Heterodimeric antibodies that bind to CD3 and tumor antigens

Abstract

To provide novel heterodimeric antibodies.SOLUTION: The heterodimeric antibodies comprise a) a CD3 binding domain, b) a monovalent CD123 binding domain, and c) a first Fc domain and a second Fc domain. The CD123 binding domain comprises a variable heavy chain domain comprising a specific sequence and a variable light chain domain comprising a specific sequence. Also provided are pharmaceutical compositions comprising the heterodimeric antibodies for cancer therapy.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 62 / 085,117, filed on November 26, 2014 U.S. Provisional Patent Application No. 62 / 084,908, filed on November 26, 2014[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Often referred to as cs, it exhibits remarkable diversity in the antibody variable region (Fv), allowing for the detection of any molecule. Since it becomes possible to create an Fv that effectively recognizes it, it is possible to create a double singularity. A typical approach is to introduce a novel variable region into an antibody.

[0003] Several alternative antibody formulations targeting bispecificity are being explored (Chames& Baty,2009,mAbs 1[6]:1-9;Holliger&Hudson, 2005,Nature Biotechnology 23[9]:1126-113 6; Kontermann, mAbs 4(2):182(2012), all of them (as expressly incorporated herein by reference). Initially, bispecific antibodies are single It was created by fusing two cell lines that produce monoclonal antibodies (Mi Stein et al., 1983, Nature 305:537-540). profit The hybrid hybridoma or quadroma reliably produces bispecific antibodies. However, these represent only a small population, and large-scale purification is required to isolate the desired antibodies. The engineering solution to this was the use of antibody fragments to create bispecific compounds. Therefore, since such fragments lack the complex quaternary structure of the full-length antibody, the variable light chain and Variable heavy chains can be ligated within a single genetic construct. (Bispecific antibodies, single-stranded bispecific antibodies) Antibody cleavage in many different forms, including antibodies, tandem scFv type, and Fab2 bispecific. Fractions have been prepared (Chames & Baty, 2009, mAbs 1[6]:1-9 ;Holliger&Hudson,2005,Nature Biotechnolo gy 23[9]:1126–1136; expressly incorporated herein by reference) These forms can be expressed at high levels in bacteria, and their small size makes them preferable. Although they may have the advantage of superior permeability, they are rapidly eliminated in vivo, and their production and This may present manufacturing obstacles related to stability. The main reason for these drawbacks is that antibody fragments are typically To maintain a long half-life in serum (i.e., neonatal Fc receptor FcRn), or to purify Larger sizes that serve as binding sites for (i.e., protein A and protein G) Its size, high stability, and ability to bind to various Fc receptors and ligands, The antibody lacks a constant region possessing the relevant functional properties.

[0004] More recent studies have shown that by modifying the double bond into a full-length antibody-like form, fragment-based Attempts have been made to address the drawbacks of the bisingularity (Wu et al., 2007, Na ture Biotechnology 25

[11] :1290-1297; U.S. Patent Application No. 12 / 477,711; Michaelson et al., 2009 mAbs 1[2]:128-141; PCT / U.S. Patent Application Publication No. 2008 / 074 Specification No. 693; Zuo et al., 2000, Protein Engineer ing 13[5]:361-367; U.S. Patent Application No. 09 / 865,198; Shen et al.,2006,J Biol Chem 281

[16] :107 06-10714;Lu et al.,2005,J Biol Chem 280

[0020] :19665-19672;PCT / United States Patent Application Publication No. US2005 / 0254 Specification No. 72 (expressly incorporated herein by reference). These forms are primarily F Because it has a c region, it overcomes some of the problems of the bispecificity of antibody fragments. These forms One notable drawback is the construction of a new antigen-binding site at the top of the homodimer constant chain. Therefore, the binding to a new antigen is always divalent.

[0005] For a number of antigens that are of interest as simultaneous targets in a bispecific form for therapeutic purposes, the desired The binding is monovalent, not divalent. In many immune receptors, cell activation occurs through monovalent binding interactions. This is achieved by crosslinking. The mechanism of crosslinking is typically by antibody / antigen immune complexes. Alternatively, it may be mediated through effector cells for the association of target cells. For example, FcγR Low affinity Fc gamma receptors (F) such as IIa, FcγRIIb, and FcγRIIIa cγR) monovalently binds to the antibody Fc region. Monovalent binding suppresses the expression of these FcγRs. Although it does not activate cells, the receptor is involved in immune complex formation or cell-to-cell contact. They cross-link and cluster on the cell surface, triggering activation. They mediate cell killing. In the case of receptors involved, such as FcγRIIIa on natural killer (NK) cells... Receptor crosslinking and cell activation are processes that allow effector cells to meet target cells in a highly active manner. This occurs when combining (Bowles & Weiner, 2005, J Immunol Methods 304:88-99 (explicitly invoked by reference). Similarly, B details The inhibitory receptor FcγRIIb on the cell surface forms an immune complex with the cell surface B cell receptor (BCR). It downregulates B cell activation only when it associates with soluble IgG and BCR. Therefore, it is a mechanism mediated by the formation of immune complexes with the same recognized antigen (Heym in 2003, Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature Reviews Immu nology 10:328-343; (incorporated by reference in its entirety). As another example , CD3 activation of T cells occurs only when the associated T cell receptor (TCR) associates with MHC loaded with antigen on antigen-presenting cells in highly active cell synapses (Kuhns et al., 2006, Immunity 24:133-139). Of course, non-specific bivalent cross-linking of CD3 using anti-CD3 antibodies induces cytokine storms and toxicity (Perruche et al., 2009, J Immunol 183[2]:953-61; Chatenoud & Bluestone, 2 007, Nature Reviews Immunology 7:622-632; incorporated by reference in its entirety). Therefore, in practical clinical applications, the preferred mode of co-association of CD3 for killing re-directed target cells is a monovalent bond that results in activation only when associating with the co-associated target (Kuhns et al., 2006, Immunity 24:133-139). 007, Nature Reviews Immunology 7:622-632; incorporated by reference in its entirety). Therefore, in practical clinical applications, the preferred mode of co-association of CD3 for killing re-directed target cells is a monovalent bond that results in activation only when associating with the co-associated target cells. CD38, also known as cyclic ADP-ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The types of functional effects among blood cells are attributed to CD38-mediated signaling[[ID=*]] [[ID=*]]

[0006] [[ID=*]] including lymphocyte proliferation, cytokine release, regulation of the development and survival of B and myeloid cells, and induction of dendritic cell maturation. CD38 is expressed in non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and myeloid cells, and dendritic cells, and its expression is often associated with disease progression (incorporated by reference in its entirety). CD38 is expressed in non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T-acute lymphoblastic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AM) L), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia. Control in numerous hematopoietic malignancies, including hematopoietic malignancy (CML), and in various hematopoietic malignancy-derived cell lines. It cannot be controlled. On the other hand, most undifferentiated pluripotent stem cells in the hematopoietic system are CD38 negative. Despite recent advances in the discovery and development of anti-inflammatory drugs, numerous tumors, including CD38-expressing tumors, remain. This form of cancer still has a poor prognosis. Therefore, treating such forms of cancer There is a demand for improved methods for this purpose.

[0007] B cell antigen CD19 (also known as CD19, B cell surface antigen B4, and Leu-12) Human pan- It is a B cell surface marker. CD19 promotes the proliferation and survival of mature B cells. It associates with CD21 in a complex on the cell surface. It also associates with CD81 and Leu- It associates with 13 and enhances B cell receptor (BCR) signaling. Together with BCR, CD 19 is an intrinsic antigen receptor induction important for B cell clonal proliferation and humoral immunity. It modulates signaling thresholds. In cooperation with CD21, it is involved in the adaptive and innate immune systems. Connect them. When activated, the cytoplasmic end of CD19 becomes phosphorylated, and Src-fa This results in binding by Millie kinase and recruitment of PI-3 kinase. Since it is expressed in NHL cells and some leukemias, it is used against lymphoid cancers. It is an interesting target for immunotherapy.

[0008] Several antibodies or antibody conjugates that target CD19 are being tested in preclinical trials for the treatment of cancer. These have been evaluated in trials or clinical trials. These anti-CD19 antibodies or antibody conjugates However, it is not limited to MT-103 (single-stranded bispecific CD19 / CD3 antibody; H offman et al,2005 Int J Cancer 115:98-10 4;Schlereth et al,2006 Cancer Immunol Im munother 55:503-514), CD19 / CD16 bispecific antibody (Sc hlenzka et al,2004 Anti-cancer Drugs 15: 915-919;Kipriyanov et al,2002 J Immunol 169:137-144), BU12-saporin (Flavell et al, 199 5 Br J Cancer 72:1373-1379), and anti-CD19-idal Bicine (Rowland et al, 1993 Cancer Immunol Im (Munother 55:503-514) (All references are explicitly invoked.) These are some examples.

[0009] CD123 (also known as interleukin-3 receptor α (IL-3Rα)) is dendritic It is expressed in cells, monocytes, eosinophils, and basophils. CD123 is also involved in hematopoietic stem cells. Cell / progenitor cells determine the majority of myeloid lineages (CD13+, CD14+, CD33+, CD1 It is constitutively expressed by (5low) and by some CD19+ cells. It is absent from the cells. [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, bispecific products derived from antibody fragments act as biophysical and pharmacokinetic barriers. While affected by this, a drawback of those constructed in full-length antibody-like form is that they are the primary target antigens. In the absence of [unclear], it multivalently associates with simultaneous target antigens, resulting in nonspecific activation and potentially toxicity. This invention brings about a novel bispecific antibody that is specific to CD3 and CD38. This will solve the problem by implementing it. [Means for solving the problem]

[0011] Therefore, in one aspect, the present invention is a) a first monomer and i) a first heavy chain 1) First variable heavy chain domain; 2) First steady heavy chain containing the first Fc domain; 3 ) Includes scFv variable light chain domain, scFv linker and scFv variable heavy chain domain , including scFv covalently bonded to the C-terminus of the Fc domain using a domain linker. a) a first monomer containing the first heavy chain and a) a second variable heavy chain domain and a second Fc domain A second monomer containing a second heavy chain, including a second steady heavy chain containing a second heavy chain; c) variable light chain domer The present invention provides a heterodimer antibody comprising a common light chain containing a yin and a constant light chain domain.

[0012] In a further embodiment, the present invention relates to a) a first monomer, i) a first heavy chain, 1) First variable heavy chain domain; 2) First constant heavy chain domain containing the first Fc domain; and 3) Covalently bonded to the C-terminus of the first Fc domain using a domain linker a) a first monomer containing a first heavy chain containing a first variable light chain domain; b) a second monomer i) a second variable heavy chain domain; ii) a second steady heavy chain domain containing a second Fc domain main; and iii) the second variable heavy chain domain using the domain linker The second Fc domain contains a third variable heavy chain domain covalently bonded to its C-terminus. A monomer and a common light chain including a variable light chain domain and a constant light chain domain, We provide a ronidimer antibody.

[0013] In a further embodiment, the present invention relates to a) a first monomer, i) a first heavy chain, 1) First variable heavy chain domain; 2) First CH1 domain and first Fc domain 1) First steady heavy chain; 3) scFv variable light chain domain, scFv linker and scFv variable It includes a dyscalar chain domain, and a domain linker is used to connect the C-terminus of the CH1 domain and the The first heavy chain includes scFv covalently bonded to the N-terminus of the Fc domain of 1. a) monomer and a second constant domain including a second variable heavy chain domain and a second Fc domain A second monomer containing a second heavy chain, and a variable light chain domain and a constant light chain domain. This invention provides a heterodimer antibody containing a common light chain including the main chain.

[0014] In a further embodiment, the present invention relates to a) a first monomer, i) a first heavy chain, 1) First variable heavy chain domain; 2) First constant heavy chain domain containing the first Fc domain; and 3) The second variable light chain domain uses a domain linker to connect the first steady heavy chain A covalent bond is formed between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain. a) a first monomer containing a first heavy chain containing a first variable light chain domain; b) second A monomer comprising i) a second variable heavy chain domain; ii) a second Fc domain The constant heavy chain domain; and iii) the second variable heavy chain domain is a domain linker A third variable heavy chain domain is covalently bonded to the C-terminus of the second Fc domain. a second monomer containing; c) a common light chain containing a variable light chain domain and a constant light chain domain This invention provides a heterodimer antibody containing [a specific compound / component].

[0015] In a further embodiment, the present invention relates to a) a first monomer, i) a first heavy chain, 1) First variable heavy chain domain; 2) First CH1 domain and first Fc domain 1) First steady heavy chain; 3) scFv variable light chain domain, scFv linker and scFv variable It includes a dyscalar chain domain, and a domain linker is used to connect the C-terminus of the CH1 domain and the The first heavy chain includes scFv covalently bonded to the N-terminus of the Fc domain of 1. a) a monomer and a) a second monomer containing a second Fc domain and c) a variable light chain domain The present invention provides a heterodimer antibody comprising a light chain containing a constant light chain domain.

[0016] In some embodiments, the first and second Fc domains are S364K / E357Q:L36 8D / K370S;L368D / K370S:S364K;L368E / K370S:S 364K;T411T / E360E / Q362E:D401K;L368D / K370S Select from the group consisting of :S364K / E357L and K370S:S364K / E357Q It has a set of selectable amino acid substitutions. Furthermore, it has a variable heavy chain domain and a variable light chain domain. In binds to the first target tumor antigen (TTA), and scFv binds to the second TTA or human CD. It is coupled to 3. In some embodiments, TTA is CD19, CD20 and CD123 Selected from the following group.

[0017] In further embodiments, the present invention relates to H1.32_L1.47, H1.89_L1.47, H Regarding 1.90_L1.47, H1.33_L.1.47, and H1.31_L1.47 The CDR and / or variable domain and / or scFv sequence shown in the drawing The present invention provides an anti-CD3 antigen-binding domain. The present invention also provides nucleic acid compositions and expression vector compositions. Provides materials and host cells.

[0018] In a further embodiment, the present invention relates to a) a first monomer, i) a first Fc domain; ii) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain Including anti-CD3 covalently bound to the N-terminus of the Fc domain using a domain linker. a) a first monomer containing scFv; b) a second monomer, i) a heavily variable domain; and (ii) a second monomer containing a heavy chain containing a heavy chain constant domain containing a second Fc domain; c) A light chain comprising a variable light chain domain and a variable light constant domain, anti-CD3 scF v is anti-CD H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47 and anti-CD3 H1.33_L1.47 (sequence number XX) We provide heterodimer antibodies selected from the following groups: heavily variable domain and lightly variable domain. The main focus is TTA (not limited to CD19, CD20, CD38, and CD123). It binds to (including).

[0019] In a further embodiment, the present invention comprises a) sequence RASWSVSYIH (sequence number XX) vlCDR1, vlCDR2 having sequence ATSNLAS (sequence number XX), and A variable light chain domain containing vlCDR3 having the sequence QQWTHNPPT (sequence number XX) and b) vhCDR1 having sequence SYNMH (sequence number XX), sequence AIYPGNGAT vhCDR2 having SYSQKFQG (sequence number XX), and sequence SYYMGGDW Anti-CD2 containing a variable heavy chain domain including vhCDR3 having YFDV (SEQ ID NO: XX) Provides 20 antibody-binding domains. In some embodiments, the anti-CD20 antibody-binding domain is It has the sequence C2B8 H1.202_L1.113.

[0020] In a further embodiment, the present invention comprises a) sequence RASSSVSYIH (sequence number XX) vlCDR1, vlCDR2 having sequence ATSNLAS (sequence number XX), and A variable light chain domain containing vlCDR3 having the sequence QQWTSNPPT (sequence number XX) and b) vhCDR1 having sequence SYNMH (sequence number XX), sequence AIYPGNGDT vhCDR2 having SYNQKFQG (sequence number XX), and sequence STYYGGDW Anti-CD2 containing a variable heavy chain domain including vhCDR3 having YFNV (SEQ ID NO: XX) It provides 20 antibody-binding domains.

[0021] In some embodiments, the anti-CD20 antibody-binding domain has the C2B8_H1L1 sequence. .

[0022] In a further embodiment, the present invention relates to a) a first monomer, i) a first Fc domain; ii) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain Including anti-CD3 covalently bound to the N-terminus of the Fc domain using a domain linker. a) a first monomer containing scFv; b) a second monomer, i) a heavily variable domain; and (ii) a second monomer containing a heavy chain containing a heavy chain constant domain containing a second Fc domain; c) A light chain comprising a variable light chain domain and a variable light constant domain, and a variable heavy chain and a light The chain contains the C2B8 H1.202_L1.113 or C2B8_H1L1 binding domain. This invention provides heterodimer antibodies that form a heterodimer antibody.

[0023] In a further embodiment, the present invention relates to a) a first monomer, i) a first Fc domain; ii) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain Including anti-CD3 covalently bound to the N-terminus of the Fc domain using a domain linker. a) a first monomer containing scFv; b) a second monomer, i) a heavily variable domain; and (ii) a second monomer containing a heavy chain containing a heavy chain constant domain containing a second Fc domain; c) A heterodimer antibody comprising a light chain containing a variable light chain domain and a variable light constant domain. This provides a variable domain, which is bound to CD123 and 7G3_H1.1 It may have the sequence 09_L1.47.

[0024] In further embodiments, the present invention relates to XENP15049, XENP15051; XENP1 5050, XENP13676, XENP14696, XENP15629, XENP1 5053, XENP15630, XENP15631, XENP15632, XENP1 5633, XENP15634, XENP15635, XENP15636, XENP1 5638, XENP15639, XENP13677, XENP14388, XENP1 4389, XENP14390, XENP14391, XENP14392, XENP1 4393, XENP16366, XENP16367, XENP16368, XENP1 6369, XENP16370, XENP16371, XENP16372, XENP1 It consists of 6373, XENP16375, XENP16376, and XENP16377. Provides heterodimer antibodies selected from a group. Nucleic acids, expression vectors, and host cells are In addition to methods for producing these proteins and treating patients who have them, It will be provided. [Brief explanation of the drawing]

