Heterodimeric antibodies that bind to CD3 and tumor antigens

Abstract

To provide novel heterodimeric antibodies for treating B-cell lymphoma or leukemia.SOLUTION: A heterodimeric antibody comprises an anti-CD123 Fab-Fc heavy chain sequence 1 having a specific sequence, an anti-CD3 scFv-Fc heavy chain sequence 2 having a specific sequence, and an anti-CD123 light chain sequence having a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed under 35 U.S.C. §§ 119(e) and 120 on May 8, 2015. U.S. Provisional Patent Application No. 62 / 159,111, filed November 4, 2015 No. 62 / 251,005, filed November 4, 2015. U.S. Provisional Patent Application No. 62 / 250,971, filed November 11, 2015 No. 14 / 952,714 filed on November 25, 2015 and U.S. Patent Application No. PCT / US Patent Application Publication No. 2015 / 062772 filed on the same day (these All are incorporated herein by reference in their entirety, with specific reference to the figures, descriptions and claims therein. (which is expressly incorporated herein by reference in its entirety). [Background technology]

[0002] Antibody-based therapeutics treat a variety of diseases, including cancer and autoimmune / inflammatory disorders However, there are still many unknowns about this class of drugs, particularly their clinical efficacy. Improvements are still needed to enhance efficacy. One approach is to incorporate additional and novel antigen-binding sites into antibody-based drugs, thereby allowing the binding of a single immunoglobulin. The goal is to modify the phosphomolecule so that it can co-associate with two different antigens. Such non-natural or alternative antibody formats that associate with The remarkable diversity of antibody variable regions (Fv) allows the This allows the creation of Fvs that essentially recognize each other, making it possible to create bispecific antibodies. A typical approach to this is to introduce novel variable regions into the antibody.

[0003] Several alternative antibody formats 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 (Expressly incorporated herein by reference). Initially, bispecific antibodies were developed based on the idea that each antibody contained a single It was produced by fusing two cell lines that produce monoclonal antibodies (Mi Stein et al., 1983, Nature 305:537-540). profit The hybrid hybridomas or quadromas produced bispecific antibodies. However, they represent only a minority population, and large-scale purification is required to isolate the desired antibodies. An engineering solution to this is the use of antibody fragments to create bispecific antibodies. Such fragments lack the complex quaternary structure of full-length antibodies, and therefore contain only the variable light chain and The variable heavy chains can be linked in a single genetic construct. Bispecific antibodies, single-chain bispecific Antibody fragments in many different forms, including antibodies, tandem scFvs, and Fab2 bispecifics The fragments 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 formats are preferred because they can be expressed at high levels in bacteria and because of their small size. Although they may have the advantage of good permeability, they are rapidly cleared in vivo and their production and These drawbacks are primarily due to the fact that antibody fragments typically maintain a long half-life in serum (i.e., neonatal Fc receptor FcRn) or Larger groups serve as binding sites for the synthesis of proteins (i.e., Protein A and Protein G). Its properties, including size, high stability, and binding to various Fc receptors and ligands, The main drawback is the lack of antibody constant regions that have the functional properties of the antibody.

[0004] More recent work has demonstrated the feasibility of fragment-based antibody binding by engineering double bonds into full-length antibody-like formats. Attempts have been made to address the shortcomings of bispecifics (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 / US 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 / US Patent Application Publication No. 2005 / 025472 (Expressly incorporated herein by reference). These formats primarily contain the Fc region. These formats overcome some of the drawbacks of antibody fragment bispecifics because they have a wide range of functions. One notable drawback is that it requires the construction of a new antigen-binding site on top of the homodimeric constant chain. The key point is that binding to new antigens is always bivalent.

[0005] For multiple antigens that are interesting as simultaneous targets in a therapeutic bispecific format, it is desirable Binding is monovalent, not bivalent. For many immune receptors, cell activation occurs through monovalent binding interactions. The cross-linking mechanism is typically achieved by antibody / antigen immune complexes. or mediated through effector cell to target cell engagement. For example, FcγR Low affinity Fc gamma receptors (F Monovalent binding occurs when antibodies expressing these FcγRs bind to the Fc region of the antibody. However, upon immune complex formation or cell-cell contact, the receptor They are cross-linked and clustered on the cell surface, causing activation and mediating cell killing. Receptors involved in this, such as FcγRIIIa on natural killer (NK) cells, Receptor cross-linking and cell activation allow effector cells to meet target cells in a highly active manner. This occurs when the two proteins are combined (Bowles & Weiner, 2005, J Immunol Methods 304:88-99, expressly incorporated by reference). The inhibitory receptor FcγRIIb on cells binds to immune complexes with the cell surface B cell receptor (BCR). downregulates B cell activation only when associated with soluble IgG and the BCR. The mechanism is mediated by immune complex formation with the same antigen recognized by the host (Heym an 2003, Immunol Lett 88[2]:157-161;Smith and Clatworthy,2010,Nature Reviews Immu (Likely a misreading of the text, but likely a misreading of the text, possibly a misreading of the text) CD3 activation of T cells results in highly active intercellular signaling of their associated T cell receptors (TCRs). In B. napus, this occurs only when antigens on antigen-presenting cells interact with loaded MHC (Kuhns et al., 2006, Immunity 24:133-139). Of course, nonspecific bivalent cross-linking of CD3 with anti-CD3 antibodies can result in cytokine stress. This induces inflammation and toxicity (Perruche et al., 2009, J Imm unol 183[2]:953-61;Chatenoud&Bluestone,2 007,Nature Reviews Immunology 7:622-632; (Expressly incorporated by reference). Therefore, in practical clinical use, the redirected The preferred mode of CD3 co-engagement for killing of target cells is It is a monovalent bond that only brings about activation.

[0006] CD38, also known as cyclic ADP-ribose hydrolase, contains a long C-terminal extracellular It is a type II transmembrane glycoprotein with a cytoplasmic domain and a short N-terminal cytoplasmic domain. Among blood cells, functional effects include lymphocyte proliferation, cytokine release, B and myeloid lineages CD38-mediated signaling, including regulation of cell development and survival, and induction of dendritic cell maturation CD38 is a key regulator of non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BLL), and leukemia (LE). Myeloma (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's lymphoma (HL), and chronic myeloid leukemia (CML) It is expressed in many hematopoietic malignancies, including CML, and in cell lines derived from various hematopoietic malignancies. On the other hand, most undifferentiated pluripotent stem cells in the hematopoietic system are CD38 negative. Despite recent advances in cancer drug discovery and development, many tumors, including CD38-expressing tumors, remain untreated. These forms of cancer still have a poor prognosis. There is a need for improved methods for

[0007] The B cell antigen CD19 (also known as CD19, B cell surface antigen B4, and Leu-12) , a human pan- expressed from early stages of pre-B cell development through terminal differentiation into plasma cells CD19 is a B cell surface marker that promotes the proliferation and survival of mature B cells. On the cell surface, it associates in a complex with CD21. It also interacts with CD81 and Leu- 13 and enhances B cell receptor (BCR) signaling. Together with the BCR, CD 19 is a unique antigen receptor-induced protein important for B cell clonal expansion and humoral immunity. It regulates signaling thresholds. In collaboration with CD21, it mediates the relationship between the adaptive and innate immune systems. Upon activation, the cytoplasmic tail of CD19 becomes phosphorylated, leading to the binding of Src-phosphorylated CD19. This results in the binding of myristate kinase and recruitment of PI-3 kinase. It is also expressed in NHL cells and some leukemias, making it a promising candidate for cancers derived from the lymphatic system. It is an interesting immunotherapy target.

[0008] Several antibodies or antibody conjugates targeting CD19 have been used in preclinical trials for the treatment of cancer. These anti-CD19 antibodies or antibody conjugates are being evaluated in experimental or clinical trials. Examples include, but are not limited to, MT-103 (single-chain bispecific CD19 / CD3 antibody; 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 expressly incorporated by reference) Examples include:

[0009] CD123 (also known as interleukin-3 receptor alpha (IL-3Rα)) is a dendritic CD123 is expressed on cells, monocytes, eosinophils, and basophils. CD123 is also expressed on committed hematopoietic stem cells. The majority of myeloid lineage (CD13+, CD14+, CD33+, CD1 5low) and constitutively expressed by some CD19+ cells. + Absent in cells. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, bispecific antibodies made from antibody fragments overcome biophysical and pharmacokinetic barriers. While the drawback of those constructed in a full-length antibody-like format is that they are unable to bind to the primary target antigen. Multivalent association with co-target antigens in the absence of a target molecule prevents nonspecific activation and potentially toxicity. The present invention provides a novel bispecific antibody specific to CD3 and CD38. By introducing this system, this problem will be solved. [Means for solving the problem]

[0011] Thus, in one aspect, the present invention provides a) a first monomer, which is i) a first heavy chain 1) a first variable heavy chain domain; 2) a first constant heavy chain comprising a first Fc domain; ) comprising an scFv variable light chain domain, an scFv linker, and an scFv variable heavy chain domain , comprising an scFv covalently linked to the C-terminus of the Fc domain using a domain linker a) a first monomer comprising a first heavy chain; and b) a second variable heavy chain domain, and a second Fc domain. and c) a second monomer comprising a second heavy chain, the second monomer comprising a second constant heavy chain comprising a variable light chain domain. The present invention provides a heterodimeric antibody comprising a common light chain comprising a constant light chain domain and a common light chain comprising a constant light chain domain.

[0012] In a further aspect, the present invention provides a) a first monomer comprising: i) a first heavy chain; 1) a first variable heavy chain domain; 2) a first constant heavy chain domain comprising a first Fc domain; and 3) covalently linked to the C-terminus of the first Fc domain using a domain linker. a) a first monomer comprising a first heavy chain comprising a first variable light chain domain; and b) a second monomer i) a second variable heavy chain domain; ii) a second constant heavy chain domain comprising a second Fc domain. and iii) said second variable heavy chain domain is linked to said a second antibody comprising a third variable heavy chain domain covalently linked to the C-terminus of the Fc domain of the second antibody; and c) a common light chain comprising a variable light chain domain and a constant light chain domain. A dimeric antibody is provided.

[0013] In a further aspect, the present invention provides a) a first monomer comprising: i) a first heavy chain; 1) a first variable heavy chain domain; 2) a first CH1 domain and a first Fc domain. 3) a first constant heavy chain comprising an scFv variable light chain domain, an scFv linker and an scFv variable light chain domain; The C-terminus of the CH1 domain and the C-terminus of the CH2 domain are connected using a domain linker. a first heavy chain comprising an scFv covalently linked to the N-terminus of the Fc domain of and b) a second constant heavy chain comprising a second variable heavy chain domain, and a second Fc domain. a second monomer comprising a second heavy chain; and c) a second monomer comprising a variable light chain domain and a constant light chain domain. The present invention provides heterodimeric antibodies comprising a common light chain and a common main chain.

[0014] In a further aspect, the present invention provides a) a first monomer comprising: i) a first heavy chain; 1) a first variable heavy chain domain; 2) a first constant heavy chain domain comprising a first Fc domain; and 3) the second variable light chain domain is linked to the first constant heavy chain domain using a domain linker. a covalent bond between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain a) a first monomer comprising a first heavy chain comprising a first variable light chain domain; and b) a second a second variable heavy chain domain; a second variable heavy chain domain; a second Fc domain; and iii) said second variable heavy domain having a domain linker. a third variable heavy chain domain covalently linked to the C-terminus of the second Fc domain using and c) a common light chain comprising a variable light domain and a constant light domain. The present invention provides a heterodimeric antibody comprising:

[0015] In a further aspect, the present invention provides a) a first monomer comprising: i) a first heavy chain; 1) a first variable heavy chain domain; 2) a first CH1 domain and a first Fc domain. 3) a first constant heavy chain comprising an scFv variable light chain domain, an scFv linker and an scFv variable light chain domain; The C-terminus of the CH1 domain and the C-terminus of the CH2 domain are connected using a domain linker. a first heavy chain comprising an scFv covalently linked to the N-terminus of the Fc domain of a) a second monomer comprising a second Fc domain; and c) a variable light chain domain and and a light chain comprising a constant light chain domain.

[0016] In some embodiments, the first and second Fc domains are S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411 E / K The scFv has a set of amino acid substitutions selected from the group consisting of: 360E / Q362E:D401K; L368D / K370S:S364K / E357L and K370S:S364K / E357Q. Furthermore, the variable heavy and variable light domains bind to a first target tumor antigen (TTA), and the scFv binds to a second TTA or human CD3. In some embodiments, the TTA is selected from the group consisting of CD19, CD20, and CD123.

[0017] In a further aspect, the present invention provides H1.32_L1.47, H1.89_L1.47, H Regarding 1.90_L1.47, H1.33_L1.47 and H1.31_L1.47 and having the CDR and / or variable domain and / or scFv sequences shown in the figures. The present invention also provides nucleic acid compositions, expression vector compositions, and the like. The present invention provides a method for producing a recombinant human ovarian cancer cell.

[0018] In a further aspect, the present invention provides a) a first monomer comprising: i) a first Fc domain; ii) an scFv variable light chain domain, an scFv linker and an scFv variable heavy chain domain and an anti-CD3 antibody covalently linked to the N-terminus of the Fc domain using a domain linker. a) a first monomer comprising an scFv; and b) a second monomer comprising: i) a heavy variable domain; and and ii) a second monomer comprising a heavy chain comprising a heavy chain constant domain comprising a second Fc domain; c) a light chain comprising a variable light domain and a variable light constant domain, v: 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 (SEQ ID NO: XX) The present invention provides a heterodimeric antibody, wherein the heavy variable domain and the light variable domain are selected from the group consisting of: The main ones are TTA (including but not limited to CD19, CD20, CD38 and CD123) (including

[0019] In a further aspect, the invention provides a method for the preparation of a medicament for the detection ... vlCDR1 having the sequence ATSNLAS (SEQ ID NO: XX), vlCDR2 having the sequence ATSNLAS (SEQ ID NO: XX), and a variable light chain domain comprising a vlCDR3 having the sequence QQWTHNPPT (SEQ ID NO: XX); b) vhCDR1 with the sequence SYNMH (SEQ ID NO: XX), the sequence AIYPGNGAT vhCDR2 with the sequence SYSQKFQG (SEQ ID NO: XX), and the sequence SYYMGGDW and an anti-CD31 comprising a variable heavy chain domain comprising a vhCDR3 having YFDV (SEQ ID NO: XX). In some embodiments, the anti-CD20 antibody binding domain is C2B8 has the sequence H1.202_L1.113.

[0020] In a further aspect, the present invention provides a method for the preparation of a medicament for the treatment of autophagic encephalopathy, comprising administering to a subject a subject the method comprising administering to a subject autophagic encephalopathy, ... vlCDR1 having the sequence ATSNLAS (SEQ ID NO: XX), vlCDR2 having the sequence ATSNLAS (SEQ ID NO: XX), and a variable light chain domain comprising a vlCDR3 having the sequence QQWTSNPPT (SEQ ID NO: XX); b) vhCDR1 with the sequence SYNMH (SEQ ID NO: XX), the sequence AIYPGNGDT vhCDR2 with the sequence SYNQKFQG (SEQ ID NO: XX), and the sequence STYYGGDW and an anti-CD31 comprising a variable heavy chain domain comprising a vhCDR3 having YFNV (SEQ ID NO: XX). 20 antibody binding domains are provided.

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

[0022] In a further aspect, the present invention provides a) a first monomer comprising: i) a first Fc domain; ii) an scFv variable light chain domain, an scFv linker and an scFv variable heavy chain domain and an anti-CD3 antibody covalently linked to the N-terminus of the Fc domain using a domain linker. a) a first monomer comprising an scFv; and b) a second monomer comprising: i) a heavy variable domain; and and ii) a second monomer comprising a heavy chain comprising a heavy chain constant domain comprising a second Fc domain; c) a light chain comprising a variable light chain domain and a variable light constant domain, The chains contain the C2B8 H1.202_L1.113 or C2B8_H1L1 binding domains. The present invention provides a heterodimeric antibody formed by the method of claim 1.

[0023] In a further aspect, the present invention provides a) a first monomer comprising: i) a first Fc domain; ii) an scFv variable light chain domain, an scFv linker and an scFv variable heavy chain domain and an anti-CD3 antibody covalently linked to the N-terminus of the Fc domain using a domain linker. a) a first monomer comprising an scFv; and b) a second monomer comprising: i) a heavy variable domain; and and ii) a second monomer comprising a heavy chain comprising a heavy chain constant domain comprising a second Fc domain; c) a heterodimeric antibody comprising a variable light domain and a light chain comprising a variable light constant domain. In this embodiment, the variable domain binds to CD123 and 09_L1.47.

[0024] In a further aspect, the present invention provides 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 6373, XENP16375, XENP16376, XENP16377, XENP1 4045, and XENP13928. The nucleic acids, expression vectors and host cells all contribute to the production and expression of these proteins. Also provided are methods for treating patients with the disease.

[0025] In a further aspect, the present invention provides 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 6373, XENP16375, XENP16376, XENP16377, XENP1 4045, and XENP13928. Six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3) The nucleic acids, expression vectors, and host cells all provide these proteins. Methods for producing the proteins and treating patients therewith are also provided.

[0026] In a further aspect, the present invention provides two sets of CDRs: XENP15049, XENP15 051;XENP15050, XENP13676, XENP14696, XENP15 629, XENP15053, XENP15630, XENP15631, XENP15 632, XENP15633, XENP15634, XENP15635, XENP15 636, XENP15638, XENP15639, XENP13677, XENP14 388, XENP14389, XENP14390, XENP14391, XENP14 392, XENP14393, XENP16366, XENP16367, XENP16 368, XENP16369, XENP16370, XENP16371, XENP16 372, XENP16373, XENP16375, XENP16376, XENP16 377, XENP14045, and XENP13928 Six CDRs (vhCDR1) from one variable region of the antigen-binding domain of a heterodimeric antibody , vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3) A second set of antibodies from the variable regions of each of the first set and other second antigen-binding domains. The nucleic acid, expression vector, and host cell are all provided herein. Also provided are methods for making these proteins and treating patients with them. do.

[0027] In a further aspect, the present invention provides two sets of vh and vl domains, XENP 15049, XENP15051;XENP15050, XENP13676, XENP 14696, XENP15629, XENP15053, XENP15630, XENP 15631, XENP15632, XENP15633, XENP15634, XENP 15635, XENP15636, XENP15638, XENP15639, XENP 13677, XENP14388, XENP14389, XENP14390, XENP 14391,XENP14392,XENP14393,XENP16366,XENP 16367, XENP16368, XENP16369, XENP16370, XENP 16371, XENP16372, XENP16373, XENP16375, XENP 16376, XENP16377, XENP14045, and XENP13928 A first antibody fragment from one variable region of the antigen-binding domain of a heterodimeric antibody selected from the group consisting of: and a second set of antigen-binding domains comprising the first set of antigen-binding domains and the second set of antigen-binding domains. The nucleic acids, expression vectors, and host cells all provide these tandem antibodies. Methods for making the proteins and treating patients with them are also provided. [Brief explanation of the drawings]

