.0 for 300 s. The concentration of the loaded protein was 15 μg / ml. The unreacted active centers on the sensor surface were quenched for 300 s in a 1 M aqueous solution of ethanolamine at pH 8.5. The baseline of the fifth step of the experiment and all subsequent steps was carried out in a dynamic buffer solution (1xKB) with the following formulation: 4.3mM Na2HPO4; 136.9mM NaCl, 1.5mM KH2PO4; 2.7mM KCl; the volume fraction of Tween 20 added was 0.1%; the mass fraction of BSA added was 0.1%; pH 7.4. After recording the baseline (step 5), the antibody was loaded onto the sensor; the concentration of the loaded antibody was 30μg / ml (step 6). Then, a third baseline was recorded (step 7). This step shows the absence of fast signal decay, indicating a specific interaction of the loaded antibody with the hPD1ex-H6F pre-immobilized on the sensor. In the next step of the association (step 8), the sensor containing the immobilized hPD1ex-H6F and the bound antibody was immersed in a hCSF1R_His antigen solution at a concentration of 100μg / ml. The signal amplification in this step is due to the presence of the antibody loaded on the sensor, the FAB fragment against the hCSF1R_His antigen.

[0390] The sensorgrams were analyzed using ForteBio Octet Data Analysisn 9.0 software. The main conclusions are shown in Table 9. Sensorgrams showing the steps of the experiment are shown in Figure 7. The results show that samples 02-006 and 02-007 show simultaneous binding to the hPD1ex-H6F and hCSF1R_His antigens.

[0391] [Table 10]

[0392] The results show that antibodies 02-006 and 02-007 show simultaneous binding to the hPD1ex-H6F and hCSF1R_His antigens. Example 10 Analysis of the molecular weight of the bispecific chimeric antibodies according to the invention using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) with mass spectrometric detection To confirm the correct assembly of the molecules, the inventors carried out an analysis of the molecular weight of the full-length bispecific chimeric antibody according to the invention. This method allows the identification of the full-length bispecific chimeric antibody according to the invention consisting of four different chains and its discrimination from by-products formed from another set of chains. These data, together with the data obtained in Example 5, suggest that the technical solution comprising replacing the pair of constant CH1-CK domains with a pair of membrane proximal domains derived from MHC or MHC-like proteins provides for the correct assembly of the bispecific chimeric antibody according to the invention.

[0393] The analysis was carried out using an Agilent 1290 Infinity II UPLC-Agilent 6530 Q-Tof chromatography-mass spectrometry combination on a BioResolve Polyphenyl RP column (2.1x50 mm, 2.7 μm particle size; a BioResolve Polyphenyl RP precolumn, 2.1x5 mm, 2.7 μm particle size, was also used). Prior to the analysis, all molecules were treated with PNGase F (Promega) enzyme to remove N-glycans. For this, the enzyme was diluted in water to a concentration of 1 unit of activity in 1 μl. Samples with a protein content of 120 μg were mixed with a buffer solution (100 mM ammonium bicarbonate pH 7.2), a solution of PNGase F was added in an enzyme:protein ratio of 1 unit:50 μg, and the mixture was incubated in a thermostat at (37.0±0.1) °C for 18 h.

[0394] For the analysis, a test solution of 10 μg was selected according to the measured protein concentration and the concentration was adjusted to 0.2 mg / ml with mobile phase A. The sample input volume was 7 μl. Prior to analysis, the chromatographic system was equilibrated with mobile phases at an initial ratio of A: 95%, B: 5% (phase A contained 0.1% formic acid solution, 0.02% trifluoroacetic acid solution in water, and phase B contained 0.1% formic acid solution, 30% acetonitrile in isopropyl alcohol) for at least 30 min until a stable pressure was achieved. The mass spectrometer was calibrated using calibration standards according to the manufacturer's guidelines.

[0395] The samples were separated in a concentration gradient of mobile phase B (5%-27% from 5-6 min after sample introduction, then 27%-37% from 6-30 min) at a column temperature of (60±1)°C and a flow rate of 0.5 ml / min, and a chromatogram was obtained at a wavelength of 280 nm.