[0025] [Figure 1A] This describes several forms of the present invention. Two forms of the "bottle opener" form are shown, namely having an anti-CD3 antigen-binding domain containing scFv and an anti-TTA antigen-binding domain containing Fab, and having these reversed. All mAb-Fv, mAb-scFv, Central-scFv, and Central-Fv forms are shown. Furthermore, both single-arm Central-scFv and single-arm Central-Fv forms are shown, where one monomer simply contains an Fc domain. A double scFv form is also shown. [Figure 1B] This describes several forms of the present invention. Two forms of the "bottle opener" form are shown, namely having an anti-CD3 antigen-binding domain containing scFv and an anti-TTA antigen-binding domain containing Fab, and having these reversed. All mAb-Fv, mAb-scFv, Central-scFv, and Central-Fv forms are shown. Furthermore, both single-arm Central-scFv and single-arm Central-Fv forms are shown, where one monomer simply contains an Fc domain. A double scFv form is also shown. [Figure 1C] Same as above [Figure 2]This represents an array of “High CD3” anti-CD3_H1.30_L1.47 constructs, including variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and scFv constructs having charged linkers (double underlined). These charged linkers may be replaced with uncharged linkers or different charged linkers as necessary, to apply to all arrays shown in the drawings. [Figure 3] This represents an array of “High-Int#1” anti-CD3_H1.32_L1.47 constructs, including variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and scFv constructs having charged linkers (double underlined). These charged linkers may be replaced with uncharged linkers or different charged linkers as necessary, to apply to all arrays shown in the drawing. [Figure 4] This represents an array of “High-Int#2” anti-CD3_H1.89_L1.47 constructs, including variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and scFv constructs having charged linkers (double underlined). These charged linkers may be replaced with uncharged linkers or different charged linkers as necessary, to apply to all arrays shown in the drawing. [Figure 5] This represents an array of “High-Int#3” anti-CD3_H1.90_L1.47 constructs, including variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and scFv constructs having charged linkers (double underlined). These charged linkers may be replaced with uncharged linkers or different charged linkers as necessary, to apply to all arrays shown in the drawing. [Figure 6] This represents an array of “Int” anti-CD3_H1.90_L1.47 constructs, including variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and scFv constructs having charged linkers (double underlined). These charged linkers may be replaced with uncharged linkers or different charged linkers as necessary, to apply to all arrays shown in the drawings. [Figure 7] This represents an array of “Low” anti-CD3_H1.31_L1.47 constructs, including variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and scFv constructs having charged linkers (double underlined). These charged linkers may be replaced with uncharged linkers or different charged linkers as necessary, to apply to all arrays shown in the drawings. [Figure 8] This represents the sequence of the High CD38:OKT10_H1.77_L1.24 construct, which includes variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and an scFv construct having a charged linker (double underlined). [Figure 9] This represents the sequence of an intermediate CD38:OKT10_H1L1.24 construct, which includes variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and an scFv construct having a charged linker (double underlined). [Figure 10] This represents the sequence of Low CD38:OKT10_H1L1 constructs, including variable weight and variable light chain domains (underlined in CDR), as well as separate vlCDR and vhCDR, and scFv constructs having charged linkers (double underlined). [Figure 11] This represents an array of XENP15331. [Figure 12] This represents an array of XENP13243. [Figure 13] This represents the array of XENP14702. [Figure 14] This represents the array XENP15426. [Figure 15] This represents an array of XENP14701. [Figure 16] This represents the array of XENP14703. [Figure 17] This represents an array of XENP13243. [Figure 18] This represents the array of XENP18967. [Figure 19] This represents an array of XENP18971. [Figure 20] This represents an array of XENP18969. [Figure 21] This represents the array of XENP18970. [Figure 22] This represents an array of XENP18972. [Figure 23] This represents an array of XENP18973. [Figure 24] This represents an array of XENP15055. [Figure 25] This represents an array of XENP13544. [Figure 26] This represents an array of XENP13694. [Figure 27] This represents the sequence of human CD3ε. [Figure 28] This shows the full-length (SEQ ID NO: 130) and extracellular domain (ECD; SEQ ID NO: 131) of the human CD38 protein. [Figure 29A] This represents useful pairs of heterodimerized mutant sets (including skew and pI mutants). [Figure 29B] This represents useful pairs of heterodimerized mutant sets (including distortion and pI mutants). [Figure 29C] This represents useful pairs of heterodimerized mutant sets (including distortion and pI mutants). [Figure 29D] This represents useful pairs of heterodimerized mutant sets (including distortion and pI mutants). [Figure 29E] This represents useful pairs of heterodimerized mutant sets (including distortion and pI mutants). [Figure 30] This represents a list of the constant regions of the allomorphic stereomutant antibodies and their respective substitutions. pI_(-) indicates a lower pI variant, while pI_(+) indicates a higher pI variant. These can be conjugated arbitrarily and independently with other heterodimerized variants of the present invention (and similarly other variant types as outlined herein). [Figure 31] This represents a useful cleavage mutant that cleaves the FcγR bond (sometimes referred to as a "knockout" or "KO" mutant). [Figure 32]Two particularly useful embodiments of the present invention are shown. [Figure 33A] This describes several charged scFv linkers that have been found to increase or decrease the pI of heterodimer antibodies that utilize one or more scFv components. A single prior art scFv linker with a single charge is referred to as "Whitlow" from Whitlow et al., Protein Engineering 6(8):989-995 (1993). It is noteworthy that this linker has been used to reduce aggregation in scFv and enhance proteolytic stability. [Figure 33B] Same as above [Figure 34] This lists modified heterodimer-distorted Fc variants along with their heterodimer yield (measured by HPLC-CIEX) and thermal stability (measured by DSC). Thermal stability that was not measured is indicated with "nd". [Figure 35] This is the expression yield of the bispecific product after purification for protein A affinity. [Figure 36] This is a cation exchange purification chromatogram. [Figure 37] This is a re-induced T-cell cytotoxicity assay (24-hour incubation, 10k RPMI8226 cells, 400k T cells). The test substance is anti-CD38 × anti-CD3 bispecific. Detection was performed using LDH. [Figure 38] This is a re-induced T-cell cytotoxicity assay (24-hour incubation, 10k RPMI8226 cells, 500k human PBMCs). The test substance is an anti-CD38 × anti-CD3 bispecific. Detection was performed using LDH. [Figure 39] This represents an array of XENP14419. [Figure 40] This represents an array of XENP14420. [Figure 41] This represents an array of XENP14421. [Figure 42] This represents an array of XENP14422. [Figure 43] This represents an array of XENP14423. [Figure 44] This was a re-induced T-cell cytotoxicity assay (96-hour incubation, 40k RPMI8226 cells, 400k human PBMCs). The test substance was anti-CD38 × anti-CD3 Fab-scFv-Fc. Detection was performed by flow cytometry, specifically by the disappearance of CD38+ cells. [Figure 45] Figure 1 shows a further analysis of the re-induced T cell cytotoxicity assay. The first row shows the mean fluorescence intensity (MFI) of the activation marker CD69 against CD4+ and CD8+ T cells, as detected by flow cytometry. The second row shows the percentage of CD4+ and CD8+ T cells that are Ki-67+, as a measure of cell proliferation. The third row shows the mean intracellular fluorescence intensity (MFI) of the granzyme B inhibitor PI-9 against CD4+ and CD8+ T cells, as detected by flow cytometry. [Figure 46] This is a mouse study design to investigate the antitumor activity of the anti-CD38 × anti-CD3 Fab-scFv-Fc bispecific compound. [Figure 47] Tumor size as measured by IVIS® as a function of time and processing. [Figure 48] This is an IVIS (registered trademark) bioluminescence image (day 10). [Figure 49] This refers to the depletion of CD38+ cells after a single administration of the test substance in cynomolgus monkeys. [Figure 50] This is T cell activation measured by the mean fluorescence intensity (MFI) of CD69 in cynomolgus monkeys (color-coded as shown in Figure 49). [Figure 51] The displayed serum level of IL-6 is the level after a single dose of the test substance. [Figure 52] This represents an array of XENP15427. [Figure 53] This represents an array of XENP15428. [Figure 54] This represents an array of XENP15429. [Figure 55] This represents the array of XENP15430. [Figure 56]This represents an array of XENP15431. [Figure 57] This represents an array of XENP15432. [Figure 58] This represents an array of XENP15433. [Figure 59] This represents an array of XENP15434. [Figure 60] This represents an array of XENP15435. [Figure 61] This represents an array of XENP15436. [Figure 62] This represents an array of XENP15437. [Figure 63] This represents an array of XENP15438. [Figure 64] This shows the binding affinity in the viacore assay. [Figure 65] This shows the purity of heterodimers during the preparation of a stable pool using fluctuating light chain, Fab-Fc, and scFv-Fc ratios. [Figure 66] This involves the depletion of human IgM and IgG2 by anti-CD38 × anti-CD3 bispecific mutants in the huPBMC mouse model. [Figure 67A] This represents a humanized anti-CD3 mutant scFv with optimized stability. Substitutions are given to the H1_L1.4 scFv sequence. Amino acid numbering is Kabat numbering. [Figure 67B] Same as above [Figure 68A] This is the amino acid sequence of the humanized anti-CD3 mutant scFv with optimized stability. CDR is underlined. For each heavy / light chain combination, four sequences are listed: (i) scFv with a C-terminal 6xHis tag, (ii) scFv alone, (iii) VH alone, and (iv) VL alone. [Figure 68B] Same as above [Figure 68C] Same as above [Figure 68D] Same as above [Figure 68E] Same as above [Figure 68F] Same as above [Figure 68G] Same as above [Figure 68H] Same as above [Figure 68I] Same as above [Figure 68J] Same as above [Figure 68K] Same as above [Figure 68L] Same as above [Figure 68M] Same as above [Figure 68N] Same as above [Figure 68O] Same as above [Figure 68P] Same as above [Figure 68Q] Same as above [Figure 68R] Same as above [Figure 68S] Same as above [Figure 68T] Same as above [Figure 68U] Same as above [Figure 68V] Same as above [Figure 68W] Same as above [Figure 68X] Same as above [Figure 68Y] Same as above [Figure 68Z] Same as above [Figure 69] This was a re-induced T-cell cytotoxicity assay (24-hour incubation, 10k RPMI8226 cells, 500k PBMCs). The test product was anti-CD38 (OKT10_H1L1, OKT10_H1.77_L1.24) × anti-CD3 Fab-scFv-Fc. Detection was performed by LDH. [Figure 70] This was a huPBL-SCID Ig depletion study. The test substance was administered at 0.03, 0.3, or 3 mg / kg on day 8 post-transplantation of PBMCs. The route of administration was intraperitoneal. Blood samples were collected on day 14 post-transplantation of PBMCs, processed into serum, and assayed for human IgM and IgG2. [Figure 71] This represents an array of XENP15049. [Figure 72] This represents an array of XENP15051. [Figure 73] This represents the array of XENP15050. [Figure 74] This represents an array of XENP13676. [Figure 75] This represents an array of XENP14696. [Figure 76] This represents an array of XENP15629. [Figure 77] This represents an array of XENP15053. [Figure 78] This represents the array of XENP15630. [Figure 79] This represents an array of XENP15631. [Figure 80] This represents an array of XENP15632. [Figure 81] This represents an array of XENP15633. [Figure 82] This represents an array of XENP15634. [Figure 83] This represents an array of XENP15635. [Figure 84] This represents an array of XENP15636. [Figure 85] This represents an array of XENP15638. [Figure 86] This represents an array of XENP15639. [Figure 87] This represents an array of XENP13677. [Figure 88] This represents an array of XENP14388. [Figure 89] This represents an array of XENP14389. [Figure 90] This represents an array of XENP14390. [Figure 91] This represents an array of XENP14391. [Figure 92] This represents an array of XENP14392. [Figure 93] This represents an array of XENP14393. [Figure 94] This represents an array of XENP16366. [Figure 95] This represents an array of XENP16367. [Figure 96] This represents an array of XENP16368. [Figure 97] This represents an array of XENP16369. [Figure 98]This represents an array of XENP16370. [Figure 99] This represents an array of XENP16371. [Figure 100] This represents an array of XENP16372. [Figure 101] This represents an array of XENP16373. [Figure 102] This represents an array of XENP16374. [Figure 103] This represents an array of XENP16375. [Figure 104] This represents an array of XENP16376. [Figure 105] This represents an array of XENP16377. [Figure 106] This represents the sequences of the CD20 and CD123 antigens. [Figure 107] This is a surface plasmon resonance (SRP) measurement of CD3 affinity. The test material was anti-CD20(C2B8_H1.202_L1.113)×anti-CD3 Fab-scFv-Fc. Human CD3δε-Fc (Sino Biological) was covalently bonded to the chip surface. The test material was passed through at 3.125, 12.5, 50, and 200 nM. [Figure 108] This is a surface plasmon resonance (SRP) measurement of CD3 affinity. The test material was anti-CD20(C2B8_H1.202_L1.113)×anti-CD3 Fab-scFv-Fc. Cynomolgus monkey CD3δε-Fc (Sino Biological) was covalently bonded to the chip surface. The test material was passed through at 3.125, 12.5, 50, and 200 nM. [Figure 109] This is a surface plasmon resonance (SPL) measurement of CD3 affinity. The test material was anti-CD20(C2B8_H1.202_L1.113)×anti-CD3 Fab-scFv-Fc. Human CD3δε-Fc (Sino Biological) was covalently bonded to the chip surface. The test material was passed through at 31.25, 125, 500, and 2000 nM. [Figure 110]This is a surface plasmon resonance (SPR) measurement of CD3 affinity. The test material was anti-CD20 (C2B8_H1.202_L1.113) × anti-CD3 Fab-scFv-Fc. Cynomolgus monkey CD3δε-Fc (Sino Biological) was covalently bonded to the chip surface. The test material was passed through at 31.25, 125, 500, and 2000 nM. [Figure 111] This is a surface plasmon resonance (SPR) measurement of CD3 affinity. The test material was anti-CD20 (C2B8_H1.202_L1.113) × anti-CD3 Fab-scFv-Fc. Cynomolgus monkey CD3δε-Fc (Sino Biological) was covalently bonded to the chip surface. The test material was passed through at 31.25, 125, 500, and 2000 nM. [Figure 112] This was a re-induced T-cell cytotoxicity assay (24-hour incubation, 10k Ramos cells, 250k PBMCs). The test product was anti-CD20(C2B8_H1.202_L1.113)×anti-CD3 Fab-scFv-Fc. Detection was performed by LDH. [Figure 113] This was a re-induced T-cell cytotoxicity assay (24-hour incubation, 20k Jeko cells, 200k PBMCs (CD19 depleted)). The test product was anti-CD20 (C2B8_H1.202_L1.113) × anti-CD3 Fab-scFv-Fc. Detection was performed by flow cytometry, specifically by the disappearance of CD19+ cells. [Figure 114] Figure 113 shows IL-6 production after 24 hours in the experiment described. [Figure 115A] This was a re-induced T-cell cytotoxicity assay (5-hour incubation, 20k Jeko cells, 500k PBMCs (CD19 depleted)). The test product was anti-CD20(C2B8_H1L1) × anti-CD3 Fab-scFv-Fc. Detection was performed by flow cytometry, specifically by the disappearance of CD19+ cells. [Figure 115B] Same as above [Figure 116A]This was a re-induced T-cell cytotoxicity assay (24-hour incubation, 20k Jeko cells, 500k PBMCs (CD19 depleted)). The test product was anti-CD20 (C2B8_H1.202_L1.113) × anti-CD3 Fab-scFv-Fc. Detection was performed by flow cytometry, specifically by the disappearance of CD19+ cells. [Figure 116B] Same as above [Figure 117] Figure 113 shows IL-6 production after 24 hours in the experiment described. [Figure 118] This was a re-induced T-cell cytotoxicity assay (24-hour incubation, 10k RPMI8226 cells, 500k PBMCs). The test product was anti-CD38 (OKT10_H1L1, OKT10_H1.77_L1.24) × anti-CD3 Fab-scFv-Fc. Detection was performed by LDH. [Figure 119] This was a huPBL-SCID Ig depletion study. The test substance was administered at a dose of 5 mg / kg on days 1 and 8 post-transplantation of PBMCs. The administration route was intraperitoneal. Blood samples were collected on day 14 post-transplantation of PBMCs, processed into serum, and assayed for human IgM and IgG2. [Figure 120] This was a huPBL-SCID Ig depletion study. The test substance was administered at 0.03, 0.3, or 3 mg / kg on day 8 post-transplantation of PBMCs. The route of administration was intraperitoneal. Blood samples were collected on day 14 post-transplantation of PBMCs, processed into serum, and assayed for human IgM and IgG2. [Figure 121] This represents the array High CD20 C2B8_H1.202_L1.113. [Figure 122] This represents the array Low CD20 C2B8_H1L1. [Figure 123] This represents the array CD123 7G3_H1.109_L1.57. [Figure 124]A matrix of possible combinations in the present invention is shown. "A" means that the CDR of the referenced CD3 sequence can be combined with the CDR of the right-hand TTA. That is, the vhCDR from the variable heavy chain CD3 H1.30 sequence and the vlCDR from the variable light chain of the CD3 L1.57 sequence can be combined with the vhCDR from the CD38 OKT10 H1.77 sequence and the vlCDR from the OKT10L1.24 sequence. "B" means that the CDR from the CD3 construct can be combined with the variable heavy and variable light chain domains from the TTA. That is, the vhCDR from the variable heavy chain CD3 H1.30 sequence and the vlCDR from the variable light chain of the CD3 L1.57 sequence can be combined with the variable heavy chain domains CD38 OKT10 H1.77 sequence and OKT10L1.24 sequence. By reversing "C", the variable heavy chain domain and variable light chain domain from the CD3 sequence are used together with the CDR of the TTA. "D" is the case where both the variable heavy chain and variable light chain from each are combined. "E" is the case where the scFv of CD3 is used together with the CDR of TTA, and "F" is the case where the scFv of CD3 is used together with the variable heavy and variable light chain domains of the TTA antigen-binding domain. [Modes for carrying out the invention]

[0026] I. Definition To help this application be better understood, several definitions are given below. These definitions are It is intended to include grammatical equivalents.

[0027] In this specification, "cleavage" means reduction or removal of activity. Therefore, for example, "F "Cleavages the cγR bond" refers to the Fc region where amino acid variants do not have specific variants. This means that it has a 50% lower initiation bond compared to the region, but 70~80~90~95~9 A decrease in activity of less than 8% is preferable, and generally, the activity is undetectable in the viacore assay. The binding level is below the effective level. Specific applications in FcγR bond cleavage are shown in Figure 16. That is the case.

[0028] When used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to FcγR Nonspecific cytotoxic cells expressing recognize the binding antibody on target cells, and subsequently the target cells This refers to a cell-mediated reaction that causes lysis. ADCC is involved in the binding of FcγRIIIa and its phase In relation to this, increased binding to FcγRIIIa leads to increased ADCC activity.

[0029] When "ADCP" or antibody-dependent cell phagocytosis is used herein, FcγR Nonspecific cytotoxic cells that express the antibody recognize the bound antibody on the target cell, followed by phagocytosis of the target cell. This refers to a cell-mediated reaction that triggers an action.

[0030] In this specification, “modification” refers to amino acid substitutions, insertions, and / or other modifications within a polypeptide sequence. This refers to a deletion or modification of a portion chemically linked to a protein. For example, modification The ornament may be a PEG structure attached to a modified carbohydrate or protein. In this specification, "amino acid modification" refers to amino acid substitutions, insertions, and / or other amino acid modifications within a polypeptide sequence. "T" means deletion. For clarity, unless otherwise specified, amino acid modifications are always DN. A is an amino acid encoded by A, for example, 20 amino acids having codons in DNA and RNA. The focus is on amino acids.

[0031] In this specification, “amino acid substitution” or “substitution” refers to a specific position within the parent polypeptide sequence. This means replacing an amino acid with a different amino acid. In particular, in some embodiments Substitution is a natural occurrence in a particular location, not present in that organism or any organism. This targets amino acids that do not naturally exist. For example, substitution E272Y is the glutamate at position 272. This refers to a mutant polypeptide in which nic acid is replaced by tyrosine, in this case the Fc mutant. To clarify, changing the nucleic acid coding sequence does not change the starting amino acid (for example, To increase the expression level of the host organism, CGG (which codes for arginine) is converted to CGA (or Proteins designed to replace (the one that codes for arginine) are called "amino This is not "acid substitution," meaning it also involves the creation of a new gene that codes for the same protein. However, if a protein has the same amino acid at the specific position where it starts, then it It is not an amino acid substitution.

[0032] When used herein, "amino acid insertion" or "insertion" refers to the parent polypeptide sequence. This refers to the addition of an amino acid sequence at a specific position within a molecule. For example, -233E or 233E. This refers to the insertion of glutamate after position 233 and before position 234. In addition, -233 ADE or A233ADE is AlaAspGl after 233rd place and before 234th place. This refers to the insertion of 'u'.

[0033] When used herein, "amino acid deletion" or "deletion" refers to the parent polypeptide sequence. This refers to the removal of an amino acid sequence at a specific position within a sequence. For example, E233- or E233 # or E233() refers to the deletion of glutamate at position 233. In addition, EDA2 33- or EDA233# is a deletion of the sequence GluAspAla that starts at position 233. To point.

[0034] "Mutant protein" or "protein variant" or "variant" is defined herein as follows: When used, it is a protein that differs from the parent protein due to at least one amino acid modification. It means quality. Protein variants refer to the protein itself, a composition containing the protein, or This may refer to the amino sequence that codes for it. Preferably, the protein variant is the parent protein Compared to the parent protein, at least one amino acid modification, for example, approximately 1 to approximately 70 It has amino acid modifications, preferably about 1 to about 5 amino acid modifications. As shown below, In some embodiments, the parent polypeptide, for example, the Fc parent polypeptide, For example, the Fc region derived from IgG1, IgG2, IgG3, or IgG4, but variant A human sequence having the "parent polypeptide," for example, the IgG1 / 2 hybrid in Figure 19, is It may also be useful. The protein variant sequences described herein are preferred over the parent protein sequence. At least approximately 80% identity, and most preferably at least approximately 90% identity, Preferably, the mutants will have at least approximately 95-98-99% identity. A protein can refer to the mutant protein itself, a composition containing a protein mutant, or an encoding of it. It can refer to the DNA sequence that does so. Therefore, "antibody variant" or "mutant antibody" is a genuine When used in detail, it refers to an antibody different from the parent antibody due to at least one amino acid modification. In this specification, "IgG variant" or "mutant IgG" is used less than Both are different from parent IgG (again, often derived from human IgG sequences) due to a single amino acid modification. This refers to an antibody, and also to "immunoglobulin variant" or "mutant immunoglobulin". When used herein, at least one amino acid modification is required for immunophilic compound This refers to an immunoglobulin sequence different from that of a column. It is referred to as "Fc variant" or "Variant Fc". As used herein, "Fc" refers to a protein containing amino acid modifications within its Fc domain. The Fc variants of this invention are defined according to the amino acid modifications that constitute them. Therefore, for example, N434S or 434S is located at position 434 relative to the parent Fc polypeptide. These are Fc variants with a substituted serine, where the numbering follows the EU index. M428L / N434S is a substitution of parent Fc polypeptide M428L and N43 This defines an Fc variant having 4S. The identity of the WT amino acids may not be specified. In this case, the above mutant is referred to as 428L / 434S. The order in which substitutions are provided is arbitrary. Yes, for example, 428L / 434S is the same Fc mutant as M428L / N434S. It is observed that, among other things, all positions of the antibody considered in this invention are particularly Unless otherwise specified, amino acid position numbering follows the EU index. Alternatively, EU indices such as Kabat or the EU numbering scheme number EU antibodies. Pointing (Edelman et al., 1969, Proc Natl Acad Sc i USA63:78-85, as referred to herein in whole). The modification is, This may involve addition, deletion, or substitution. Substitutions may involve natural amino acids, and sometimes synthetic amino acids. May contain acids. For example, see U.S. Patent No. 6,586,207; International Publication No. 98 / 4 Patent No. 8032; International Publication No. 03 / 073238; U.S. Patent Application Publication No. 2004-0214 Specification No. 988A1; International Publication No. 05 / 35727A2; International Publication No. 05 / 74524 A2; JWCin et al., (2002), Journal of th e American Chemical Society124:9026-9027 ;JWChin,&P.G.Schultz, (2002), ChemBioChe m 11:1135-1137;JWChin, et al., (2002), P ICAS United States of America 99:11020-1 1024; and L. Wang, & P.G. Schultz, (2002), Chem. Examples include 1-10 (all of which are referenced as a whole).