[0028] [Figure 1A] Several formats of the invention are depicted. Two "bottle opener" formats are depicted: one with an anti-CD3 antigen-binding domain comprising an scFv and an anti-TTA antigen-binding domain comprising a Fab, and the other with these reversed. mAb-Fv, mAb-scFv, Central-scFv, and Central-Fv formats are all depicted. While they are depicted as having anti-CD3 as an scFv, as discussed herein, any Fv sequence can be separated and combined, and the anti-CD3 and anti-TTA domains of mAb-Fv, mAb-scFv, central-scFv, and central-Fv can be switched. Additionally, "single-arm" formats, where one monomer contains only the Fc domain, are depicted: both single-arm Central-scFv and single-arm Central-Fv. Dual-scFv formats are also depicted. [Figure 1B] Same as above [Figure 1C] Same as above [Figure 2] Figure 1 shows the sequence of the "High CD3" anti-CD3_H1.30_L1.47 construct, which includes the variable heavy and variable light domains (CDRs underlined) and individual vl and vh CDRs, and an scFv construct with a charged linker (double underlined). This charged linker may be replaced with an uncharged or different charged linker, as is the case for all sequences represented in the figures. [Figure 3] Figure 1 depicts the sequence of the "High-Int#1" anti-CD3_H1.32_L1.47 construct, which includes variable heavy and variable light domains (CDRs underlined) and individual vl and vh CDRs, and an scFv construct with a charged linker (double underlined). This charged linker may be replaced with an uncharged or different charged linker, as is the case for all sequences depicted in the figures. [Figure 4] Figure 1 depicts the sequence of the "High-Int#2" anti-CD3_H1.89_L1.47 construct, which includes variable heavy and variable light domains (CDRs underlined) and individual vl and vh CDRs, and an scFv construct with a charged linker (double underlined). This charged linker may be replaced with an uncharged or different charged linker, as is the case for all sequences depicted in the figures. [Figure 5] Figure 1 depicts the sequence of the "High-Int#3" anti-CD3_H1.90_L1.47 construct, which includes variable heavy and variable light domains (CDRs underlined) and individual vl and vh CDRs, and an scFv construct with a charged linker (double underlined). This charged linker may be replaced with an uncharged or different charged linker, as is the case for all sequences depicted in the figures. [Figure 6]Figure 1 depicts the sequence of the "Int" anti-CD3_H1.90_L1.47 construct, which includes the variable heavy and variable light domains (CDRs underlined) and individual vl and vh CDRs, and an scFv construct with a charged linker (double underlined). This charged linker may be replaced with an uncharged or different charged linker, as is the case for all sequences depicted in the figures. [Figure 7] Figure 1 depicts the sequence of the "Low" anti-CD3_H1.31_L1.47 construct, which includes the variable heavy and variable light domains (CDRs underlined) and individual vl and vh CDRs, and an scFv construct with a charged linker (double underlined). This charged linker may be replaced with an uncharged or different charged linker, as is the case for all sequences depicted in the figures. [Figure 8] 1 depicts the sequence of the High CD38:OKT10_H1.77_L1.24 construct, which contains the variable heavy and variable light chain domains (CDRs underlined) and individual vl and vh CDRs, as well as an scFv construct with a charged linker (double underlined). [Figure 9] 1 depicts the sequence of the intermediate CD38:OKT10_H1L1.24 construct, which contains the variable heavy and variable light chain domains (CDRs underlined) and individual vl and vh CDRs, as well as an scFv construct with a charged linker (double underlined). [Figure 10] 1 depicts the sequence of the Low CD38:OKT10_H1L1 construct, which contains the variable heavy and variable light chain domains (CDRs underlined) and individual vl and vh CDRs, as well as an scFv construct with a charged linker (double underlined). [Figure 11] This represents the sequence of XENP15331. [Figure 12] This represents the sequence of XENP13243. [Figure 13] This represents the sequence of XENP14702. [Figure 14] This represents the sequence of XENP15426. [Figure 15] This represents the sequence of XENP14701. [Figure 16] This represents the sequence of XENP14703. [Figure 17] This represents the sequence of XENP13243. [Figure 18] This represents the sequence of XENP18967. [Figure 19] This represents the sequence of XENP18971. [Figure 20] This represents the sequence of XENP18969. [Figure 21] This represents the sequence of XENP18970. [Figure 22] This represents the sequence of XENP18972. [Figure 23] This represents the sequence of XENP18973. [Figure 24] This represents the sequence of XENP15055. [Figure 25] This represents the sequence of XENP13544. [Figure 26] This represents the sequence of XENP13694. [Figure 27] 1 shows the sequence of human CD3ε. [Figure 28] The full-length (SEQ ID NO: 130) and extracellular domain (ECD; SEQ ID NO: 131) of human CD38 protein are shown. [Figure 29A] These represent useful pairs of heterodimerization mutant sets (including skew and pI mutants). In Figure 29E, there are mutants for which there is no corresponding "monomer 2" mutant. These are pI mutants that can be used alone on one monomer or can be included, for example, on the Fab side of a bottle opener, and an appropriate charged scFv linker can be used on the second monomer that utilizes an scFv as the second antigen-binding domain. Suitable charged linkers are shown in Figure 33. [Figure 29B] Same as above [Figure 29C] Same as above [Figure 29D] Same as above [Figure 29E] Same as above [Figure 30]

[0033] Figure 1 shows a list of the constant regions of the isomeric stereovariant antibodies and their respective substitutions. pI_(-) indicates a lower pI variant, while pI_(+) indicates a higher pI variant. These may optionally and independently be combined with other heterodimerization variants of the invention (and similarly other variant types as outlined herein). [Figure 31] Useful truncation mutants (sometimes referred to as "knockout" or "KO" mutants) that ablate FcγR binding are presented. [Figure 32] Two particularly useful embodiments of the present invention are presented. [Figure 33A]

[0033] The following describes several charged scFv linkers that find use in increasing or decreasing the pI of heterodimeric antibodies that utilize one or more scFv components. (+H) positive linkers find particular use herein, particularly with the anti-CD3 vl and vh sequences shown herein. A single prior art scFv linker bearing a single charge is referred to as "Whitlow" from Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should be noted that this linker was used to reduce aggregation and enhance proteolytic stability in scFvs. [Figure 33B] Same as above [Figure 34] Figure 1 shows a list of engineered heterodimer-skewed Fc mutants along with their heterodimer yield (measured by HPLC-CIEX) and thermal stability (measured by DSC). Thermal stability not measured is indicated by "nd." [Figure 35] Bispecific expression yield after Protein A affinity purification. [Figure 36] Cation exchange purification chromatogram. [Figure 37] This was a re-induced T cell cytotoxicity assay (24-hour incubation, 10k RPMI8226 cells, 400k T cells). The test article was an anti-CD38 x anti-CD3 bispecific antibody. Detection was by LDH. [Figure 38]Redirected T cell cytotoxicity assay (24-hour incubation, 10 kJ RPMI8226 cells, 500 kJ human PBMCs). Test article was an anti-CD38 x anti-CD3 bispecific antibody. Detection was by LDH. [Figure 39] This represents the sequence of XENP14419. [Figure 40] Represents the sequence of XENP14420. [Figure 41] This represents the sequence of XENP14421. [Figure 42] This represents the sequence of XENP14422. [Figure 43] This represents the sequence of XENP14423. [Figure 44] This was a re-induced T cell cytotoxicity assay (96-hour incubation, 40kJ RPMI8226 cells, 400kJ human PBMCs). The test substance was an anti-CD38 x anti-CD3 Fab-scFv-Fc. Detection was by flow cytometry, specifically, the depletion of CD38+ cells. [Figure 45] Further analysis of the redirected T cell cytotoxicity assay described in Figure 1. The first row shows the mean fluorescence intensity (MFI) of the activation marker CD69 for 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 intracellular mean fluorescence intensity (MFI) of the granzyme B inhibitor PI-9 for CD4+ and CD8+ T cells, as detected by flow cytometry. [Figure 46] Design of a mouse study to investigate the antitumor activity of anti-CD38 x anti-CD3 Fab-scFv-Fc bispecifics. [Figure 47] Tumor size measured by IVIS® as a function of time and treatment. [Figure 48] IVIS® bioluminescence images (day 10). [Figure 49] Depletion of CD38+ cells following a single dose of the indicated test article in cynomolgus monkeys. [Figure 50] T cell activation measured by CD69 mean fluorescence intensity (MFI) in cynomolgus monkeys (color coded as in Figure 49). [Figure 51] Serum levels of IL-6 after a single dose of the indicated test article. [Figure 52] This represents the sequence of XENP15427. [Figure 53] This represents the sequence of XENP15428. [Figure 54] This represents the sequence of XENP15429. [Figure 55] This represents the sequence of XENP15430. [Figure 56] This represents the sequence of XENP15431. [Figure 57] This represents the sequence of XENP15432. [Figure 58] This represents the sequence of XENP15433. [Figure 59] This represents the sequence of XENP15434. [Figure 60] This represents the sequence of XENP15435. [Figure 61] This represents the sequence of XENP15436. [Figure 62] This represents the sequence of XENP15437. [Figure 63] This represents the sequence of XENP15438. [Figure 64] Binding affinities in Biacore assays are shown. [Figure 65] Heterodimer purity is shown among generating stable pools with varying light chain, Fab-Fc, and scFv-Fc ratios. [Figure 66] Depletion of human IgM and IgG2 by anti-CD38 x anti-CD3 bispecifics in a huPBMC mouse model. [Figure 67A] Figure 1 represents a stability-optimized humanized anti-CD3 variant scFv. Substitutions are given relative to the H1_L1.4 scFv sequence. Amino acid numbering is Kabat numbering. [Figure 67B] Same as above [Figure 68A]

[0023] Figure 1 shows the amino acid sequences of stability-optimized humanized anti-CD3 variant scFvs. CDRs are 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, 10 kJ RPMI8226 cells, 500 kJ PBMCs). The test substance was anti-CD38 (OKT10_H1L1, OKT10_H1.77_L1.24) x anti-CD3 Fab-scFv-Fc. Detection was performed by LDH. [Figure 70] This was a huPBL-SCID Ig depletion study. Test compounds were administered intraperitoneally at 0.03, 0.3, or 3 mg / kg on day 8 after PBMC transfer. Blood samples were collected on day 14 after PBMC transfer, processed to serum, and assayed for human IgM and IgG2. [Figure 71] This represents the sequence of XENP15049. [Figure 72] This represents the sequence of XENP15051. [Figure 73] Represents the sequence of XENP15050. [Figure 74] This represents the sequence of XENP13676. [Figure 75] This represents the sequence of XENP14696. [Figure 76] This represents the sequence of XENP15629. [Figure 77] This represents the sequence of XENP15053. [Figure 78] Represents the sequence of XENP15630. [Figure 79] This represents the sequence of XENP15631. [Figure 80] This represents the sequence of XENP15632. [Figure 81] This represents the sequence of XENP15633. [Figure 82] This represents the sequence of XENP15634. [Figure 83] This represents the sequence of XENP15635. [Figure 84] This represents the sequence of XENP15636. [Figure 85] This represents the sequence of XENP15638. [Figure 86] This represents the sequence of XENP15639. [Figure 87] This represents the sequence of XENP13677. [Figure 88] This represents the sequence of XENP14388. [Figure 89] This represents the sequence of XENP14389. [Figure 90] This represents the sequence of XENP14390. [Figure 91] This represents the sequence of XENP14391. [Figure 92] This represents the sequence of XENP14392. [Figure 93] This represents the sequence of XENP14393. [Figure 94] Represents the sequence of XENP16366. [Figure 95] Represents the sequence of XENP16367. [Figure 96] Represents the sequence of XENP16368. [Figure 97] This represents the sequence of XENP16369. [Figure 98] Represents the sequence of XENP16370. [Figure 99] This represents the sequence of XENP16371. [Figure 100] Represents the sequence of XENP16372. [Figure 101] This represents the sequence of XENP16373. [Figure 102] Represents the sequence of XENP16374. [Figure 103] This represents the sequence of XENP16375. [Figure 104] The CDR, vh, and vl sequences of the anti-CD20 Fab arm are shown in Figure 121. [Figure 105] Represents the sequence of XENP16377. [Figure 106] The sequences of the CD20 and CD123 antigens are shown. [Figure 107] Surface plasmon resonance measurement of CD3 affinity. The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Human CD3δε-Fc (Sino Biological) was covalently bound to the chip surface. The test compound was passed over at 3.125, 12.5, 50, and 200 nM. [Figure 108]Surface plasmon resonance assay for CD3 affinity. The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Cynomolgus monkey CD3δε-Fc (Sino Biological) was covalently bound to the chip surface. The test compound was passed over at 3.125, 12.5, 50, and 200 nM. [Figure 109] Surface plasmon resonance measurement of CD3 affinity. The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Human CD3δε-Fc (Sino Biological) was covalently bound to the chip surface. The test compound was passed over at 31.25, 125, 500, and 2000 nM. [Figure 110] Surface plasmon resonance assay for CD3 affinity. The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Cynomolgus monkey CD3δε-Fc (Sino Biological) was covalently bound to the chip surface. The test compound was passed over at 31.25, 125, 500, and 2000 nM. [Figure 111] Surface plasmon resonance assay for CD3 affinity. The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Cynomolgus monkey CD3δε-Fc (Sino Biological) was covalently bound to the chip surface. The test compound was passed over at 31.25, 125, 500, and 2000 nM. [Figure 112] This was a re-induced T cell cytotoxicity assay (24-hour incubation, 10 kJ Ramos cells, 250 kJ PBMCs). The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Detection was by LDH. [Figure 113]Redirected T cell cytotoxicity assay (24-hour incubation, 20k Jeko cells, 200k PBMCs (CD19 depleted)). The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Detection was by flow cytometry, specifically, depletion of CD19+ cells. [Figure 114] IL-6 production after 24 hours in the experiment described in Figure 113. [Figure 115A] This was a re-induced T cell cytotoxicity assay (5-hour incubation, 20k Jeko cells, 500k PBMCs (CD19 depleted)). The test compound was anti-CD20 (C2B8_H1L1) x anti-CD3 Fab-scFv-Fc. Detection was by flow cytometry, specifically, the depletion of CD19+ cells. [Figure 115B] Same as above [Figure 116A] Redirected T cell cytotoxicity assay (24-hour incubation, 20k Jeko cells, 500k PBMCs (CD19 depleted)). The test compound was anti-CD20 (C2B8_H1.202_L1.113) x anti-CD3 Fab-scFv-Fc. Detection was by flow cytometry, specifically, depletion of CD19+ cells. [Figure 116B] Same as above [Figure 117] IL-6 production after 24 hours in the experiment described in Figure 113. [Figure 118] This was a re-induced T cell cytotoxicity assay (24-hour incubation, 10 kJ RPMI8226 cells, 500 kJ PBMCs). The test substance was anti-CD38 (OKT10_H1L1, OKT10_H1.77_L1.24) x anti-CD3 Fab-scFv-Fc. Detection was performed by LDH. [Figure 119] This is a huPBL-SCID Ig depletion study. The test article was administered at 5 mg / kg on days 1 and 8 after PBMC transfer. The route of administration was intraperitoneal. Blood samples were collected on day 14 after PBMC transfer, processed to serum, and assayed for human IgM and IgG2. [Figure 120] This was a huPBL-SCID Ig depletion study. Test compounds were administered intraperitoneally at 0.03, 0.3, or 3 mg / kg on day 8 after PBMC transfer. Blood samples were collected on day 14 after PBMC transfer, processed to serum, and assayed for human IgM and IgG2. [Figure 121]

[00130] Figure 33 depicts the sequence of High CD20 C2B8_H1.202_L1.113. The charged linker depicted is (+H), although other charged or uncharged linkers such as those depicted in Figure 33 can be used. [Figure 122] Figure 33 depicts the sequence of Low CD20 C2B8_H1L1. The charged linker depicted is (+H), although other charged or uncharged linkers such as those depicted in Figure 33 can be used. [Figure 123] Figure 33 depicts the sequence of CD123 7G3_H1.109_L1.57. The charged linker depicted is (+H), although other charged or uncharged linkers such as those depicted in Figure 33 can be used. [Figure 124]A matrix of possible combinations in the present invention is shown. "A" means that the CDRs of the referenced CD3 sequence can be combined with the CDRs of TTA on the right. That is, the vhCDRs from the variable heavy chain CD3 H1.30 sequence and the vlCDRs from the variable light chain of the CD3 L1.57 sequence can be combined with the vhCDRs from the CD38 OKT10 H1.77 sequence and the vlCDRs from the OKT10L1.24 sequence. "B" means that the CDRs from the CD3 construct can be combined with the variable heavy and variable light chain domains from TTA. That is, the vhCDRs from the variable heavy chain CD3 H1.30 sequence and the vlCDRs 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 the OKT10L1.24 sequence. By reversing "C," the variable heavy and variable light chain domains from the CD3 sequence are used together with the CDRs of TTA. "D" is when both the variable heavy and variable light chains from each are combined. "E" is when a CD3 scFv is used with the TTA CDRs, and "F" is when a CD3 scFv is used with the variable heavy and variable light domains of the TTA antigen-binding domain. All of these combinations can be made in a bottle-opener format, for example, with any of the scaffold formats shown in Figure 162, or in alternative formats such as the mAb-Fv, mAb-scFv, Central-scFv, Central-Fv, or dual-scFv formats of Figure 1, including scaffolds of the formats shown in Figures 131 and 132. However, formats that would involve bivalent binding of CD3 are generally not preferred. That is, "A" (CD3 CDR x TTA CDR) can be added to the bottle opener sequence (including those in Figure 162 or different heterodimerization variants) or to the mAb-scFv scaffold of Figure 132, central-scFv, mAb-Fv format, or central-Fv format. [Figure 125] FIG. 1 is a schematic diagram of an anti-CD123×anti-CD3 Fab-scFv-Fc bispecific. [Figure 126]1 is a table showing mutants engineered to increase the affinity and stability of 7G3_H1L1. [Figure 127] 1 is a table showing the properties of the final affinity- and stability-optimized humanized variants of 7G3. [Figure 128] Binding of XENP14045 (anti-CD123 x anti-CD3) bispecific binding to the CD123-positive AML cell line KG-1a. [Figure 129] This is the re-induction of T cell cytotoxicity (RTCC) of XENP14045, which kills KG-1a cells. [Figure 130] RTCC of XENP14045 with KG-1a cells using various ratios of effector to target (E:T) cells demonstrating "sequential killing" by XENP14045-generated T cells. [Figure 131] Drug serum levels of 2 mg / kg XENP14045 given IV to C57BL / 6 mice. The bispecific half-life was 6.2 days. [Figure 132] Killing of CD123+ blood basophils and plasmacytoid dendritic cells (PDC) in cynomolgus monkeys given a single IV dose of 0.01, 0.1, or 1 mg / kg XENP14045. [Figure 133] Killing of CD123+ basophils and plasmacytoid dendritic cells (PDC) in the bone marrow of cynomolgus monkeys given a single IV dose of 0.01, 0.1, or 1 mg / kg XENP14045. [Figure 134] T cell redistribution following a single IV dose of XENP14045 in cynomolgus monkeys. [Figure 135] CD69 induction on T cells after a single IV dose of XENP14045 in cynomolgus monkeys. [Figure 136A] The sequences of the present invention are shown with the CDR regions underlined. [Figure 136B] Same as above [Figure 136C] Same as above [Figure 137]Heterodimer purity during stable pool production using various light chain, Fab-Fc, and scFv-Fc ratios (top). Heterodimer purity for various conditions of pool F2 (bottom). [Figure 138] SEC showing highly pure XENP14045 cell line material after two-step purification. [Figure 139] Represents T cell-mediated killing of CD123+ cells. [Figure 140] Represents a bispecific mechanism for recruiting cytotoxic T cells that kill AML stem cells and blasts. [Figure 141] Figure 1 shows efficient production of XENP14045 bispecifics. [Figure 142] Figure 1 shows that the XENP14045 bispecific antibody binds to human AML with a KD of 8.1 nM to human CD3. [Figure 143] Figure 1 shows that the XENP14045 bispecific antibody is cross-reactive with primate cells and has a KD of 5.7 nM for cynomolgus monkey CD3. [Figure 144] 1 shows that anti-CD123×anti-CD3 kills human AML cell lines. [Figure 145] 1 shows that anti-CD123×anti-CD3 kills human AML cell lines. [Figure 146] 1 shows the long half-life of the bispecific in mice. [Figure 147] Single dose administration in monkeys is shown. [Figure 148] Depletion of monkey CD123+ cells in blood basophils. Basophil gate, flow cytometry, CD20- CD16+ CD14- CD4- CD8- FceR1+. [Figure 149] Depletion of bone marrow basophils using the same gating is shown. [Figure 150] 1 shows repeated administration to deplete CD123+ cells in monkeys. [Figure 151]Depletion of CD123+ cells in monkeys. Basophil gate, flow cytometry: CD20- CD16+ CD14- CD4- CD8- FceR1+. Plasmacytoid dendritic cell gate, flow cytometry: CD20- CD16- CD14- CD4- CD8- CD303+. [Figure 152] Depletion in monkey bone marrow, same gating as in Figure 151. [Figure 153] Figure 153 shows that CD123+ cell depletion correlates with T cell redistribution and activation. [Fig. 154] Figure 154 shows that CD123+ cell depletion correlates with T cell redistribution and activation. [Figure 155] Figure 155 shows that CD123+ cell depletion correlates with T cell redistribution and activation. [Figure 156]

[00109] Figures 125A-C present data related to the difficulty of humanizing anti-CD123 murine sequences, as described in Example 3. Figures 125A-C show the loss of affinity due to humanization (primarily due to vH) when 13760 is the Fab of the H0L0 starting murine antibody, 13763 is the first humanized vH candidate, and 13761 has humanized heavy and light chain Fabs. Figure 125D shows the approximately 10-fold loss in RTCC potency as a result of humanization. [Figure 157] Figure 1 shows the results of the first round of humanization, which generated 108 mutants containing LDA, target, and back-substitutions that were affinity screened in Fab format on a Biacore CD123 chip, and the stability of neutral and higher affinity mutants was screened with DSF ("Library 1"). [Figure 158] 1 shows the increase in Tm discussed in Example 3. [Figure 159A] Figure 159 shows the results of converting the Fab into a bottle opener format using scFv and Fab against CD3 as described in detail. Figure 159A shows the binding assay and Figure 159B shows the RTCC assay. [Figure 159B] Same as above [Figure 160] Results from "round 2" of humanization as outlined in Example 3 are shown. Note that XENP13967 is equivalent to XENP14045 on the CD123 side. 13967 has a different CD3 scFv as shown in the sequence. [Figure 161] 1 shows the results of the round 2 Tm assay of Example 3. [Figure 162A] The sequences of some useful bottle opener-format scaffolds without the Fv sequence are shown (e.g., scFv and Fab-side vh and vl). As will be understood by those of skill in the art and outlined below, these sequences can be used with any of the vh and vl pairs outlined herein, where one monomer comprises an scFv (optionally including a charged scFv linker) and the other monomer comprises a Fab sequence (e.g., the vh is attached to the "Fab-side heavy chain" and the vl is attached to the "constant light chain"). The scFv can be anti-CD3 or anti-TTA, and the Fab can be other. That is, any of the Fv sequences outlined herein for CD3, CD123, CD38, CD19, or CD20 can be incorporated into these scaffolds in any combination. Note that these bottle opener scaffolds find use in the Central-scFv format of Figure 1B, where an additional second Fab (vh-CH1 and vl-constant light chain) with the same antigen binding as the first Fab is added to the N-terminus of the "bottle opener" scFv. [Figure 162B] Same as above [Figure 162C] Same as above [Figure 162D] Same as above [Figure 163]The sequences of the mAb-scFv scaffolds used in the present invention are shown, to which the Fv sequences of the present invention are added. As will be understood by those skilled in the art and outlined below, these sequences can be used with any of the vh and vl pairs outlined herein. One monomer contains both the Fab and scFv (optionally including a charged scFv linker), while the other monomer contains the Fab sequence (e.g., the vh is attached to the "Fab heavy chain" and the vl is attached to the "constant light chain"). Monomer 1 is the Fab-scFv pI negative side, and contains the heterodimerization mutant L368D / K370S, the homogenous pI mutants N208D / Q295E / N384D / Q418E / N421D, and the truncation mutants E233P / L234V / L235A / G236del / S267K (all relative to IgG1). Monomer 2 is the scFv pI positive side and contains the heterodimerization mutant 364K / E357Q, however, other pairs of skewed mutants can be used instead, particularly [S364K / E357Q:L368D / K370S]; [L368D / K370S:S364K]; [L368E / K370S:S364K]; [T411E / K360E / Q362E:D401K]; [L368D / K370S:S364K / E357L], and [K370S:S364K / E357Q]. DETAILED DESCRIPTION OF THE INVENTION

[0029] I. Definition In order that this application may be more fully understood, some definitions are set forth below. Such definitions are , and grammatical equivalents are intended to be encompassed.