[0396] Data was processed in PMi Intact software. The names of the chains contained in the bispecific chimeric antibodies are shown in Table 10.

[0397] [Table 11]

[0398] Mass spectrometry results for antibodies 02-004, 02-006, 02-009 (most representative) are shown in Table 11. Figure 8 also shows an image of the deconvoluted mass spectrum of peaks 4-6 of the total ion current chromatogram for antibody 02-004, with the peaks for the remaining antibodies appearing similarly.

[0399] For each antibody, the table shows the mass of material in the corresponding chromatogram peak; the peaks were further annotated to obtain the identification of the chains contained in a given mass fragment. The mass distribution was similar in all samples: the major peak was at 146 kDa, and the minor mass peaks were at approximately 23 kDa (corresponding to the mass of a single light chain) and approximately 74 kDa (corresponding to a heterodimer of heavy and light chains). The major peaks were annotated based on the theoretical value of the average mass.

[0400] Peak 4 of antibody 02-004 was not annotated by the software because the settings included the assumption that all N-terminal glutamines were in the pyro form (hence the mass of pyroglutamine was 18 Da smaller than that of glutamine), thus the mass obtained for peak 4 corresponds to the mass of the molecule annotated in peak 5, where one of the N-terminal glutamines is in the non-pyro form.

[0401] Peaks 5 and 6 of the 02-004 antibody have three masses corresponding to the full-length molecule and the two lysine-free form of the full-length molecule (missing one C-terminal cysteine ​​and two C-terminal lysines). For antibodies 02-006 and 02-009, the mass distribution was in a similar manner.

[0402] [Table 12-1]

[0403] [Table 12-2]

[0404] [Table 12-3]

[0405] [Table 12-4]

[0406] Taken together, these results so far show that the mass of the full-length bispecific chimeric antibody is consistent with that of a molecule composed of four different chains, and that the preparation contains low molecular weight fragments, although these are in small amounts.

[0407] Example 11 Confirmation of correct assembly of the bispecific chimeric antibody according to the invention using proteolysis and mass spectrometry of the resulting fragments To directly verify the correct pairing of the light and heavy chains, the bispecific chimeric antibodies according to the invention were cleaved by GingisKHAN protease (Genovis), whose recognition site is located in the antibody hinge region (...KSCDK / THTCPPCP...). Such proteolysis results in the destruction of the full-length antibody into an Fc fragment, a Fab fragment, and a Fab-like fragment that contains the α3 domain of the CD1b protein, which replaces the β2-microglobulin and the CK and CH1 domains. The fragments of the bispecific molecules were analyzed after proteolysis using tandem electrophoresis under non-reducing conditions; the resulting fragments were also subjected to mass spectrometry.

[0408] The proteolysis reaction mixture contained 40 μg of bispecific antibody and 40 units of GingisKHAN enzyme (ratio of 1 enzyme unit:1 μg protein) in a buffer consisting of 100 mM Tris-HCl, pH 8.0, 1 mM cysteine ​​in a volume of 60 μl. The reaction was carried out for 1 h in a thermostat at (37.0±0.1) °C and stopped by adding iodoacetamide to a concentration of 10 mM. For electrophoresis, a buffer containing SDS (to a concentration of 1% SDS) was added to the resulting sample and SDS gel electrophoresis was performed under non-reducing conditions.

[0409] Figure 9 shows the results of SDS gel electrophoresis. After treatment of bispecific chimeric antibodies (02-004 to 02-009) with GingisKHAN protease, three large fragments were formed, which were observed in the electropherogram in the region of 40 to 50 kDa and showed different mobility in polyacrylamide gels. Monospecific molecules with and without (01-011) a dimerization unit based on the membrane-proximal domain of the MHC-like protein CD1b (prorugolimab, ocrelizumab) were also treated with GingisKHAN protease (Figure 9B, lanes 3, 4, 5). As a result of comparing the electrophoretic mobility formed as a result of proteolysis of the fragments, it was concluded that the fragment showing the lowest mobility level corresponds to the Fc fragment, the intermediate mobility corresponds to the Fab fragment, and the fragment showing the highest mobility level corresponds to the Fab-like fragment containing the α3 domain of the CD1b protein replacing the β2 microglobulin and the CK and CH1 domains. In the case of antibodies 02-006 to 02-009, the electropherograms showed additional fragments formed as a result of non-specific cleavage of the variable domain of the anti-CSF1R fragment. To determine the exact mass of the fragments formed as a result of proteolysis, we performed mass spectrometry.