[0035] As used herein, “protein” means at least two covalent bonds It refers to combined amino acids, and includes proteins, polypeptides, oligopeptides, and peptides. It contains peptidyl groups, which are natural amino acids and peptide bonds, or synthetic peptide mimetic compounds. The structure, or "analog," may include, for example, peptoids (Simon et al. .,PNAS USA 89(20):9367(1992), the whole is referenced. (Used). Amino acids are natural or synthetic (for example, It may be any of the amino acids (that are not encoded by DNA). For example, h Mophenylalanine, citrulline, ornithine, and noreleucine are intended for use in this invention. It is considered a synthetic amino acid, and both D- and L-(R or S) amino acids are designed as such. The method may also be used. The variants of the present invention were developed, for example, by Schultz and colleagues. The technology described, for example, is not limited to Cropp & Shultz, 2004, Tren ds Genet.20(12):625-30,Anderson et al.,2 004,Proc Natl Acad Sci USA101(2):7566-71 , Zhang et al., 2003, 303(5656):371-3, and Ch in et al., 2003, Science 301(5635):964-7(Su Incorporated using the method described by (all of which are referenced as a whole) Modifications may include the use of synthetic amino acids. Furthermore, the polypeptide may have one or more sides Synthetic derivatization of chains or terminals, glycosylation, PEGylation, and cyclic mutations. permutation, cyclization, linker to other molecules, protein or protein This may include fusion to a quality domain and the addition of a peptide tag or label.

[0036] When used herein, "residue" refers to a location in a protein and its associated location. It means the identification of amino acids. For example, asparagine 297 (Asn297 or N29) (Also known as 7) is the residue at position 297 in the human antibody IgG1.

[0037] "Fab" or "Fab area" as used herein refers to VH, CH1, VL , and means polypeptides containing the CL immunoglobulin domain. Fab refers to this domain The region may be referred to by itself, or by a full-length antibody, antibody fragment, or Fab fusion protein. This region may also be referred to in relation to the Fv, or Fv fragment, or Fv region. When used herein, a polypeptide comprising the VL and VH domains of a single antibody is referred to. To taste. As those skilled in the art will understand, these generally consist of two chains.

[0038] When used herein, "IgG subclass modification" or "isotype modification" refers to , one amino acid of one IgG isotype in differently aligned IgG isotypes This refers to amino acid modification that converts to the corresponding amino acid. For example, IgG1 converts to tyrosine Since it also contains IgG2, the F296Y substitution in IgG2 is at EU position 296. Phenylanine is thought to be an IgG subclass modification.

[0039] When used herein, "unnatural modification" refers to amino acid modifications that are not isotypes. It tastes like this. For example, since neither IgG contains serine at position 434, IgG1, I Substitution 434S in gG2, IgG3, or IgG4 (or their hybrids) This is considered an unnatural modification.

[0040] When used herein, "amino acids" and "amino acid identification" refer to DNA and R It refers to one of the 20 naturally occurring amino acids encoded by NA.

[0041] When used herein, the "effector function" refers to the Fc region of an antibody and the Fc receptor. This refers to biochemical events resulting from interactions with ligands. Effector functions are limited While not explicitly defined, it includes ADCC, ADCP, and CDC.

[0042] When used herein, "IgG Fc ligand" refers to an Fc / Fc ligand complex. A molecule derived from any organism that binds to the Fc region of an IgG antibody, preferably a p It means lipeptide. Fc ligand is not limited to FcγRI, FcγRII FcγRIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor It contains Staphylococcus protein A, Streptococcus protein G, and virus FcγR. c ligands are also a family of Fc receptors homologous to FcγR. Contains homolog (FcRH) (Davis et al., 2002, Immunolog Medical Reviews 190:123-136, cited in its entirety. ). Fc ligands may include undiscovered molecules that bind to Fc. Specific IgG Fc ligands Gand is an FcRn and Fcγ receptor. "Fc ligand" as used herein When it is applied, any organism that binds to the Fc region of the antibody forms an Fc / Fc ligand complex. This refers to molecules derived from, preferably polypeptides.

[0043] "Fcγ receptor," "FcγR," or "FcqammaR" are used herein. When this occurs, it means any member of the family of proteins that bind to the Fc region of an IgG antibody. It is encoded by the FcγR gene. In humans, this family is not limited to However, FcγRI(CD64), for example, isoforms FcγRIa, FcγRIb, FcγRIc;FcγRII(CD32), for example, isoform FcγRIIa( (including allotypes H131 and R131), FcγRIIb (FcγRIIb-1 and (including FcγRIIb-2), and FcγRIIc; and FcγRIII (CD) 16) For example, isoform FcγRIIIa (allotypes V158 and F158) (including) and FcγRIIIb (allotype FcγRIIb-NA1 and FcγRII (Contains b-NA2) (Jefferis et al., 2002, Immunol L (ett 82:57-65, as used by reference in whole) and any undiscovered Contains human FcγR or FcγR isoform or allotype. FcγR is While not limited to humans, mice, rats, rabbits, and monkeys, it can originate from any organism. It is also permissible. Mouse FcγR is not limited to FcγRI(CD64), FcγR II (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16) -2) Along with any undiscovered mouse FcγR or FcγR isoform or Includes allotypes.

[0044] When used herein, "FcRn" or "neonatal Fc receptor" refers to an IgG antibody. This refers to proteins that bind to the Fc region, and at least in part, they are controlled by the FcRn gene. FcRn is used in humans, mice, rats, rabbits, and other animals. It may be derived from any organism including the ru. Functional FcR as is known in the art. The n protein contains two polypeptides, often referred to as the heavy chain and the light chain. It is β-2-microglobulin, and its heavy chain is encoded by the FcRn gene. Unless otherwise specified in the details, FcRn or FcRn protein consists of FcRn heavy chain and β- This refers to a complex of 2-microglobulin. It enhances binding to the FcRn receptor, and in some cases... Various FcRn variants used to further extend the serum half-life are shown in the caption for Figure 83. It will be done.

[0045] When used herein, "parent polypeptide" refers to a product that has been modified to create a variant. It refers to the initiation polypeptide. The parent polypeptide is a naturally occurring polypeptide. Alternatively, it may be a variant or modified version of a naturally occurring polypeptide. Chido refers to the polypeptide itself, a composition containing the parent polypeptide, or an amine encoding it. It may also refer to an anoacid sequence. Therefore, "pro-immunoglobulin" as used herein At that time, it means an unmodified immunoglobulin polypeptide that is modified to create a mutant. Furthermore, when used herein, the "parent antibody" is modified to produce a mutant antibody. This refers to an unmodified antibody. "Parent antibody" is a known, commercially available recombinant antibody as outlined below. The inclusion of the body is noteworthy.

[0046] When "Fc" or "Fc region" or "Fc domain" is used herein, A polypeptide containing the constant region of an antibody, excluding the initial constant region immunoglobulin domain, and In some cases, it means part of the hinge. Therefore, Fc is IgA, IgD, and Ig The last two constant-region immunoglobulin domains of G, as well as the last of IgE and IgM. The three constant-region immunoglobulin domains, as well as the flexible N-terminus of these domains. It means hinge. In the case of IgA and IgM, Fc may contain the J chain. In the case of IgG, The Fc domain consists of immunoglobulin domains C-gamma 2 and C-gamma 3 (Cγ2 and Cγ2). γ3), and the lower hinge region between C-gamma 1 (Cγ1) and C-gamma 2 (Cγ2) It includes. The boundary of the Fc region may differ, but the human IgG heavy chain Fc region is usually residue C226. Alternatively, it is defined as including P230 to its carboxyl terminus, and the numbering is assigned to Kabat. It follows the EU index as described. In some embodiments, it is described more thoroughly later. To enable this, amino acid modifications are performed, for example, on one or more FcγR receptors or FcRn receptors. This is done to the Fc region in order to modify the bonding.

[0047] In this specification, "multiple constant region" refers to the CH1-hinge-CH2-CH3 portion of the antibody. ru.

[0048] In this specification, "Fc fusion protein" or "immunoadhesin" refers to the term used herein. As described above, the binding site to the target protein, etc., is generally different to other proteins (as specified herein). As described, the protein contains an Fc region that is optionally linked (via a linker). This means that, in some cases, one monomer of a heterodimer antibody contains an antibody heavy chain (scFv (either containing or further containing a light chain), the other monomer has a mutant Fc domain It is an Fc fusion product containing both antibody and ligand. In some embodiments, these are "half antibody - half Fusion proteins are also called "fusion bodies."

[0049] As used herein, "position" refers to the location within a protein sequence. The positions may be numbered sequentially, or in an established format, for example, E when numbering antibodies. You may also follow the U index.

[0050] When used herein, "target antigen" is specifically determined by the variable region of a given antibody. This refers to the molecule to which it is bound. The target antigen is a protein, carbohydrate, lipid, or other compound. It may also be a physical substance. A very large number of suitable target antigens are listed below.

[0051] "Strandedness" refers to the heterodimer of the present invention as used herein. In relation to antibody monomers, heterodimerization mutations are similar to two strands of "matching" DNA. The body is incorporated into each monomer such that it preserves the "matching" ability to form a heterodimer. For example, if some pI variants modify monomer A (e.g., increase the pI ), the conformational variant, which is a "charge pair" that is also available, does not interfere with the pI variant. For example, a charge variant that increases the pI is placed on the same "chain" or "monomer" to preserve both functionalities. Similarly, in the case of "distorted" variants present in a pair of sets as will be more fully outlined later, one of ordinary skill in the art will consider the pI in terms of determining whether the pI separation is similarly maximized using the distorted pI depending on which chain or monomer of the pair the set is incorporated into. When considering the "target cell" as used herein, it means a cell that expresses the target antigen. When considering the "variable region" as used herein, it means a region of an immunoglobulin that contains one or more Ig domains and is substantially encoded by any of the Vk, Vλ, and / or VH genes that make up the κ locus, λ locus, and the heavy chain immunoglobulin locus, respectively. "Wild type or WT" as used herein means an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0052] The antibodies of the present invention are generally isolated or recombinant. "Isolated" when used to describe the various polypeptides disclosed herein means identified and separated

[0053] from the components with which it is naturally associated. from the components with which it is naturally associated. from the components with which it is naturally associated. from the components with which it is naturally associated.

[0054] from the components with which it is naturally associated. from the components with which it is naturally associated. from the components with which it is naturally associated.

[0055] from the components with which it is naturally associated. from the components with which it is naturally associated. Poly This refers to peptides. Typically, isolated polypeptides undergo at least one purification step. It will be prepared by [method]. "Isolated antibodies" will have different antigen specificities. This refers to antibodies that are substantially free of other antibodies. "Recombinant" means that the antibody has undergone recombinant nuclear synthesis within an exogenous host cell. This means that it is manufactured using acid technology.

[0056] "Specific binding" or "specific binding" to a specific antigen or epitope. "Specific" means a binding that is measurably different from a nonspecific interaction. Specific binding is, for example, a control molecule that has a similar structure but generally does not possess binding activity. It can be measured by comparing the bonding of the molecules with that of the child molecules. For example, specific Target binding can be confirmed by competition with a control molecule similar to the target.

[0057] Specific binding to a particular antigen or epitope is, for example, at least about 10⁻⁴M , at least approximately 10-5M, at least approximately 10-6M, at least approximately 10-7M, less Both are approximately 10-8M, at least approximately 10-9M, alternatively, at least approximately 10-10M, less At least 10-11M, at least 10-12M, or higher, antigen It has KD (KD refers to the dissociation rate of a specific antibody-antigen interaction) for the epitope. This can be indicated by antibodies that bind to an antigen. Typically, antibodies that bind specifically to an antigen or an antigen are shown to be antibodies that bind to an antigen or an antigen. Compared to the control molecule for the pitope, the concentrations were 20, 50, 100, 500, 1000, and 5, This would result in a KD ratio of 000x, 10,000x, or even higher.

[0058] Furthermore, specific binding to a particular antigen or epitope, for example, compared to a control, can be observed. At least 20 times, 50 times, 100 times, 500 times, 1000 times, 5,0 KA against antigens or epitopes with a 10,000, or higher ratio. This can be indicated by antibodies that have Ka (KA or Ka is a specific antibody-antigen interaction). (Refers to the speed of meeting.)

[0059] II. Overview Bispecific antibodies that co-associate CD3 and tumor antigen targets allow T cells to target tumor cells. They are designed and used to attack and re-induce substances to dissolve. For example, CD3 Examples include BiTE and DART forms, which monovalently associate with tumor antigens. CD3 While methods targeting this area show considerable promise, there are also common side effects associated with such therapies. The use of cytokines often leads to toxic cytokine release syndrome, and the associated cytokines This is due to the production of the anti-CD3 binding domain of the bispecific antibody, which associates with all T cells. Therefore, a subset of CD4 T cells with high cytokine production is recruited. Furthermore, CD 4. T cell subsets, through recruitment and proliferation, can potentially lead to immunosuppression and long-term tumor suppression. This includes regulatory T cells that may have a negative impact on control. Furthermore, these forms are F It lacks a c-domain and exhibits an extremely short serum half-life in patients.

[0060] While methods targeting CD3 are showing considerable promise, there are also risks associated with such therapies. Common side effects often lead to toxic cytokine release syndrome, and related side effects It involves itokine production. The anti-CD3 binding domain of the bispecific antibody associates with all T cells. As a result, a CD4 T cell subset with high cytokine productivity is mobilized. Further more, the CD4 T cell subset includes regulatory T cells such that mobilization and proliferation potentially result in immunosuppression and may have a negative impact on long-term tumor suppression. One such approach that may reduce cytokine production and perhaps reduce CD4 T cell activation is to reduce the

[0061] affinity of the anti-CD3 domain for CD3. Thus, in some embodiments, the invention provides an antibody construct comprising an anti-CD3 antigen-binding domain that is a "potent" or "high- affinity" binder for CD3 (e.g., one example is the heavy and light variable domains represented as H1.30_L1.47 (optionally including a charged linker as needed)), and that also binds to CD38. In other embodiments, the invention provides an antibody construct comprising an anti-CD3 antigen-binding domain that is a "weak" or "lower-affinity" binder for CD3.

[0062] Additional embodiments provide an antibody construct comprising an anti-CD3 antigen-binding domain having an intermediate or "moderate" affinity for CD3 and that also binds to CD38. It should be understood that the "high, moderate, low" anti-CD3 sequences of the invention can be used in various heterodimerization formats. While (and explanatory texts are expressly incorporated herein by reference) may be used in ru.

[0063] Therefore, the present invention provides heterodimer antibodies that bind to two different antigens, for example Then, antibodies bind to two different target antigens, generally target tumor antigens (TTAs), as described below. They are "bispecific" in that they meet. These heterodimer antibodies target these target antigens, Monovalent (for example, a single antigen-binding domain such as a variable heavy chain and variable light chain domain pair) (or bivalent (having two antigen-binding domains, each independently binding to the antigen)) It can bind in either way. The heterodimer antibody of the present invention is similarly outlined below. The "pI mutant" is coupled to the telodimer, which enables simple purification from the homodimer of the telodimer. The formation of heterodimers across homodimers, as well as is sufficiently outlined in the text, is "distorted." Based on the use of different monomers having the amino acid substitution (skew). Heterodimer of the present invention In the case of bispecific antibodies of the body, the present invention generally produces heterodimeric proteins. The Fc domain, which is self-organizable within the producing cell, is modified or mutant. Depending on the method used and for creating and purifying such heterodimer proteins Yes, they are.

[0064] III. Antibodies The present invention relates to two different antigens, for example, CD3 and a target tumor antigen, for example, CD20. It binds to CD38 and CD123, and is used in the production of bispecific antibodies, which are generally therapeutic antibodies. Regarding this matter, as will be discussed below, the term "antibody" is commonly used. The antibodies for which a pathway can be found include conventional antibodies, as well as antibody derivatives, fragments, and the antibodies described herein. It can take several forms, including imitations, as described herein.

[0065] Conventional antibody structural units typically contain tetramers. Each tetramer typically consists of two pairs of identical tetramers. It consists of one polypeptide chain, and each pair has one "light" chain (typically about 25 kDa). (having a molecular weight) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa) ) has. Human light chains are classified into κ light chains and λ light chains. The present invention is not limited to However, it has several subclasses, including IgG1, IgG2, IgG3, and IgG4. This refers to the IgG class. Therefore, "isotype" is used in this specification. At that time, immunoglobulins are defined by the chemical and antigenic characteristics of their constant regions. This means one of the subclasses. The therapeutic antibody is also isotype and / or It should be understood that this may include hybrids of subclasses. For example, U.S. patent As shown in the specification of publication No. 2009 / 0163699 (as referred to by reference) Furthermore, the present invention encompasses modifications of the pI of the IgG1 / G2 hybrid.

[0066] The amino-terminal portion of each chain is generally referred to as the "Fv domain" in the art and herein. These are called the "Fv region" or "Fv region," and consist of approximately 100-110 cells primarily involved in antigen recognition. It includes a variable region of amino acids beyond that. In the variable region, an antigen-binding site is formed. Therefore, three loops converge in each of the V domains of the heavy and light chains. Each of the loops is complementary. This is called the sex-determining region (hereinafter referred to as "CDR"), and changes in the amino acid sequence occur there. The most pronounced characteristic is dynamism. "Variable" means that a specific segment of the variable region is sequenced differently between antibodies. This refers to the fact that it varies widely. The variability within the variable region is not uniformly distributed. In contrast, the V region is a relative proportion of the framework region (FR) consisting of 15 to 30 amino acids. It consists of a relatively invariant continuous region, each 9-15 amino acid lengths or longer. It is separated by a shorter region of extreme variability, which is called the "hyper-variable region."

[0067] Each VH and VL has FR1-CDR1-FR2-C from the amino terminus to the carboxyl terminus. Three highly variable regions ("complementarity determination regions") are aligned in the order of DR2-FR3-CDR3-FR4. It consists of a "region," a "CDR," and four FRs.

[0068] The hypervariable region is generally the amino acid residues 24-34 (LCDR1: "L") within the light chain variable region. (This indicates the light chain), around 50-56 (LCDR2), and around 89-97 (LCDR3) , 31-35B (HCDR1: "H" indicates heavy chain), 50-65 (H CDR2), and amino acid residues around 95-102 (HCDR3) (Kabat e t al.,SEQUENCES OF PROTEINS OF IMMUNOLOG ICAL INTEREST,5th Ed.Public Health Servi ce,National Institutes of Health,Bethesd a, Md. (1991)), as well as residues that form hypervariable loops (e.g., light Residues 26-32 (LCDR1), 50-52 (LCDR2), and 91 within the chain variable region ~96 (LCDR3), and 26~32 (HCDR1) and 53~55 (H) within the heavy chain variable region. CDR2), and 96-101 (HCDR3); Chothia and Lesk ( This includes the 1987) J.Mol.Biol.196:901~917. CDRs will be discussed later.

[0069] Throughout this specification, the Kabat numbering system will be used to identify residues within the variable domain (approximately the light chain). When referring to residues 1-107 in the variable region and residues 1-113 in the heavy chain variable region, When the EU numbering system is in the Fc region, it is commonly used (for example, Kabat et al., see above (1991).

[0070] The present invention provides a number of different CDR sets. In this case, the "complete CDR set" is , 3 variable light CDRs and 3 variable heavy CDRs, for example, vlCDR1, vlCDR2 This includes vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These are each It may be part of a larger variable light chain domain or a variable heavy chain domain. Furthermore, Honmei As will be more thoroughly outlined in the detailed document, the variable heavy chain domain and variable light chain domain are heavy chain And when light chains are used (for example, when Fab is used), separate polypeptide chains In the case of the above, or scFv sequences, they can exist on a single polypeptide chain.

[0071] CDRs contribute to antigen binding, or more specifically, the formation of the antibody epitope binding site. An "epitope" is a specific feature within the variable region of an antibody molecule, also known as a paratope. This refers to a determinant that interacts with a specific antigen-binding site. An epitope is an amino acid or sugar side chain. A group of molecules that typically possesses specific structural and charge properties. An antigen may have two or more epitopes.

[0072] Epitopes are amino acid residues that are directly involved in binding (also known as the immune-dominant components of epitopes). (and so on) other amino acid residues not directly involved in binding, such as specific antigen-binding peptides It may also contain amino acid residues that are effectively blocked by thiosulfate. In other words, amino acids The residues are located within the footprint of the specific antigen-binding peptide.

[0073] Epitopes can be either three-dimensional or linear. Three-dimensional epitopes This is due to spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues within the polypeptide chain. These are epitopes. Stereostructural or non-stereostructural epitopes are affected by the presence of a denaturing solvent. Below, we distinguish them in that the connection with the former is lost, but the connection with the latter is not. That's good too.

[0074] Epitopes typically have at least three, more generally, in a specific spatial arrangement. It contains at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope are , a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to its target antigen (e.g.) For example, this can be seen in "binning."