[0030] As used herein, "cleavage" refers to the reduction or elimination of activity. "Abrupts cγR binding" means that the amino acid variant in the Fc region is different from the Fc region without the specific variant. This means that the initial bond is 50% lower than in the 70-80-90-95-9 A loss of activity of less than 8% is preferred, and generally activity is detectable in the Biacore assay. The specific application of cleavage of FcγR binding is shown in FIG. That is why.

[0031] "ADCC" or "antibody-dependent cellular cytotoxicity" as used herein refers to the ability of FcγR Nonspecific cytotoxic cells expressing the α-antibody recognize the bound antibody on the target cell, and subsequently activate the target cell. ADCC refers to a cell-mediated reaction that results in lysis. ADCC is associated with binding to FcγRIIIa. In this regard, increased binding to FcγRIIIa results in increased ADCC activity.

[0032] "ADCP" or antibody-dependent cellular phagocytosis, as used herein, refers to the process by which FcγRs are activated. Nonspecific cytotoxic cells expressing ATP recognize the bound antibody on the target cell and subsequently phagocytose the target cell. It refers to a cell-mediated reaction that causes an effect.

[0033] As used herein, a "modification" refers to an amino acid substitution, insertion, and / or deletion within a polypeptide sequence. means a deletion or modification to a moiety chemically linked to a protein. The decoration may be a modified carbohydrate or a PEG structure attached to a protein. As used herein, "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion within a polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications are always expressed as DNA fragments. Amino acids coded by A, e.g., the 20 amino acids with codons in DNA and RNA Targets amino acids.

[0034] As used herein, an "amino acid substitution" or "substitution" refers to an amino acid at a particular position within a parent polypeptide sequence. In particular, in some embodiments, The substitution is not naturally occurring at the particular position, not naturally occurring either in that organism or any organism. For example, the substitution E272Y replaces glutamic acid at position 272. The term "Fc variant" refers to a variant polypeptide in which phosphate is replaced with tyrosine, in this case an Fc variant. For clarity, altering the nucleic acid coding sequence does not alter the starting amino acid (e.g., To increase the expression level in the host organism, CGG (encoding arginine) was replaced with CGA (or The protein is designed to have the amino acid sequence (which encodes for arginine) replaced by the amino acid sequence (which encodes for arginine). It is not a "substitution of an amino acid," i.e., it does not involve the creation of a new gene that encodes the same protein. Regardless, if a protein has the same amino acid at a particular position where it starts, it It is not an amino acid substitution.

[0035] An "amino acid insertion" or "insertion," as used herein, refers to an amino acid sequence that is inserted into a parent polypeptide sequence. For example, -233E or 233E refers to the insertion of glutamic acid after position 233 and before position 234. In addition, -233 ADE or A233ADE is AlaAspGl after position 233 and before position 234 Refers to the insertion of u.

[0036] "Amino acid deletion" or "deletion," as used herein, refers to an amino acid sequence that is a repeat of a parent polypeptide sequence. For example, E233- or E233 # or E233() refers to a deletion of glutamic acid at position 233. In addition, EDA2 33- or EDA233# indicates a deletion of the sequence GluAspAla starting at position 233. Point.

[0037] "Mutant protein" or "protein variant" or "variant" as used herein When used, a protein that differs from the parent protein by virtue of at least one amino acid modification A protein variant refers to the protein itself, a composition comprising the protein, or It may refer to the amino acid sequence that encodes it. Preferably, the protein variant is a variant of the parent protein. At least one amino acid modification compared to the parent protein, e.g., about 1 to about 70 amino acids compared to the parent protein. and preferably about 1 to about 5 amino acid modifications, as follows: In some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, comprises the human wild-type sequence: For example, an Fc region derived from IgG1, IgG2, IgG3, or IgG4, but a variant A human sequence having the formula: The protein variant sequences herein preferably have the same structure as the parent protein sequence. Preferably at least about 80% identical, and most preferably at least about 90% identical, more preferably More preferably, the mutant protein will have at least about 95 to 98 to 99% identity. The protein may be the mutant protein itself, a composition containing the protein mutant, or a compound encoding it. Thus, "antibody variant" or "variant antibody" may refer to a DNA sequence that encodes a variant of the antibody of the present invention. As used herein, an antibody that differs from a parent antibody by virtue of at least one amino acid modification. Therefore, "IgG variant" or "variant IgG" as used herein means at least They differ from the parent IgG (again, often derived from a human IgG sequence) by a single amino acid modification. and "immunoglobulin variant" or "variant immunoglobulin" means an antibody comprising: As used herein, a parent immunoglobulin sequence differs from a parent immunoglobulin sequence by virtue of at least one amino acid modification. "Fc variant" or "variant Fc" means an immunoglobulin sequence that differs from that of the corresponding Fc variant. As used herein, refers to a protein that contains an amino acid modification within the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that compose them. Thus, for example, N434S or 434S represents a nucleotide at position 434 relative to the parent Fc polypeptide. Fc variants having a substitution serine, where numbering is according to the EU index. , M428L / N434S is the substitution M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may be unspecified. The above mutant is referred to as 428L / 434S. The substitutions can be provided in any order. i.e., an Fc variant in which, for example, 428L / 434S is the same as M428L / N434S. For any position contemplated by the present invention with respect to an antibody, Unless otherwise noted, amino acid position numbering follows the EU index. EU indexes such as Kabat or EU numbering schemes number EU antibodies. Pointing (Edelman et al., 1969, Proc Natl Acad Sc i USA 63:78-85, herein incorporated by reference in its entirety). The substitutions may be additions, deletions, or substitutions. The substitutions may be natural amino acids, and in some cases synthetic amino acids. See, for example, U.S. Pat. No. 6,586,207; WO 98 / 44444; No. 8032; WO 03 / 073238; U.S. Patent Application Publication No. 2004-0214988A1; International Publication No. 05 / 35727A2 Fret; International Publication No. 05 / 74524A2 Pamphlet; JWChin et al., (2002), Journal of the American Chemistry cal Society124:9026-9027;JWChin,&P.GS Chultz, (2002), ChemBioChem 11:1135-1137;J .W.Chin, et al.,(2002), PICAS United Stat es of America 99:11020-11024; and L. Wang, & P.G. Schultz, (2002), Chem. 1-10 (see all in their entirety) (Incorporated by reference herein).

[0038] As used herein, a "protein" herein refers to a molecule that is bound to at least two covalent bonds. means a compound amino acid, a protein, a polypeptide, an oligopeptide, and a peptide Peptidyl groups may be derived from natural amino acids and peptide bonds, or from synthetic peptidomimetics. These may include structures or "analogs," such as peptoids (Simon et al. ., PNAS USA 89(20):9367(1992), incorporated by reference in its entirety. Amino acids may be naturally occurring or synthetic (e.g., (amino acids that are not encoded by DNA) Diphenylalanine, citrulline, ornithine and noreleucine are not contemplated by the present invention. These are considered synthetic amino acids and are available in both D- and L- (R or S) designed amino acids. The variants of the present invention may be used, for example, as developed by Schultz and colleagues. techniques, including but 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 (all of which are incorporated by reference in their entireties). Modifications may include the use of synthetic amino acids. Chain or terminal synthetic derivatization, glycosylation, PEGylation, circular mutation permutation, cyclization, linker to other molecules, protein or These may include fusion to a protein domain and addition of a peptide tag or label.

[0039] "Residue," as used herein, refers to a position in a protein and its associated This refers to the identification of amino acids, e.g., asparagine 297 (Asn297 or N29 7) is the residue at position 297 in the human antibody IgG1.

[0040] "Fab" or "Fab region" as used herein refers to the VH, CH1, VL Fab refers to a polypeptide containing a CL immunoglobulin domain. The domain may be referred to alone or in combination with a full length antibody, antibody fragment or Fab fusion protein. This region may also be referred to in the context of "Fv" or "Fv fragment" or "Fv region." As used herein, a polypeptide comprising the VL and VH domains of a single antibody is meant. As will be appreciated by those skilled in the art, these generally consist of two chains.

[0041] "IgG subclass modified" or "isotype modified" as used herein , one amino acid of one IgG isotype is changed in the aligned IgG isotypes. For example, IgG1 is modified to convert tyrosine and and IgG2, and therefore, the F296Y substitution at EU position 296 in IgG2 Phenylanine is considered an IgG subclass modifier.

[0042] "Non-naturally occurring modification" as used herein means a non-isotypic amino acid modification. For example, since neither IgG contains serine at position 434, IgG1, I The substitution 434S in IgG2, IgG3, or IgG4 (or hybrids thereof) It is considered a non-natural modification.

[0043] "Amino acid" and "amino acid identity" as used herein refer to DNA and R It refers to one of the 20 naturally occurring amino acids encoded by NA.

[0044] "Effector functions," as used herein, refer to the functions of the Fc region of an antibody and an Fc receptor or Effector function refers to the biochemical events that result from the interaction of a protein with a target protein or ligand. These include, but are not limited to, ADCC, ADCP, and CDC.

[0045] "IgG Fc ligand" as used herein refers to an Fc / Fc ligand complex. Any biologically derived molecule, preferably polyclonal antibody, that binds to the Fc region of an IgG antibody, forming a Fc ligands include, but are not limited to, FcγRI, FcγRII, , FcγRIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor , staphylococcal protein A, streptococcal protein G, and viral FcγR. The c ligands also contain Fc receptors, a family of Fc receptors that are homologous to FcγRs. homologue (FcRH) (Davis et al., 2002, Immunolog ical Reviews 190:123-136, incorporated by reference in its entirety. Fc ligands may also include undiscovered molecules that bind to Fc. The ligands are FcRn and Fcγ receptors. "Fc ligand" as used herein Any biological entity that binds to the Fc region of an antibody, forming an Fc / Fc ligand complex when It refers to a molecule, preferably a polypeptide, derived from

[0046] "Fcγ receptor," "FcγR," or "FcqammaR" are used herein. When used herein, it refers to any member of a family of proteins that bind to the Fc region of an IgG antibody. and are encoded by FcγR genes. In humans, this family includes, but is not limited to: However, FcγRI (CD64), including the isoforms FcγRIa, FcγRIb, and and FcγRIc; FcγRII (CD32), e.g., the isoform FcγRIIa ( allotypes H131 and R131), FcγRIIb (FcγRIIb-1 and and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD 16), e.g., the isoform FcγRIIIa (allotypes V158 and F158) ) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRII b-NA2) (Jefferis et al., 2002, Immunol L Ett 82:57-65, incorporated by reference in its entirety), along with any undiscovered FcγRs include human FcγRs or FcγR isoforms or allotypes of the following: From any organism, including but not limited to, human, mouse, rat, rabbit, and monkey Mouse FcγR may include, but 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.

[0047] "FcRn" or "neonatal Fc receptor" as used herein refers to a receptor for IgG antibodies. It refers to a protein that binds to the Fc region and is encoded, at least in part, by the FcRn gene. FcRn is expressed in, but not limited to, human, mouse, rat, rabbit, and rhesus monkeys. As is known in the art, functional FcRs may be derived from any organism containing FcRs. The n protein contains two polypeptides, often referred to as the heavy chain and the light chain. The heavy chain is encoded by the FcRn gene. Unless otherwise specified in the text, FcRn or FcRn protein refers to an FcRn heavy chain and a β- It refers to a complex of IgG1 and IgG2-microglobulin, which enhances binding to the FcRn receptor and may Various FcRn mutants that can be used to further extend serum half-life are shown in the legend to Figure 83. will be done.

[0048] A "parent polypeptide," as used herein, refers to a polypeptide that is subsequently modified to produce a variant. A parent polypeptide refers to a starting polypeptide that is to be synthesized. or may be a variant or modified version of a naturally occurring polypeptide. The peptide may be the polypeptide itself, a composition containing the parent polypeptide, or an amino acid that encodes it. Thus, the term "parent immunoglobulin" as used herein may refer to a specific amino acid sequence. refers to an unmodified immunoglobulin polypeptide that is modified to produce a variant, A "parent antibody," as used herein, also refers to a compound that is modified to generate a variant antibody. "Parent antibody" refers to a known, commercially available, recombinantly produced antibody as outlined below. It should be noted that the body is included.

[0049] "Fc" or "Fc region" or "Fc domain" as used herein means a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain, Sometimes it refers to a portion of the hinge. Thus, Fc refers to IgA, IgD, and Ig The last two constant region immunoglobulin domains of G, and the last of IgE and IgM The three constant region immunoglobulin domains of For IgA and IgM, Fc may include the J chain. For IgG, The Fc domain consists of immunoglobulin domains C gamma 2 and C gamma 3 (Cγ2 and C γ3), and the lower hinge region between C gamma 1 (Cγ1) and C gamma 2 (Cγ2) Although the boundaries of the Fc region might vary, the human IgG heavy chain Fc region typically comprises residues C226 or P230 to its carboxyl terminus, and numbering is based on Kabat According to the EU Index as described below. In some embodiments, As described above, the amino acid modifications may be directed to, for example, one or more FcγR or FcRn receptors. Modifications are made to the Fc region to alter binding of the antibody.

[0050] As used herein, "heavy constant region" refers to the CH1-hinge-CH2-CH3 portion of an antibody. do.

[0051] As used herein, an "Fc fusion protein" or "immunoadhesin" refers to a protein comprising a fusion protein, a fusion protein, or a fusion protein of interest, as defined herein. As described above, the binding moieties for the target protein are generally attached to different proteins (as defined herein). a protein comprising an Fc region linked (optionally via a linker moiety) as described Optionally, one monomer of the heterodimeric antibody comprises an antibody heavy chain (e.g., scFv). the other monomer contains a variant Fc domain and In some embodiments, these "half antibody-half fusions" are Fc fusions comprising the antibody and a ligand. The "fused proteins" are called "fusion bodies."

[0052] "Position" as used herein means a position in the sequence of a protein. Positions may be numbered sequentially or in accordance with established formats, such as E when numbering antibodies. You can also follow the U index.

[0053] A "target antigen," as used herein, is an antigen that is specifically targeted by the variable region of a given antibody. The target antigen may be a protein, carbohydrate, lipid, or other compound. A large number of suitable target antigens are described below.

[0054] "Strandedness" as used herein refers to the heterodimer of the present invention. In the context of antibody monomers, heterodimerization mutations occur similarly to two strands of "matching" DNA. in each monomer so that the body preserves its "matching" ability to form heterodimers. For example, some pI variants are incorporated into monomer A (e.g., When the nucleotide sequence is altered, the steric variants that are also available are "charge pairs" and the pI changes. Charge variants that do not interfere with the structure, e.g., increase the pI, are on the same "chain" or "monomer." Similarly, the pairing of In the case of "skewed" variants present in a set, one skilled in the art will determine which of the pairs will be incorporated. Whether chain or monomer, pI separation is maximized similarly with skewed pIs. The pI is then examined to determine whether the

[0055] "Target cell," as used herein, means a cell that expresses a target antigen.

[0056] "Variable region" as used herein refers to the kappa locus, lambda locus, and and any of the Vk, Vλ, and / or VH genes that make up the heavy chain immunoglobulin locus. of an immunoglobulin containing one or more Ig domains substantially encoded by either It means the area.

[0057] As used herein, "wild type or WT" refers to a naturally occurring gene, including allelic variations. A WT protein refers to an amino acid sequence or nucleotide sequence that is intentionally modified. It has an unmodified amino acid sequence or nucleotide sequence.

[0058] The antibodies of the present invention are generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, identified and isolated and / or recovered from the cell or cell culture in which it is expressed. An isolated polypeptide usually has undergone at least one purification step. An "isolated antibody" is one that is prepared by the same method as other antibodies having different antigen specificities. "Recombinant" refers to an antibody that is substantially free of exogenous antibodies. It means made using acid techniques.

[0059] "Specific binding" or "specifically binds" to a particular antigen or epitope By "specific" is meant binding that is measurably different from non-specific interactions. Specific binding can be achieved, for example, by binding to a control molecule, which is a molecule of similar structure that generally does not have binding activity. This can be measured by checking the binding of the molecule compared to the binding of the molecule. Specific binding can be confirmed by competition with a control molecule that is similar to the target.

[0060] Specific binding to a particular antigen or epitope is, for example, at least about 10 M , at least about 10-5M, at least about 10-6M, at least about 10-7M, or less at least about 10-8M, at least about 10-9M, alternatively, at least about 10-10M, or at least about 10-11 M, at least about 10-12 M, or higher, antigen or has a KD for the epitope (KD refers to the dissociation rate of a particular antibody-antigen interaction) Typically, an antibody that specifically binds to an antigen can be expressed by an antibody that specifically binds to the antigen or enzyme. 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5, 0,000-fold, 10,000-fold, or more, higher KD.

[0061] Specific binding to a particular antigen or epitope can also be measured by, for example, increasing the antigen binding activity of an antigen compared to a control. At least 20x, 50x, 100x, 500x, 1000x, 5,0 00-fold, 10,000-fold, or more higher KA or KA against an antigen or epitope or Ka (KA or Ka is the specific antibody-antigen interaction Binding affinity is commonly measured using a Biacore assay. do.

[0062] II. Overview Bispecific antibodies that co-engage CD3 and tumor antigen targets allow T cells to target tumor cells. These antibodies are designed and used to attack and redirect the CD3 and tumor antigen monovalently associated with CD3. While approaches targeting these drugs have shown considerable promise, common side effects of such treatments remain. Use of steroids often leads to toxic cytokine release syndrome and associated cytokine The anti-CD3 binding domain of the bispecific antibody associates with all T cells. From these cells, a highly cytokine-producing CD4 T cell subset is recruited. 4 T cell subsets whose recruitment and expansion potentially result in immunosuppression and long-term tumor suppression. These include regulatory T cells, which may have a negative effect on immune regulation. Without the c domain, it exhibits an extremely short serum half-life in patients.

[0063] While CD3 targeting has shown considerable promise, there are many limitations to such treatments. Common side effects often result in toxic cytokine release syndrome and associated symptoms. The anti-CD3 binding domain of the bispecific antibody associates with all T cells. This results in the recruitment of highly cytokine-producing CD4 T cell subsets. Furthermore, the CD4 T cell subset, whose recruitment and proliferation potentially leads to immunosuppression, These include regulatory T cells, which may have a negative impact on tumor suppression. One such method may reduce the number of CD4 T cells present in the blood and possibly reduce CD4 T cell activation. The method relies on reducing the affinity of the anti-CD3 domain for CD3.

[0064] Thus, in some embodiments, the present invention provides a "potent" or "high affinity" antibody to CD3. A "compatible" binder (e.g., a heavy and a light variable domain (optionally including a charged linker as needed), and CD3 8. Therefore, the present invention provides anti-CD3 antibodies that are "lighter" or "lower affinity" binders for CD3. Further embodiments provide an antibody construct comprising a CD38 antigen-binding domain. Anti-CD3 antigen-binding domains with intermediate or "moderate" affinity for CD3 The antibody constructs are provided, each comprising a domain. Binding affinity is determined using a Biacore assay. Generally measured.

[0065] The "high, medium, and low" anti-CD3 sequences of the present invention can be used in a variety of heterodimerization formats. It should be understood that the heterodimer "bottom" is also possible in most of the disclosures herein. While the "ball opener" format is used, these variable heavy and variable light arrangements, as well as sc Fv sequences (and Fab sequences containing these variable heavy and variable light sequences) can be expressed in other formats, For example, the one shown in Figure 2 of International Publication No. WO 2014 / 145806 (the The drawings, formats and descriptions used in this specification are expressly incorporated herein by reference. It is possible.