[0410] Prior to mass spectrometry, immediately after adding iodoacetamide, the samples were transferred onto a Zeba (MWCO 7 kDa) column in 50 mM ammonium bicarbonate, pH (7.6±0.2) according to the manufacturer's guidelines; PNGase F was then added to the samples at a ratio of 1 enzyme unit:50 μg protein and the samples were incubated in a thermostat at (37.0±0.1) °C for 18 h.

[0411] Mass spectrometry of the resulting fragments was carried out according to the method described in Example 10. The RP UHPLC results with mass spectrometry detection are shown in Table 12. Antibodies 02-006 and 02-008 showed fragments of 11,424 Da and 37,035 Da out of a total of 48,459 Da corresponding to the mass of a Fab-like fragment containing the α3 domain of the CD1b protein replacing the β2 microglobulin and the CK and CH1 domains and the variable fragments of the light and heavy chains for CSF1R. Antibodies 02-007 and 02-009 showed fragments of 11,424 Da and 36,062 Da out of a total of 47,486 Da corresponding to the mass of a Fab fragment containing the variable fragments of the light and heavy chains for CSF1R, which is in agreement with the data obtained after SDS gel electrophoresis of these samples and confirms the presence of a putative further proteolysis in the variable domain for CSF1R (FIG. 9).

[0412] [Table 13-1]

[0413] [Table 13-2]

[0414] [Table 13-3]

[0415] [Table 13-4]

[0416] [Table 13-5]

[0417] [Table 13-6]

[0418] Masses of 97811.8 Da and 97693 Da for sample 02-009 correspond to molecules with one truncated Fab-like fragment. These results show that all bispecific chimeric antibodies according to the invention were destroyed after proteolysis into Fc fragments, Fab fragments and fragments corresponding in mass to β2 microglobulin and to a Fab-like fragment comprising the α3 domain of the CD1b protein replacing the CK and CH1 domains, and demonstrate that the technical solution based on the replacement of the CH1-CK constant domain pair with a membrane proximal domain pair derived from an MHC or MHC-like protein provides for the correct assembly of the bispecific chimeric antibodies according to the invention.

[0419] Example 12 Examination of the effect of amino acid substitutions in dimerization units based on the membrane proximal domains of MHC-like proteins on the thermal stability of chimeric antibodies The inventors further tested various substitutions in the dimerization unit and their effect on the purity and stability of the resulting chimeric antibody. A monospecific format was tested, which is an antibody consisting of two heavy chains and two light chains, in which the heavy chain CH1 and light chain CK domains are replaced by the α3 domains of β2 microglobulin and CD1b protein, respectively (forward orientation of the dimerization unit). The sequences of the VH and VL variable domains were obtained from the antibody prorugolimab (CJSC Biocad). The mutations shown in Table 13 were introduced into the sequences of the α3 domains of β2 microglobulin and CD1b protein. The sequence of the α3 domain of CD1b protein further includes the C-terminal extension shown in Table 13. Table 13 shows the number of the position where the mutation was introduced from the start of the dimerization unit, and indicates the correspondence of the positions numbered according to the EU numbering of antibody chain amino acids (Edelman GM et al., Proc. Natl. Acad. Sci. USA 63 (1969), pp. 78-85; Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Shown below is the numbering of the position where the substitution was introduced from the start of the dimerization unit; the position of the substitution in the hinge region follows the EU numbering.