[0075] The carboxyl terminus of each chain defines a constant region primarily involved in effector function. Kabat et al. collected a very large number of primary sequences of the variable regions of the heavy and light chains. Based on the degree of sequence preservation, Kabat et al. used CDR and frameworks to select individual primary sequences. They were classified into two categories, and a list was created (SEQUENCES OF IMMUNOLOGY CAL INTEREST,5th Ed.,NIH publication,No. 91-3242, EAKabat et al. (The whole is referenced) (See reference).

[0076] The IgG subclass of immunoglobulins includes several immunoglobulin mei in its heavy chain. The term "immunoglobulin (Ig) domain" in this specification refers to a distinct tertiary structure. This refers to the region of immunoglobulins that it possesses. What is interesting in this invention is the steady-state weight (CH) It is a heavy chain domain that includes the domain and hinge domain. In relation to IgG antibodies, Ig Each G isotype has three CH regions. Therefore, the "CH" associated with IgG... The domain is as follows: "CH1" is an EU domain as described in Kabat. This refers to the 118th to 220th positions according to DEX. "CH2" is as described in Kabat. This refers to a ranking between 237th and 340th in the EU index, and "CH3" is listed in Kabat. This refers to a ranking between 341st and 447th according to the EU index, as shown herein and described below. As such, the pI mutant is one of the CH region and hinge region, which will be discussed later. It is possible for more than this to exist.

[0077] The sequences shown herein begin in the CH1 region, i.e., at position 118, and if specified It should be noted that the variable region is excluded. For example, the first part of sequence number 2 Even though a mino acid is listed as position "1" in the sequence listing, according to the EU numbering system, it is located at position 118 in the CH1 region. It corresponds to the position.

[0078] Another type of Ig domain in the heavy chain is the hinge region. In this specification, “hinge” or The "hinge region," "antibody hinge region," or "immunoglobulin hinge region" is an antibody A flexible polypeptide containing amino acids between its first and second constant domains. This means that, structurally, the CH1 domain of IgG terminates at EU position 220, and the The CH2 domain starts at EU position 237 of the residue. Therefore, in the case of IgG, the antibody hyphen The range includes cells from position 221 (IgG1 D221) to 236 (IgG1 G236). The numbering as defined herein is based on the EU index as described in Kabat. In some embodiments, for example, a lower hinge is included in relation to the Fc region. This “lower hinge” generally refers to position 226 or 230. Thus, pI mutants can also be generated in the hinge region.

[0079] The light chain generally consists of a variable light chain domain (which includes the light chain CDR and is combined with the variable heavy chain domain). Two regions: the Fv region (which forms the Fv region) and the constant light chain region (often referred to as CL or Cκ). Includes two domains.

[0080] Another region that will be further replaced, as outlined later, is the Fc region.

[0081] Therefore, the present invention provides different antibody domains. The technology described herein As is well known in the field, the heterodimeric antibody of the present invention has different domains in the heavy chain and light chain. These include, and they may overlap. These domains are not limited to However, Fc domain, CH1 domain, CH2 domain, CH3 domain, hinged domain N, a dual steady-state domain (CH1-hinge-Fc domain or CH1-hinge-CH2-C H3), variable heavy chain domain, variable light chain domain, light constant domain, FAb domain and Includes the scFv domain.

[0082] Therefore, the "Fc domain" is the -CH2-CH3 domain, optionally hinged It includes the main. The heavy chain contains a variable heavy chain domain and a constant domain, which is CH2-C The light chain contains a CH1-optional hinge-Fc domain including H3. The light chain is a variable light chain and Includes light constant domains.

[0083] Some embodiments of the present invention include at least one scFv domain, which is naturally Although they do not exist, the variable heavy chain domains and are generally linked together by the scFv linker. Contains variable light chain domains. Prepared by recombinant technology as shown herein. Several suitable scFv linkers exist that include conventional peptide bonds.

[0084] Linker peptides are mainly composed of the following amino acid residues: Gly, Ser, Ala, or Th It may contain r. The linker peptide holds two molecules together so that they retain the desired activity. So, the lengths are sufficient to connect them in a way that assumes an accurate three-dimensional structure relative to each other. It is necessary to have. In one embodiment, the linker is about 1 to 50 amino acids long, preferably about The length is 1 to 30 amino acids. In one embodiment, a linker with a length of 1 to 20 amino acids may also be used. Often, in some embodiments, the use of approximately 5 to 10 amino acids is observed. Useful linkers For example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS Glycine-serine containing n (where n is an integer of at least 1 (and generally 3-4)) Polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible polymers. Examples of linkers include polyethylene glycol (PE). Alternatively, although not limited to these, polyethylene glycol (PE) is also an option. G) Polypropylene glycol, polyoxyalkylene, or polyethylene glycol Various non-protein polymers, including copolymers of phosphorus and polypropylene glycol, are available. It may be found to have a use as a car, that is, a use as a linker.

[0085] Other linker sequences may include any sequence of any length from the CL / CH1 domain. However, not all residues in the CL / CH1 domain (for example, the first residue of the CL / CH1 domain) (5-12 amino acid residues). The linker is an immunoglobulin light chain, e.g., Cκ or Cκ. It may originate from λ. The linker is, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα 2. May be derived from any isotype of immunoglobulin heavy chain, including Cδ, Cε, and Cμ. The linker sequence also contains other proteins such as Ig-like proteins (e.g., TCR, F cR, KIR), sequences derived from hinge regions, and other native sequences derived from other proteins. You may come.

[0086] In some embodiments, the linker is located in any two domains (which are outlined together herein). It is a "domain linker" used to link (things). Any suitable linker While these are available, in many embodiments, for example, (GS)n, (GSGGS)n, ( GGGGS)n, and (GGGS)n(n is an integer of at least 1 (and generally 3-4) ~5) along with a glycine-serine polymer containing two domains, each domain Recombination with sufficient length and flexibility to allow the organism to maintain its biological function Any peptide sequence that enables adhesion is used. Depending on the case, attention may also be paid to the "chain state". As will be outlined later, the charged domain linker is a part of the scFv linker. It can be used when applied in a specific manner.

[0087] In some embodiments, the scFv linker is a charged scFv linker, and the majority of them are shown in Figure As shown in 33. Therefore, the present invention relates to the fraction of pI between the first and second monomers. To facilitate separation, a charged scFv linker is further provided. That is, positive or negative Either (or both in the case of scaffolds using scFv on different monomers) By incorporating a charged scFv linker, the monomer containing the charged linker becomes an Fc domain. It is possible to change pI without causing further changes within these charges. The linker can be replaced in any scFv having a standard linker. Furthermore, those skilled in the art will know As can be understood, the charged scFv linker is based on the desired change in pI. Therefore, it is used for precise "chains" or monomers. For example, as discussed herein... To produce a triple F-type heterodimer antibody, each of the desired antigen-binding domains The first pI of the Fv region is calculated, and one is selected to create the scFv. Furthermore, either a positive or negative linker is selected depending on the pI.

[0088] Similarly, to enhance the pI separation of monomers in the present invention, a charged domain linker may also be used. Therefore, in any embodiment of this specification where a linker is used, Figure 3 You can use the items included in item 3.

[0089] In some embodiments, the antibody is full-length. As used herein, "full-length antibody" refers to a heterodimer. To enable either the formation of a compound or the purification of a heterodimer from a homodimer, A variable region in the Fc domain that includes one or more modifications as outlined herein This refers to the structure that constitutes the natural biological form of an antibody, including the constant region. Full-length antibodies are Generally, it includes Fab and Fc domains, and in addition, as generally shown in the drawings, It may have extra antigen-binding domains such as scFv.

[0090] In one embodiment, the pI is modified to produce a heterodimer. An antibody is an antibody fragment as long as it contains at least one constant domain. Other antibody fragments that can do this are CH1, CH2, CH3, and hin of the present invention which modify pI. It comprises a fragment having one or more of the following domains: di and CL domains. For example, an Fc fusion is Fc regions (CH2 and CH3) fused to another protein, and optionally the hinge region It is a fusion product of (including). Many Fc fusion products are known in the art, and the heterogeneity of the present invention This can be improved by adding a dimerizing mutant. In the present invention, CH1;CH1,CH2 antibody fusion including CH3;CH2;CH3;CH2 and CH3;CH1 and CH3 It is possible to manufacture these items, and any or all of these are described herein. Using any combination of ronimerization mutants, the hinge region is arbitrarily constructed. It is possible.

[0091] In particular, the form shown in Figure 1 is an antibody commonly referred to as a "heterodimer antibody," and The protein self-organizes into heterodimeric Fc domains, with at least two related Fc molecules. It means having a column.

[0092] Chimeric and humanized antibodies In some embodiments, the antibody is a mixture from different species, e.g., a chimeric antibody and / or This could be a humanized antibody. Generally, both "chimeric antibodies" and "humanized antibodies" are two This refers to antibodies that combine regions derived from the above species. For example, "chimeric antibodies" have traditionally been... It includes a variable region derived from mouse (or possibly rat) and a constant region derived from human. "Humanized antibodies" generally refer to antibodies that have a variable domain framework region that is found in human antibodies. This refers to non-human antibodies that have been replaced with other antibodies. Generally, in humanized antibodies, all antibodies except CDRs are human. Encoded by polynucleotides derived from or within its CDR It is identical to the antibody. It is partially or entirely encoded by nucleic acids derived from non-human organisms. The CDR is transplanted into a β-sheet framework of the human antibody variable region to produce antibodies. Its specificity is determined by the transplanted CDR. The production of such antibodies is, for example, international Publication No. 92 / 11018, Jones, 1986, Nature 321:522-5 25, Verhoeyen et al., 1988, Science 239:1534 -1536 (all referenced as a whole). First port Selected acceptor framework to restore lost affinity in the structure Often, a "reverse mutation" of the residue to the corresponding donor residue is required (US 5530). U.S. Patent No. 101, U.S. Patent No. 5585089, U.S. Patent No. 5693761 Book, U.S. Patent No. 5,693,762, U.S. Patent No. 6,180,370, U.S. Patent U.S. Patent No. 5859205, U.S. Patent No. 5821337, U.S. Patent No. 60542 U.S. Patent No. 97, U.S. Patent No. 6,407,213 (all are invoked by reference as a whole). Humanized antibodies also, ideally, target the constant region of immunoglobulins (typically human immunoglobulins). This will include at least a portion of the constant region of robulin, and therefore typically human This will include the Fc region. Humanized antibodies are also used in mice with genetically modified immune systems. It can be prepared using [the specified method]. Roque et al., 2004, Biotechnol. Prog.20:639-654 (as a whole, referenced). Non-human antibodies Various techniques and methods for catalysis and reshaping are well known in the art (Tsu rushita&Vasquez,2004,Humanization of Mon oclonal Antibodies,Molecular Biology of B Cells, 533-545, Elsevier Science (USA), See the references cited therein (all of which are referenced as a whole). The method of humanization is not limited, but see Jones et al., 1986, Nature. 321:522-525;Riechmann et al.,1988;Natur e 332:323-329; Verhoeyen et al., 1988, Sci. nce,239:1534-1536;Queen et al.,1989,Proc. Natl Acad Sci,USA 86:10029-33;He et al. ,1998,J.Immunol.160:1029-1035;Carter et al. al.,1992,Proc Natl Acad Sci USA 89:4285- 9,Presta et al.,1997,Cancer Res.57(20):4 593-9;Gorman et al.,1991,Proc.Natl.Acad. Sci.USA88:4181-4185;O'Connor et al.,1998 ,Protein Eng 11:321-8 (all are referenced as a whole) This includes methods described in (ru). Humanization methods, or methods for reducing the immunogenicity of the variable region of a non-human antibody. Another method for doing this is described in Roguska et al., 1994, Proc. Natl. A cad.Sci.USA 91:969-973 (as referred to in whole) This may include a surface retreatment method as described. In one embodiment, the parent antibody is the Art of the Art. Humanization and affinity maturation are performed as is well known. A method based on a structure such as that described in Patent Application No. 11 / 004,590 may be used. . To humanize and / or affinity mature the antibody variable region, but not limited to, Wu et al.,1999,J.Mol.Biol.294:151-162;Baca et al.,1997,J.Biol.Chem.272(16):10678-1 0684;Rosok et al.,1996,J.Biol.Chem.271(3 7):22611-22618;Rader et al.,1998,Proc.Na tl.Acad.Sci.USA 95:8910-8915;Krauss et a l.,2003,Protein Engineering 16(10):753-7 A selection of methods, including those described in 59 (all of which are referenced collectively). The following methods may be used. These methods are not limited to, but include the specification of U.S. Patent Application No. 09 / 810,510. Book;Tan et al.,2002,J.Immunol.169:1119-112 5;De Pascalis et al.,2002,J.Immunol.169: Including the methods described in 3076-3084 (all of which are referenced collectively). Other humanization methods may involve transplanting only a portion of the CDR.

[0093] IV. Heterodimal antibodies Therefore, in some embodiments, the present invention relates to heterodimer Fc domains and heterodimers. Two different heavy chain mutants will self-assemble to form a dimeric antibody. This invention provides heterodimer antibodies that depend on the use of sequences.

[0094] The present invention enables binding to two or more antigens or ligands, for example, bispecific binding. This study focuses on novel constructs for providing heterodimeric antibodies that enable the synthesis of heterodimeric antibodies. Merged antibody constructs are formed when two Fc domains of the antibody's heavy chain, for example, assemble to form a "dimer". This is based on the self-assembly properties of the two "monomers". As will be thoroughly discussed below, Dimer antibodies are produced by modifying the amino acid sequence of each monomer. Therefore In general, the present invention promotes the formation of heterodimers and / or homodimers. To simplify the purification of telodimers, different constant regions of each chain are used. The goal is to create heterodimer antibodies that can co-associate with antigens using several methods dependent on no-acid variants. Let's assume that.

[0095] In this way, the present invention provides a bispecific antibody. The topic generally involves simultaneously binding to two different antigens, bringing the different antigens into close proximity, and developing new functions. There is a demand for "bispecific" antibodies that offer both sexual properties and new therapeutic options. Generally speaking, these Antibodies are produced by incorporating the genes in their respective heavy and light chains into the host cell. This generally yields the desired heterodimer (AB) and two homodimers (A -A and BB (without issues on the light chain heterodimer) are formed. However, Furthermore, a major obstacle in the formation of bispecific antibodies is the transition from homodimer antibodies to heterodimer antibodies. Purify and / or bias heterodimer formation against homodimer formation. The difficulty lies in doing so.

[0096] Several mechanisms exist that can be used to produce the heterodimer of the present invention. Furthermore, As understood by the industry, combining these mechanisms leads to advanced heterodimerization. Therefore, amino acid variants that result in the production of heterodimers are These are called "heterodimizing mutants." As will be discussed later, heterodimerizing mutants These are stereotypical mutants (for example, "knobs and holes" as shown below). or "distortion" mutants and "charge pair" mutants (see below) and homo from heterodimers May contain a "pI variant" that enables the purification of dimers. International Publication No. 2014 / 14580 Issue 6 (the entire issue, and the considerations regarding "heterodimerized mutants," are as follows) Specifically, (as referred to herein) a mechanism useful for heterodimerization "Knob and Hole" ("KIH"); sometimes referred to as "distortion" variant in this specification (International Publication No. (See the discussion in issue 2014 / 145806), as noted in International Publication No. 2014 / 145806. The “electrostatic manipulation” or “charge pair” described in International Publication No. 2014 / 145806, p I variant, and as outlined in International Publication No. 2014 / 145806 and below Includes more common Fc variants.

[0097] In this invention, there are several basic mechanisms that can facilitate the purification of heterodimer antibodies. On the other hand, since each monomer has a different pI, it depends on the use of pI mutants, A- This enables isoelectric point purification of A, AB, and BB dimer proteins. Alternatively, some Scaffold formats, such as the "Triple F" format, also enable size-based separation. As will be outlined further, it is also possible to "distort" the formation of heterodimers relative to homodimers. It is possible. Therefore, between steric heterodimerized mutants and pI or charge pair mutants From the combinations, specific applications in the present invention can be found.

[0098] Generally, the specific application embodiments of the present invention increase the pI difference between two monomers. When coupled with the pI mutant, it promotes heterodimerization rather than homodimerization. It depends on the set of mutants, including distorted mutants.

[0099] In addition, as will be outlined more thoroughly later, depending on the form of the heterodimer antibody, pI The mutant may be contained within the constant and / or Fc domain of the monomer, or within the charged You can use a linker (either a domain linker or an scFv linker). In other words, scaffolds that utilize scFv such as the triple F format are further refined for purification purposes. This may include a charged scFv linker (either positive or negative) that provides a pI boost. As will be understood by those skilled in the art, with only a charged scFv linker, further pI adjustment While several triple F forms without accompanying are useful, the present invention particularly involves one or both monomers. It provides pI variants and / or similarly charged domain linkers. Furthermore, By further modifying amino acids with the intention of enhancing functionality, changes in pI, for example, Fc FcRn and KO variants may also be obtained.

[0100] In this invention, pI is used as a separation mechanism that enables the purification of heterodimeric proteins. Amino acid variants can be introduced into one or both monomeric polypeptides; i.e. The pI of one monomer (simply referred to herein as "monomer A") is separated from monomer B. It can be modified so that, or the pI of monomer A is increased and the pI of monomer B is increased By lowering the coefficient, both monomers A and B can be modified. This will be outlined in more detail later. The change in pI of either or both monomers is due to the removal or addition of charged residues. (For example, a neutral amino acid can be positively or negatively charged at an amino acid residue, for example, glycy (It is replaced from n to glutamic acid), and the charged residue is changed from positive or negative charge to the opposite charge. By changing the charge (from aspartic acid to lysine), or by changing a charged residue to a neutral residue, This can be done by changing (for example, charge loss: from lysine to serine). Several of these variants are shown in the diagram.

[0101] Thus, this embodiment of the present invention allows for the separation of heterodimers from homodimers. This provides a sufficient change in pI to bring about at least one of the monomers. To be understood by those skilled in the art, and as will be further considered later, this is "wild." The "type" heavy chain constant region and the mutant region modified to increase or decrease pI (w By using tA-+B or wtA--B, or by increasing one of the regions This can be achieved by reducing the other region (A+-B- or A-B+). .

[0102] Therefore, generally speaking, components of some embodiments of the present invention are amino acid substitutions ("pI variants"). By incorporating a pI substitution (or "pI substitution") into one or both monomers, the dimeric protein By changing the isoelectric point (pI) of at least one monomer of the chlorine (if not both), These are amino acid variants within the constant region of an antibody, targeted at forming a "pI antibody." As shown herein, the separation of a heterodimer from two homodimers is performed by two monomers This can be achieved when the body's pI differs by a minimum of 0.1 units in pH, and also when it differs by 0.2, 0.3, Applications of the present invention can be found in all cases where the value is 0.4, 0.5 or greater.

[0103] As will be understood by those skilled in the art, in order to obtain good separation, each or both monomers The number of pI mutants included is, in part, related to the initiation pI of the component in the triple F form, and the objective. This will depend on the scFv and Fab start pI. In other words, which monomer To modify or determine the "direction" (e.g., more positive or more negative), two indicators The Fv sequence of the target antigen is calculated, and the determination is made from there. Therefore, different Fv will have different starting pIs used in this invention. As outlined herein, pI is defined as the total pI difference of each monomer being at least about 0.1L Designed to have an OG of 0.2 to 0.5 (as outlined herein). .

[0104] Furthermore, some embodiments will be understood by those skilled in the art and will be outlined herein. Therefore, heterodimers can be separated from homodimers based on size. For example, as shown in Figure 1. As such, several forms separate heterodimers and homodimers based on size. Make it possible.

[0105] To achieve heterodimerization, the pI mutant is used by utilizing the constant region of the heavy chain. If possible, a more modular approach to designing and purifying bispecific proteins, including antibodies. A method is provided. Therefore, in some embodiments, heterodimerized mutants (distortion) Since the individual antibody (including purified heterodimerized mutants) is not included in the variable region, Each body needs to be modified. Furthermore, in some embodiments, immunity is caused by the pI mutant. The possibility of epidemicogenicity occurring is such that pI changes without introducing significant immunogenicity, Importing the I variant from a different IgG isotype significantly reduced it. However, a further challenge to be addressed is the high human sequence content, for example, non-human sequences at any specific location. This involves elucidating low pI constant domains that involve minimizing or avoiding T residues.

[0106] Possible side effects of modifying this pI include prolongation of serum half-life and FcRn binding. There is also an enhancement of the combination. That is, the specification of U.S. Patent Application No. 13 / 194,904 (the whole thing) As described in (referenced), (those found in the antibody and Fc fusion) By reducing the pI of the antibody constant domain (including), serum retention is extended in vivo. This can result. These pI variants for extending the serum half-life are also used for purification. It promotes the change in pI.

[0107] Furthermore, the ability to remove, minimize, or distinguish homodimers when they are present. Because this is important, the pI variant of the heterodimerized mutant is used in the analysis of bispecific antibodies. It is noteworthy that this will bring further benefits to the quality control process. Therefore, the ability to reliably test the reproducibility of heterodimer antibody production is crucial.

[0108] Heterodimizing mutants This invention enables the formation of heterodimers and / or purification from homodimers. Heterodimer proteins, including heterodimer antibodies in various forms, utilizing heterodimer variants. To provide quality.