[0066] Thus, the present invention provides heterodimeric antibodies that bind to two different antigens, e.g. For example, antibodies can bind to two different target antigens, commonly target tumor antigens (TTAs), as described below. These heterodimeric antibodies are "bispecific" in that they bind to their target antigens. Monovalent (e.g., a single antigen-binding domain is present, such as a variable heavy and variable light domain pair) bivalent (there are two antigen-binding domains that bind to each antigen independently) The heterodimeric antibodies of the present invention may bind to either the heterodimeric or heterodimeric antibody as also outlined below. Conjugated with a "pI mutant" that allows simple purification of heterodimers from homodimers. "Skewing" heterodimer formation over homodimers as thoroughly outlined by The heterodimers of the present invention are based on the use of different monomers with "skew" amino acid substitutions. In the case of bispecific antibodies, the present invention generally involves the use of a method for producing heterodimeric proteins. of a modified or mutant Fc domain capable of self-assembly within the producing cell. Depending on the use and the methods for making and purifying such heterodimeric proteins, There are.

[0067] III. Antibodies The present invention utilizes two different antigens, e.g., CD3 and a target tumor antigen, e.g., CD19, Bispecific antibodies that bind to CD20, CD38, and CD123 and are generally therapeutic antibodies As discussed below, the term "antibody" is used generically. Antibodies that find use in the present invention include conventional antibodies, as well as the antibody derivatives described herein, It may take several forms as described herein, including fragments and mimetics.

[0068] The structural unit of a conventional antibody typically comprises a tetramer. Each tetramer typically comprises two pairs of identical Each pair consists of a single polypeptide chain, with one "light" chain (typically about 25 kDa) a single "heavy" chain (typically having a molecular weight of about 50-70 kDa) 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. The IgG class is 356 (D or E) and 358 (L or It is noted that the present invention has different allotypes with polymorphisms in the nucleotide sequences (M). The sequence uses the 356D / 358M allotype, but other allotypes are included herein. That is, any sequence containing an IgG1 Fc domain included herein may be Instead of the 6D / 358M allotype, one can have 356E / 358L.

[0069] In addition, many of the sequences herein have at least one amino acid at position 220 replaced with a serine. It has two cysteines, which generally are " scFv monomer” side, but it is “Fab monomer” to reduce disulfide formation. These exchanged cysteines can be on the cysteine-containing side or both. (C220S) or both are specifically included within the sequences herein.

[0070] Therefore, "isotype" as used herein refers to the structure of their constant regions. Any of the subclasses of immunoglobulins defined by their biological and antigenic characteristics It means that the therapeutic antibody may also be a hybrid of isotypes and / or subclasses. It should be understood that the present invention may include, for example, U.S. Patent Application Publication No. 2009 / 016 As shown in US Pat. No. 3,699 (incorporated by reference), the present invention provides an IgG1 / G This involves altering the pI of the two hybrids.

[0071] The amino-terminal portion of each chain is commonly referred to in the art and herein as an "Fv domain." The Fv region is composed of approximately 100 to 110 amino acids, which are primarily involved in antigen recognition. The variable region contains a variable region of 1 or more amino acids that form the antigen-binding site. Therefore, three loops are collected in each of the V domains of the heavy and light chains. The CDRs are called sex-determining regions (hereinafter referred to as "CDRs"), where variations in the amino acid sequence occur. The most notable difference is that certain segments of the variable region vary in sequence among antibodies. This refers to the fact that the variability within a variable region is not uniformly distributed. Instead, the V region is composed of a ratio of 15 to 30 amino acids called the framework region (FR). It consists of a series of relatively invariant regions, each of which is 9-15 amino acids long or longer. These are separated by shorter regions of extreme variability called "hypervariable regions."

[0072] Each VH and VL has, from the amino terminus to the carboxy terminus, FR1-CDR1-FR2-C The three hypervariable regions ("complementarity determining regions") are arranged in the order DR2-FR3-CDR3-FR4. It consists of a nucleotide sequence ("CDR") and four FRs.

[0073] The hypervariable region generally consists of amino acid residues 24 to 34 within the light chain variable region (LCDR1: "L " indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) and 31 to 35B in the heavy chain variable region (HCDR1: "H" indicates heavy chain), 50 to 65B ( HCDR2), and amino acid residues 95 to 102 (HCDR3) (Kabat et al.,SEQUENCES OF PROTEINS OF IMMUNOLO GICAL INTEREST,5th Ed.Public Health Serv ice,National Institutes of Health,Bethes da, Md. (1991)), and / or residues forming hypervariable loops (e.g., Residues 26-32 (LCDR1), 50-52 (LCDR2), and 9 in the light chain variable region 1 to 96 (LCDR3), and 26 to 32 (HCDR1), 53 to 55 ( HCDR2), and 96–101 (HCDR3); Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. The CDRs of are described below.

[0074] As will be appreciated by those skilled in the art, the exact numbering and arrangement of the CDRs may vary. However, the numbering system may vary. It will be understood that the present disclosure of a sequence includes the disclosure of the associated CDRs. Thus, the disclosure of each variable heavy region includes a vhCDR (e.g., vhCDR1, vhCD The disclosure of each variable light region is vlCDR (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0075] Throughout this specification, the Kabat numbering system is used to refer to residues within the variable domain (approximately 100, ... When referring to the amino acid sequence of the heavy chain variable region (residues 1-107 of the amino acid sequence of the heavy chain variable region, and residues 1-113 of the heavy chain variable region), The EU numbering system is commonly used for the Fc region (e.g., Kabat et al. al., supra (1991)).

[0076] The present invention provides a number of different sets of CDRs, where a "complete set of CDRs" is , three variable light CDRs and three variable heavy CDRs, e.g., vlCDR1, vlCDR2 , vlCDR3, vhCDR1, vhCDR2 and vhCDR3, which respectively , may be part of a larger variable light chain domain or variable heavy chain domain. As more fully outlined in the specification, the variable heavy and light chain domains are and if a light chain is used (e.g., if a Fab is used), a separate polypeptide The sequences may be present on multiple chains, or in the case of scFv sequences, on a single polypeptide chain.

[0077] CDRs contribute to forming the antigen-binding, or more specifically, epitope-binding, site of an antibody. An "epitope" is a specific region within the variable region of an antibody molecule known as a paratope. An epitope refers to a determinant that interacts with a specific antigen-binding site. A group of molecules that usually have specific structural characteristics as well as specific charge characteristics. An antigen may have more than one epitope.

[0078] An epitope is a molecule that contains amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope). and other amino acid residues not directly involved in binding, e.g., specific antigen-binding peptides. In other words, the amino acid The residues are present within the footprint of a specific antigen-binding peptide.

[0079] Epitopes can be either conformational or linear. Conformational epitopes is formed by spatially juxtaposed amino acids from different segments of the linear polypeptide chain Linear epitopes are formed by adjacent amino acid residues in a polypeptide chain. Conformational or non-conformational epitopes are epitopes that are induced by the presence of denaturing solvents. Below, we distinguish between the two in that the connection with the former is lost, but the connection with the latter is not lost. Good too.

[0080] Epitopes are typically at least three, more commonly, in a unique spatial arrangement. contains at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope , a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to a target antigen (e.g. This can be seen in the "binning" (for example).

[0081] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected a large number of primary sequences of the variable regions of heavy and light chains. Based on the degree of sequence conservation, Kabat et al. classified individual primary sequences into CDR and framework regions. The list was compiled by classifying them into SEQUENCES OF IMMUNOLOGY CAL INTEREST,5th Ed.,NIH publication,No. 91-3242, E.A. Kabat et al. (incorporated by reference in its entirety ) for more information.

[0082] The IgG subclass of immunoglobulins contains several immunoglobulin domains in the heavy chain. As used herein, "immunoglobulin (Ig) domain" refers to a domain that has a distinct tertiary structure. Of interest in the present invention is the constant weight (CH) region of an immunoglobulin. The heavy chain domain includes the ATP domain and the hinge domain. Each G isotype has three CH regions. The domain is: "CH1" is the EU Internet address as described in Kabat "CH2" refers to the number 118-220 in the index. It refers to the 237th to 340th place in the EU index, and "CH3" is listed in Kabat. The EU Index, as set forth herein and as described below, refers to a ranking of 341-447. As discussed below, the pI variants may be located in one of the CH and hinge regions. There can be more than one.

[0083] The sequences presented herein are from the CH1 region, i.e., starting at position 118, and are It should be noted that the variable region is not included except for the first address of SEQ ID NO:2. Although the amino acid is represented as position "1" in the sequence listing, it is located at 118 in the CH1 region according to the EU numbering. Corresponds to rank.

[0084] Another type of Ig domain in the heavy chain is the hinge region. "Hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to a region of an antibody A flexible polypeptide comprising amino acids between the first and second constant domains of Structurally, the CH1 domain of IgG ends at EU position 220, and The CH2 domain starts at residue EU position 237. Therefore, in the case of IgG, the antibody The range includes positions 221 (D221 of IgG1) to 236 (G236 of IgG1). As defined herein, numbering is based on the EU index as described in Kabat In some embodiments, for example, in the context of an Fc region, a lower hinge is included. The "lower hinge" generally refers to position 226 or 230. As shown, pI variants can also be made in the hinge region.

[0085] The light chain generally comprises a variable light chain domain (which includes the light chain CDRs and which, together with the variable heavy chain domain, The Fv region is formed by a constant light chain region (often called CL or Cκ). Contains two domains.

[0086] Another region that is subject to further substitutions, as outlined below, is the Fc region.

[0087] Thus, the present invention provides different antibody domains. As is known in the art, the heterodimeric antibodies of the present invention may contain different domains within the heavy and light chains. These domains include, but are not limited to: However, the Fc domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, heavy constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-C H3), a variable heavy domain, a variable light domain, a light constant domain, a FAb domain, and Contains an scFv domain.

[0088] Thus, an "Fc domain" refers to a -CH2-CH3 domain, optionally a hinge domain, In embodiments herein, when an scFv is attached to an Fc domain, In this case, it is the C-terminus of the scFv construct that is attached to the hinge of the Fc domain, e.g., It is generally attached to the sequence EPKS, which is the beginning of the hinge. It comprises a human nucleotide sequence and a constant domain, which comprises CH1-optionally CH2-CH3. The light chain contains a variable light domain and a light constant domain. , variable heavy chain, scFv linker, and variable light chain domains. In most constructs and sequences, the C-terminus of the variable light chain is connected to the N-terminus of the scFv linker. and its C-terminus is attached to the N-terminus of the variable heavy chain (N-vh-linker-vl -C), but it can be switched (N-vl-linker-vh-C).

[0089] Some embodiments of the invention comprise at least one scFv domain, which is naturally occurring. However, the variable heavy chain domains and the variable heavy chain domains are generally linked together by an scFv linker. As shown herein, recombinantly produced There are several suitable scFv linkers that can be used, including conventional peptide bonds.

[0090] The linker peptide is primarily composed of the following amino acid residues: Gly, Ser, Ala, or T. The linker peptide may comprise a linker peptide that connects the two molecules together so that they retain the desired activity. long enough to link in such a way that they assume the correct conformation relative to each other, In one embodiment, the linker should have a length of about 1 to 50 amino acids, preferably In one embodiment, a linker of 1 to 20 amino acids in length is used. In some embodiments, the use of about 5 to about 10 amino acids is recognized. For example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGG S) Glycine-seriate 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. Alternatively, flexible linkers include, but are not limited to, polyethylene glycol (P EG), polypropylene glycol, polyoxyalkylene, or polyethylene glycol Various non-proteinaceous polymers, including copolymers of polyethylene glycol and polypropylene glycol, have been shown to It may find use as a linker, i.e., it may find use as a linker.

[0091] Other linker sequences may include any sequence of any length of the CL / CH1 domain. but not all residues in the CL / CH1 domain (e.g., the most The linker is a nucleotide sequence of an immunoglobulin light chain, e.g., Cκ or The linker can be derived from, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, C Derived from immunoglobulin heavy chains of any isotype, including α2, Cδ, Cε, and Cμ The linker sequence may also be used to bind other proteins such as Ig-like proteins (e.g., TCR, FcR, KIR), sequences from hinge regions, and other naturally occurring sequences from other proteins. It may be derived from.

[0092] In some embodiments, the linker may be any two domains (outlined herein together). Any suitable linker is a "domain linker" used to link the While many embodiments may use, for example, (GS), (GSGGS), ( (GGGS)n, and (GGGS)n, where n is an integer of at least 1 (and generally 3 to 4 Each of the two domains has a glycine-serine polymer containing Recombination with sufficient length and flexibility to allow the gene to retain its biological function. Any peptide sequence that allows for attachment may be used. In terms of the linker structure, as outlined below, the charged domain linker is a functional part of the scFv linker. When used in the embodiment, it can be used.

[0093] In some embodiments, the scFv linker is a charged scFv linker, the majority of which is shown in FIG. 33. Thus, the present invention provides a method for determining the pI distribution between the first and second monomers. To facilitate separation, a charged scFv linker is further provided, i.e., either positively or negatively charged. Either (or both in the case of scaffolds using scFvs on different monomers) By incorporating a charged scFv linker, the monomer containing the charged linker can form an Fc domain. It is possible to change the pI without causing further changes in the The linker can be substituted into any scFv with a standard linker. As can be seen, the charged scFv linker can be chosen based on the desired change in pI. For example, as discussed herein, Thus, to generate a triple F-format heterodimeric antibody, each of the desired antigen-binding domains is The initial pI of the Fv region in Also depending on the pI, either a positive or negative linker is selected.

[0094] Similarly, charged domain linkers may also be used to enhance the pI separation of the monomers of the present invention. Therefore, in any embodiment herein where a linker is used, 3 can be used.

[0095] In some embodiments, the antibody is full length. A "full length antibody" herein refers to a heterodimer. specific for the formation of heterodimers or the purification of heterodimers from homodimers. and a variable region in the Fc domain that contains one or more modifications as outlined herein. A full-length antibody refers to the structure that constitutes the natural biological form of an antibody, including the amino acid sequence and constant regions. , generally comprising Fab and Fc domains, and in addition, generally as shown in the figures , scFv, or the like.

[0096] In one embodiment, the polypeptides may be modified to produce heterodimers, such as by modifying the pI. An antibody is an antibody fragment as long as it contains at least one constant domain capable of binding to the antibody. Other antibody fragments that can be used include the CH1, CH2, CH3, and hindpeptides of the present invention that have been modified to have altered pI. For example, Fc fusions include fragments having one or more of the Fc, CL, and CL domains. Fc region (CH2 and CH3, optionally including the hinge region) fused to another protein Many Fc fusions are known in the art, and the heterodimers of the present invention are fusions of This can be improved by adding merization mutants. In the present invention, CH1; CH1, CH2 and and CH3; CH2; CH3; CH2 and CH3; and antibody fusions comprising CH1 and CH3. any or all of which may be prepared using the heterologous vectors described herein. Any combination of dimerization mutants can be used, optionally with a hinge region. This can be done.

[0097] In particular, the format shown in Figure 1 is an antibody commonly referred to as a "heterodimeric antibody," Proteins containing at least two related Fc sequences self-assembled into heterodimeric Fc domains. This means that it has a column.

[0098] Chimeric and Humanized Antibodies In some embodiments, the antibodies are mixtures of antibodies from different species, e.g., chimeric antibodies and / or In general, both "chimeric antibodies" and "humanized antibodies" are defined as antibodies that are It refers to an antibody that combines regions derived from the above species. For example, "chimeric antibodies" have traditionally been It contains variable regions of mouse (or rat, if desired) origin and constant regions of human origin. "Humanized antibodies" generally refer to antibodies in which the variable domain framework regions have been modified to conform to sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody except for the CDRs is replaced with human or encoded by a polynucleotide derived from a human or human genome, except within its CDRs. are identical to antibodies encoded in part or in whole by nucleic acid derived from a non-human organism. The CDRs are grafted into the beta-sheet framework of a human antibody variable region to generate an antibody; The specificity of the antibody is determined by the grafted CDR. Publication No. 92 / 11018 Pamphlet, Jones, 1986, Nature 321 :522-525, Verhoeyen et al., 1988, Science 23 9:1534-1536 (all of which are incorporated by reference in their entirety). To restore the affinity lost in the initial transplant construct, the selected acceptor fragment was "Backmutation" of framework residues to the corresponding donor residues is often required (U.S. Pat. No. 6,233,162). Patent No. 5530101, U.S. Patent No. 5585089, U.S. Patent No. 5693 761, U.S. Patent No. 5,693,762, U.S. Patent No. 6,180,370 No. 5,859,205, No. 5,821,337, U.S. Pat. No. 6,054,297, U.S. Pat. No. 6,407,213 (all incorporated by reference in their entirety). Humanized antibodies also optimally contain immunoglobulin constant regions (typically The antibody will contain at least a portion of a human immunoglobulin constant region, and therefore Typically, a human Fc region is included. Humanized antibodies also utilize a genetically engineered immune system. The gene can be generated using mice carrying the gene. Roque et al., 2004, Biote Chnol. Prog. 20:639-654 (incorporated by reference in its entirety). Techniques and methods for humanizing and reshaping human antibodies are well known in the art. There is (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies,Molecular Biolo gy of B Cells,533-545,Elsevier Science(U SA), and the references cited therein (all of which are incorporated by reference in their entirety). )). Humanization methods include, but are not limited to, those described in Jones et al., 1986, Nature 321:522-525;Riechmann et al.,1988 ;Nature 332:323-329;Verhoeyen et al.,198 8,Science,239:1534-1536;Queen et al.,198 9,Proc Natl Acad Sci,USA 86:10029-33;He et al.,1998,J.Immunol.160:1029-1035;Cart er et al.,1992,Proc Natl Acad Sci USA 89 :4285-9,Presta et al.,1997,Cancer Res.57 (20):4593-9;Gorman et al.,1991,Proc.Natl .Acad.Sci.USA88:4181-4185;O'Connor et al ., 1998, Protein Eng 11:321-8 (all in their entirety) (Incorporated by reference herein). Other methods to reduce virulence are described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973 (incorporated by reference in its entirety) This may include a surface retreatment method such as those described in

[0099] In certain embodiments, the antibodies of the invention are derived from a particular germline heavy chain immunoglobulin gene. heavy chain variable regions from these and / or light chains from specific germline light chain immunoglobulin genes For example, such antibodies may be "the product of" particular germline sequences. " or "derived therefrom" may also include human antibodies comprising heavy or light chain variable regions. or may consist of. "is the product of" human germline immunoglobulin or A human antibody "derived from" therefore has the amino acid sequence of a human antibody derived from a human germline Compared with the amino acid sequence of immunoglobulins, the sequence is closest to the sequence of a human antibody (i.e., Identified by selecting human germline immunoglobulin sequences with the highest identity (%) "is the product of" or a particular human germline immunoglobulin sequence. Human antibodies "derived therefrom" may be derived from, for example, naturally occurring somatic mutations or site-specific mutations. Contains amino acid differences compared to the germline sequence due to the deliberate introduction of mutations However, humanized antibodies are typically derived from human germline immunoglobulin genes. The amino acid sequence is at least 90% identical to the amino acid sequence encoded by the clone, and the germline immunoglobulin amino acid sequence of the species (e.g., mouse germline sequence) When compared, they contain amino acid residues that identify the antibody as being derived from a human sequence. In some cases, humanized antibodies are antibodies encoded by germline immunoglobulin genes. The amino acid sequence and the amino acid sequence are at least 95, 96, 97, 98, or 99% or more They may also be at least 96%, 97%, 98%, or 99% identical. Humanized antibodies derived from specific human germline sequences are derived from human germline immunoglobulins. The amino acid sequence encoded by the α-glucan gene differs by only 10 to 20 amino acids. (before the introduction of any distortions, pIs, and truncation mutants herein; i.e., (i.e., the number of variants is generally low prior to the introduction of the variants of the present invention). Antibodies are composed of five or more amino acid sequences encoded by germline immunoglobulin genes. or even may only exhibit no more than 4, 3, 2, or 1 amino acid difference (and Prior to the introduction of any distortion, pI, and truncation mutants herein; i.e., mutant numbers are generally low before the introduction of the mutants of the present invention).

[0100] In one embodiment, the parent antibody has been affinity matured as known in the art. The methods for affinity maturation include those described, for example, in U.S. Patent Application No. 11 / 004,590. Structure-based methods such as those described above may also be used. To achieve sexual maturity, the method of, but not limited to, Wu et al., 1999, J. Mol. B iol.294:151-162;Baca et al.,1997,J.Biol. Chem.272(16):10678-10684;Rosok et al.,19 96,J.Biol.Chem.271(37):22611-22618;Rader et al.,1998,Proc.Natl.Acad.Sci.USA 95:8 910-8915;Krauss et al.,2003,Protein Engineering Engineering 16(10):753-759 (all incorporated by reference in their entirety) Selection-based methods may be used, including but not limited to those described in U.S. Patent Application No. 09 / 810,510; Tan et al., 2002, J. Immunol.169:1119-1125;De Pascalis et al. , 2002, J. Immunol. 169:3076-3084 (all references taken as a whole) Other humanization methods, including those described in (which are incorporated by reference), involve the removal of only a portion of the CDRs. This may involve transplantation.

[0101] IV. Heterodimeric Antibodies Thus, in some embodiments, the present invention provides a heterodimeric Fc domain and a heterodimeric Fc domain. Fc of two different heavy chain variants that will self-assemble to form a dimeric antibody The heterodimeric antibodies provided depend on the use of the sequences.