[0420] [Table 14-1]

[0421] [Table 14-2]

[0422] Figure 10 shows the results of non-reducing SDS gel electrophoresis of the produced antibodies. Samples of prorugolimab (classical IgG1) and 01-011 (sample with initial dimerization unit based on the membrane proximal domain of MHC-like protein, see Example 5) were applied as controls on lanes 1 and 2, respectively (Figures 10A and 10B). The results show that the mobility of the large fragments of all modified samples corresponds to the full-length molecule (see lanes 3-8 in Figure 10A, lanes 3-4 in Figure 10B), thus indicating that the introduced modifications do not lead to the assembly of the full-length molecule. SE HPLC (Table 14) shows that some modifications lead to a decrease in monomer content and an increase in the number of small fragments (antibodies 03-001, 03-003, 03-005, 03-006, 03-007); however, some of them showed an increase in monomer percentage (03-002, 03-004, 03-008). Furthermore, we analyzed the aggregation temperature by dynamic light scattering of the resulting antibodies; the results are also shown in Table 14.

[0423] [Table 15]

[0424] The results show that antibody variants that showed an increased relative monomer content by SE HPLC further showed an increased aggregation temperature compared to unmodified variants of the MHC-like protein-based dimerization unit.

Claims

1. A bivalent, bispecific chimeric antibody, the antibody comprising: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen; wherein the first light chain comprises a light chain variable domain and a light chain constant domain; wherein the first heavy chain comprises a heavy chain variable domain and an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and a third (CH3) heavy chain constant domain; b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen; wherein the second light chain comprises a light chain variable domain and a constant domain; The constant domain is selected from the group consisting of the first membrane proximal domain of the MHC (major histocompatibility complex) or the first membrane proximal domain of an MHC-like protein, wherein the second heavy chain comprises: a heavy chain variable domain, a constant domain selected from the group consisting of the second membrane proximal domain of the MHC (major histocompatibility complex) or the second membrane proximal domain of an MHC-like protein, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains. Includes; wherein said first membrane proximal domain of an MHC or MHC-like protein and said second membrane proximal domain of an MHC or MHC-like protein form a heterodimer therebetween that is stabilized by a disulfide bond; wherein the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are modified to contact each other with their surfaces to form a bivalent, bispecific chimeric antibody, and said modification in the heavy chain CH3 domain is a substitution that facilitates heterodimerization.

2. The bivalent, bispecific chimeric antibody comprising:

2. the first membrane proximal domain of MHC the first membrane-proximal domain of MHC class I (major histocompatibility complex class I), the first membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the first membrane proximal domain of MHC class I, or Modified variants of the first membrane proximal domain of MHC class II may be selected from the group comprising: the second membrane proximal domain of MHC the second membrane-proximal domain of MHC class I (major histocompatibility complex class I), the second membrane-proximal domain of MHC class II (major histocompatibility complex class II), a modified variant of the second membrane proximal domain of MHC class I, or Modified variants of the second membrane-proximal domain of MHC class II may be selected from the group comprising: Modified variant refers to a variant containing a substitution with a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from said first and second membrane proximal domains of the MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, each resulting in an increase in thermodynamic stability Tm of more than 1°C compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, that result in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; The bivalent, bispecific chimeric antibody of claim 1.

3. 3. The bivalent, bispecific chimeric antibody of claim 2, wherein the MHC class II is selected from the group of HLA-DM, HLA-DO, HLA-DP, HLA-DQ or HLA-DR.

4. 3. The bivalent, bispecific chimeric antibody of claim 2, wherein the MHC class I is selected from the group of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G.

5. the first membrane proximal domain of an MHC-like protein the first membrane-proximal domain of CD1 (cluster of differentiation 1); the first membrane-proximal domain of HFE (hemochromatosis protein), a modified variant of the first membrane proximal domain of CD1, or Modified variants of the first membrane-proximal domain of HFE may be selected from the group comprising: the second membrane proximal domain of an MHC-like protein the second membrane-proximal domain of CD1 (cluster of differentiation 1); the second membrane-proximal domain of HFE (hemochromatosis protein), a modified variant of the second membrane proximal domain of CD1, or Modified variants of the second membrane-proximal domain of HFE may be selected from the group comprising: Modified variant refers to a variant containing a substitution with a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from said first and second membrane proximal domains of the MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, each resulting in an increase in thermodynamic stability Tm of more than 1°C compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, that result in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; The bivalent, bispecific chimeric antibody of claim 1.