[0109] Several suitable pairs of heterodimerized distortion mutant sets exist. These mutants are: They exist as a "set" of "pairs." That is, one set of a pair is incorporated into the first monomer. Then, the other set of the pair is incorporated into the second monomer. These sets are not necessarily Without behaving as a "knobs in holes" variant, There is a one-to-one correspondence between residues on one monomer and residues on the other monomer, that is, These pairs promote the formation of heterodimers and prevent the formation of homodimers. By forming interfaces between monomers, hundreds of heterodimers spontaneously form under biological conditions. The fraction is as expected: 50% (25% homodimer A / A: 50% heterodimer A / B) It is noteworthy that it becomes possible to achieve over 90% homodimer (B / B) instead of 25%. It should.

[0110] Stereomorphs In some embodiments, heterodimer formation can be promoted by the addition of stereomutants. In other words, by modifying the amino acids within each heavy chain, different heavy chains can associate and form the same Fc group. It is more likely to form a heterodimer structure than a homodimer with a mino acid sequence. . Suitable stereomutations are shown in Figure 29.

[0111] One mechanism generally supports the formation of heterodimers and opposes the formation of homodimers. In this field, the term "nob and ho" refers to the modification of amino acids that creates steric effects. It is referred to as "Lu" and can be used at will, but this is sometimes used in US patent applications No. 61 / 596,846, Ridgway et al., Protein E Engineering 9(7):617(1996);Atwell et al. J.Mol.Biol.1997 270:26; US Patent No. 8,216,805 As stated in the book (all of which are incorporated herein by reference): This is referred to as "knob and hole." In the same diagram, several "monomer A" depend on "knob and hole." -The monomer B pair has been identified. Furthermore, Merchant et al., Natu As described in re Biotech.16:677(1998), these "knobs and h The "ru" mutation distorts the formation of heterodimers, and therefore combines with the disulfide bond. They can be combined.

[0112] Further mechanisms that have applications in the fabrication of heterodimers are found in Gunasekara n et al., J. Biol. Chem. 285(25):19637(2010) As described herein (which is incorporated by reference in its entirety), it is referred to as “electrostatic control.” This may occur. This may be referred to as a “charge pair” in this specification. In this embodiment, Static electricity is used to distort the formation of heterodimers. Those skilled in the art will understand. These may also have an effect on pI, and therefore on purification. Therefore, in some cases, they can be considered pI mutants. However, if these are heterozygous Because they were created to promote merging and were not used as purification tools, These are classified as "stereotypical mutants." These are not limited to D221R / P228R. / K409R (for example, these are "monomer-compatible sets") and paired with D221E / P22 8E / L368E and C220R / E224R / P228R / K409R and their paired C22 Includes 0E / P228E / 368E.

[0113] Further monomer A and monomer B variants can be selectively and independently produced in any quantity. , other variants, for example, the pI variant outlined herein or U.S. Patent Application Publication No. 201 Other stereomorphs shown in Figure 37 of Specification No. 2 / 0149876 (the drawings and descriptions) And the sequence numbers are expressly invoked herein by reference. Cut.

[0114] In some embodiments, the stereovariates outlined herein are arbitrarily and independently selected. Along with any pI variant (or other variants, such as Fc variants, FcRn variants, etc.) It can be incorporated into one or both monomers, and can be included independently and optionally. Alternatively, it can be excluded from the proteins of the present invention.

[0115] A list of preferred distortion variants can be found in Figure 29, and Figure 34 shows in particular many embodiments. Here are some useful pairs. In many embodiments, but not limited to, S364K / E357 Q:L368D / K370S;L368D / K370S:S364K;L368E / K3 70S:S364K;T411T / E360E / Q362E:D401K;L368D / Includes K370S:S364K / E357L and K370S:S364K / E357Q Pairs are particularly used. From a nomenclature standpoint, "S364K / E357Q:L368D The pair " / K370S" has one monomer having the double mutant set S364K / E357Q. This means that the other party has the double mutant set L368D / K370S.

[0116] pI (isoelectric point) mutants in heterodimers Generally, as understood by those skilled in the art, pI variants fall into two common categories. , in other words, substances that increase the pI of proteins (basicity change) and substances that increase the pI of proteins There are substances that cause a decrease (acidification). As described herein, all of these mutants A combination can be provided in which one monomer is wild-type or significantly different from wild-type. It may also be a mutant that does not exhibit pI, or the other may be more basic or more acidic Either of these is possible. Alternatively, each monomer may change, becoming more basic or more acidic. There are some that become like this.

[0117] Preferred combinations of pI mutants are shown in Figure 30, which are outlined herein and shown in the drawings. As such, these changes are shown for IgG1, but not for all isotypes. Furthermore, isotype hybrids can be modified in this way. The heavy chain constant domain is IgG If the value is between 2 and 4, R133E and R133Q can also be used.

[0118] Light chain mutants of antibody heterodimers In the case of antibody-based heterodimers, for example, at least one monomer is added to the heavy chain domain. If a light chain is present, pI variants can also be created within the light chain. By reducing the pI of the light chain... The amino acid substitutions used for this purpose are not limited to K126E, K126Q, and K145E. , K145Q, N152D, S156E, K169E, S202E, K207E and light One example is the addition of the peptide DEDE at the C-terminus of the chain. This category is based on a constant lambda light chain. The changes in - are R108Q, Q124E, K126Q, N138D, K145T and This includes one or more substitutions in Q199E. Furthermore, it is also possible to increase the pI of the light chain. .

[0119] Isotype variants Furthermore, many embodiments of the present invention involve converting one IgG isotype to another IgG isotype. It relies on the "incorporation" of pI amino acids at specific positions into the ip, and by doing so This reduces or eliminates the possibility of unwanted immunogenicity being introduced into the mutant. Some of these are U.S. Patent Application Publication No. 2014 / 0370013 (as referenced herein) As shown in Figure 21 (which is used in reference), that is, for various reasons including high effector functionality Therefore, IgG1 is a common isotype for therapeutic antibodies. However, IgG1 The polysteady region of has a higher pI than the polysteady region of IgG2 (8.10 vs. 7.31). By introducing an IgG2 residue at a specific position in the IgG1 backbone, the resulting The monomer pI decreases (or increases), and in addition, the serum half-life is prolonged. For example, I IgG1 has glycine at position 137 (pI 5.97), and IgG2 has glutamic acid (pI 3 .22) has. Incorporating glutamate results in the p of the resulting protein. This will affect I. As will be discussed later, several amino acid substitutions are generally considered mutants. It is required to have a significant effect on the antibody's pI. However, as will be discussed later, It is noteworthy that even changes in the IgG2 molecule can enable the extension of the serum half-life. It should be done.

[0120] In other embodiments, to reduce the overall charge state of the resulting protein (For example, by changing amino acids with a higher pI to amino acids with a lower pI) ), or, as will be explained further later, to adapt to structures for stability, etc. It induces non-isotype amino acid changes.

[0121] Furthermore, by modifying pI in both the heavy steady domain and the light steady domain, Significant changes can be observed in each monomer of the telodimer. As discussed herein, By making the pI values ​​of the two monomers differ by at least 0.5, ion exchange chromatography can be performed. Separation by phi or isoelectric focusing electrophoresis, or by other methods sensitive to isoelectric focusing. It could be possible.

[0122] Calculation of pI The pI of each monomer is the pI of the mutant heavy chain constant domain and the mutant heavy chain constant domain and The pI of the entire monomer, including the fusion partner, may depend on this. Therefore, in some embodiments, The change in pI is shown in Figure 19 of U.S. Patent Application Publication No. 2014 / 0370013. The calculation is performed using the chart, based on the mutant heavy chain constant domain. In general, the choice of which monomer to modify depends on the Fv and scaffold region. It is determined by the specific pI. Alternatively, the pI of each monomer can be compared.

[0123] pI variants also result in better FcRn in vivo binding. If pI variants reduce monomeric pI, they improve serum retention in vivo. This could have the additional benefit of being able to do so.

[0124] Although still under investigation, it is believed that FcRn binds to FcRn within endosomes at pH 6. Since it is isolated, the Fc region is thought to have a longer half-life in vivo (G hetie and Ward,1997 Immunol Today.18(12) (592-598, as used in whole). Next, the endosomal compartment is detailed Fc is reused on the cell surface. When that compartment opens to the extracellular space, a higher p of approximately 7.4 is observed. H induces the reverse release of Fc into the bloodstream. In mice, Dall' Acqua et al. However, Fc mutants with enhanced binding to FcRn at pH 6 and pH 7.4 were found in serum concentrations And it was shown that it actually reduces the half-life to the same level as wild-type Fc (Dall'Acq ua et al.2002, J.Immunol.169:5171-5180, entire (Used by reference as follows). The increase in affinity of Fc to FcRn at pH 7.4 is This is thought to prevent the back release of Fc into the bloodstream. Therefore, the half-life of Fc in vivo Fc mutations that extend the range ideally increase binding to FcRn at lower pH levels. While strengthening, it also enables the release of Fc at higher pH levels. The amino acid histidine is 6 The charge state is changed within the pH range of 0 to 7.4. Therefore, Fc / FcRn complex It is not surprising to find the His residue at a crucial position during the fusion process.

[0125] In recent years, antibodies with variable regions that have lower isoelectric points have also been found to have longer serum half-lives. It has been suggested that this is possible (Igawa et al., 2010 PEDS.23) 5):385-392, as a whole (referred to by reference). However, regarding this mechanism Understanding remains limited. Furthermore, the variable region differs between antibodies. Reduced pI And if the constant region variant has an extended half-life, then the antibody will be as described herein. This will provide a more modular approach to improving pharmacokinetic properties.

[0126] Further Fc variants for enhanced functionality In addition to pI amino acid variants, binding to one or more FcγR receptors is also possible, though not limited to them. It can be created for various reasons, including sex modification and modification of binding to the FcRn receptor. There are several types of Fc amino acid modifications.

[0127] Therefore, the protein of the present invention includes pI variants and stereovariates, as specified herein. The mutants may include amino acid modifications, including the dimerized mutants outlined. Each set of mutants is independent. And may be included selectively or excluded from any particular heterodimer protein. It is possible.

[0128] FcγR mutant Therefore, useful tools can be created to modify the binding of one or more FcγR receptors. Several Fc substitutions exist. Substitutions that result in bond strengthening and bond weakening can be useful. For example, enhanced binding to Fc RIIIa is generally associated with ADCC (Antibody-Dependent Cell-Induced Criticality). Nonspecific cytotoxic cells expressing FcγR recognize the bound antibody on the target cell, and subsequently... It is known to lead to an increase in cell-mediated reactions that cause the lysis of target cells. Similarly, reduced binding to FcγRIIb (an inhibitory receptor) is also advantageous under certain conditions. It is possible. The amino acid substitutions for which applications are found in the present invention are as described in U.S. Patent Application No. 11 / 1. U.S. Patent Application No. 24,620 (especially Figure 41), U.S. Patent Application No. 11 / 174,287, U.S. Patent Application No. 11 / 396,495, U.S. Patent Application No. 11 / 538,406 The specification (all of these, in whole and in particular, the variants disclosed therein) This includes those listed in (and expressly incorporated herein by reference). While not limited to specific mutants for which a path can be found, 236A, 239D, and 239E are examples of such mutants. , 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E Examples include / 330L, 243A, 243L, 264A, 264V, and 299T.

[0129] Furthermore, U.S. Patent Application No. 12 / 341,769 (which is referenced in its entirety herein) As specifically disclosed in (more referenced), it is not limited to, but includes 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L and 259I / 308 In addition to F / 428L, it is used to enhance binding to the FcRn receptor and prolong the serum half-life. Further Fc substitutions exist for which a path can be found.

[0130] Tear mutant Similarly, another category of functional mutants is "FcγR cleavage mutant" or "Fc-knotted mutant." These are variants of the "Kout (FcKO or KO)" variant. In these embodiments, some therapeutic In the process, to avoid additional mechanisms of action, one or more or all Fcγ receptors (For example, FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) It is desirable to reduce or eliminate the normal binding of the Fc domain. For example, For example, in many embodiments, particularly in the use of bispecific antibodies that monovalently bind to CD3 To remove or significantly reduce ADCC activity, the binding of FcγRIIIa is cleaved. It is generally desirable to cleave, where one of the Fc domains cleaves one or more Fcγ receptors. This includes mutants. These cleavage mutants are shown in Figure 31, each of which was included independently and arbitrarily. In a preferred embodiment, G236R / L328R, E233 may be included or excluded. P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G2 36del / S239K / A327G, E233P / L234V / L235A / G236 del / S267K / A327G and E233P / L234V / L235A / G236 A cleavage mutant selected from the group consisting of del is used. The cleavage mutants referenced herein It is noteworthy that the isoform cleaves the FcγR bond but generally does not cleave the FcRn bond. It is.

[0131] Combinations of heterodimers and Fc mutants As will be understood by those skilled in the art, the enumerated (including distorted and / or pI variants) All heterodimerized mutants retain their "chain state" or "monomer compartment." As long as it is possible, it can be combined arbitrarily, selectively, and independently in any way. Furthermore, this All of their mutants can be combined into any of the heterodimerization forms.

[0132] In the case of pI variants, embodiments in which specific uses are found are shown in the drawings, while purification is facilitated. Therefore, other combinations are made according to the basic rules that modify the pI difference between the two monomers. It can be manufactured.

[0133] Furthermore, either of the heterodimerized mutants (distortion and pI) is generally considered in this specification. As explained, Fc cleavage mutants, Fc mutants, and FcRn mutants are all independently and arbitrarily selected. They are combined in a specific way.

[0134] Useful forms of the present invention As will be understood by those skilled in the art and will be discussed more thoroughly later, the heterodimer fusion of the present invention Synthetic proteins can generally take on a wide variety of three-dimensional configurations, as shown in Figure 1. The diagram shows a "single-ended" three-dimensional configuration, where one "arm" of the molecule is... One type of anomaly exists, and a different anomaly exists for the other "arm". Other drawings are This represents a "dual-ended" configuration, where at least one specific type of molecule is present at the "top" of the molecule. A property exists, and one or more different specificities exist at the "bottom" of the molecule. Therefore, the present invention This refers to novel immunoglobulin compositions that co-associate with different first and second antigens. Let's assume that.

[0135] As will be understood by those skilled in the art, the heterodimer form of the present invention has different bond values ​​and It may have bivalent and bispecific properties. That is, the heterodimer antibody of the present invention is bivalent and bispecific. This is possible, and here one of the target tumor antigens (e.g., CD3) is bound by the first binding domain. Combined with the other target tumor antigen (e.g., CD20, CD19, CD38, CD123, etc.), ) is bound by a second binding domain. Heterodimeric antibodies are also trivalent and bivalent. Specificity is possible, where the first antigen is bound by two binding domains, and the second antigen... It is joined by a second binding domain. As outlined herein, potential side effects To reduce its effectiveness, when CD3 is one of the target antigens, CD3 is bound exclusively in a monovalent form. It is preferable that this be done.

[0136] In this invention, an anti-CD3 antigen is combined with an anti-target tumor antigen (TTA) antigen-binding domain. A combined domain is used. As will be understood by those skilled in the art, the drawings (especially Figures 2-7, Anti-CD3 CDR, anti-CD3 variable light chain (see Figure 68) as shown in either of the above. Use either the main and variable heavy chain domains, or the Fab or scFv collection. Similarly, any of the anti-TTA antigen-binding domains, for example, anti-CD38, anti-CD38, can be used. 20. The anti-CD19 and anti-CD123 antigen-binding domains are shown in one of the figures. Una CDR, variable light chain and variable heavy chain domains, Fab and scFv in any combination Therefore, whether or not they can be used in combination arbitrarily and independently, can.

[0137] Bottle opener type One heterodimer scaffold for which a specific application can be found in the present invention is shown in Figure 1A, A In the form of a "triple F" or "bottle opener" scaffold as shown in B. Yes. In this embodiment, one of the heavy chains of the antibody is a single-stranded Fv (as defined later, "sc"). The other heavy chain has a "regular" FAb form, which includes variable heavy and light chains. Yes, it exists. This structure has a rough visual resemblance to a bottle opener, so it is easy to understand. In detail, it is referred to as the "triple F" format (scFv-FAb-Fc) or the "bottle opener" format. This can sometimes happen (see Figure 1). The two chains are, as will be explained more thoroughly later, Hetian Constant regions that promote the formation of ronidimer antibodies (e.g., Fc domain, CH1 domain and It is coupled by using amino acid variants in the (or hinge region).

[0138] The "Triple F" configuration of the present invention has several obvious advantages. Furthermore, antibody analogs that rely on two scFv constructs have stability and aggregation problems. These are common, and in this invention, they are mitigated by the addition of "standard" heavy and light chain pairs. It is possible. Furthermore, for forms that depend on two heavy chains and two light chains, heavy chains and light In cases of incorrect chain pairing (for example, pairing of heavy chain 1 with light chain 2), there are no problems.

[0139] Many of the embodiments outlined herein are generally (but often) charged (t) containing variable heavy chain and variable light chain domains covalently linked using an scFv linker. It depends on the bottle opener form containing a first monomer, which includes scFv, in this case scF v is at the N-terminus of the first Fc domain, usually (as outlined herein, uncharged or (which can be either charged) are covalently linked via a domain linker. The second monomer in the Ner form is a heavy chain, and the composition further contains a light chain.

[0140] In general, in many preferred embodiments, scFv is a domain that binds to CD3. The Fabs of the heavy and light chains bind to the TTA of the other. Furthermore, the Fc domain of the present invention is Generally, distortion mutants (for example, a particularly useful distortion mutant is S364K / E357Q:L36) 8D / K370S;L368D / K370S:S364K;L368E / K370S:S 364K;T411T / E360E / Q362E:D401K;L368D / K370S Select from the group consisting of :S364K / E357L and K370S:S364K / E357Q If selected, the set of amino acid substitutions shown in Figures 29 and 34, optional selections Selectively cleaved mutants (including those shown in Figure 31), and arbitrarily charged scFv linkers. (Including those shown in Figure 33), and the heavy chain includes pI variants (including those shown in Figure 30). Includes (mu).

[0141] The present invention relates to the case where the anti-CD3 scFv sequence is as shown in Figures 2 to 7 and Figure 68. It provides a bottle opener.

[0142] The present invention relates to the case where the anti-CD38 sequence is as shown in the drawings, including Figures 8-10, and the CD38 anti-CD38 sequence. This provides a bottle opener form having a proto-binding domain.

[0143] The present invention relates to the CD20 antigen-binding domain when the anti-CD20 sequence is as shown in the drawing. It provides a bottle opener type.

[0144] The present invention relates to the CD19 antigen-binding domain when the anti-CD19 sequence is as shown in the drawing. It provides a bottle opener type.

[0145] The present invention relates to the CD123 antigen-binding domain when the anti-CD123 sequence is as shown in the drawings. It provides a bottle opener type that has a nozzle.

[0146] mAb-Fv format One heterodimer scaffold for which a specific application can be found in the present invention is shown in Figure 1. This is in mAb-Fv format. In this embodiment, this format is an "extra" variable heavy chain domain. C-terminal attachment to one monomer and C-terminal attachment of the "extra" variable light chain domain to the other monomer. By relying on the use of terminal attachment, a third antigen-binding domain is formed, where two The monomer's Fab portion binds to TTA, while the "extra" scFv domain binds to CD3. do.

[0147] In this embodiment, the first monomer has a first variable light chain domain that uses a domain linker. The first variable heavy chain domain and the first Fc domain are covalently bonded to the C-terminus of the first Fc domain. The first heavy chain contains a first constant heavy chain domain which includes an Fc domain. This includes a second variable heavy chain domain of a second constant heavy chain domain containing a second Fc domain and A third variable heavy chain is covalently bonded to the C-terminus of the second Fc domain using a domain linker. It includes a domain. The two variable domains attached to the C-terminus bind to the scFv of CD3. To form two identical Fabs that bond to the TTA, A common light chain, including variable light chain domains and constant light chain domains, which associate with the heavy chain, is further used. With respect to many of the embodiments herein, these structures are as desired herein. As described, this includes distortion mutants, pI mutants, truncation mutants, and additional Fc mutants. nothing.

[0148] The present invention relates to the case where the anti-CD3 scFv sequence is as shown in Figures 2 to 7 and Figure 68. Provides mAb-Fv format.

[0149] The present invention provides an mAb-Fv format in which the anti-CD38 sequence is shown in Figures 8-10. .

[0150] The present invention relates to an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the drawing. Provides mAb-Fv format.

[0151] The present invention relates to an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the drawing. Provides mAb-Fv format.

[0152] The present invention relates to an anti-CD123 sequence having a CD123 antigen-binding domain as shown in the drawing. It provides the mAb-Fv format.

[0153] The present invention provides an mAb-Fv format that includes cleavage mutants as shown in Figure 31.

[0154] The present invention proposes an mAb-Fv format that includes distorted variants as shown in Figures 29 and 34. To provide.