[0102] The present invention provides antibodies capable of binding to more than one antigen or ligand, e.g., bispecific antibodies. The present invention is directed to novel constructs for providing heterodimeric antibodies that allow for the synthesis of heterodimeric antibodies. A dimeric antibody construct is one in which two Fc domains of the heavy chains of an antibody, e.g., assemble to form a "dimer." As will be discussed more fully below, Dimeric antibodies are created by modifying the amino acid sequence of each monomer. Thus, the present invention generally provides methods for promoting heterodimer formation and / or inhibiting homodimer formation. To allow for easier purification of the heterodimer, different constant region sequences for each chain were used. We aimed to generate heterodimeric antibodies that can co-associate with antigens in several ways depending on amino acid mutations. Let's call it an elephant.

[0103] Thus, the present invention provides bispecific antibodies. The problem is generally that a molecule binds to two different antigens simultaneously, bringing the different antigens into close proximity and creating a new function. There is a demand for "bispecific" antibodies that offer novel therapeutic potential. Antibodies are produced by including genes for each heavy and light chain in a host cell. This generally results in the production of the desired heterodimer (AB) as well as two homodimers (A -A and BB (which do not involve the issue of light chain heterodimerization) are formed. However, a major obstacle in the formation of bispecific antibodies is the conversion of homodimeric antibodies to heterodimeric antibodies. Purify and / or bias heterodimer formation against homodimer formation The difficulty is in doing so.

[0104] There are several mechanisms available for generating the heterodimers of the present invention. As understood by practitioners, the combination of these mechanisms results in a high degree of heterodimerization. Therefore, the amino acid mutations that result in the production of heterodimers are As discussed below, heterodimerization mutants is a steric variant (e.g., "knobs and holes" below). or "skew" mutants and "charge pair" mutants described below) and homodimers from heterodimers. It may contain "pI variants" that allow for purification of dimers. WO 2014 / 14580 6 pamphlet (see the entire pamphlet and the discussion of "heterodimerization mutants" at Specifically, the following (which is incorporated herein by reference) describes heterodimerization: A useful mechanism is the "knob and hole" ("KIH"; sometimes referred to herein as the "skewed" variant). (See discussion in WO 2014 / 145806), WO 20 "Electrostatic steering" or "charge pairs" as described in the pamphlet of International Publication No. 14 / 145806 pI variants described in WO 2014 / 145806 and WO 201 Further common Fc variants as outlined in the brochure No. 4 / 145806 and below. Includes variants.

[0105] In the present invention, there are several basic mechanisms that can facilitate the purification of heterodimeric antibodies. On the other hand, since each monomer has a different pI, depending on the use of pI mutants, A- This allows for isoelectric purification of A, AB, and BB dimeric proteins. Scaffold formats, such as the "triple F" format, also allow for size-based separation. Furthermore, as outlined, it is also possible to "skew" heterodimer formation relative to homodimers. Therefore, the heterodimerization mutants and the pI or charge pair mutants can be combined. Combinations find particular use in the present invention.

[0106] In general, specific application embodiments of the present invention increase the pI difference between the two monomers. Conjugated with a pI mutant, it promotes heterodimerization over homodimerization. , which depends on the set of mutants including distortion mutants.

[0107] In addition, as will be more fully outlined below, depending on the format of the heterodimeric antibody, the pI The variants may be contained within the constant and / or Fc domains of the monomer, or may be included in the charge linker. A linker (either a domain linker or an scFv linker) can be used. That is, scaffolds utilizing scFvs, such as triple F formats, can be further refined for purification purposes. The scFv may contain a charged linker (either positive or negative) that provides a pI boost. As will be appreciated by those skilled in the art, additional pI adjustments may be made with only charged scFv linkers. Although several triple F forms without the 'F' are useful, the present invention is particularly directed to the use of 'F' in one or both of the monomers. pI variants towards the nucleotide sequence, and / or similarly charged domain linkers are provided. Further amino acid modifications can be made to alter the pI, e.g., Fc , FcRn and KO mutants may also be obtained.

[0108] In the present invention, pI is utilized as a separation mechanism to enable the purification of heterodimeric proteins. The amino acid mutation can be introduced into one or both of the monomeric polypeptides; i.e. The pI of one of the monomers (referred to herein simply as "monomer A") is increased from that of monomer B. or to increase the pI of monomer A and decrease the pI of monomer B. Both monomers A and B can be modified, lowering the Additionally, changes in the pI of either or both monomers can be achieved by removing or adding charged residues. by (e.g., neutral amino acids are converted to positively or negatively charged amino acid residues, e.g., glycine (replacement of glutamic acid from guanine) and changing charged residues from positive or negative to opposite charge. By changing the charge (aspartic acid to lysine) or by changing a charged residue to a neutral residue This can be achieved by changing the lysine to a serine (e.g., loss of charge). Some of these variants are shown in the figures.

[0109] Thus, this embodiment of the invention is capable of separating heterodimers from homodimers. providing sufficient pI change in at least one of the monomers to allow As will be appreciated by those skilled in the art, and as will be further discussed below, this is a "wild The heavy chain constant region is a "type" heavy chain constant region and a variant region (w) that has been modified to increase or decrease the pI. By using both tA-+B or wtA--B, or by increasing one region, This can be done by increasing the area and decreasing the other area (A+-B- or A-B+). .

[0110] Thus, in general, components of some embodiments of the present invention may contain amino acid substitutions ("pI variants"). The dimeric protein can be formed by incorporating a nucleotide sequence (or "pI substitution") into one or both monomers. By changing the isoelectric point (pI) of at least one (if not both) monomer of the protein, These are amino acid mutations within the antibody constant region that are intended to form "pI antibodies." As shown herein, the separation of the heterodimer from the two homodimers is achieved by separating the two monomers. This can be achieved when the pI of the body differs by at least 0.1 pH units, and is 0.2, 0.3, 0.4, and 0.5 or greater all find use in the present invention.

[0111] As will be appreciated by those skilled in the art, to obtain good separation, it is desirable to have each or both monomers The number of pI variants included will depend in part on the starting pI of the components, e.g., in a triple F format, the desired The starting pI of the scFv and Fab will depend on the starting pI of either monomer. To modify or determine the "direction" (e.g., more positive or negative), two indicators The Fv sequence of the target antigen is calculated and the determination is made therefrom. Accordingly, different Fvs will have different starting pIs for use in the present invention. As outlined herein, the pI is determined such that the total pI difference for each monomer is at least about 0.1 l. og (preferably 0.2-0.5 as outlined herein) .

[0112] Furthermore, as will be appreciated by those skilled in the art and as outlined herein, some embodiments In this case, heterodimers can be separated from homodimers based on size. For example, as shown in FIG. Some formats allow for the separation of heterodimers and homodimers based on size, as shown in Make it possible.

[0113] To achieve heterodimerization, pI variants are used by using the constant region of the heavy chain. If this is the case, more modular approaches for designing and purifying bispecific proteins, including antibodies, are possible. Thus, in some embodiments, heterodimerization mutants (skewed and purified heterodimerization mutants) are not contained within the variable region, so individual antibodies Furthermore, in some embodiments, immunity due to pI variants may be increased. The potential for immunogenicity is due to the pI being varied so that the pI is altered without introducing significant immunogenicity. The effect of importing I variants from different IgG isotypes was significantly reduced. Therefore, a further problem to be solved is to obtain a high human sequence content, e.g., a non-human sequence at any particular position. The goal is to elucidate low pI constant domains with minimization or avoidance of amino acid residues.

[0114] Potential side effects of modifying this pI include prolonging serum half-life and improving FcRn binding. There are also enhancements for cases such as those described in U.S. Patent Application Serial No. 13 / 194,904 (the entirety of which is incorporated herein by reference). (as found in fusions of antibodies and Fc), as described in Prolonged serum retention in vivo by lowering the pI of antibody constant domains (including These pI variants for extending serum half-life may also result in increased cleavage time for purification. This promotes a change in the pI of the

[0115] Additionally, the ability to either eliminate, minimize, or distinguish between homodimers when they exist. Since the pI of heterodimerization mutants is important, the pI of heterodimerization mutants is important for the analysis and characterization of bispecific antibodies. It should be noted that this provides additional benefits in the quality control process. Similarly, the ability to reliably test the reproducibility of heterodimeric antibody production is important.

[0116] Heterodimerization mutants The present invention provides heterodimers that allow for the formation and / or purification of heterodimers from homodimers. Heterodimeric proteins, including heterodimeric antibodies, in various formats utilizing dimeric variants. Provide quality.

[0117] There are several sets of suitable pairwise heterodimerization-skewed mutants. These mutants are The nucleotides are present in "sets" of "pairs." That is, one set of the pair is incorporated into the first monomer. The other set of the pair is incorporated into a second monomer. without behaving as a "knobs-in-holes" mutant. There is a one-to-one correspondence between residues on one monomer and residues on the other monomer, i.e., These pairs of pairs promote heterodimer formation and prevent homodimer formation. By forming an interface between the monomers, one hundred heterodimers can form spontaneously under biological conditions. The fraction was 50% of the expected (25% homodimer A / A: 50% heterodimer A / B: It is noteworthy that the homodimer B / B ratio is greater than 90% rather than 25%. It should be.

[0118] Stereovariants In some embodiments, heterodimer formation can be promoted by the addition of a steric variant. That is, by modifying the amino acids within each heavy chain, different heavy chains can be associated to produce the same Fc antigen. It is more likely to form heterodimer structures than homodimers with the amino acid sequence. Suitable stereovariants are included in Figure 29.

[0119] One mechanism generally favors heterodimer formation and opposes homodimer formation. The term "knob and hole" is used in the art to refer to amino acid modifications that create steric effects such as While this is sometimes referred to as "the 'rule'" and can be used optionally, it is sometimes referred to as "the 'rule'" in U.S. Patent Application No. No. 61 / 596,846, Ridgway et al., Protein En engineering 9(7):617(1996);Atwell et al.,J Mol. Biol. 1997 270:26; U.S. Pat. No. 8,216,805 (all of which are incorporated herein by reference in their entireties) In the figure, several "monomer A- A pair of monomers, B and B, has been identified. As described in Biotech. 16:677 (1998), these "knob and hose" The "al" mutation distorts the formation of heterodimers, thus combining with disulfide bonds. It can be combined.

[0120] A further mechanism that finds use in generating heterodimers is described by Gunasekara et al. n et al., J. Biol. Chem. 285(25):19637(2010) (which is incorporated herein by reference in its entirety) This may be referred to herein as a "charge pair." In this embodiment, Electrostatics are used to distort the formation of heterodimers. As such, these may also have an effect on pI and therefore purification. Therefore, they can sometimes be considered pI variants. They were engineered to promote merization and were not used as purification tools. are classified as "stereovariants." These include, but are not limited to, D221R / P228R / K409R (e.g., these are the "monomer matched set") and the pair D221E / P22 C22 paired with 8E / L368E and C220R / E224R / P228R / K409R Includes 0E / P228E / 368E.

[0121] The additional monomer A variant and the monomer B variant may optionally and independently be present in any amount. , other variants, such as the pI variants outlined herein or U.S. Patent Application Publication No. 201 Other stereovariants shown in Figure 37 of JP 2002 / 0149876 (the drawings and legends thereof) and SEQ ID NOs. are expressly incorporated herein by reference). Cut.

[0122] In some embodiments, the stereovariants outlined herein are optionally and independently: With any pI variant (or other variants, e.g., Fc variants, FcRn variants, etc.) can be incorporated into one or both monomers and can be independently optionally included or can be excluded from the proteins of the present invention.

[0123] A list of suitable distortion variants can be found in Figure 29, and Figure 34 shows some particularly useful pairs in many embodiments, including, but not limited to, S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411 E / K The set of pairs comprising 360E / Q362E:D401K;L368D / K370S:S364K / E357L and K370S:S364K / E357Q is particularly used. In terms of nomenclature, the pair "S364K / E357Q:L368D / K370S" means that one of the monomers has the double mutant set S364K / E357Q and the other has the double mutant set L368D / K370S.

[0124] pI (isoelectric point) variants in heterodimers Generally, as will be appreciated by those skilled in the art, there are two general categories of pI variants: i.e., those that increase the pI of a protein (basic changes) and those that decrease the pI of a protein As described herein, all of these variants One monomer may be wild-type or significantly different from the wild-type. The variant may not exhibit the same pI, while the other may be either more basic or more acidic. Alternatively, each monomer may undergo a change, becoming either more basic or more acidic. There are some that become like this.

[0125] Preferred combinations of pI variants are shown in Figure 30. As shown, these changes are shown for IgG1, but are not limited to all isotypes, including In addition, isotype hybrids can be modified in this manner. When the ratio is 2 to 4, R133E and R133Q can also be used.

[0126] In one embodiment, for example, in a bottle opener format, a preferred combination of pI variants is The combination contains one monomer containing the 208D / 295E / 384D / 418E / 421D variants. Negative Fab side (N208D / Q295E / N384 compared to human IgG1) D / Q418E / N421D) and a positively charged scFv linker containing (GKPGS) However, as will be understood by those skilled in the art, As shown, the first monomer contains a CH1 domain including position 208. In constructs that do not contain one domain (e.g., in a double scFv format, for example), Heterodimeric Fc fusion proteins that do not utilize a CH1 domain in one of the main domains A preferred negative pI mutant Fc set is the 295E / 384D / 418E / 421D mutant (Q295E / N384D / Q418E / N421D compared to human IgG1) include.

[0127] Light chain variants of antibody heterodimers In the case of antibody-based heterodimers, for example, at least one monomer is attached to a heavy chain domain. In the case of a light chain in addition to the ribonucleotides, pI variants can also be made within the light chain. Amino acid substitutions for this purpose include, but are not limited to, K126E, K126Q, and K145E. , K145Q, N152D, S156E, K169E, S202E, K207E and light This category is based on the constant lambda light chain. The changes in the α-amino acid sequence are R108Q, Q124E, K126Q, N138D, K145T and and Q199E. In addition, the pI of the light chain can be increased. .

[0128] Isotype variants Additionally, many embodiments of the present invention provide for the detection of IgG from one IgG isotype to another. It relies on the "incorporation" of pI amino acids at specific positions into the This reduces or eliminates the possibility of introducing unwanted immunogenicity into the variants. Some are described in U.S. Patent Application Publication No. 2014 / 0370013, incorporated herein by reference. 21 of the patent application (incorporated herein). That is, for various reasons, including high effector function, Therefore, IgG1 is a common isotype for therapeutic antibodies. The heavy constant region of IgG1 has a higher pI than the heavy constant region of IgG2 (8.10 vs. 7.31) By introducing IgG2 residues into specific positions of the IgG1 backbone, the resulting The pI of the monomer is decreased (or increased) and in addition exhibits an increased serum half-life. IgG1 has glycine at position 137 (pI 5.97), while IgG2 has glutamic acid (pI 3 22). The incorporation of glutamic acid increases the p As discussed below, several amino acid substitutions generally affect the This is required to significantly affect the pI of the antibody. It is noteworthy that even changes in the IgG2 molecule can extend the serum half-life. should be.

[0129] In other embodiments, to reduce the overall charge state of the resulting protein, (e.g., by changing a higher pI amino acid to a lower pI amino acid) ), or to accommodate structures for stability etc., as will be explained further below. Non-isotypic amino acid changes are made.

[0130] In addition, by modifying the pI of both the heavy and light constant domains, Significant changes can be seen in each monomer of the heterodimer. As discussed herein, By making the pI of the two monomers differ by at least 0.5, ion exchange chromatography can be performed. Separation by electrophoresis or isoelectric focusing, or other methods sensitive to the isoelectric point, is It may be possible.

[0131] pI calculation The pI of each monomer is calculated by multiplying the pI of the variant heavy chain constant domain by the pI of the variant heavy chain constant domain and .... The pI of the fusion partner and the total monomer content may depend on the pI of the fusion partner. The change in pI is shown in Figure 19 of US Patent Application Publication No. 2014 / 0370013. The calculated values ​​are based on the variant heavy chain constant domains using the chart provided in the present specification. As shown in Fig. 1, which monomers are modified generally depends on the Fv and scaffold regions. Alternatively, the pI of each monomer can be compared.

[0132] pI variants that also result in better FcRn in vivo binding If pI mutants lower the pI of the monomer, they may improve serum retention in vivo. This may have the additional benefit of

[0133] Although still under investigation, it is thought that FcRn binds to FcRn at pH 6 in endosomes, resulting in FcRn-mediated endosomal transport. The Fc region is thought to have a longer half-life in vivo due to the sequestering of hetie and Ward,1997 Immunol Today.18(12) :592-598, incorporated by reference in its entirety). The endosomal compartment then When the compartment is opened to the extracellular space, a higher p of approximately 7.4 is released. H induces reverse release of Fc into the blood. In mice, Dall' Acqua et al. However, Fc mutants with enhanced binding to FcRn at pH 6 and pH 7.4 were detected at serum concentrations and showed that it actually reduced the half-life to the same level as wild-type Fc (Dall'Acq ua et al.2002, J.Immunol.169:5171-5180, entire The increase in affinity of Fc for FcRn at pH 7.4 is This is thought to inhibit the release of Fc back into the blood. Fc mutations that extend the FcRn binding time would ideally increase binding to FcRn at lower pH. The amino acid histidine enhances the binding of Fc to the ATP-binding domain while also allowing the release of Fc at higher pH. It changes its charge state within the pH range of 0.0 to 7.4. It is not surprising to find His residues at key positions in the complex.

[0134] Recently, antibodies with variable regions with lower isoelectric points have also been shown to have longer serum half-lives. It has been suggested that this may be possible (Igawa et al., 2010 PEDS.23( 5):385-392, which is incorporated by reference in its entirety. It remains poorly understood. Furthermore, the variable regions differ between antibodies. and constant region variants with extended half-life, as described herein, of antibodies. This will provide a more modular approach to improving pharmacokinetic properties.

[0135] Additional Fc variants for added functionality In addition to pI amino acid variants, other amino acid variants include, but are not limited to, binding to one or more FcγR receptors. Active molecules can be engineered for a variety of reasons, including altering the affinity of the molecule, altering the binding to the FcRn receptor, etc. There are several Fc amino acid modifications that can be used.

[0136] Thus, the proteins of the present invention are referred to herein as nucleotides, including pI variants and conformational variants. Each set of variants may contain amino acid modifications, including dimerization variants as outlined. and may optionally be included or excluded from any particular heterodimeric protein. It is possible.

[0137] FcγR mutants Thus, useful antibodies that can be engineered to modify binding to one or more FcγR receptors are There are several Fc substitutions. Substitutions that result in enhanced binding and those that result in decreased binding may be useful. For example, enhanced binding to Fc RIIIa is generally associated with ADCC (antibody-dependent cellular cytotoxicity). Nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently It is known that the activation of ATP leads to an increase in the cell-mediated response (cell-mediated response) that causes lysis of the target cells. Similarly, reduced binding to FcγRIIb (an inhibitory receptor) may be advantageous in some circumstances as well. The amino acid substitutions that find use in the present invention can be No. 24,620 (particularly FIG. 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 which is incorporated herein by reference in its entirety and in particular with respect to the variants disclosed therein. (which are expressly incorporated herein by reference). Specific variants that find use include, but are not limited to, 236A, 239D, 239E , 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V and 299T.

[0138] Additionally, U.S. Patent Application Serial No. 12 / 341,769 (incorporated herein by reference in its entirety) As specifically disclosed in U.S. Pat. No. 4,344,999, the disclosure of which is incorporated by reference herein, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L and 259I / 308 F / 428L, which is used to enhance binding to the FcRn receptor and extend serum half-life There are further Fc substitutions that have been discovered.

[0139] truncation mutants Similarly, another category of functional mutants is the "FcγR truncation mutants" or "Fc knot mutants." In these embodiments, some therapeutic In order to avoid additional mechanisms of action in the process, one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) It is desirable to reduce or eliminate the normal binding of the Fc domain. For example, in many embodiments, in particular in the use of bispecific antibodies that bind monovalently to CD3. In order to eliminate or significantly reduce ADCC activity, FcγRIIIa binding is disrupted. It is generally desirable to cleave the Fc domains, where one of the Fc domains is cleaved by one or more Fcγ receptors. These truncation mutants are shown in Figure 31, each of which is independent and optionally included. In a preferred embodiment, G236R / L328R, E233 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 del. The truncation mutants referred to herein are selected from the group consisting of It is noteworthy that the variants disrupt FcγR binding but generally do not disrupt FcRn binding. It is.

[0140] Heterodimer and Fc variant combinations As will be appreciated by those skilled in the art, the listed (including distortions and / or pI variants) ) All of the heterodimerization mutants retain their "chain state" or "monomer compartment." They can be optionally and independently combined in any manner as long as they are compatible with the intended purpose. All of these variants can be combined into any of the heterodimerization formats .

[0141] In the case of pI variants, embodiments that find particular use are shown in the figures, while others facilitate purification. Therefore, other combinations can be made following the basic rule of modifying the pI difference between the two monomers. It can be manufactured.

[0142] In addition, any of the heterodimerization variants (skewed and pI) are generally as outlined herein. As described above, the Fc truncation mutant, Fc mutant, and FcRn mutant are independently and arbitrarily selected. are combined selectively.