6. 6. The bivalent, bispecific chimeric antibody of claim 5, wherein the CD1 is selected from the group of CD1a, CD1b, CD1c, CD1d or CD1e.

7. 2. The bivalent, bispecific chimeric antibody of claim 1, wherein the variable fragment of the second light chain (VL) is separated from the first membrane proximal domain of an MHC or MHC-like protein by a linker of 1 to 25 amino acids in length, and / or the variable fragment of the second heavy chain (VH) is separated from the second membrane proximal domain of an MHC or MHC-like protein by a linker of 1 to 25 amino acids in length.

8. a) the CH3 domain of one heavy chain is modified such that on the surface of the CH3 domain of one heavy chain that contacts the surface of the CH3 domain of the other heavy chain in said bivalent, bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, resulting in the formation of a knob on the surface of the CH3 domain of one heavy chain that can fit into a hole on the surface of the CH3 domain of the other heavy chain; and b) the CH3 domain of the other heavy chain is modified such that on the surface of the CH3 domain of the second heavy chain that contacts the surface of the CH3 domain of the first heavy chain in said bivalent bispecific antibody, an amino acid residue is substituted with an amino acid residue having a smaller side chain volume, resulting in the formation of a hole on the surface of the CH3 domain of the second heavy chain that can accommodate a knob on the interface of the CH3 domain of said first heavy chain; wherein the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W); and wherein the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The bivalent, bispecific chimeric antibody of claim 1.

9. 2. The bivalent, bispecific chimeric antibody of claim 1, wherein the constant domain of the first light chain of the antibody is selected from CK or CL.

10. 2. The bivalent, bispecific chimeric antibody of claim 1, wherein the CH3 domains of the antibody are further modified by the introduction of cysteine ​​(C) as an amino acid into the corresponding positions of each CH3 domain so that disulfide bridges can form between both CH3 domains.

11. 9. The bivalent, bispecific chimeric antibody of claim 8, wherein the CH3 domain of one heavy chain is modified to form a knob and the CH3 domain of the other heavy chain is modified to form a hole, or vice versa.

12. 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; or the CH3 domain of one heavy chain has amino acid substitutions Y349C / T366S / L368A / Y407, and the CH3 domain of the other heavy chain has amino acid substitutions S354C / T366W; A bivalent, bispecific chimeric antibody according to claim 11.

13. the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are the α2 domain of MHC II and the β2 domain of MHC II, respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are the β2 domain of MHC II and the α2 domain of MHC II, respectively, and form a heterodimer therebetween; The bivalent, bispecific chimeric antibody of claim 1.

14. the α2 domain of MHC II has an amino acid sequence selected from the group of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38, and the β2 domain of MHC II has an amino acid sequence selected from the group of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39; or, The β2 domain of MHC II has an amino acid sequence selected from the group of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39, and the α2 domain of MHC II has an amino acid sequence selected from the group of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO:

38. A bivalent, bispecific chimeric antibody according to claim 13.

15. the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are modified variants of the α2 domain of MHC II and the β2 domain of MHC II, respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are modified variants of the β2 domain of MHC II and the α2 domain of MHC II, respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are the α3 domain of MHC I and β2 microglobulin (β2M), respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are β2 microglobulin (β2M) and the α3 domain of MHC I, respectively, and form a heterodimer therebetween; or, Modified variant refers to a variant containing a substitution with a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from said first and second membrane proximal domains of the MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, each resulting in an increase in thermodynamic stability Tm of more than 1°C compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, that result in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; The bivalent, bispecific chimeric antibody of claim 1.

16. The α3 domain of MHC I has an amino acid sequence selected from the group of SEQ ID NOs: 1-29, and β2 microglobulin (β2M) has the amino acid sequence of SEQ ID NO: 46; or, The β2 microglobulin (β2M) has the amino acid sequence of SEQ ID NO: 46, and the α3 domain of MHC I has an amino acid sequence selected from the group of SEQ ID NOs: 1 to 29.