[0155] mAb-scFv One heterodimer scaffold for which a specific application can be found in the present invention is shown in Figure 1. This is in mAb-Fv format. In this embodiment, this format is in which scFv is added to one of the monomers. By relying on the use of C-terminal attachment, a third antigen-binding domain is formed, where two The Fab portion of the monomer binds to TTA, and the "extra" scFv domain binds to CD3. They combine. Therefore, the first monomer consists of the scFv variable light chain domain and the scFv linker. The first has an scFv covalently bonded to the C-terminus, which includes an scFv variable heavy chain domain. Includes heavy chains (including variable heavy chain domains and constant domains). In this embodiment, TTA The variable light chain domains associate with the heavy chain to form two identical Fabs that bind together. A common light chain containing a constant light chain domain is further used. In many of the embodiments described herein In relation to these constructs, distorted variants, pI, as desired and described herein, are available. This includes mutants, truncated mutants, and additional Fc mutants.

[0156] The present invention relates to the case where the anti-CD3 scFv sequence is as shown in Figures 2 to 7 and Figure 68. Provides mAb-Fv format.

[0157] The present invention provides an mAb-Fv format in which the anti-CD38 sequence is shown in Figures 8-10. .

[0158] The present invention relates to an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the drawing. Provides mAb-Fv format.

[0159] The present invention relates to an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the drawing. Provides mAb-Fv format.

[0160] The present invention relates to an anti-CD123 sequence having a CD123 antigen-binding domain as shown in the drawing. It provides the mAb-Fv format.

[0161] The present invention provides an mAb-Fv format that includes cleavage mutants as shown in Figure 31.

[0162] The present invention proposes an mAb-Fv format that includes distorted variants as shown in Figures 29 and 34. To provide.

[0163] Central scFv One heterodimer scaffold for which a specific application can be found in the present invention is shown in Figure 1. This is in Central-scFv format. In this embodiment, this format is inserted sc By relying on the use of the Fv domain, a third antigen-binding domain is formed, where 2 The Fab portion of the monomer binds to TTA, and the "extra" scFv domain binds to CD3. They bind. The scFv domain is between the Fc domain and one of the CH1-Fv regions of the monomer. The insertion provides a third antigen-binding domain.

[0164] In this embodiment, one monomer is an scFv variable light chain domain, an scFv linker Along with the scFv containing the scFv variable heavy chain domain, the first variable heavy chain domain, CH It contains a first heavy chain including one domain and an Fc domain. scFv is a heavy constant domain A domain linker is used between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain. They are covalently bonded. In this embodiment, two identical Fabs are formed that bond to the TTA. Therefore, a common light chain, which includes variable light chain domains and constant light chain domains that associate with the heavy chain, is further developed. Used for. With respect to many of the embodiments herein, these structures are used as herein. Desired and described, distorted mutants, pI mutants, truncated mutants, and additional Fc mutants. This includes, among others.

[0165] The present invention relates to the case where the anti-CD3 scFv sequence is as shown in Figures 2 to 7 and Figure 68. Provides Central-scFv format.

[0166] The present invention relates to a Central-scFv type anti-CD38 sequence as shown in Figures 8-10. Provide the formula.

[0167] The present invention relates to an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the drawing. Provides Central-scFv format.

[0168] The present invention relates to an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the drawing. Provides Central-scFv format.

[0169] The present invention has a CD123 antigen-binding domain such that the anti-CD123 sequence is represented by v. It provides the Central-scFv format.

[0170] The present invention relates to a Central-scFv format including a cleavage mutant as shown in Figure 31. provide.

[0171] The present invention includes Central-sc, which contains distorted variants as shown in Figures 29 and 34. Provides Fv format.

[0172] Central-Fv format One heterodimer scaffold for which a specific application can be found in the present invention is shown in Figure 1. This is the Central-Fv format. In this embodiment, the format is the inserted scFv By relying on the use of the domain, a third antigen-binding domain is formed, where two The monomer's Fab portion binds to TTA, while the "extra" scFv domain binds to CD3. The scFv domain is inserted between the Fc domain and the monomer's CH1-Fv region. This provides a third antigen-binding domain, where each monomer has a component of scFv. (For example, one monomer contains a variable heavy chain domain, and the other contains a variable light chain domain.) .

[0173] In this embodiment, one monomer has a first variable heavy chain domain, a CH1 domain and Includes a first heavy chain containing an Fc domain and additional variable light chain domains. Light chain domain This is because a dendritic is formed between the C-terminus of the CH1 domain of the hemistable domain and the N-terminus of the first Fc domain. Covalent bonding occurs using the main linker. The other monomer has a first variable heavy chain domain, C The first heavy chain, including the H1 domain and Fc domain, and additional variable heavy chain domains Includes. The light chain domain is the C-terminus of the CH1 domain of the heavy constant domain and the first Fc domain. It is covalently bonded to the N-terminus of using a domain linker.

[0174] In this embodiment, two identical Fabs are associated with the TTA to form two identical Fabs. Furthermore, a common light chain containing variable light chain domains and constant light chain domains is used. With respect to many of the embodiments in this specification, these structures are desired and described herein. This includes distorted mutants, pI mutants, truncated mutants, and additional Fc mutants.

[0175] The present invention relates to the case where the anti-CD3 scFv sequence is as shown in Figures 2 to 7 and Figure 68. Provides Central-Fv format.

[0176] The present invention relates to an anti-CD38 sequence in Central-Fv format as shown in Figures 8-10. provide.

[0177] The present invention relates to an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the drawing. Provides Central-Fv format.

[0178] The present invention relates to an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the drawing. Provides Central-Fv format.

[0179] The present invention relates to an anti-CD123 sequence having a CD123 antigen-binding domain as shown in the drawing. It provides the Central-Fv format.

[0180] The present invention provides a Central-Fv format that includes cleavage mutants as shown in Figure 31. do.

[0181] The present invention relates to Central-Fv, which includes distorted variants as shown in Figures 29 and 34. Provides the format.

[0182] Single-arm Central-scFv One heterodimer scaffold for which a specific application can be found in the present invention is shown in Figure 1. It is a single-arm Central-scFv type. In this embodiment, one monomer is One monomer contains only the Fc domain, while the other monomer uses the inserted scFv domain. This forms a second antigen-binding domain. In this form, the Fab portion is TTA Either scFv is coupled to CD3 and scFv is coupled to CD3, or vice versa. The main component is inserted between the Fc domain and one of the CH1-Fv regions of the monomer.

[0183] In this embodiment, one monomer is an scFv variable light chain domain, an scFv linker Along with the scFv containing the scFv variable heavy chain domain, the first variable heavy chain domain, CH It contains a first heavy chain including one domain and an Fc domain. scFv is a heavy constant domain A domain linker is used between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain. They are covalently bonded. The second monomer contains an Fc domain. In this embodiment, Fab forms To achieve this, a light chain containing variable light chain domains and constant light chain domains that associate with the heavy chain is formed. They are used in many of the embodiments described herein. With respect to many of the embodiments described herein, these structures are used in this specification. Desired and described, distorted mutants, pI mutants, truncated mutants, and additional Fc mutations. Includes the body, etc.

[0184] The present invention relates to the case where the anti-CD3 scFv sequence is as shown in Figures 2 to 7 and Figure 68. Provides a single-arm central-scFv format.

[0185] The present invention relates to a single-arm central-anti-CD38 array as shown in Figures 8-10. Provides the scFv format.

[0186] The present invention relates to an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the drawing. It provides a single-arm Central-scFv format.

[0187] The present invention relates to an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the drawing. It provides a single-arm Central-scFv format.

[0188] The present invention relates to an anti-CD123 sequence having a CD123 antigen-binding domain as shown in the drawing. It provides a single-arm Central-scFv format.

[0189] The present invention relates to a single-arm Central-sc including a truncated mutant as shown in Figure 31. Provides Fv format.

[0190] The present invention relates to a single-arm Centr including a distorted variant as shown in Figures 29 and 34. Provides the al-scFv format.

[0191] Dual scFv format The present invention also relates to a dual scFv format known in the art and shown in Figure 1. To provide.

[0192] The present invention relates to the case where the anti-CD3 scFv sequence is as shown in Figures 2 to 7 and Figure 68. Provides a dual scFv format.

[0193] The present invention provides an anti-CD38 sequence in a dual scFv format as shown in Figures 8-10. .

[0194] The present invention relates to an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the drawing. Provides a dual scFv format.

[0195] The present invention relates to an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the drawing. Provides a dual scFv format.

[0196] The present invention relates to an anti-CD123 sequence having a CD123 antigen-binding domain as shown in the drawing. It provides a dual scFv format.

[0197] The present invention provides a double scFv format including a cleavage mutant as shown in Figure 31.

[0198] The present invention proposes a double scFv format including distorted variants as shown in Figures 29 and 34. To provide.

[0199] target antigen The bispecific antibody of the present invention has two different antigen-binding domains, namely CD3. Those that bind to a target tumor antigen (generally monovalent), and those that bind to a target tumor antigen (referred to herein as "TTA"). It has (sometimes referred to as) suitable target tumor antigens, but is not limited to CD. 20, CD38, CD123; ROR1, ROR2, BCMA; PSMA; SSTR2; SSTR5, CD19, FLT3, CD33, PSCA, ADAM17, CEA, Her 2, EGFR, EGFR-vIII, CD30, FOLR1, GD-2, CA-IX, T rop-2, CD70, CD38, Mesoserine, EphA2, CD22, CD79b, G PNMB, CD56, CD138, CD52, CD74, CD30, CD123, RON Examples include ERBB2 and EGFR.

[0200] The "triple F" format is particularly useful when targeting two (or more) different antigens. It is advantageous (as outlined herein, this targeting is monovalent and divalent, depending on the form) (Any combination of bindings is possible). Therefore, immunoglobulins in this specification are preferred Each monomer co-associates with two target antigens. The specificity of each monomer is listed herein. A bispecific form that is even more useful for applications involving an anti-CD3 binding domain is shown in Figure. As shown in 1.

[0201] A particularly preferred application of the heterodimer antibodies described herein is one in which they monovalently associate with each target antigen. This is a co-target pair when it is advantageous or important. Such antigens are, for example, immune complex forms. These may be immune receptors that are activated in adulthood. The cellular activation of numerous immune receptors is simply, Typically, this occurs through crosslinking by antibody / antigen immune complexes, or through the association of target cells. It is mediated through effector cells. Some immune receptors, such as CD3 receptors on T cells, are involved. In Gunal signaling receptors, activation only occurs upon association with co-associated targets, which is inconsistent with clinical settings. Specific cross-linking is important because it can induce cytokine storms and toxicity. Activation is performed using immunoglobulins as described herein to therapeutically interact with such antigens in a non-multivalent manner. By valence association, it occurs only in the microenvironment of the primary target antigen, and is generated simply in response to crosslinking. The ability to target two different antigens with different binding titers is novel and useful in this invention. This is the form in which the target may be therapeutically advantageous or necessary for monovalent co-association. Examples of antigens, though not limited to them, include immune-activating receptors such as CD3, FcγR, and TL. Toll-like receptors (TLRs) such as R4 and TLR9, cytokines, chemokines, etc. Examples include itokine receptors and chemokine receptors. In many embodiments, antigen receptors One of the bonding sites is bonded to CD3, and in some embodiments, it is an scFv-containing monomer. be.

[0202] Not limited to, but both cytokines and membrane-binding factors (including transmembrane receptors) The following is a list of target antigens containing soluble factors: 17-IA, 4-1BB, 4Dc, 6- Keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RI B ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, AD AMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, AL K, ALK-1, ALK-7, α-1-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-ID, ASPARTIC, atrial natriuretic factor, AV / B3 INT Glyn, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulant (Bly S), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK , Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFG F, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 B MP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BM P-6 Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2) , BMPR, BMPR-IA(ALK-3), BMPR-IB(ALK-6), BRK- 2. RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, cylinder Syn, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3) C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cA MP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cateph Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin シンO, カテプシンS, カテプシンV, カテプシンX / Z / P, CBL, CCI, CCK 2. CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15 ,CCL16,CCL17,CCL18,CCL19,CCL2,CCL20,CCL2 1. CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CC L28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 1 0. CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CC R5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD1 1c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD 21. CD22, CD23, CD25, CD27L, CD28, CD29, CD30, C D30L, CD32, CD33(p67ンパクquality), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD5 5. CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), C D89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGM P, CINC, Clostridium botulinum toxin, ウ Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG -2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6 , CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, C XCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-related Antigen, DAN, DCC, DcR3, DC-SIGN, Degradation-promoting factor, des(1-3)- IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, deoxyribonucle Aze, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, E DA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1 EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET- 1. Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation Protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-1 9, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3(Vgr-2), GDF-5(BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3) ), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF , GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, G ITR, Glucagon, Glut 4, Glycoprotein IIb / IIIa (GPIIb / II Ia) GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, haptic (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB enzyme Envelope glycoprotein, HCMV)gH Envelope glycoprotein, HCMV UL, Blood growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu(ErbB-2), Her3(ErbB-3), Her4(ErbB-4), simple Herpesvirus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, High molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV III B gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM HRG, Hrk, human cardiac myosin, human cytomegalovirus (human cyt Omegalovirus (HCMV), Human Growth Hormone (HGH), HVEM, I -309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN g, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IG FBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2 R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18 R, IL-23, interferon (INF)-α, INF-β, INF-γ, inhibitor Insulin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikre In L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Ke Latinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP(TGF-1) ), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, L efty, Lewis Y antigen, Lewis Y-related antigen, LFA-1, LFA-3, Lfo, LIF LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin β-receptors, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MC AM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEA SES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP , MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-1 1, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP- 24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian duct inhibitor, Mug, MuSK, N AIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, Nepri Lysin, neurotrophin-3, -4, or -6, neuroturin, nerve cell compound long factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG- 3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, P BSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, PE CAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, embryo Disc alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin Prorelaxin, Protein C, PS, PSA, PSCA, Prostate-Specific Membrane Antigen (PSM) A), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RAN TES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory multinucleated virus Respiratory syncytial virus (RSV)F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, S100, S CF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SI GIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, S PARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-7 2 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, T ERT, TfR, TGF, TGF-α, and TGF -β, TGF-β Panactivator, TGF-β RI(ALK-5), TGF-β RI I, TGF-β RIIb, TGF-β RIII, TGF-β1, TGF-β2, and TG F-β3, TGF-β4, TGF-β5, glycoprotein, Ck-1, and Ck-1 Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2 TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF- RII、TNFRSF10A(TRAIL R1 Apo-2、DR4)、TNFRSF 10B(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B )、TNFRSF10C(TRAIL R3 DcR1、LIT、TRID)、TNFR SF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RA NK ODF R、TRANCER)、TNFRSF11B(OPG OCIF、TR1 )、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI) 、 、 TNFRSF13C(BAFF R)、TNFRSF14(WHAT ATAR、Hve A, LIGHT R, TR2, TNFRSF16(NGFR p75NTR), TNF RSF17(BCMA) TNFRSF18(GITR AITR) TNFRSF19 (TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSF1 A(TNF RI CD120a、p55-60)、TNFRSF1B(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFCR), TNFRSF4(OX40 ACT35 , TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1B B CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTR AIL R2 TNFRH2), TNFRST23(DcTRAIL R1TNFRH1 ), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL -1) TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWE AK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL 2), TNFSF13B(BAFF BLYS, TALL1, THANK, TNFSF2 0), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (T L1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (T NF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TN FSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fasri Gandr (Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand C) D70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB) Ligand (CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAI LR, TRAIL-R1, TRAIL-R2, TRANCE, Transferring Receptor Body, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor Related antigen expression: Lewis Y-related carbohydrates, TWEAK, TXB2, Ung, uPAR, uPA R-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE -Cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR -3(flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA- 4. VNR integrin, Von Willebrand factor, WIF-1, WNT1, WNT 2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, W NT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL 2. XCR1, XCR1, XEDAR, XIAP, XPD, and hormones and growth Proteins, subunits, domains, motifs, and / or receptors for factors that belong to the receptor. Alternatively, substantially any antigen including an epitope may be labeled by immunoglobulins as herein. It may be targeted.

[0203] Examples of antigens that can be specifically targeted by the immunoglobulins of the present invention include, but are not limited to, Although not performed, CD20, CD19, Her2, EGFR, EpCAM, CD3, FcγR IIIa (CD16), FcγRIIa (CD32a), FcγRIIb (CD32b) Toll-like receptors (TLRs) such as FcγRI (CD64), TLR4, and TLR9 , IL-2, IL-5, IL-13, IL-12, IL-23, and TNFα and other Cytokine receptors such as itokines, IL-2R, chemokines, chemokine receptors, V Examples include growth factors such as EGF and HGF. The present invention forms a bispecific antibody. Therefore, antibodies can be produced against any combination of these antigens, that is, Each of these antigens may be optionally and independently included from the bispecific antibodies according to the present invention. Alternatively, they can be excluded.

[0204] A particularly preferred combination in bispecific antibodies is the antigen-binding domain to CD3. , as well as selected from domains that bind to CD19, CD20, CD38, and CD123. These are antigen-binding domains, and their sequences are shown in the diagram.

[0205] The nucleic acid of the present invention The present invention further provides nucleic acid compositions encoding the bispecific antibodies of the present invention. As understood by this, nucleic acid compositions are in the form of heterodimeric proteins and scans. It will depend on the fold. Therefore, for example, the form is, for example, a triple F form (for example) For example, the first amino acid monomer containing the Fc domain and scFv, and the second containing the heavy chain and light chain. If a sequence of three amino acids is required as two amino acid monomers, then for expression, three amino acids Nucleic acid sequences can be incorporated into one or more expression vectors. Similarly, some forms (e.g., Figure) In the dual scFv format disclosed in 1, only two nucleic acids are required, and furthermore, These can be inserted into one or two expression vectors.

[0206] As is known in the art, the nucleic acids encoding the components of the present invention are, in the art Known expression levels of host cells used to produce the heterodimer antibody of the present invention It can be incorporated into a promoter. Generally, nucleic acids have several regulatory elements (promoters, multiple (The starting point, selectable markers, ribosome binding sites, inducing factors, etc.) are operably linked. The expression vector can be extrachromosomal or embedded.

[0207] Next, the nucleic acids and / or expression vectors of the present invention have applications in numerous embodiments. along with mammalian cells (e.g., CHO cells), mammals, bacteria, yeast, insects and / or This involves several different types of host cells, including fungal cells, as are well known in the art. It will be transformed.

[0208] In some embodiments, the nucleic acid encoding each monomer and the light chain are optionally encoded Nucleic acids, when applicable according to their form, generally have different or the same promoter. It is contained within a single expression vector under control. In the embodiment of the specific application of the present invention, Each of these two or three nucleic acids is contained on a different expression vector. and U.S. Patent Application No. 62 / 025,931 (as incorporated herein by reference) As shown, different vector ratios can be used to drive heterodimer formation. Yes, it is possible. In other words, surprisingly, proteins consist of a first monomer: a second monomer: a light chain (hetero). In many embodiments of this specification, which have three polypeptides containing a dimeric antibody, 1 While they are included in a 1:2 ratio, these are not the ratios that yield the best results. Error! The reference is See "Not Found".

[0209] The heterodimer antibody of the present invention is a host containing an expression vector, as is well known in the art. It is produced by culturing cells. Once produced, ion exchange chromatography Conventional antibody purification steps, including the tepp, are performed. As discussed herein, at least Also, having two monomers with a pI of 0.5 different, ion exchange chromatography and These can be separated by isoelectric focusing electrophoresis or other methods sensitive to isoelectric focusing. That is, each monomer has a different isoelectric point (pI), and each monomer has a different pI, and is heterodimeric By including pI substitutions that modify the plant to have different pIs, a "triple F" heterozygous can be created. This facilitates the isoelectric point purification of dimers (e.g., using anion exchange columns or cation exchange columns). These substitutions also remove any contaminants from the double scFv-Fc and mAb homogeneous compounds after purification. To assist in the measurement and monitoring of mers (e.g., IEF gels, cIEF, and IEX for analysis). column).

[0210] treatment The compositions of the present invention, once prepared, find applications in several fields. CD20 CD38 and CD123 are all derived from numerous hematopoietic malignancies and various hematopoietic malignancies. Uncontrolled in cell lines, therefore, the heterodimer antibody of the present invention is effective against cancer, for example, limited This does not apply to all B-cell lymphomas and leukemias, for example, but not limited to non-Hodgkin NHL lymphoma, Burkitt lymphoma (BL), multiple myeloma (MM), B-chronic lymphoma Lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T-cell lymphocyte TCL (Toxic Myeloid Leukemia), Acute Myeloid Leukemia (AML), Hairy Cell Leukemia (HCL), Hodgkin's disease Lymphoma (HL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, and chronic bone It has been found to have applications in the treatment of myelic leukemia (CML).

[0211] Therefore, the heterodimer composition of the present invention has found applications in the treatment of these cancers. It will be done.