[0143] Useful forms of the invention As will be understood by those skilled in the art and discussed more fully below, the heterodimeric fusion proteins of the present invention Synthetic proteins can adopt a wide variety of conformations, as generally shown in Figure 1. The drawing represents a "single-end" configuration, where one "arm" of the molecule There is one specificity and a different specificity for the other "arm." represents a "dual-end" configuration, where there is at least one specific There is a specificity and one or more different specificities at the "bottom" of the molecule. The present invention relates to novel immunoglobulin compositions that co-associate with different first and second antigens. Let's say.

[0144] As will be appreciated by those skilled in the art, the heterodimeric forms of the present invention may be formed by combining heterodimeric groups of different valencies and The heterodimeric antibodies of the present invention may be bivalent and bispecific. wherein one target tumor antigen (e.g., CD3) is bound by a first binding domain. The other target tumor antigen (e.g., CD20, CD38, CD123, etc.) is bound to the second Heterodimeric antibodies can also be trivalent and bispecific. wherein the first antigen is bound by two binding domains and the second antigen is bound by a second binding domain. The binding domain reduces potential side effects as outlined herein. Therefore, when CD3 is one of the target antigens, CD3 is bound exclusively monovalently. is preferred.

[0145] In the present invention, an anti-CD3 antigen combined with an anti-target tumor antigen (TTA) antigen binding domain is As will be appreciated by those skilled in the art, the figures (particularly Figures 2-7 and and Figure 68). Using any collection of main and variable heavy chain domains, Fab and scFv Similarly, any of the anti-TTA antigen binding domains, e.g., anti-CD38, anti-CD 20, anti-CD19 and anti-CD123 antigen binding domains are shown in any of the figures. Any combination of such CDRs, variable light and variable heavy domains, Fab and scFv whether or not they can be used in combination optionally and independently in the context of It is possible.

[0146] Bottle opener format One heterodimeric scaffold that finds particular use in the present invention is shown in FIG. in the "triple F" or "bottle opener" scaffold format as shown in A and B. In this embodiment, one heavy chain of the antibody is a single-chain Fv ("scFv" as defined below). Fv"), and the other heavy chain is in the "standard" FAb format containing a variable heavy chain and a light chain. This structure is referred to herein as a bottle opener due to its rough visual similarity to a bottle opener. This is referred to as the "triple F" format (scFv-FAb-Fc) or "bottle opener" format. The two chains may be heterodimerized, as explained more fully below (see Figure 1). Constant regions (e.g., Fc domain, CH1 domain and / or The binding is achieved by the use of amino acid variants in the amino acid sequence (or hinge region).

[0147] The "Triple F" format of the present invention has several distinct advantages. Furthermore, antibody analogs that rely on two scFv constructs have been shown to have stability and aggregation problems. These are often mitigated by the addition of "standard" heavy and light chain pairings in the present invention. Furthermore, for formats that rely on two heavy chains and two light chains, In the case of incorrect pairing of chains (eg, heavy chain 1 paired with light chain 2), no problem exists.

[0148] Many of the embodiments outlined herein generally involve (in many cases, but not all) charged The scFv comprises variable heavy and variable light domains covalently linked using a scFv linker (as described above). The first monomer, containing the scFv, is reliant on a bottle opener format, in which case the scFv is Fvs contain a first Fc domain at the N-terminus, usually (as outlined herein) uncharged or The domains are covalently linked via a domain linker (which can be either neutral or charged). The second monomer of the polynucleotide format is a heavy chain and the composition further comprises a light chain.

[0149] Generally, in many preferred embodiments, the scFv is a domain that binds to CD3, The Fabs of the heavy and light chains bind to the other TTA.

[0150] Additionally, the Fc domains of the present invention generally comprise skewed mutants (e.g., particularly useful skewed mutants are S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411 E / K360E / Q362E:D401K;L368D / K370S:S364K / E357L and K370S:S364K / E357Q), optionally truncation mutants (including those shown in Figure 31), optionally charged scFv linkers (including those shown in Figure 33), and the heavy chain comprises pI mutants (including those shown in Figure 30).

[0151] In some embodiments, any of the vh and vl sequences presented herein (CD All of the antibodies depicted in the figure, including those specific for 20, CD38, and CD123, are The anti-CD3 scFv sequences shown in the figure (including the vh and vl sequences) can be used. This can be used to add the "Fab side" to the bottle opener skeleton format in Figure 162. Anti-CD3 sequences that find particular use in these embodiments include the scaffold shown in Figure 162. Anti-CD3 H1.30_L1.47 and anti-CD3 H1.3 are attached as scFvs. 2_L1.47, anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.4 7, anti-CD3 H1.33_L1.47, and anti-CD3 H1.31_L1.47. do.

[0152] The present invention relates to an anti-CD3 scFv sequence as shown in Figures 2 to 7 and 68, A bottle opener format including any combination with the skeleton format of FIG. 162 is provided. 2 to 7 and 68. Can be used as the ab side.

[0153] The present invention relates to a CD38 antibody in which the anti-CD38 sequence is as shown in the drawings, including Figures 8 to 10. As described above, each of the v The h and vl anti-CD38 sequences can be on either the Fab or scFv side. , linked as one of the antigen-binding domains in a bottle opener format, including those in FIG. 162 When the anti-CD38 sequence is on the Fab side, any of the anti-CD3 scFvs shown in the figure can be used. Sequences can also be used, particularly those attached as the scFv side of the scaffold shown in Figure 162. anti-CD3 H1.30_L1.47, anti-CD3 H1.32_L1.47, and anti-CD3 H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3 H1.33_ L1.47, and anti-CD3 H1.31_L1.47.

[0154] The present invention relates to a CD20 antigen-binding domain in which the anti-CD20 sequence is as shown in the figures. As described above, the respective vh and vl anti-C The D20 sequence can be on either the Fab or scFv side and is shown in Figure 162. The antigen-binding domains can be linked as one of the bottle opener-style antigen-binding domains comprising: When the CD20 sequence is on the Fab side, any of the anti-CD3 scFv sequences shown in the figure can be used. In particular, the anti-CD3 H1 antibody can be attached as the scFv side of the scaffold shown in Figure 162. .30_L1.47, anti-CD3 H1.32_L1.47, anti-CD3 H1.89_L1 .47, anti-CD3 H1.90_L1.47, anti-CD3 H1.33_L1.47, and and anti-CD3 H1.31_L1.47.

[0155] The present invention relates to the CD123 antigen-binding domain, where the anti-CD123 sequence is as shown in the figures. As described above, each of the vh and vl The anti-CD123 sequence can be on either the Fab or scFv side, see Figure 162 The antigen-binding domains can be linked together as one of the bottle opener-style antigen-binding domains, including When the anti-CD123 sequence is on the Fab side, any of the anti-CD3 scFv sequences shown in the figure can be used. In particular, an anti-CD40 antibody attached as the scFv side of the scaffold shown in Figure 162 can be used. 3 H1.30_L1.47, anti-CD3 H1.32_L1.47, anti-CD3 H1.8 9_L1.47, anti-CD3 H1.90_L1.47, anti-CD3 H1.33_L1.4 7, and anti-CD3 H1.31_L1.47.

[0156] mAb-Fv format One heterodimeric scaffold that finds particular use in the present invention is shown in FIG. In this embodiment, this format includes an "extra" variable heavy chain domain. C-terminal attachment of the variable light chain domain to one monomer and the "extra" variable light chain domain to the other monomer. Relying on the use of C-terminal attachment to form the third antigen-binding domain, where The two monomeric Fab domains bind to TTA, and the "extra" scFv domain binds to CD3. Combine.

[0157] In this embodiment, the first monomer comprises a first variable light chain domain linked to the first monomer using a domain linker. a first variable heavy chain domain and a first Fc domain covalently linked to the C-terminus of the first Fc domain a first heavy chain (vh1-CH) comprising a first constant heavy chain domain comprising an Fc domain of 1-hinge-CH2-CH3-[optional linker]-vl2). The second monomer is a second variable heavy chain domain and a second constant heavy chain domain containing two Fc domains; a third variable heavy chain domain covalently linked to the C-terminus of the second Fc domain using a linker; (vj1-CH1-hinge-CH2-CH3-[optional linker]-vh2 The two C-terminally attached variable domains form an scFv that binds to CD3 (e.g., (However, it is less preferred to have bivalent CD3 binding.) In this embodiment, T The variable light chain domain associates with the heavy chain to form two identical Fabs that bind to the TA and a common light chain comprising a constant light chain domain. For the most part, these constructs may contain distortion mutants, as desired and described herein. These include pI mutants, truncation mutants, additional Fc mutants, and the like.

[0158] The present invention relates to the anti-CD3 scFv sequences shown in Figures 2 to 7 and 68. The mAb-Fv format is provided.

[0159] The present invention provides mAb-Fv formats in which the anti-CD38 sequences are shown in Figures 8-10. .

[0160] The present invention relates to a method for producing a CD20 antigen-binding domain comprising administering to a subject an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the figures. The mAb-Fv format is provided.

[0161] The present invention relates to a method for producing a CD19 antigen-binding domain comprising administering to a subject an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the figures. The mAb-Fv format is provided.

[0162] The present invention relates to a method for producing a compound having an anti-CD123 sequence having a CD123 antigen binding domain as shown in the figures. The present invention provides a mAb-Fv format that

[0163] The present invention provides mAb-Fv formats that include truncation mutants such as those shown in FIG.

[0164] The present invention provides mAb-Fv formats that include distortion mutants such as those shown in Figures 29 and 34. Provide.

[0165] mAb-scFv One heterodimeric scaffold that finds particular use in the present invention is shown in FIG. In this embodiment, the format is a mAb-scFv format in which the scFv is attached to one of the monomers. forming a third antigen-binding domain by relying on the use of a C-terminal attachment of v, The two monomeric Fab portions bind to TTA, and the "extra" scFv domain binds to CD3 Thus, the first monomer binds to the scFv variable light chain domain in either orientation. , a C-terminally covalently attached scFv comprising an scFv linker and an scFv variable heavy chain domain A first heavy chain (including a variable heavy chain domain and a constant domain) having an Fv (vh 1-CH1-hinge-CH2-CH3-[optional linker]-vh2-scFv linker Car-vl2 or vh1-CH1-hinge-CH2-CH3-[optional linker] In this embodiment, two identical scFvs that bind to TTA are A variable light domain and a constant light domain that associate with the heavy chain to form a single Fab Further, a common light chain comprising the following structures is used: Constructs may include distortion mutants, pI mutants, truncation mutants, and the like, as desired and described herein. , additional Fc variants, etc.

[0166] The present invention relates to the anti-CD3 scFv sequences shown in Figures 2 to 7 and 68. The mAb-Fv format is provided.

[0167] The present invention provides mAb-Fv formats in which the anti-CD38 sequences are shown in Figures 8-10. .

[0168] The present invention relates to a method for producing a CD20 antigen-binding domain comprising administering to a subject an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the figures. The mAb-Fv format is provided.

[0169] The present invention relates to a method for producing a CD19 antigen-binding domain comprising administering to a subject an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the figures. The mAb-Fv format is provided.

[0170] The present invention relates to a method for producing a compound having an anti-CD123 sequence having a CD123 antigen binding domain as shown in the figures. The present invention provides a mAb-Fv format that

[0171] The present invention provides mAb-Fv formats that include truncation mutants such as those shown in FIG.

[0172] The present invention provides mAb-Fv formats that include distortion mutants such as those shown in Figures 29 and 34. Provide.

[0173] Central scFv One heterodimeric scaffold that finds particular use in the present invention is shown in FIG. In this embodiment, the format is a Central-scFv format containing an inserted scFv. By relying on the use of an Fv domain to form a third antigen-binding domain, The two monomeric Fab domains bind to TTA, and the "extra" scFv domain binds to CD3. The scFv domain binds to the Fc domain and one of the CH1-Fv regions of the monomer. The insertion provides a third antigen-binding domain.

[0174] In this embodiment, one monomer comprises an scFv variable light domain, an scFv linker and and an scFv comprising a first variable heavy chain domain, CH The antibody comprises a first heavy chain comprising an Fc domain (and an optional hinge) and an Fc domain. scFv consists of a heavy constant domain, a C-terminus of the CH1 domain, and an N-terminus of the first Fc domain. are covalently linked with an optional domain linker between them (vh1-CH1-[optional [optional linker including hinge]-vh2-scFv linker-vl2-[optional linker including hinge] -CH2-CH3 or the opposite orientation for scFv, vh1-CH1-[optional linker]-vl2-scFv linker-vh2-[optional linker including hinge] The other monomer is a standard Fab. In this embodiment, TT The variable light domain and heavy chain domain associate to form two identical Fabs that bind to A. A common light chain comprising a constant light chain domain and a constant light chain domain is further used. With respect to these constructs, as desired and described herein, distortion mutants, p I mutants, truncation mutants, additional Fc mutants, etc.

[0175] The present invention relates to the anti-CD3 scFv sequences shown in Figures 2 to 7 and 68. The Central-scFv format is provided.

[0176] The present invention relates to a method for treating CD38 in the form of a central scFv, in which the anti-CD38 sequence is as shown in Figures 8 to 10. Provide the formula.

[0177] The present invention relates to a method for producing a CD20 antigen-binding domain comprising administering to a subject an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the figures. The Central-scFv format is provided.

[0178] The present invention relates to a method for producing a CD19 antigen-binding domain comprising administering to a subject an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the figures. The Central-scFv format is provided.

[0179] The present invention relates to a method for producing a compound having an anti-CD123 sequence having a CD123 antigen binding domain as shown in the figures. We provide a Central-scFv format that

[0180] The present invention provides a Central-scFv format containing truncation mutants as shown in Figure 31. provide.

[0181] The present invention provides a Central-sc gene that includes distortion variants such as those shown in Figures 29 and 34. Provides Fv format.

[0182] Central-Fv format One heterodimeric scaffold that finds particular use in the present invention is shown in FIG. In this embodiment, the format is a Central-Fv format containing an inserted scFv By relying on the use of domains to form a third antigen-binding domain, where two The monomeric Fab portion binds to TTA, and the "extra" scFv domain binds to CD3 The scFv domain is inserted between the Fc domain and the CH1-Fv region of the monomer. This provides a third antigen-binding domain, where each monomer has components of an scFv. (e.g., one monomer contains a variable heavy chain domain and the other contains a variable light chain domain) .

[0183] In this embodiment, one monomer comprises a first variable heavy chain domain, a CH1 domain and It comprises a first heavy chain containing an Fc domain and an additional variable light chain domain. The heavy constant domain is bound to the C-terminus of the CH1 domain and the N-terminus of the first Fc domain. Covalently linked using a main linker (vh1-CH1-[optional linker]-v The other monomer contains the first variable heavy chain domain, CH1 domain. A first heavy chain containing a main and an Fc domain and an additional variable heavy chain domain ( vh1-CH1-[optional linker]-vh2-hinge-CH2-CH3). The main amino acid sequence is the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain. They are covalently linked between them using a domain linker.

[0184] In this embodiment, the heavy chains associate to form two identical Fabs that bind to TTA. A common light chain is further used, which comprises a variable light domain and a constant light domain. For many of the embodiments herein, these constructs are desirable and described herein. These include distortion mutants, pI mutants, truncation mutants, additional Fc mutants, etc.

[0185] The present invention relates to the anti-CD3 scFv sequences shown in Figures 2 to 7 and 68. Provides Central-Fv format.

[0186] The present invention relates to a method for treating CD38 in a manner similar to that described above, in which the anti-CD38 sequence is in the form of a Central-Fv, as shown in Figures 8 to 10. provide.

[0187] The present invention relates to a method for producing a CD20 antigen-binding domain comprising administering to a subject an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the figures. Provides Central-Fv format.

[0188] The present invention relates to a method for producing a CD19 antigen-binding domain comprising administering to a subject an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the figures. Provides Central-Fv format.

[0189] The present invention relates to a method for producing a compound having an anti-CD123 sequence having a CD123 antigen binding domain as shown in the figures. We provide the Central-Fv format.

[0190] The present invention provides Central-Fv formats that include truncation mutants such as those shown in Figure 31. do.

[0191] The present invention provides Central-Fv including distortion mutants as shown in Figures 29 and 34. Provide a format.

[0192] Single-arm Central-scFv One heterodimeric scaffold that finds particular use in the present invention is shown in FIG. In this embodiment, one of the monomers is a single-arm Central-scFv format. One monomer contains only the Fc domain, while the other monomer uses an inserted scFv domain. In this format, the Fab portion is linked to the TTA The scFv domain binds either to CD3 and the scFv domain binds to CD4 or vice versa. The domain is inserted between the Fc domain and the CH1-Fv region of one of the monomers.

[0193] In this embodiment, one monomer comprises an scFv variable light domain, an scFv linker and and an scFv comprising a first variable heavy chain domain, CH scFvs contain a first heavy chain containing a heavy constant domain and an Fc domain. A domain linker is used between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain. The second monomer comprises an Fc domain. In this embodiment, the Fab domain is formed. Further, a light chain comprising a variable light domain and a constant light domain is associated with the heavy chain to form a For many of the embodiments herein, these constructs are used in distortion mutants, pI mutants, truncation mutants, additional Fc mutations as desired and described Including the body.

[0194] The present invention relates to the anti-CD3 scFv sequences shown in Figures 2 to 7 and 68. A single-arm central-scFv format is provided.

[0195] The present invention relates to a method for treating an inflammatory bowel disease in which the anti-CD38 sequence is a single-arm Central- Provided in scFv format.

[0196] The present invention relates to a method for producing a CD20 antigen-binding domain comprising administering to a subject an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the figures. A single-arm Central-scFv format is provided.

[0197] The present invention relates to a method for producing a CD19 antigen-binding domain comprising administering to a subject an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the figures. A single-arm Central-scFv format is provided.

[0198] The present invention relates to a method for producing a compound having an anti-CD123 sequence having a CD123 antigen binding domain as shown in the figures. It provides a single-arm Central-scFv format that

[0199] The present invention provides a single-arm Central-sc protein containing a truncation mutant as shown in Figure 31. Provides Fv format.

[0200] The present invention provides single-arm Centrifugal forceps containing distortion mutants as shown in Figures 29 and 34. al-scFv format is provided.

[0201] Dual scFv format The present invention also relates to dual scFv formats known in the art and as shown in FIG. to provide.

[0202] The present invention relates to the anti-CD3 scFv sequences shown in Figures 2 to 7 and 68. A dual scFv format is provided.

[0203] The present invention provides a dual scFv format in which the anti-CD38 sequences are shown in Figures 8-10. .

[0204] The present invention relates to a method for producing a CD20 antigen-binding domain comprising administering to a subject an anti-CD20 sequence having a CD20 antigen-binding domain as shown in the figures. A dual scFv format is provided.

[0205] The present invention relates to a method for producing a CD19 antigen-binding domain comprising administering to a subject an anti-CD19 sequence having a CD19 antigen-binding domain as shown in the figures. A dual scFv format is provided.

[0206] The present invention relates to a method for producing a compound having an anti-CD123 sequence having a CD123 antigen binding domain as shown in the figures. A dual scFv format is provided.

[0207] The present invention provides dual scFv formats that include truncation mutants as shown in FIG.

[0208] The present invention provides dual scFv formats containing skewed variants as shown in Figures 29 and 34. Provide.

[0209] The present invention provides dual scFv formats comprising pI variants and / or charged scFv linkers. (Generally, one of the monomers is Q295E / N384D / Q418E / N4 81D and the other contains a positively charged scFv linker, or they both contain the opposite. containing a charged scFv linker).

[0210] target antigen The bispecific antibodies of the present invention have two different antigen-binding domains, i.e., a CD3-binding domain and a CD4-binding domain. those that bind to target tumor antigens (generally monovalent), and those that bind to target tumor antigens (referred to herein as "TTAs"). Suitable target tumor antigens include, but are 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, mesothelin, EphA2, CD22, CD79b, G PNMB, CD56, CD138, CD52, CD74, CD30, CD123, RON , ERBB2, and EGFR.

[0211] The "Triple F" format is particularly useful when targeting two (or more) different antigens. (As outlined herein, this targeting can be monovalent and bivalent, depending on the format. (It may be any combination of bonds). Thus, the immunoglobulins herein are preferably Preferably, the specificity of each monomer is selected from the list herein and co-associates with two target antigens. Further useful bispecific formats for use with anti-CD3 binding domains may be selected. Shown in Figure 1.

[0212] A particularly preferred application of the heterodimeric antibodies herein is that they bind monovalently to each target antigen. Such antigens are co-targeting pairs where the antigens are advantageous or important. The cellular activation of many immune receptors may simply be Typically by cross-linking obtained by antibody / antigen immune complexes or by target cell association Some immune receptors, such as the CD3 receptor on T cells, mediate the activation of effector cells. For signaling receptors, activation only upon engagement with co-associated targets is not a clinical scenario. This is important because specific cross-linking can induce cytokine storms and toxicity. Activation can be achieved by using the immunoglobulins herein to bind therapeutically relevant antigens in a monovalent, rather than multivalent, manner. By selectively associating with the primary target antigen, the antigen is generated solely in response to cross-linking and is present only in the microenvironment of the primary target antigen. The ability to target two different antigens with different valencies is a novel and useful feature of the present invention. This is an embodiment of the present invention. Targets where monovalent co-association may be therapeutically advantageous or necessary Examples of antigens include, but are not limited to, immune activating receptors, such as CD3, FcγR, TL Toll-like receptors (TLRs), such as TLR4 and TLR9, cytokines, chemokines, and serotonin receptors (SCRs). In many embodiments, antigen binding proteins include cytokine receptors, and chemokine receptors. One of the binding sites binds to CD3, and in some embodiments, it is an scFv-containing monomer. be.