16. A bivalent, bispecific chimeric antibody according to claim 15.

17. the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are a modified variant of the α3 domain of MHC I and a modified variant of β2 microglobulin (β2M), respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of MHC and the second membrane proximal domain of MHC are a modified variant of β2 microglobulin (β2M) and a modified variant of the α3 domain of MHC I, respectively, and form a heterodimer therebetween; Modified variant refers to a variant containing a substitution with a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from said first and second membrane proximal domains of the MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, each resulting in an increase in thermodynamic stability Tm of more than 1°C compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, that result in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; The bivalent, bispecific chimeric antibody of claim 1.

18. 18. The bivalent, bispecific chimeric antibody of claim 17, wherein the modified variant of β2 microglobulin (β2M) has an amino acid sequence selected from SEQ ID NO: 47 or SEQ ID NO:

48.

19. the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are the α3 domain of CD1 and β2 microglobulin (β2M), respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are β2 microglobulin (β2M) and the α3 domain of CD1, respectively, and form a heterodimer therebetween; The bivalent, bispecific chimeric antibody of claim 1.

20. The α3 domain of CD1 has an amino acid sequence selected from the group of SEQ ID NOs: 40 to 44, and β2 microglobulin (β2M) has the amino acid sequence of SEQ ID NO: 46; or, The β2 microglobulin (β2M) has the amino acid sequence of SEQ ID NO: 46, and the α3 domain of CD1 has an amino acid sequence selected from the group of SEQ ID NOs: 40 to 44.

20. The bivalent, bispecific chimeric antibody of claim 19.

21. the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are a modified variant of the α3 domain of CD1 and a modified variant of β2 microglobulin (β2M), respectively, and form a heterodimer therebetween; Modified variant refers to a variant containing a substitution with a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from said first and second membrane proximal domains of the MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, each resulting in an increase in thermodynamic stability Tm of more than 1°C compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, that result in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; The bivalent, bispecific chimeric antibody of claim 1.

22. 22. The bivalent, bispecific chimeric antibody of claim 21 , wherein the modified variant of the α3 domain of CD1 has an amino acid sequence selected from the group of SEQ ID NOs: 49-56 and the modified variant of β2 microglobulin (β2M) has an amino acid sequence selected from SEQ ID NO: 47 or SEQ ID NO:

48.

23. the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are a modified variant of β2 microglobulin (β2M) and a modified variant of the α3 domain of CD1, respectively, and form a heterodimer therebetween; Modified variant refers to a variant containing a substitution with a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from said first and second membrane proximal domains of the MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, each resulting in an increase in thermodynamic stability Tm of more than 1°C compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, that result in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; The bivalent, bispecific chimeric antibody of claim 1.

24. 24. The bivalent, bispecific chimeric antibody of claim 23, wherein said modified variant of β2 microglobulin (β2M) has an amino acid sequence selected from SEQ ID NO: 47 or SEQ ID NO: 48, and said modified variant of the α3 domain of CD1 has an amino acid sequence selected from the group of SEQ ID NOs: 49-56 or SEQ ID NO:

109.

25. the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are the α3 domain of HFE and β2 microglobulin (β2M), respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are the α3 domain of β2 microglobulin (β2M) and HFE, respectively, and form a heterodimer therebetween; The bivalent, bispecific chimeric antibody of claim 1.

26. The α3 domain of HFE has the amino acid sequence of SEQ ID NO: 45, and the β2 microglobulin (β2M) has the amino acid sequence of SEQ ID NO: 46; or, The β2 microglobulin (β2M) has the amino acid sequence of SEQ ID NO: 46, and the α3 domain of HFE has the amino acid sequence of SEQ ID NO:

45.

26. The bivalent, bispecific chimeric antibody of claim 25.

27. the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are a modified variant of the α3 domain of HFE and a modified variant of β2 microglobulin (β2M), respectively, and form a heterodimer therebetween; or, the first membrane proximal domain of an MHC-like protein and the second membrane proximal domain of an MHC-like protein are a modified variant of β2 microglobulin (β2M) and a modified variant of the α3 domain of HFE, respectively, and form a heterodimer therebetween; Modified variant refers to a variant containing a substitution with a cysteine ​​(C) that forms a disulfide bridge between the chains of the heterodimer produced from said first and second membrane proximal domains of the MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, each resulting in an increase in thermodynamic stability Tm of more than 1°C compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, that result in a greater than 5% reduction in the amount of aggregates at concentrations greater than 10 mg / ml compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; or, Modified variants refer to variants that contain one or more substitutions at various positions in the membrane proximal domain of an MHC or MHC-like protein, respectively, resulting in the removal of a glycosylation site compared to the wild-type membrane proximal domain of an MHC or MHC-like protein; The bivalent, bispecific chimeric antibody of claim 1.