[0212] Antibody composition for in vivo administration The antibody preparations used in accordance with the present invention are available for storage in the form of lyophilized preparations or aqueous solutions. In this state, antibodies having the desired purity can be optionally supplied to pharmaceutically acceptable carriers and excipients. It is prepared by mixing it with a stabilizer (Remington's Pharma Ceutical Sciences 16th edition,Osol,A.Ed

[1980] ). Acceptable carriers, excipients, or stabilizers are permitted in the amount and concentration used. It is non-toxic to the recipient at a certain degree, and does not contain phosphates, citrates, or other organic acids. Buffers; antioxidants including ascorbic acid and methionine; preservatives (e.g., octade ammonium sildimethylbenzyl chloride; hexamethonium chloride; benzalkonium chloride , benzethonium chloride; phenol, butyl or benzyl alcohol; methyl or This includes alkylparabens such as propylparaben; catechol; resorcinol; cyclohex Sanol; 3-pentanol; and m-cresol; low molecular weight (less than approximately 10 residues) Lipeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as lyvinylpyrrolidone; glycine, glutamine, asparagine, histamine Amino acids such as thidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, e.g. For example, glucose, mannose, or dextrin; chelating agents such as EDTA; sucrose Sugars such as rosin, mannitol, trehalose, or sorbitol; sodium, etc. Salt-forming counterions; metal complexes (e.g., Zn-protein complexes); and / or tweezers Non-components such as Pluronic (trademark), Pluronic (trademark), or polyethylene glycol (PEG). Contains ionic surfactants.

[0213] The formulations herein may also include two or more activations as needed for specific signs during treatment. The mixture may also contain compounds, preferably those having complementary activities that do not have harmful effects on each other. For example, it may be desirable to provide antibodies with other specificities. Or, Furthermore, this composition contains cytotoxic agents, cytokines, growth inhibitors and / or small molecules. It may contain an antagonist. Such molecules may preferably coexist in an amount effective for the intended purpose. ru.

[0214] The active ingredient also contains colloidal drug delivery systems (e.g., liposomes, albumin micros). (Fairs, microemulsions, nanoparticles and nanocapsules) or macroemulsions For example, microcapsules prepared by coacervation technology or interfacial polymerization. For example, hydroxymethylcellulose or gelatin-microcapsules and por It may also be encapsulated in methyl methacrylate microcapsules. Such a technique is Rem ington's Pharmaceutical Sciences 16th ed. This is disclosed in tion, Osol, A. Ed. (1980).

[0215] Preparations intended for in vivo administration should be sterile or nearly sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0216] A sustained-release formulation may be prepared. A suitable example of a sustained-release formulation is a solid hydrophobic polymer containing an antibody. One example is a semipermeable matrix, where the matrix is ​​used in molded products, such as films. Or it is in the form of microcapsules. Examples of sustained-release matrices include polyester, and Hydrogel (for example, poly(2-hydroxyethyl methacrylate), or poly( Vinyl alcohol, polylactic acid (U.S. Patent No. 3,773,919), L-glucose Copolymer of tamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate Lactic acid-glycolic acid copolymer, for example, Leupron Depot (trademark) (lactic acid-glycolic acid copolymer). Injectable microspheres consisting of licoleic acid copolymer and leuprolide acetate, and Examples include poly-D-(-)-3-hydroxybutyric acid. Ethylene vinyl acetate and milk While polymers such as acid-glycolic acid allow for molecular release over more than 100 days, A specific type of hydrogel releases proteins for a shorter period of time.

[0217] When encapsulated antibodies remain in the body for a long period, they change as a result of being exposed to water at 37°C. It may become symmetric or aggregate, leading to decreased biological activity and possible changes in immunogenicity. For stabilization, rational strategies can be devised that depend on the mechanisms involved. For example, the aggregation mechanism. It was discovered that this is the formation of intermolecular S-S bonds through thio-disulfide exchange. For synthesis and stabilization, the sulfhydryl residues are modified, and the mixture is freeze-dried from an acidic solution to control the moisture content. By controlling the process, using appropriate additives, and developing specific polymer matrix compositions, And it can be achieved.

[0218] Mode of administration The antibodies and chemotherapeutic agents of the present invention can be administered by known methods, for example, intravenously as a bolus. Therefore, or through continuous infusion over a long period, it can be administered intramuscularly, intraperitoneally, intracerebrospinal fluid, subcutaneously, and jointly. The drug is administered to the subject via internal, intrabursal, subarachnoid, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred.

[0219] Treatment methods The present invention provides a therapeutic method that offers a positive therapeutic response to a disease or condition. It is used. "Positive therapeutic response" is an improvement in a disease or condition, and / or disease. Alternatively, improvement in symptoms related to the condition is intended. For example, if the treatment response is positive. , the following improvements in the disease, namely (1) a reduction in the number of neoplastic cells; (2) reduction in neoplastic cell death (3) Increase in tumor cells; (5) Inhibition of tumor growth (i.e., a certain degree of slow growth) (6) Slowing down, preferably stopping; (7) Increase in patient survival rate; and (8) Related to disease or condition This refers to one or more partial reliefs from a series of related symptoms.

[0220] A positive therapeutic response in any given disease or condition is not specific to that disease or condition. Tumor response can be determined by standardized response criteria. Tumor response is assessed using magnetic resonance imaging (M RI scan, X-ray imaging, computed tomography (CT) scan, bone scanning Canal imaging, endoscopy, and tumor biopsy sampling including bone marrow aspiration (BMA) Screening techniques such as counting of tumor cells in circulation are used to determine tumor morphology. Changes (i.e., total tumor burden, tumor size, etc.) can be evaluated.

[0221] In addition to these positive therapeutic responses, subjects receiving treatment also experienced symptoms related to the disease. You can experience the beneficial effects of such improvements.

[0222] Improvement in the disease may be characterized as complete remission. "Complete remission" is a term used to describe the symptoms of myeloma. In the example, any past abnormal X-ray examination, bone marrow, and cerebrospinal fluid (CSF) or Along with the normalization of abnormal monoclonal proteins, there is no clinically detectable disease. This is intended to be the case.

[0223] Such a response may occur for at least 4 to 8 weeks, or sometimes as follows, after treatment according to the method of the present invention. It can last for 6-8 weeks. Alternatively, improvement in the disease can be classified as partial remission. It is permissible. "Partial remission" may last for 4-8 weeks, or 6-8 weeks, and new lesions may not appear. In the absence of any measurable tumor burden (i.e., malignant cells present in the subject), In terms of the number, or the amount of large tumors or abnormal monoclonal proteins measured) A reduction of at least approximately 50% is intended.

[0224] The treatment according to the present invention includes a "therapeutic effective dose" of the drug used. The "therapeutic effective dose" is determined by the following: This refers to the effective dose and duration required to achieve the desired treatment outcome.

[0225] The effective therapeutic dose depends on factors such as the individual's medical condition, age, sex, and weight, as well as the individual's response to the drug. The effective therapeutic dose may vary depending on the ability to induce the desired response. or in cases where the therapeutically beneficial effects of the antibody portion outweigh any toxic or harmful effects. be.

[0226] In tumor treatment, the "effective dose" is also measured by its ability to stabilize disease progression. It may be determined. The ability of cancer-inhibiting compounds to predict efficacy in human tumors in animals. It can be evaluated positively in terms of modeling.

[0227] Alternatively, this property of the composition inhibits cell growth or induces apoptosis. The compound's capabilities are evaluated by testing them in a known in vitro assay performed by a skilled practitioner. It may be done. The therapeutically effective dose of the therapeutic compound may reduce tumor size, or instead It may alleviate the symptoms in the subject. A person skilled in the art would know the size of the subject and the subject's symptoms. The amount to be determined is based on factors such as the severity and the specific composition or route of administration selected. This will allow us to determine it.

[0228] The administration plan is adjusted to produce the desired optimal response (e.g., therapeutic response). For example, as indicated in emergency situations regarding treatment, a single bolus may be administered, or several doses. The dose may be administered over time, or the dose may be proportionally reduced or increased. i. Parenteral compositions are formulated in dose-unit form for ease of administration and dose uniformity. This is also acceptable. When used herein, the dose unit form is a single unit for the subject to be treated. This refers to physically separated units suitable as a dose, and each unit consists of the required pharmaceutical carrier and It contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in relation to the above. .

[0229] The specifications for the dosage unit form of the present invention are (a) the inherent properties of the active compound and the achieved (b) For the purpose of treating specific therapeutic effects, and for the treatment of susceptibility in the individual, such activation Designated by the specific limitations in the relevant technical field for compounding compounds, and directly relating to them It depends on.

[0230] The effective dose and administration plan for the bispecific antibody used in this invention are determined to treat The appropriate treatment may depend on the disease or condition and may be determined by a person skilled in the art.

[0231] The exemplary, non-limiting range of the therapeutically effective dose of the bispecific antibody used in this invention is approximately 0.1-100 mg / kg, for example, approximately 0.1-50 mg / kg, for example, approximately 0.1-20 mg g / kg, for example, approximately 0.1-10 mg / kg, for example, approximately 0.5, for example, approximately 0.3, approximately 1, Or approximately 3 mg / kg. In another embodiment, the antibody is administered in doses of 1 mg / kg or more, e.g. For example, a dose of 1-20 mg / kg, for example, a dose of 5-20 mg / kg, for example, 8 mg / kg It is administered in the following dosage.

[0232] Medical professionals with ordinary skills in the relevant technical field may obtain the required pharmaceutical composition. The efficacy can be easily determined and prescribed. For example, a physician or veterinarian can easily determine the dosage and prescribe medicine. The level at which the drug used in the composition is administered is required to obtain the desired therapeutic effect. You can start at a lower level and gradually increase the dose until the desired effect is achieved. .

[0233] In one embodiment, the bispecific antibody is 10-500 mg / kg, for example, 200-400 It is administered by infusion at a weekly dose of mg / kg. Such administration is, for example, 1 to 8 times, for example, 3 The administration may be repeated up to 5 times. The administration should be over a period of 2 to 24 hours, for example, 2 to 12 hours. This may be done by continuous infusion.

[0234] In one embodiment, when it is necessary to reduce side effects, including toxicity, bispecific antibodies are used, for example It is administered by slow, continuous infusion over a long period of time, such as more than 24 hours.

[0235] In one embodiment, the bispecific antibody is 250 mg to 2000 mg, for example, 300 mg. Weekly doses of 500mg, 700mg, 1000mg, 1500mg, or 2000mg. The dosage is administered up to 8 times, for example, 4 to 6 times. The administration interval is 2 to 24 hours, for example, 2 to 12 hours. It may be carried out by continuous infusion over an intervening period. Such a regimen may be, for example, 6 months. The treatment may be repeated once or more times as needed, after 12 months. The dosage is determined by the amount of the active ingredient in the blood at the time of administration. The amount of the compound is, for example, obtained by taking a biological sample and targeting the antigen-binding region of the bispecific antibody. By measuring using anti-idiotype antibodies that transform into idiotypes, the determination or regulation can be made. That's fine.

[0236] In further embodiments, the bispecific antibody is used for 2 to 12 weeks, for example, 3 to 10 weeks, for example It is administered once a week for 4 to 8 weeks.

[0237] In one embodiment, the bispecific antibody is administered once a week for a period of, for example, 6 months or more. It is administered as part of maintenance therapy.

[0238] In one embodiment, the bispecific antibody is injected once and then radioactively It is administered by a regimen that includes the injection of bispecific antibodies compounded with isotopes. The treatment can be repeated, for example, after 7 to 9 days.

[0239] As a non-limiting example, treatment according to the present invention may be a single treatment or 24, 12, 8, 6, 4, or Approximately 0.1 to 100 per day, using divided doses every two hours or any combination thereof. mg / kg, for example, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60 The daily dose of antibody is 70, 80, 90, or 100 mg / kg, at the start of treatment. After, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, Small At least once, or instead, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 At least once in weeks 2, 13, 14, 15, 16, 17, 18, 19, or 20, They may also be provided as any combination thereof.

[0240] In some embodiments, the bispecific antibody molecule is used as one or more additional therapeutic agents, for example It is used in combination with chemotherapeutic agents. A non-exclusive example of a DNA-damaging chemotherapeutic agent is topoisomerase I Inhibitors (e.g., irinotecan, topotecan, camptothecin and their analogues) (e.g., metabolites and doxorubicin); topoisomerase II inhibitors (e.g., etopo (e.g., melphalan, ku) Lorambucil, Busulfan, Thiotepa, Ifosfamide, Carmustine, Lomustine, Semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA interfering substances (e.g., cisplatin, oxaliplatin) (e.g., bryo) and carboplatin; DNA interfering agents and free radical generators, e.g., bryo Mycin; and nucleoside mimetics (e.g., 5-fluorouracil, capesic) Bin, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pe Examples include antstatins and hydroxyureas.

[0241] As chemotherapeutic agents that disrupt cell replication, paclitaxel, docetaxel, and related products are used. Similar compounds; vincristine, vinblastine, and related analogues; thalidomide, lenalidomide D, and related analogues (e.g., CC-5013 and CC-4047); protein ch Rosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteaso NF-κB inhibitors (e.g., bortezomib); NF-κB inhibitors including IκB kinase inhibitors It binds to proteins that are overexpressed in cancer, thereby downregulating cell replication. Antibodies (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab); and proteins known to be upregulated, overexpressed, or activated in cancer Other inhibitors of the enzyme (whose inhibition downregulates cell replication) are also available.

[0242] In some embodiments, the antibody of the present invention is used with Velcade® (bortezomib). It can be used before, during, or after treatment.

[0243] All cited references are explicitly invoked herein by reference in their entirety. It will be done.

[0244] While specific embodiments of the present invention are described for illustrative purposes, there are a great many variations in detail. However, this can be done without departing from the present invention as described in the attached claims. This will be understood by those skilled in the art. [Examples]

[0245] Examples illustrating the present invention are provided below. These examples illustrate the present invention in an optional manner. This does not mean limiting to any specific application or operating theory of the present invention. The numbering of the steady-state region locations follows the EU index, such as Kabat. et al., 1991, Sequences of Proteins of Im Munological Interest,5th Ed.,United Stat es Public Health Service,National Institute (Utes of Health, Bethesda; referenced as a whole) Those skilled in the art with respect to antibodies will know that this practice involves discontinuous attachment within a specific region of the immunoglobulin sequence. It consists of a number and a normalized reference to a conserved position within the immunoglobulin family. They will understand what makes it possible. Therefore, defined by the EU index. The position of any given immunoglobulin does not necessarily correspond to its continuous sequence. It will become.

[0246] General and specific scientific and technological information is provided in U.S. Patent Application Publication No. 2015 / 0307629. , U.S. Patent Application Publication No. 2014 / 0288275 and International Publication No. 2014 / 1 No. 45806 (All of these, in whole and in particular the technology outlined therein, refer to (Explicitly referenced by) outlined in [reference].

[0247] Examples Example 1: Alternative format Production of bipolar bodies A schematic diagram of the anti-CD38 × anti-CD3 dual singularity is shown in Figure 1. The amino acid sequences as alternative forms of the isomorphs are listed in Figures 39 to 43. The DNA that codes for the three strands required is synthesized by gene synthesis (Blue Heron Prepared using Biotechnology (Bothell, Wash.), standard fraction Subcloning was performed into the expression vector pTT5 using subbiological techniques. Site-directed mutagenesis was achieved. From(QuikChange,Stratagene,Cedar Creek,Tex. ) or substitution introduced using either additional gene synthesis and / or subcloning. For expression, DNA was introduced into HEK293E cells, and the resulting protein was then processed. Rothene A affinity (GE Healthcare) and cation exchange chromatography The supernatant was purified using [a specific method]. The yield after protein A affinity purification is shown in Figure 35. Cation exchange chromatography purification is performed using a 50 mM MES wash / equilibrium buffer (p (H6.0) and elution buffer of 50mM MES (pH6.0 + 1M NaCl linear gradient) Using a HiTrap SP HP column (GE Healthcare) with the following properties: The process was performed (see Figure 36 for the chromatogram).

[0248] Reinduced T cell cytotoxicity Re-induction of CD38+RPMI8266 myeloma cell line with anti-CD38 × anti-CD3 bispecific cells Induced T-cell cytotoxicity (RTCC) was characterized in vitro. 10k RP MI8266 cells were incubated with 500k human PBMCs for 24 hours. TCC was measured by LDH fluorescence as shown in the figure (see Figure 37).

[0249] Example 2 Reinduced T cell cytotoxicity Anti-CD38 × anti-CD3 Fab-scFv-Fc dual singularity, CD38 + RPMI8 In vitro studies on re-induced T-cell cytotoxicity (RTCC) in 266 myeloma cell lines Characterized the cells. 40k RPMI8266 cells were compared with 400k human PBMCs for 96 hours. The samples were incubated for a period of time. RTCC was measured by flow cytometry as shown in the figure. (See Figure 44). CD4+ and CD8+T of CD69, Ki-67, and PI-9. Cellular expression was also characterized by flow cytometry, which is shown in Figure 45.

[0250] Mouse model of antitumor activity - Day 23, each of the four groups consisting of 5 NOD severe combined immunodeficiency γ (NSG) mice , 5 x 10 6 RPMI8226TrS tumor cells (multiple myeloma, luciferase-producing) The (represented) was transplanted via intravenous tail vein injection. On day 0, 10 × 10 units were placed in the peritoneal cavity of the mice. 6 of Human PBMCs were transplanted. After transplantation of the PBMC on day 0, the test substance was transferred weekly (on days 0 and 7), as shown in the figure. Administer by intraperitoneal injection at the dose level indicated in 4. The study design is further summarized in Figure 46. To monitor tumor growth, use an in vivo imaging system (IVIS®) to fully understand the process. Flux / mouse was monitored by measuring XmAb13551 and XmAb Both 15426 compounds showed substantial antitumor effects (see Figures 47 and 48).

[0251] Experiments in cynomolgus monkeys Cynomolgus monkeys were administered a single dose of a dual-specific anti-CD38 × anti-CD3 compound. (Anti-RSV × anti-C) D3 bispecificity controls were also included. The dose level was 20 μg / kJ (in the three independent studies). XmAb13551 at g (n=2), XmAb15426 at 0.5 mg / kg (n=3) 3 mg / kg of XmAb14702 (n=3), or 3 mg / kg of XmAb132 The result was 45 (anti-RSV × anti-CD3 control, n=3). The anti-CD38 × anti-CD3 bispecific group was 45. This rapidly depleted CD38+ cells in the peripheral blood (see Figure 49). Anti-CD38 × anti-CD The 3-dual singularity resulted in T cell activation, as measured by CD69 expression (Figure 50). (See reference). Serum levels of IL-6 were also measured (see Figure 51). Compared with XmAb13551. In general, XmAb15426 prolongs the duration of CD38+ cell depletion and T cell activation and It is noteworthy that this was accompanied by a decrease in IL-6 production levels.

[0252] XmAb15426 and XmAb14702 are 0.5 mg / kg and 3 mg respectively. The tests were conducted with single doses of / kg. Both antibodies showed good tolerability at these higher doses. This was consistent with moderate levels of IL-6 found in serum from treated monkeys. Furthermore, moderate XmAb15426, which has a certain degree of CD3 affinity, can be used at 0.5 mg / kg to treat CD38+ cells. This was compared to the original high-affinity XmAb13551 administered at 2, 5, or 20 μg / kg. This resulted in more effective depletion. Depletion with XmAb15426 was the best in prior tests. It was more persistent compared to the XmAb13551 in larger quantities (7 days vs. 2 days, respectively). In particular, the target Cell depletion was more pronounced with XmAb15426, but T cell activation (CD69, CD25 and PD1 derivations are even more effective than the 20 μg / kg XmAb13551 group. In monkeys treated with XmAb15426 administered at 5 times the normal dose, the levels were significantly lower. XmAb14702, which has low CD3 affinity, activates CD38+ cells and T cells. It had little effect on [the target].