[0213] Both cytokines and membrane-bound factors (including transmembrane receptors), including but not limited to: The following list of target antigens includes 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 Inte Glycine, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulatory substance (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, Bombe 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, cathepsin Cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin Cathepsin O, cathepsin S, cathepsin V, cathepsin 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 protein), 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, u 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-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)- IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, deoxyribonucleic acid 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 Factor 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, hapten Enzyme (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 Herpes simplex virus (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 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, kallikrein In L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, 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 receptor, 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 inhibitory factor, Mug, MuSK, N AIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, Nepri Lysin, neurotrophin-3, -4, or -6, neurturin, neuronal growth factor receptors 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, PDGF, PDK-1, PE CAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, embryo Platelet 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 syncytial 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, T ERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, 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 (Fas linker) Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand C D70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB Ligands (CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAI LR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor Body, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor Related antigen expression: Lewis Y-related carbohydrate, 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 hormone and growth Proteins, subunits, domains, motifs, and / or proteins belonging to receptors for factors or virtually any antigen containing an epitope can be targeted by the immunoglobulins herein. It may also be targeted.

[0214] Exemplary antigens that can be specifically targeted by the immunoglobulins of the present invention include, but are not limited to, Not expressed on the surface of cells, but CD20, CD19, Her2, EGFR, EpCAM, CD3, and 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 Cytokines, cytokine receptors such as IL-2R, chemokines, chemokine receptors, V Examples of the growth factors that form the bispecific antibodies of the present invention include growth factors such as EGF and HGF. Therefore, it is possible to generate antibodies against any combination of these antigens. Each of these antigens may be optionally and independently included in the bispecific antibodies according to the invention. or can be excluded.

[0215] A particularly preferred combination in a bispecific antibody is an antigen-binding domain for CD3 , as well as domains that bind to CD19, CD20, CD38, and CD123. The antigen-binding domains are shown in the figures.

[0216] Nucleic acids of the present invention The present invention further provides nucleic acid compositions encoding the bispecific antibodies of the present invention. As understood by the present invention, the nucleic acid composition is useful for the formation and scanning of heterodimeric proteins. So, for example, if the form is a triple F-form (e.g. For example, a first amino acid monomer containing an Fc domain and an scFv, a second amino acid monomer containing a heavy chain and a light chain, If three amino acid sequences are required as the two amino acid monomers, three The nucleic acid sequence may be incorporated into one or more expression vectors. In the dual scFv format as disclosed in , only two nucleic acids are required, and They can be inserted into one or two expression vectors.

[0217] As is known in the art, nucleic acids encoding the components of the present invention can be prepared by methods known in the art. The expression vectors used in the production of the heterodimeric antibodies of the present invention are known. Generally, the nucleic acid contains a number of regulatory elements (promoter, operably linked to a target gene (e.g., a nucleic acid sequence ... Expression vectors can be extrachromosomal or integrating vectors.

[0218] The nucleic acids and / or expression vectors of the invention, in turn, find use in a number of embodiments. mammalian, bacterial, yeast, insect and / or A number of different types of host cells, including bacterial or fungal cells, are well known in the art. is transformed into

[0219] In some embodiments, the nucleic acid encoding each monomer and the optional nucleic acid encoding the light chain are The acids are generally under the control of different or the same promoters, if applicable, depending on the format. In an embodiment of the present invention, the Each of these two or three nucleic acids is contained on a different expression vector. and U.S. Patent Application No. 62 / 025,931, which are incorporated herein by reference. As shown, different vector ratios can be used to drive heterodimer formation. That is, surprisingly, the protein can be made up of a first monomer: a second monomer: a light chain (heterodimer). In the case of many embodiments herein having three polypeptides comprising a monomeric antibody, While included in a 1:2 ratio, these are not the ratios that produce the best results. See Not issued.

[0220] The heterodimeric antibodies of the present invention can be expressed in host cells containing expression vectors, as is well known in the art. It is produced by culturing cells. Once produced, it is purified by ion exchange chromatography. Conventional antibody purification steps are performed, including at least the steps discussed herein. The pI of the two monomers differs by at least 0.5, allowing for the separation of the two monomers by ion exchange chromatography or may be capable of separation by isoelectric focusing or other methods that are sensitive to the isoelectric point. That is, the isoelectric point (pI) of each monomer is calculated based on the pI of each monomer. By including pI substitutions that alter the nucleotide sequence to have a different pI, a "triple F" heterodimer can be obtained. Isoelectric purification of dimers becomes easy (e.g., anion exchange column, cation exchange column) These substitutions also prevent any contaminating dual scFv-Fc and mAb homodimers after purification. Aids in measuring and monitoring cleavage (e.g., IEF gels, cIEF, and analytical IEX) column).

[0221] treatment Once prepared, the compositions of the present invention find use in several applications. , CD38, and CD123 are all expressed in numerous hematopoietic tumors and tumors derived from various hematopoietic tumors. The heterodimeric antibodies of the present invention are not regulated in cell lines, and therefore may be useful in treating cancer, e.g. All B-cell lymphomas and leukemias, including but not limited to non-Hodgkin lymphomas and leukemias, NHL, Burkitt's lymphoma (BL), multiple myeloma (MM), B-chronic lymphoma B-CLL, B and T acute lymphoblastic leukemia (ALL), T-cell lymphoma Acute myeloid leukemia (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's disease lymphoma (HL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, and chronic bone marrow disease It finds use in the treatment of myeloid leukemia (CML).

[0222] Thus, the heterodimeric compositions of the present invention find use in the treatment of these cancers. will be done.

[0223] Antibody Compositions for In Vivo Administration The formulations of antibodies used in accordance with the present invention may be in the form of lyophilized formulations or aqueous solutions for storage. In this embodiment, the antibody having the desired purity is optionally pharmaceutically acceptable carrier, excipient, or is prepared by mixing with a stabilizer (Remington's Pharmac eutical Sciences 16th edition, Osol, A.Ed.

[1980] Acceptable carriers, excipients, or stabilizers are used at the dosages and concentrations non-toxic to the recipient and free of phosphates, citrates, and other organic acids buffers; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl sulfate); dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, Benzethonium chloride; phenol, butyl or benzyl alcohol; methyl or Alkylparabens such as propylparaben; catechol; resorcinol; cyclohexyl 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) poly Peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; poly Hydrophilic polymers such as vinylpyrrolidone; glycine, glutamine, asparagine, histidine amino acids such as arginine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as maltose, mannitol, trehalose, or sorbitol; Salt-forming counterions; metal complexes (e.g., Zn-protein complexes); and / or Tweens (trademark), Pluronic (trademark) or non-isopropyl alcohols such as polyethylene glycol (PEG) Contains anionic surfactants.

[0224] The formulations herein may also contain two or more active ingredients as needed for the particular indication being treated. The compounds may contain complementary activities, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to provide antibodies with other specificities. Alternatively or additionally, the composition may comprise a cytotoxic agent, a cytokine, a growth inhibitory agent and / or a small molecule. Such molecules are preferably present together in amounts effective for the intended purpose. do.

[0225] The active ingredient may also be administered in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, etc.). emulsions, microemulsions, nanoparticles and nanocapsules) or macroemulsions microcapsules prepared, for example, by coacervation techniques or interfacial polymerization hydroxymethylcellulose or gelatin-microcapsules and polycapsules, respectively. The compound may be encapsulated in tri-(methyl methacrylate) microcapsules. Such a technique is described in Rem ington's Pharmaceutical Sciences 16th ed. ition, Osol, A. Ed. (1980).

[0226] Formulations to be used for in vivo administration should be sterile or nearly sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0227] Sustained release formulations may also be prepared. Suitable sustained release formulations include solid hydrophobic polymers containing the antibody. The matrix may be in the form of a shaped article, e.g., a film, or a semipermeable matrix of a polymer. Examples of sustained release matrices include polyester, Hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly( vinyl alcohol), polylactic acid (U.S. Pat. No. 3,773,919), L-glucan Copolymer of glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene vinyl acetate degradable lactic acid-glycolic acid copolymers, e.g., Leupron Depot™ (lactic acid-glycolic acid copolymers); injectable microspheres consisting of cholic acid copolymer and leuprolide acetate), and and poly-D-(-)-3-hydroxybutyric acid. Ethylene vinyl acetate and milk While polymers such as glycolic acid allow for release of molecules for over 100 days, Certain hydrogels release proteins for shorter periods of time.

[0228] Encapsulated antibodies undergo changes as a result of exposure to moisture at 37°C when they remain in the body for extended periods. may undergo degradation or aggregation, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies for stabilization can be devised depending on the mechanism involved. For example, aggregation mechanisms It was discovered that the formation of an intermolecular S--S bond was due to thio-disulfide exchange. In this case, stabilization is achieved by modifying sulfhydryl residues, lyophilizing from an acidic solution, and controlling the water content. By controlling the polymer matrix, using appropriate additives, and developing specific polymer matrix compositions, This can be achieved.

[0229] Mode of administration The antibodies and chemotherapeutic agents of the present invention can be administered by known methods, for example, by intravenous administration as a bolus. By continuous infusion over a long period of time, it can be administered intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, or intraarticularly. Administered to subjects by intravenous, intrasynovial, intrathecal, oral, topical, or inhalation routes Intravenous or subcutaneous administration of the antibody is preferred.

[0230] Treatment modality The methods of the present invention provide a method of treatment that provides a positive therapeutic response to a disease or condition. A "positive therapeutic response" is defined as an improvement in a disease or condition and / or a reduction in the severity of the disease or condition. or an improvement in symptoms associated with the condition. For example, a positive treatment response , the following improvements in disease: (1) reduction in the number of neoplastic cells; (2) neoplastic cell death. (3) inhibition of neoplastic cell survival; (5) inhibition of tumor growth (i.e., some degree of slowing) (6) increasing patient survival; and (7) reducing or eliminating the risk of developing a disease or condition. This refers to one or more of the following: partial relief from one or more related symptoms.

[0231] A positive therapeutic response in any given disease or condition is determined by the specificity of that disease or condition. Tumor response can be assessed by standardized response criteria. Radiographic imaging, computed tomography (CT) scans, bone scans Cancer imaging, endoscopy, and tumor biopsy samples, including bone marrow aspirates (BMA) Tumor morphology was assessed using screening techniques such as enumeration of tumor cells and circulating tumor cells. Changes (ie, total tumor burden, tumor size, etc.) can be assessed.

[0232] In addition to these positive treatment responses, treated subjects experienced significant improvements in disease-related symptoms. may experience the beneficial effects of improvements in

[0233] The improvement in disease may be characterized as a complete response. In cases where there are any previous abnormal radiographic tests, bone marrow, and cerebrospinal fluid (CSF) or Normalization of the abnormal monoclonal protein with the absence of clinically detectable disease It is intended that

[0234] Such a response may occur for at least 4 to 8 weeks, or sometimes as long as 4 to 8 weeks, after treatment according to the methods of the present invention. This may last for 6 to 8 weeks. Alternatively, the improvement in disease may be classified as a partial remission. A "partial response" is defined as the absence of new lesions, which may last 4-8 weeks or 6-8 weeks. In the absence of any measurable tumor burden (i.e., the number of malignant cells present in the subject), the number or amount of abnormal monoclonal protein measured A reduction of at least about 50% is contemplated.

[0235] Treatment according to the present invention includes a "therapeutically effective amount" of the agent used. It refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.

[0236] The therapeutically effective amount depends on factors such as the individual's condition, age, sex, and weight, as well as the individual's ability to tolerate the drug. The therapeutically effective amount may also vary depending on the ability of the antibody or or antibody moiety where any toxic or detrimental effects of the antibody moiety are outweighed by the therapeutically beneficial effects. be.

[0237] A "therapeutically effective amount" in tumor therapy is also measured by its ability to stabilize the progression of the disease. The ability of a compound to inhibit cancer may be determined by animal models that are predictive of efficacy in human tumors. It may also be evaluated in a model system.

[0238] Alternatively, this property of the composition inhibits cell growth or induces apoptosis. The potency of the compounds is evaluated by testing them in in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may reduce tumor size, or alternatively The subject's symptoms may be alleviated. Those skilled in the art will appreciate that the subject's size, the severity of the subject's symptoms, and the amount of the drug administered may be varied. Such amounts will be determined based on factors such as severity and the particular composition or route of administration selected. This will allow us to determine the

[0239] Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, or several doses may be administered as indicated in the emergency of a therapeutic situation. Divided doses may be administered over time, or the dose may be proportionally reduced or increased. Parenteral compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a dosage unit administered to a subject to be treated. It refers to physically discrete units suited as unit doses, each unit containing the required pharmaceutical carrier. containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in relation to do.

[0240] The specifications for the dosage unit forms of the present invention are (a) the inherent properties of the active compound and the desired effect achieved. (b) the specific therapeutic effect to be achieved, and (b) the treatment of a susceptibility in an individual, The limitations inherent in the art of formulating compounds are dictated by and directly affect Depends on.

[0241] Effective doses and administration regimens for the bispecific antibodies used in the present invention are The amount of treatment to be administered will depend on the disease or condition being treated and may be determined by one skilled in the art.

[0242] An exemplary, non-limiting range for a therapeutically effective amount of a bispecific antibody used in the present invention is about 0.1 to 100 mg / kg, for example, about 0.1 to 50 mg / kg, for example, about 0.1 to 20 mg g / kg, for example about 0.1 to 10 mg / kg, for example about 0.5, for example about 0.3, about 1, or about 3 mg / kg. In another embodiment, the antibody is administered at a dose of 1 mg / kg or more, e.g. For example, a dose of 1 to 20 mg / kg, for example, a dose of 5 to 20 mg / kg, for example, 8 mg / kg is administered at a dose of

[0243] A medical practitioner having ordinary skill in the art will be able to determine the effectiveness of the required pharmaceutical composition. Effective amounts can be readily determined and prescribed. For example, a physician or veterinarian may The level of administration of the agent used in the composition required to obtain the desired therapeutic effect. May be started at a lower level and gradually increased until the desired effect is achieved .

[0244] In one embodiment, the bispecific antibody is administered at a concentration of 10-500 mg / kg, for example 200-400 mg / kg. The dose is administered by injection at a weekly dose of 1 mg / kg. Such administration may be, for example, 1 to 8 times, for example 3 The administration may be repeated up to 5 times over a period of 2 to 24 hours, for example 2 to 12 hours. It may be administered by continuous infusion.

[0245] In one embodiment, bispecific antibodies are useful in cases where reduced side effects, including toxicity, are required, e.g. It is administered by slow continuous infusion over an extended period of time, for example, more than 24 hours.

[0246] In one embodiment, the bispecific antibody is administered in a concentration of 250 mg to 2000 mg, for example 300 mg, Weekly doses of 500mg, 700mg, 1000mg, 1500mg, or 2000mg The dose is administered up to 8 times, for example 4 to 6 times. The doses are administered over a 2 to 24 hour period, for example 2 to 12 hours. Such a regimen may be administered by continuous infusion over a period of, for example, six months or more. The dose may be repeated one or more times as needed, for example, after 12 months. The amount of the compound is measured, for example, by collecting a biological sample and targeting the antigen-binding region of the bispecific antibody. The idiotypic characteristics of the serotonin receptor are determined or regulated by measuring the serotonin receptor using an anti-idiotypic antibody that stimulates the serotonin receptor. That's fine.

[0247] In a further embodiment, the bispecific antibody is cultured for 2 to 12 weeks, for example 3 to 10 weeks, e.g. It is usually given once a week for 4 to 8 weeks.

[0248] In one embodiment, the bispecific antibody is administered as little as once a week for a period of six months or more. It is administered as a maintenance treatment.

[0249] In one embodiment, the bispecific antibody is administered by a single injection of the bispecific antibody followed by radioactive and infusion of an isotope-conjugated bispecific antibody. The treatment may be repeated, for example, after 7 to 9 days.

[0250] By way of non-limiting example, treatment according to the present invention may be administered in a single dose or in a series of 24, 12, 8, 6, 4, or Approximately 0.1 to 100 mg / day in divided doses every 2 hours, or in any combination. mg / kg, e.g., 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 start of treatment is as a daily dose of antibody at 70, 80, 90 or 100 mg / kg. 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, On the 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, or 40th day At least once, or instead of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 At least once at weeks 2, 13, 14, 15, 16, 17, 18, 19, or 20 or any combination thereof.

[0251] In some embodiments, the bispecific antibody molecule may contain one or more additional therapeutic agents, e.g. Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin, and their analogs) or metabolites, and doxorubicin; topoisomerase II inhibitors (e.g., etoposide alkylating agents (e.g., melphalan, chlorhexidine, thiazolinone, thiazolinone, thiazolinone, thiazolinone); Lorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, Semustine, streptozocin, dacarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA interfering substances (e.g., cisplatin, oxaliplatin) DNA interfering agents and free radical generators, e.g., bleomycin, and carboplatin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, vin, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, anticoagulants, ...

[0252] Chemotherapeutic agents that disrupt cell replication include paclitaxel, docetaxel, and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide and related analogs (e.g., CC-5013 and CC-4047); protein kinase inhibitors (e.g., imatinib mesylate and gefitinib); NF-κB inhibitors, including inhibitors of IκB kinases (e.g., bortezomib); 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 include other inhibitors of enzymes whose inhibition down-regulates cell replication.

[0253] In some embodiments, the antibodies of the invention are directed against VELCADE® (bortezomib). It can be used before, during, or after treatment.

[0254] All cited references are expressly incorporated herein by reference in their entirety. will be done.

[0255] While specific embodiments of the invention have been described for purposes of illustration, numerous variations in detail may be realized. can be made without departing from the invention as set forth in the appended claims. will be understood by those skilled in the art. [Example]

[0256] To illustrate the present invention, examples are provided below. These examples are provided to illustrate the present invention in any It is not intended to be limiting to any particular application or theory of operation of any of the methods contemplated by this invention. The numbering for the constant region positions is according to the EU index of Kabat (Kabat et al., 1991, Sequences of Proteins of Immu. nological interest,5th Ed.,United States Public Health Service,National Institute es of Health, Bethesda, incorporated by reference in its entirety). Those skilled in the art will appreciate that this convention is not contiguous within a particular region of an immunoglobulin sequence. It consists of a set of sequences that allow for normalized reference to conserved positions in the immunoglobulin family. Therefore, as defined by the EU Index, The positions of any given immunoglobulin do not necessarily correspond to its sequential sequence. do.

[0257] General and specific science and technology are described in U.S. Patent Application Publication No. 2015 / 0307629. , U.S. Patent Application Publication No. 2014 / 0288275 and International Publication No. 2014 / 1 Pamphlet No. 45806 (all of which are outlined in their entirety and in particular The technique is outlined in (which is expressly incorporated by reference).

[0258] Example Example 1: Alternative Formats Bispecific production A schematic diagram of the anti-CD38 x anti-CD3 bispecific antibody is shown in Figure 1. The amino acid sequences of the variant alternatives are listed in Figures 39-43. DNA encoding the three strands required for the Blue Heron gene was synthesized using gene synthesis ( (Biotechnology, Bothell, Wash.) and standard The fragment was subcloned into the expression vector pTT5 using molecular biology techniques. From(QuikChange,Stratagene,Cedar Creek,Tex. ) or additional gene synthesis and subcloning were used to introduce the substitutions. For expression, the DNA was transfected into HEK293E cells, and the resulting protein was then purified using a PCR-based PCR method. Protein A affinity (GE Healthcare) and cation exchange chromatography The yield after Protein A affinity purification is shown in Figure 35. Cation exchange chromatography purification was performed using a wash / equilibration buffer of 50 mM MES (p H6.0) and elution buffer of 50 mM MES (pH 6.0 + 1M NaCl linear gradient The experiment was carried out using a HiTrap SP HP column (GE Healthcare) with (See Figure 36 for chromatogram).

[0259] Redirected T cell cytotoxicity Anti-CD38 × anti-CD3 bispecific antibody was used to detect CD38 + Reinduction of RPMI8266 myeloma cell line The induced T cell cytotoxicity (RTCC) was characterized in vitro. MI8266 cells were incubated with 500k human PBMCs for 24 hours. TCC was measured by LDH fluorescence as indicated (see Figure 37).

[0260] Example 2 Redirected T cell cytotoxicity Anti-CD38 × anti-CD3 Fab-scFv-Fc bispecific antibody was injected into CD38 + RPMI8 In vitro redirected T cell cytotoxicity (RTCC) of 266 myeloma cell lines 40k RPMI8266 cells were incubated with 400k human PBMCs for 96 hours. RTCC was measured by flow cytometry as indicated. (See Figure 44). CD69, Ki-67, and PI-9 CD4+ and CD8+ T Cellular expression was also characterized by flow cytometry and is shown in FIG.