28. 28. The bivalent, bispecific chimeric antibody of claim 27, wherein the modified variant of β2 microglobulin (β2M) has an amino acid sequence selected from SEQ ID NO: 47 or SEQ ID NO:

48.

29. 2. The bivalent, bispecific chimeric antibody of claim 1, wherein the variable domain of the first light chain and the variable domain of the second light chain are identical.

30. The Fc fragment belongs to IgG. the Fc fragment isotype is selected from the group of human IgG1, IgG2, or IgG4; The bivalent, bispecific chimeric antibody of claim 1.

31. Substitutions are further introduced into said Fc fragment monomers, resulting in the absence of ADCC, CDC and / or ADCP properties in the bivalent, bispecific antibody. LALA substitutions (L234A and L235A) are further introduced into the Fc fragment monomer; or, the substitution E345R is further introduced into said Fc fragment monomer; The bivalent, bispecific chimeric antibody of claim 1.

32. and further introducing substitutions into said Fc fragment monomer that result in prolonged antibody activity. YTE substitutions (M252Y, S254T and T256E) are further introduced into the Fc fragment monomer. The bivalent, bispecific chimeric antibody of claim 1.

33. specifically binds to CD20 and CD3; or specifically binds to BCMA and CD3; or specifically binds to PD-L1 and CD47; or specifically binds to coagulation factor 9 (FIX) and coagulation factor 10 (FX); or specifically binds to GD2 and CD3; or specifically binds to AXL and CD3; or specifically binds to PD-L1 and TGF-beta, The bivalent, bispecific chimeric antibody of claim 1.

34. said first membrane proximal domain of the MHC or MHC-like protein and said second membrane proximal domain of the MHC or MHC-like protein form a heterodimer therebetween that is stabilized by a disulfide bond due to a mutation or mutations in the first and / or second membrane proximal domains that form an S—S bond (disulfide cysteine ​​bridge) between the first and second membrane proximal domains of the MHC or MHC-like protein; or, the first membrane proximal domain of the MHC or MHC-like protein and the second membrane proximal domain of the MHC or MHC-like protein form a heterodimer therebetween, stabilized by a disulfide bond due to one or more (1 to 10) amino acids at the C-terminus forming an S-S bond (disulfide bond, cysteine ​​bridge) between the first membrane proximal domain of the MHC or MHC-like protein and the hinge, and the extension of the first membrane proximal domain of the MHC or MHC-like protein by a terminal Cys at the C-terminus; The bivalent, bispecific chimeric antibody of claim 1.

35. 35. The bivalent, bispecific chimeric antibody of claim 34, wherein the extension of the first membrane proximal domain of an MHC or MHC-like protein is a GSC.

36. An isolated nucleic acid encoding the bivalent, bispecific chimeric antibody of claim 1.

37. An expression vector comprising the nucleic acid described in claim 36.

38. 38. A method for producing a host cell for producing the bivalent, bispecific chimeric antibody of claim 1, comprising the step of transforming the cell with the vector of claim 37.

39. A host cell for producing the bivalent, bispecific chimeric antibody of claim 1, comprising the nucleic acid of claim 36.

40. a) infecting the host cell with an expression vector comprising nucleic acid molecules encoding the first light chain and the first heavy chain of said bispecific chimeric antibody, an expression vector comprising nucleic acid molecules encoding the second light chain and the second heavy chain of said bispecific chimeric antibody; Transforming with b) culturing said host cells under conditions suitable for the synthesis of said bivalent, bispecific, chimeric antibody; and c) isolating said bivalent, bispecific antibody from the cell culture 2. A method for producing the bivalent, bispecific chimeric antibody of claim 1, comprising:

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