[0253] These results suggest that T cell activation is regulated by attenuating CD3 affinity. This represents a promising method for improving the therapeutic window of bispecific antibodies that associate with T cells. This is demonstrated. This method shows antigen sink clearance with targets such as CD38. To overcome sink clearance, improve tolerance and administer higher doses. This expands the set of antigens suitable for targeted T-cell immunotherapy. It has the potential to do so by reducing the affinity for CD3. While 15426 effectively depletes CD38+ cells, its high-affinity counterpart, Xm This study demonstrates minimizing the CRS effect observed at doses equivalent to those of Ab13551. Furthermore, the present invention provides the following: [1] a) The first monomer, i) The first heavy chain, 1) The first variable heavy chain domain; 2) A first constant heavy chain containing a first Fc domain; and 3) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain The scF contains a domain linker which is covalently bonded to the C-terminus of the Fc domain. v, The first heavy chain including containing the first monomer; b) A second variable heavy chain domain and a second steady heavy chain including a second Fc domain, Including a second monomer containing a second heavy chain; c) Common light chain including variable light chain domain and constant light chain domain Includes, The first and second Fc domains are S364K / E357Q:L368D / K 370S;L368D / K370S:S364K;L368E / K370S:S364K ;T411T / E360E / Q362E:D401K;L368D / K370S:S36 Selected from the group consisting of 4K / E357L and K370S:S364K / E357Q. Having a set of amino acid substitutions, The first variable heavy chain domain and the variable light chain domain are the first target tumor antigen (TT) Combine with A), The second variable heavy chain domain and the variable light chain domain bind to the first TTA. ,and The aforementioned scFv binds to human CD3 (sequence number XX). Heterodimer antibody. [2] The aforementioned scFv is sequence number XX(scFv13551), sequence number XX(scFv15 426), sequence number XX(scFv13423) and sequence number XX(scFv1470 A heterodimer according to [1] having a polypeptide sequence selected from the group consisting of (2) antibody. [3] The first variable heavy chain domain and the variable light chain domain are CD19, CD20 and A TTA selected from the group consisting of and CD123, as described in [1] or [2] Heterodimer antibody. [4] a) The first monomer, i) The first heavy chain, 1) The first variable heavy chain domain; 2) A first constant heavy chain domain containing a first Fc domain; and 3) Covalently bonded to the C-terminus of the first Fc domain using a domain linker First variable light chain domain The first heavy chain including A first monomer containing; b) A second monomer, i) Second variable heavy chain domain; ii) A second constant heavy chain domain containing a second Fc domain; and iii) The second variable heavy chain domain uses a domain linker to connect the second Fc domain A third variable heavy chain domain covalently bonded to the main C-terminus. A second monomer containing; c) Common light chain including variable light chain domain and constant light chain domain Includes, The first and second Fc domains are S364K / E357Q:L368D / K 370S;L368D / K370S:S364K;L368E / K370S:S364K ;T411T / E360E / Q362E:D401K;L368D / K370S:S36 Selected from the group consisting of 4K / E357L and K370S:S364K / E357Q. Having a set of amino acid substitutions, The first variable heavy chain domain and the variable light chain domain bind to the first TTA. The second variable heavy chain domain and the variable light chain domain bind to the TTA, The second variable light chain domain and the third variable heavy chain domain bind to CD3. Heterodimer antibody. [5] The aforementioned scFv is sequence number XX(scFv13551), sequence number XX(scFv15 426), sequence number XX(scFv13423) and sequence number XX(scFv1470 The heterodimer described in [4] having a polypeptide sequence selected from the group consisting of (2) antibody. [6] The first variable heavy chain domain and the variable light chain domain are CD19, CD20 and A TTA selected from the group consisting of and CD123, as described in [4] or [5] Heterodimer antibody. [7] a) The first monomer, i) The first heavy chain, 1) The first variable heavy chain domain; 2) A first constant heavy chain containing a first CH1 domain and a first Fc domain; Bini 3) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain It includes and uses a domain linker to link the C-terminus of the CH1 domain and the first Fc domain scFv covalently bonded to the N-terminus of yin The first heavy chain including A first monomer containing; b) A second variable heavy chain domain and a second steady heavy chain including a second Fc domain , a second monomer containing a second heavy chain; c) Common light chain including variable light chain domain and constant light chain domain Includes, The first and second Fc domains are S364K / E357Q:L368D / K 370S;L368D / K370S:S364K;L368E / K370S:S364K ;T411T / E360E / Q362E:D401K;L368D / K370S:S36 Selected from the group consisting of 4K / E357L and K370S:S364K / E357Q. Having a set of amino acid substitutions, The first variable heavy chain domain and the variable light chain domain bind to the first TTA. The second variable heavy chain domain and the variable light chain domain bind to the TTA, The aforementioned scFv binds to human CD3. Heterodimer antibody. [8] The aforementioned scFv is sequence number XX(scFv13551), sequence number XX(scFv15 426), sequence number XX(scFv13423) and sequence number XX(scFv1470 The heterodimer described in [7] having a polypeptide sequence selected from the group consisting of (2) antibody. [9] The first variable heavy chain domain and the variable light chain domain are CD19, CD20 and A TTA selected from the group consisting of and CD123, as described in [7] or [8] Heterodimer antibody.

[10] a) The first monomer, i) The first heavy chain, 1) The first variable heavy chain domain; 2) A first constant heavy chain domain containing a first Fc domain; and 3) The second variable light chain domain uses a domain linker to connect the first steady heavy chain A covalent bond is formed between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain. The first variable light chain domain The first heavy chain including A first monomer containing; b) A second monomer, i) Second variable heavy chain domain; ii) A second constant heavy chain domain containing a second Fc domain; and iii) The second variable heavy chain domain uses a domain linker to connect the second Fc domain A third variable heavy chain domain covalently bonded to the main C-terminus. A second monomer containing; c) Common light chain including variable light chain domain and constant light chain domain Includes, The first and second Fc domains are S364K / E357Q:L368D / K 370S;L368D / K370S:S364K;L368E / K370S:S364K ;T411T / E360E / Q362E:D401K;L368D / K370S:S36 Selected from the group consisting of 4K / E357L and K370S:S364K / E357Q. Having a set of amino acid substitutions, The first variable heavy chain domain and the variable light chain domain bind to the first TTA. The second variable heavy chain domain and the variable light chain domain bind to the TTA, The second variable light chain domain and the third variable heavy chain domain bind to human CD3. ru, Heterodimer antibody.

[11] The aforementioned scFv is sequence number XX(scFv13551), sequence number XX(scFv15 426), sequence number XX(scFv13423) and sequence number XX(scFv1470 The heterodimer described in

[10] having a polypeptide sequence selected from the group consisting of (2) body antibodies.

[12] The first variable heavy chain domain and the variable light chain domain are CD19, CD20 and A TTA selected from the group consisting of and CD123 is connected to

[10] or

[11] . A heterodimer antibody.

[13] a) The first monomer, i) The first heavy chain, 1) The first variable heavy chain domain; 2) A first constant heavy chain containing a first CH1 domain and a first Fc domain; Bini 3) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain It includes and uses a domain linker to link the C-terminus of the CH1 domain and the first Fc domain scFv covalently bonded to the N-terminus of yin A first monomer containing a first heavy chain; b) A second monomer containing a second Fc domain; c) Light chains including variable light chain domains and constant light chain domains Includes, The first and second Fc domains are S364K / E357Q:L368D / K 370S;L368D / K370S:S364K;L368E / K370S:S364K ;T411T / E360E / Q362E:D401K;L368D / K370S:S36 Selected from the group consisting of 4K / E357L and K370S:S364K / E357Q. Having a set of amino acid substitutions, The first variable heavy chain domain and the variable light chain domain bind to the first antigen. The scFv binds to the second antigen. Heterodimer antibody.

[14] The aforementioned scFv is sequence number XX(scFv13551), sequence number XX(scFv15 426), sequence number XX(scFv13423) and sequence number XX(scFv1470 The heterodimer described in

[13] having a polypeptide sequence selected from the group consisting of (2) body antibodies.

[15] The first variable heavy chain domain and the variable light chain domain are CD19, CD20 and A TTA selected from the group consisting of and CD123 is connected to

[13] or

[14] . A heterodimer antibody.

[16] a) vlCDR1 having sequence GSSTGAVTTSNYAN (sequence number XX), sequence vlCDR2 with GTNKRAP (sequence number XX), and sequence ALWYSNHWV A variable light chain domain containing vlCDR3 having (Sequence ID XX); b) vhCDR1 having sequence TYAMN (sequence number XX), sequence RIRSKANNY vhCDR2 having ATYYADSVKG (sequence number XX), and sequence HGNFGD A variable heavy chain domain containing vhCDR3 having SYVSWFAY (sequence number XX) and An anti-CD3 antibody-binding domain containing this domain.

[17] The anti-CD3 antibody-binding domain described in

[16] is an scFv.

[18] The variable light chain domain has sequence L1.47 (sequence number XX), and the variable heavy chain The antibody according to

[16] or

[17] , having the main component sequence H1.32 (sequence number XX). CD3 antibody-binding domain.

[19] The scFv has the sequence H1.32_L1.47 (sequence number XX) as described in

[18] The anti-CD3 antibody binding domain.

[20]

[19] A nucleic acid composition encoding scFv. [twenty one] An expression vector comprising the nucleic acid composition described in

[20] . [twenty two]

[21] Host cells containing the expression vector described above. [twenty three] a) vlCDR1 having sequence GSSTGAVTTSNYAN (sequence number XX), sequence vlCDR2 with GTNKRAP (sequence number XX), and sequence ALWYSNHWV A variable light chain domain containing vlCDR3 having (Sequence ID XX); b) vhCDR1 having sequence TYAMN (sequence number XX), sequence RIRSKYNNYATYYADSVKG (sequence number XX) vhCDR2 has column number XX, and sequence HGNFGDEYVSWFAY (sequence number XX) A variable heavy chain domain containing vhCDR3 and An anti-CD3 antibody-binding domain containing this domain. [twenty four] The anti-CD3 antibody-binding domain described in

[23] is an scFv. [twenty five] The variable light chain domain has sequence L1.47 (sequence number XX), and the variable heavy chain The antibody described in

[23] or

[24] , having the main element sequence H1.89 (sequence number XX). CD3 antibody-binding domain.

[26] The scFv has the sequence H1.89_L1.47 (sequence number XX) as described in

[23] The anti-CD3 antibody binding domain.

[27]

[26] A nucleic acid composition encoding scFv as described above.

[28] An expression vector comprising the nucleic acid composition described in

[27] .

[29]

[28] Host cells containing the expression vector described.

[30] a) vlCDR1 having sequence GSSTGAVTTSNYAN (sequence number XX), sequence vlCDR2 with GTNKRAP (sequence number XX), and sequence ALWYSNHWV A variable light chain domain containing vlCDR3 having (Sequence ID XX); b) vhCDR1 having sequence TYAMN (sequence number XX), sequence RIRSKYNNY vhCDR2 having ATYYADSVKG (sequence number XX), and sequence HGNFGDPYVSWF A variable heavy chain domain containing vhCDR3 having AY (sequence number XX) and An anti-CD3 antibody-binding domain containing this domain.

[31] The anti-CD3 antibody-binding domain described in

[30] is an scFv.

[32] The variable light chain domain has sequence L1.47 (sequence number XX), and the variable heavy chain The antibody according to

[30] or

[31] , having the main element sequence H1.90 (sequence number XX) CD3 antibody-binding domain.

[33] The scFv has the sequence H1.90_L1.47 (sequence number XX) as described in

[30] The anti-CD3 antibody binding domain.

[34]

[33] A nucleic acid composition encoding scFv as described above.

[35] An expression vector comprising the nucleic acid composition described in

[34] .

[36]

[35] Host cells containing the expression vector described above.

[37] a) vlCDR1 having sequence GSSTGAVTTSNYAN (sequence number XX), sequence GTNKRAP (sequence number XX) vlCDR2 having number XX, and vl having sequence ALWYSNHWV (sequence number XX) A variable light chain domain containing CDR3; b) vhCDR1 having sequence TYAMN (sequence number XX), sequence RIRSKYNNY vhCDR2 having ATYYADSVKG (sequence number XX), and sequence HGNFGDSYVSWF A variable heavy chain domain containing vhCDR3 having DY (sequence number XX) and An anti-CD3 antibody-binding domain containing this domain.

[38] The anti-CD3 antibody-binding domain described in

[37] is an scFv.

[39] The variable light chain domain has sequence L1.47 (sequence number XX), and the variable heavy chain The antibody according to

[37] or

[38] , having the main component sequence H1.33 (sequence number XX). CD3 antibody-binding domain.

[40] The scFv has the sequence H1.33_L1.47 (sequence number XX) as described in

[38] . The anti-CD3 antibody binding domain.

[41]

[38] A nucleic acid composition encoding scFv as described above.

[42] An expression vector comprising the nucleic acid composition described in

[41] .

[43]

[42] Host cells containing the expression vector described.

[44] a) vlCDR1 having sequence GSSTGAVTTSNYAN (sequence number XX), sequence vlCDR2 with GTNKRAP (sequence number XX), and sequence ALWYSNHWV A variable light chain domain containing vlCDR3 having (Sequence ID XX); b) vhCDR1 having sequence TYAMS (sequence number XX), sequence RIRSKYNNY vhCDR2 having ATYYADSVKG (sequence number XX), and sequence HGNFGD A variable heavy chain domain containing vhCDR3 having SYVSWFAY (sequence number XX) and An anti-CD3 antibody-binding domain containing this domain.

[45] The anti-CD3 antibody-binding domain described in

[44] is an scFv.

[46] The variable light chain domain has sequence L1.47 (sequence number XX), and the variable heavy chain The antibody described in

[44] or

[45] , having the main element sequence H1.31 (sequence number XX). CD3 antibody-binding domain.

[47] The scFv has the sequence H1.31_L1.47 (sequence number XX) as described in

[46] The anti-CD3 antibody binding domain.

[48]

[47] A nucleic acid composition encoding scFv as described above.

[49] An expression vector comprising the nucleic acid composition described in

[48] .

[50]

[49] Host cells containing the expression vector described.

[51] a) The first monomer, i) the first Fc domain; and ii) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain It contains an anti-CD compound covalently bonded to the N-terminus of the Fc domain using a domain linker. 3 scFv A first monomer containing; b) A second monomer, i) Heavy chain variable domain; and ii) Heavy chain constant domain containing a second Fc domain A second monomer containing a heavy chain; c) Light chains including variable light chain domains and variable light constant domains Includes, The aforementioned anti-CD3 scFv is anti-CD3H1.32_L1.47 (sequence number XX), anti-C D3H1.89_L1.47 (sequence number XX), anti-CD3H1.90_L1.47 (sequence number) Select from the group consisting of (number XX) and anti-CD3H1.33_L1.47 (sequence number XX). And The heavily variable domain and the lightly variable domain bind to the TTA. Heterodimer antibody.

[52] The heterodimer antibody according to

[0051] , wherein the TTA is selected from the group consisting of CD19, CD20, and CD123.

[53] a) vlCDR1 having sequence RASWSVSYIH (sequence number XX), sequence ATSN vlCDR2 with LAS (sequence number XX), and sequence QQWTHNPPT (sequence number A variable light chain domain containing vlCDR3 having (number XX); b) vhCDR1 having sequence SYNMH (sequence number XX), sequence AIYPGNGAT vhCDR2 having SYSQKFQG (sequence number XX), and sequence SYYMGGDW A variable heavy chain domain containing vhCDR3 having YFDV (SEQ ID NO: XX) An anti-CD20 antibody-binding domain containing this domain.

[54] The variable light chain domain has the sequence C2B8L1.113 (sequence number XX), and the The variable heavy chain domain has sequence C2B8H1.202 (sequence number XX) as described in

[53] . The anti-CD20 antibody binding domain.

[55]

[53] A nucleic acid composition encoding the binding domain described above.

[56] An expression vector comprising the nucleic acid composition described in

[55] .

[57]

[56] Host cells containing the expression vector described above.

[58] a) vlCDR1 having sequence RASSSVSYIH (sequence number XX), sequence ATSN vlCDR2 with LAS (sequence number XX), and sequence QQWTSNPPT (sequence number XX). A variable light chain domain containing vlCDR3 having (number XX); b) vhCDR1 having sequence SYNMH (sequence number XX), sequence AIYPGNGDT vhCDR2 having SYNQKFQG (sequence number XX), and sequence STYYGGDW A variable heavy chain domain containing vhCDR3 having YFNV (SEQ ID NO: XX) An anti-CD20 antibody-binding domain containing this domain.

[59] The variable light chain domain has sequence C2B8L1 (sequence number XX), and the variable heavy chain The anti-CD20 anti- Body-binding domain.

[60] A nucleic acid composition encoding the binding domain described in

[58] .

[61] An expression vector comprising the nucleic acid composition described in

[60] .

[62]

[61] Host cells containing the expression vector described above.

[63] a) The first monomer, i) the first Fc domain; and ii) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain It contains an anti-CD compound covalently bonded to the N-terminus of the Fc domain using a domain linker. 3 scFv is included in the first monomer; b) A second monomer, i) Heavy chain variable domain; and ii) Heavy chain constant domain containing a second Fc domain A second monomer containing a heavy chain; c) Light chains including variable light chain domains and variable light constant domains Includes, The aforementioned variable light chain domain is vlC having the sequence RASSSVSYIH (sequence number XX). DR1, vlCDR2 with sequence ATSNLAS (sequence number XX), and sequence QQW Includes vlCDR3 having TSNPPT (sequence number XX), and The variable heavy chain domain has vhCDR1 having sequence SYNMH (sequence number XX), vhCDR2 having column AIYPGNGDTSYNQKFQG (sequence number XX), and Includes vhCDR3 having sequence STYYGGDWYFNV (sequence number XX), Heterodimer antibody.

[64] a) The first monomer, i) the first Fc domain; and ii) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain It contains an anti-CD compound covalently bonded to the N-terminus of the Fc domain using a domain linker. 3 scFv A first monomer containing; b) A second monomer, i) Heavy chain variable domain; and ii) Heavy chain constant domain containing a second Fc domain A second monomer containing a heavy chain; c) Light chains including variable light chain domains and variable light constant domains Includes, The aforementioned variable light chain domain is vlC having the sequence RASSSVSYIH (sequence number XX). DR1, vlCDR2 with sequence ATSNLAS (sequence number XX), and sequence QQW Includes vlCDR3 having TSNPPT (sequence number XX), and The variable heavy chain domain has vhCDR1 having sequence SYNMH (sequence number XX), vhCDR2 having column AIYPGNGDTSYNQKFQG (sequence number XX), and Includes vhCDR3 having sequence STYYGGDWYFNV (sequence number XX), Heterodimer antibody.

[65] a) The first monomer, i) the first Fc domain; and ii) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain It contains an anti-CD compound covalently bonded to the N-terminus of the Fc domain using a domain linker. 3 scFv A first monomer containing; b) A second monomer, i) Heavy chain variable domain; and ii) Heavy chain constant domain containing a second Fc domain A second monomer containing a heavy chain; c) Light chains including variable light chain domains and variable light constant domains Includes, The aforementioned variable light chain domain is sequence KSSQSLLNTGNQKNYLT(sequence number XX) vlCDR1 has sequence WASTRES (sequence number XX), vlCDR2 has sequence WASTRES (sequence number XX), It includes vlCDR3 having sequence QNDYSYPYT(sequence number XX), and the above The variable heavy chain domain is vhCDR1 with sequence DYYMK (sequence number XX), sequence DI vhCDR2 having IPSNGATFYNQKFKG (sequence number XX), and sequence S Includes vhCDR3 having HLLRASWFAY (Sequence ID XX), Heterodimer antibody.

[66] XENP15049, XENP15051;XENP15050, XENP13676 , XENP14696, XENP15629, XENP15053, XENP15630 , XENP15631, XENP15632, XENP15633, XENP15634 , XENP15635, XENP15636, XENP15638, XENP15639 , XENP13677, XENP14388, XENP14389, XENP14390 , XENP14391, XENP14392, XENP14393, XENP16366 , XENP16367, XENP16368, XENP16369, XENP16370 , XENP16371, XENP16372, XENP16373, XENP16375 A heterodimer selected from the group consisting of XENP16376 and XENP16377. antibody.

[67] XENP15049, XENP15051;XENP15050, XENP13676 , XENP14696, XENP15629, XENP15053, XENP15630 , XENP15631, XENP15632, XENP15633, XENP15634 , XENP15635, XENP15636, XENP15638, XENP15639 , XENP13677, XENP14388, XENP14389, XENP14390 , XENP14391, XENP14392, XENP14393, XENP16366 , XENP16367, XENP16368, XENP16369, XENP16370 , XENP16371, XENP16372, XENP16373, XENP16375 A heterodimer selected from the group consisting of XENP16376 and XENP16377. A nucleic acid composition containing three nucleic acids that encode antibodies.

[68] An expression vector composition comprising three expression vectors, each containing nucleic acid, Therefore, the three expression vectors are XENP15049, XENP15051;XE NP15050, XENP13676, XENP14696, XENP15629, XE NP15053, XENP15630, XENP15631, XENP15632, XE NP15633, XENP15634, XENP15635, XENP15636, XE NP15638, XENP15639, XENP13677, XENP14388, XE NP14389, XENP14390, XENP14391, XENP14392, XE NP14393, XENP16366, XENP16367, XENP16368, XE NP16369, XENP16370, XENP16371, XENP16372, XE NP16373, XENP16375, XENP16376 and XENP16377 An expression vector composition encoding a heterodimeric antibody selected from the following group.

[69] A host cell containing the nucleic acid composition described in

[67] .

[70]

[68] Host cells containing the expression vector composition described above.

[71] A method for producing a heterodimeric antibody as described in

[66] , and

[69] or

[70] The steps include culturing the host cells described above under conditions in which the antibody is expressed, and recovering the antibody. A method that includes the steps to do so.

[72] A method for treating cancer, wherein the heterodimer antibody described in

[66] is required A method comprising the step of administering to a patient.

Claims

1. a) Variable heavy chain domains including vhCDR1 of SEQ ID NO: 422, vhCDR2 of SEQ ID NO: 423, and vhCDR3 of SEQ ID NO: 424, and b) Variable light chain domains including vlcDr1 of SEQ ID NO: 426, vlcDr2 of SEQ ID NO: 427, and vlcDr3 of SEQ ID NO: 428 The CD20-binding domain of the antibody, which includes this domain.

2. a) A first nucleic acid encoding a variable heavy chain domain as described in claim 1, and b) A second nucleic acid encoding the variable light chain domain described in claim 1. A nucleic acid composition containing the following:

3. An expression vector composition comprising the nucleic acid composition described in claim 2, a) A first expression vector comprising the first nucleic acid, and b) A second expression vector containing the second nucleic acid. An expression vector composition containing the following:

4. A host cell comprising the expression vector composition described in claim 3.

5. A method for producing the CD20-binding domain of an antibody according to claim 1, comprising culturing the host cell according to claim 4 under conditions in which the CD20-binding domain of the antibody is expressed, and recovering the CD20-binding domain of the antibody.

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