[0261] Mouse model of antitumor activity On day -23, each of four groups of five NOD severe combined immunodeficiency gamma (NSG) mice , 5 x 10 6 RPMI8226TrS tumor cells (multiple myeloma, luciferase-expressing On day 0, mice were intraperitoneally injected with 10 × 10 6 of Human PBMCs were transplanted. After PBMC transplantation on day 0, the test substance was administered weekly (days 0 and 7). The study design is further summarized in Figure 46. Tumor growth was monitored using an in vivo imaging system (IVIS®). The flux was monitored by measuring the flux / mouse. Both 15426 demonstrated substantial anti-tumor effects (see Figures 47 and 48).

[0262] Cynomolgus monkey studies Cynomolgus monkeys were given a single dose of anti-CD38 x anti-CD3 bispecific. A D3 bispecific control was also included. The dose level was 20 μg / kg (in three independent studies). g of XmAb13551 (n = 2), 0.5 mg / kg of XmAb15426 (n = 3) , 3 mg / kg XmAb14702 (n=3), or 3 mg / kg XmAb132 45 (anti-RSV x anti-CD3 control, n=3). Anti-CD38 x anti-CD3 bispecific , rapidly depleted CD38+ cells in peripheral blood (see Figure 49). The 3 bispecifics resulted in T cell activation as measured by CD69 expression (Figure 50 Serum levels of IL-6 were also measured (see Figure 51). Overall, XmAb15426 extended the duration of CD38+ cell depletion and enhanced T cell activation and It is noteworthy that the patients had reduced levels of IL-6 production.

[0263] XmAb15426 and XmAb14702 were administered at 0.5 mg / kg and 3 mg, respectively. Both antibodies were well tolerated at these higher doses. This was consistent with the moderate levels of IL6 observed in serum from treated monkeys. XmAb15426, which has CD3 affinity of about 100%, inhibited CD38+ cells at 0.5 mg / kg. compared with the parent high affinity XmAb13551 administered at 2, 5 or 20 μg / kg Depletion by XmAb15426 was more effective than the maximum effective dose in previous studies. The effect was more sustained compared to XmAb13551 (7 days vs. 2 days, respectively). Cell depletion was more pronounced with XmAb15426, but not with T cell activation (CD69, CD25 and PD1 induction) was 2 times greater than in the 20 μg / kg XmAb13551 group. It was significantly lower in monkeys treated with XmAb15426 at a 5-fold higher dose. XmAb14702, which has low CD3 affinity, inhibits CD38+ cells and T cell activation. had little effect on

[0264] These results suggest that CD3 affinity may be attenuated to regulate T cell activation. This is a promising approach to improve the therapeutic window of T cell-engaging bispecific antibodies. This method is shown to be effective in stimulating the clearance of target antigens such as CD38. Improve tolerability and administer higher doses to overcome sink clearance Expanding the set of antigens that are suitable for targeted T cell immunotherapy by enabling We have demonstrated that XmAb1 has the potential to inhibit CD3 by reducing its affinity for CD3. While 5426 effectively depletes CD38+ cells, its high-affinity counterpart, XmA It has been shown to minimize the CRS effects seen at doses equivalent to b13551.

[0265] Example 3 Development of CDRs against CD123 The starting point for CDR development for the humanized antibody Fab human CD123 was ATCC HB-1 The variable and variable sequences of the 7G3 murine antibody, designated herein as "7G3 H0L0," from 2009 However, the first humanized region (H1_L1; sequence shown in Figure 136) column) resulted in a significant loss of affinity (5-fold as shown in Figure 156B and C). This loss in affinity is largely due to the lack of affinity shown for the H1_L0 construct. a portion is due to heavy chain humanization (e.g., a first humanized heavy chain with a murine light chain), The H1_L1 construct showed a significant 10-fold loss of CD123-expressing KG When tested against 1a cells, RTCC (T cell cytotoxicity assay) was observed, as shown in Figure 156D. This was consistent with a 10-fold loss in efficacy (reinduction of toxicity).

[0266] Therefore, two rounds of affinity / stability optimization were performed. The first round (shown in Figure 157) The library (denoted "Library 1") contains 108 variants containing LDA, target, and back-substitutions. These were then screened on a CD123 chip in Fab format (human CH from IgG1). Affinity screening was performed with a humanized variable heavy chain domain fused to 1) and neutral The stability of higher affinity mutants was screened with DSF.

[0267] As shown in Figure 158, the Tm of the initial H1L1 variant was significantly higher compared to the starting H0L0. The results for the increased and further variants of the H1L1 parent are shown in FIG.

[0268] The round 1 mutants were then subjected to sc immunoprecipitation against CD3 as further outlined herein. Fv and developed Fab were used to incorporate into the bottle opener format, and then Fig. 159 The antibodies were tested in the KG-1a binding assay and the RTCC assay as shown in The first round of optimization improved the affinity and potency of the mutants, but further optimization is required It was said that.

[0269] The second round, "Round 2," shown in Figure 160, is the mouse level of H0L0. This resulted in restoration of binding affinity and RTCC activity. 045 has improved affinity compared to the first humanized sequence (H1L1; + H1L1). 21-fold improvement), and also a 2-fold increase in activity relative to the parent murine antibody (7G3;H0L0). It is important to note that XENP13967 is equivalent to XENP14045 on the CD123 side. 13967 has a different CD3 scFv as shown in the sequence.

[0270] Round 2 optimization also resulted in increased stability as measured by Tm. Figure 161 shows the results of the Tm assay for XENP13967 (as well as XENP1404 5) +5C for the first chimera (e.g., variable heavy and light chain mouse sequences), 13967 / 14045 is a +4C improvement compared to the original H1L1 variant. In addition, during the second round, the possible substitutions The deamidation site (-NS motif) was removed from the light chain CDR1.

[0271] Example 4 Development of CDRs for CD20 Two anti-CD20 Fabs were analyzed in a CD20 x CD3 dual antibody for binding affinity and efficacy. Both XENP13677 and XENP13676 were investigated in a specific format. Based on tuximab, the 13677 variant is significantly more potent than the 13676 variant This demonstrates a potency similar to that of the parent rituximab antibody, with CD20 affinity similar to that of the parent rituximab antibody. Antibodies of different sexes were administered in a cynomolgus monkey study to compare their in vivo properties. However, due to the higher potency of the 13677 variant, the 13677 variant is 0.03 mg / kg compared to 0.3 mg / kg administered for 13676, which is lower At these doses, both antibodies significantly depleted monkey B cells. However, surprisingly, the significantly more potent 13677 actually The lower dose showed a more rapid recovery of B cells. On the other hand, both antibodies showed approximately the same In conclusion, the lower affinity variant 13676 induced a higher level of IL6 release than expected. Unexpectedly, this represents a more favorable treatment profile, while maintaining similar levels of IL6. This causes a longer-term depletion of B cells. The present invention also provides the following. [1] a) a first monomer, i) a first heavy chain, 1) the first variable heavy chain domain; 2) a first constant heavy chain comprising a first Fc domain; 3) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain and a scF domain covalently linked to the C-terminus of the Fc domain using a domain linker. v a first heavy chain comprising a first monomer comprising: b) a second variable heavy chain domain, and a second constant heavy chain comprising a second Fc domain; , and a second monomer comprising a second heavy chain; c) a common light chain comprising a variable light domain and a constant light domain; wherein the first and second Fc domains comprise S364K / E357Q:L368 D / K370S;L368D / K370S:S364K;L368E / K370S:S3 64K;T411T / E360E / Q362E:D401K;L368D / K370S: Selected from the group consisting of S364K / E357L and K370S:S364K / E357Q the first variable heavy chain domain and the second variable light chain domain have a set of amino acid substitutions that are The main domain binds to a first target tumor antigen (TTA) and binds to the second variable heavy chain domain and the The variable light chain domain binds to the first TTA, and the scFv binds to human CD3 (sequence A heterodimeric antibody that binds to antibody number XX. [2] The scFv is SEQ ID NO: XX (scFv13551), SEQ ID NO: XX (scFv15 426), SEQ ID NO: XX (scFv13423) and SEQ ID NO: XX (scFv1470 2) The heterodimer according to [1], having a polypeptide sequence selected from the group consisting of antibody. [3] The first variable heavy chain domain and the variable light chain domain are selected from the group consisting of CD19, CD20 and and CD123. Heterodimeric antibodies. [4] a) a first monomer, i) a first heavy chain, 1) the first variable heavy chain domain; 2) a first constant heavy chain domain comprising a first Fc domain; and 3) covalently linked to the C-terminus of the first Fc domain using a domain linker First variable light chain domain a first heavy chain comprising a first monomer comprising: b) a second monomer, i) a second variable heavy chain domain; ii) a second constant heavy chain domain comprising a second Fc domain; and iii) the second variable heavy chain domain is linked to the second Fc domain using a domain linker; A third variable heavy chain domain covalently attached to the main C-terminus and a second monomer comprising: c) a common light chain comprising a variable light domain and a constant light domain; wherein the first and second Fc domains comprise S364K / E357Q:L368 D / K370S;L368D / K370S:S364K;L368E / K370S:S3 64K;T411T / E360E / Q362E:D401K;L368D / K370S: Selected from the group consisting of S364K / E357L and K370S:S364K / E357Q the first variable heavy chain domain and the second variable light chain domain have a set of amino acid substitutions that are The domain binds to the first TTA and binds to the second variable heavy chain domain and the variable light chain domain. The second variable light chain domain and the third variable heavy chain domain bind to the TTA and The main component is a heterodimeric antibody that binds to CD3. [5] The scFv is SEQ ID NO: XX (scFv13551), SEQ ID NO: XX (scFv15 426), SEQ ID NO: XX (scFv13423) and SEQ ID NO: XX (scFv1470 2) The heterodimer according to [4], having a polypeptide sequence selected from the group consisting of antibody. [6] The first variable heavy chain domain and the variable light chain domain are selected from the group consisting of CD19, CD20 and and CD123. Heterodimeric antibodies. [7] a) a first monomer, i) a first heavy chain, 1) the first variable heavy chain domain; 2) a first constant heavy chain comprising a first CH1 domain and a first Fc domain; 3) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain a domain linker connecting the C-terminus of the CH1 domain and the first Fc domain covalently linked scFv between the N-terminus of the a first heavy chain comprising a first monomer comprising: b) a second variable heavy chain domain, and a second constant heavy chain comprising a second Fc domain; , and a second monomer comprising a second heavy chain; c) a common light chain comprising a variable light domain and a constant light domain; wherein the first and second Fc domains comprise S364K / E357Q:L368 D / K370S;L368D / K370S:S364K;L368E / K370S:S3 64K;T411T / E360E / Q362E:D401K;L368D / K370S: Selected from the group consisting of S364K / E357L and K370S:S364K / E357Q the first variable heavy chain domain and the second variable light chain domain have a set of amino acid substitutions that are The domain binds to the first TTA and binds to the second variable heavy chain domain and the variable light chain domain. a heterodimeric antibody, wherein the scFv binds to the TTA and the scFv binds to human CD3. . [8] The scFv is SEQ ID NO: XX (scFv13551), SEQ ID NO: XX (scFv15 426), SEQ ID NO: XX (scFv13423) and SEQ ID NO: XX (scFv1470 2) The heterodimer according to [7], having a polypeptide sequence selected from the group consisting of antibody. [9] The first variable heavy chain domain and the variable light chain domain are selected from the group consisting of CD19, CD20 and and CD123. Heterodimeric antibodies.

[10] a) a first monomer, i) a first heavy chain, 1) the first variable heavy chain domain; 2) a first constant heavy chain domain comprising a first Fc domain; and 3) the second variable light chain domain is linked to the first constant heavy chain domain using a domain linker; a covalent bond between the C-terminus of the CH1 domain and the N-terminus of the first Fc domain The first variable light chain domain a first heavy chain comprising a first monomer comprising: b) a second monomer, i) a second variable heavy chain domain; ii) a second constant heavy chain domain comprising a second Fc domain; and iii) the second variable heavy chain domain is linked to the second Fc domain using a domain linker; A third variable heavy chain domain covalently attached to the main C-terminus and a second monomer comprising: c) a common light chain comprising a variable light domain and a constant light domain; wherein the first and second Fc domains comprise S364K / E357Q:L368 D / K370S;L368D / K370S:S364K;L368E / K370S:S3 64K;T411T / E360E / Q362E:D401K;L368D / K370S: Selected from the group consisting of S364K / E357L and K370S:S364K / E357Q the first variable heavy chain domain and the second variable light chain domain have a set of amino acid substitutions that are The domain binds to the first TTA and binds to the second variable heavy chain domain and the variable light chain domain. The second variable light chain domain and the third variable heavy chain domain bind to the TTA and The main antibody is a heterodimeric antibody that binds to human CD3.

[11] The scFv is SEQ ID NO: XX (scFv13551), SEQ ID NO: XX (scFv15 426), SEQ ID NO: XX (scFv13423) and SEQ ID NO: XX (scFv1470 The heterodimer according to

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

[12] The first variable heavy chain domain and the variable light chain domain are selected from the group consisting of CD19, CD20 and and CD123. Heterodimeric antibodies described above.

[13] a) a first monomer, i) a first heavy chain, 1) the first variable heavy chain domain; 2) a first constant heavy chain comprising a first CH1 domain and a first Fc domain; 3) scFv variable light chain domain, scFv linker and scFv variable heavy chain domain a domain linker connecting the C-terminus of the CH1 domain and the first Fc domain covalently linked scFv between the N-terminus of the a first heavy chain comprising a first monomer comprising: b) a second monomer comprising a second Fc domain; c) a light chain comprising a variable light domain and a constant light domain; wherein the first and second Fc domains comprise S364K / E357Q:L368 D / K370S;L368D / K370S:S364K;L368E / K370S:S3 64K;T411T / E360E / Q362E:D401K;L368D / K370S: Selected from the group consisting of S364K / E357L and K370S:S364K / E357Q the first variable heavy chain domain and the second variable light chain domain have a set of amino acid substitutions that are a heterodimeric antibody, wherein the main fragment binds to a first antigen and the scFv fragment binds to a second antigen .

[14] The scFv is SEQ ID NO: XX (scFv13551), SEQ ID NO: XX (scFv15 426), SEQ ID NO: XX (scFv13423) and SEQ ID NO: XX (scFv1470 The heterodimer according to

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

[15] The first variable heavy chain domain and the variable light chain domain are selected from the group consisting of CD19, CD20 and and CD123. Heterodimeric antibodies described above.

[16] a) vlCDR1 having the sequence GSSTGAVTTSNYAN (SEQ ID NO: XX), the sequence vlCDR2 with GTNKRAP (SEQ ID NO: XX), and the sequence ALWYSNHWV a variable light chain domain comprising a vlCDR3 having (SEQ ID NO: XX); b) vhCDR1 with sequence TYAMN (SEQ ID NO: XX), sequence RIRSKANNY ATYYADSVKG (SEQ ID NO: XX), and vhCDR2 with sequence HGNFGD a variable heavy chain domain comprising a vhCDR3 having SYVSWFAY (SEQ ID NO: XX); An anti-CD3 antibody binding domain comprising:

[17] The anti-CD3 antibody binding domain according to

[16] , which is an scFv.

[18] the variable light chain domain has the sequence L1.47 (SEQ ID NO: XX), and the variable heavy chain domain

[16] or

[17] , wherein the main sequence has the sequence H1.32 (SEQ ID NO: XX). CD3 antibody binding domain.

[19] The scFv has the sequence H1.32_L1.47 (SEQ ID NO: XX), The anti-CD3 antibody binding domain described above.

[20] A nucleic acid composition encoding the scFv described in

[19] . [twenty one]

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

[20] . [twenty two] A host cell comprising the expression vector described in

[21] . [twenty three] a) vlCDR1 having the sequence GSSTGAVTTSNYAN (SEQ ID NO: XX), the sequence vlCDR2 with GTNKRAP (SEQ ID NO: XX), and the sequence ALWYSNHWV a variable light chain domain comprising a vlCDR3 having (SEQ ID NO: XX); b) vhCDR1 with sequence TYAMN (SEQ ID NO: XX), sequence RIRSKYNNY ATYYADSVKG (SEQ ID NO: XX), and vhCDR2 with sequence HGNFGD a variable heavy chain domain comprising a vhCDR3 having EYVSWFAY (SEQ ID NO: XX); An anti-CD3 antibody binding domain comprising: [twenty four] The anti-CD3 antibody binding domain according to

[23] , which is an scFv. [twenty five] the variable light chain domain has the sequence L1.47 (SEQ ID NO: XX), and the variable heavy chain domain

[23] or

[24] , wherein the main sequence has the sequence H1.89 (SEQ ID NO: XX). CD3 antibody binding domain.

[26] The scFv has the sequence H1.89_L1.47 (SEQ ID NO: XX), The anti-CD3 antibody binding domain described above.

[27] A nucleic acid composition encoding the scFv described in

[26] .

[28]

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

[27] .

[29] A host cell containing the expression vector described in

[28] .

[30] a) vlCDR1 having the sequence GSSTGAVTTSNYAN (SEQ ID NO: XX), the sequence vlCDR2 with GTNKRAP (SEQ ID NO: XX), and the sequence ALWYSNHWV a variable light chain domain comprising a vlCDR3 having (SEQ ID NO: XX); b) vhCDR1 with sequence TYAMN (SEQ ID NO: XX), sequence RIRSKYNNY ATYYADSVKG (SEQ ID NO: XX), and vhCDR2 with sequence HGNFGD a variable heavy chain domain comprising a vhCDR3 having PYVSWFAY (SEQ ID NO: XX); An anti-CD3 antibody binding domain comprising:

[31] The anti-CD3 antibody binding domain according to

[30] , which is an scFv.

[32] the variable light chain domain has the sequence L1.47 (SEQ ID NO: XX), and the variable heavy chain domain

[30] or

[31] , wherein the main sequence has the sequence H1.90 (SEQ ID NO: XX). CD3 antibody binding domain.

[33] The scFv has the sequence H1.90_L1.47 (SEQ ID NO: XX). The anti-CD3 antibody binding domain described above.

[34] A nucleic acid composition encoding an scFv according to

[33] .

[35]

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

[34] .

[36] A host cell containing the expression vector described in

[35] .

[37] a) vlCDR1 having the sequence GSSTGAVTTSNYAN (SEQ ID NO: XX), the sequence vlCDR2 with GTNKRAP (SEQ ID NO: XX), and the sequence ALWYSNHWV a variable light chain domain comprising a vlCDR3 having (SEQ ID NO: XX); b) vhCDR1 with sequence TYAMN (SEQ ID NO: XX), sequence RIRSKYNNY ATYYADSVKG (SEQ ID NO: XX), and vhCDR2 with sequence HGNFGD a variable heavy chain domain comprising a vhCDR3 having SYVSWFDY (SEQ ID NO: XX); An anti-CD3 antibody binding domain comprising:

[38] The anti-CD3 antibody binding domain according to

[37] , which is an scFv.

[39] the variable light chain domain has the sequence L1.47 (SEQ ID NO: XX), and the variable heavy chain domain

[37] or

[38] , wherein the main sequence has the sequence H1.33 (SEQ ID NO: XX). CD3 antibody binding domain.

[40] The scFv has the sequence H1.33_L1.47 (SEQ ID NO: XX) as described in

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

[41] A nucleic acid composition encoding an scFv according to

[38] .

[42]

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

[41] .

[43] A host cell containing the expression vector described in

[42] .

[44] a) vlCDR1 having the sequence GSSTGAVTTSNYAN (SEQ ID NO: XX), the sequence vlCDR2 with GTNKRAP (SEQ ID NO: XX), and the sequence ALWYSNHWV a variable light chain domain comprising a vlCDR3 having (SEQ ID NO: XX); b) vhCDR1 with sequence TYAMS (SEQ ID NO: XX), sequence RIRSKYNNY ATYYADSVKG (SEQ ID NO: XX), and vhCDR2 with sequence HGNFGD a variable heavy chain domain comprising a vhCDR3 having SYVSWFAY (SEQ ID NO: XX); An anti-CD3 antibody binding domain comprising:

[45] The anti-CD3 antibody binding domain according to

[44] , which is an scFv.

[46] the variable light chain domain has the sequence L1.47 (SEQ ID NO: XX), and the variable heavy chain domain The antibody according to

[44] or

[45] , wherein the main sequence has the sequence H1.31 (SEQ ID NO: XX). CD3 antibody binding domain.

[47] The scFv has the sequence H1.31_L1.47 (SEQ ID NO: XX) The anti-CD3 antibody binding domain described above.

[48] A nucleic acid composition encoding an scFv according to

[47] .

[49]

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

[48] .

[50] A host cell containing the expression vector described in

[49] .

[51] a) a first monomer, i) a first Fc domain; ii) an scFv variable light chain domain, an scFv linker, and an scFv variable heavy chain domain and an anti-CD31 antibody covalently linked to the N-terminus of the Fc domain using a domain linker. 3 scFv a first monomer comprising: b) a second monomer, i) a heavy chain variable domain; and ii) a heavy chain constant domain containing a second Fc domain and a second monomer comprising a heavy chain comprising: c) a light chain comprising a variable light domain and a variable light constant domain; wherein the anti-CD3 scFv comprises anti-CD3H1.32_L1.47 (SEQ ID NO: XX) , anti-CD3H1.89_L1.47 (sequence number XX), anti-CD3H1.90_L1.47 (SEQ ID NO: XX) and anti-CD3H1.33_L1.47 (SEQ ID NO: XX) and wherein the heavy variable domain and the light variable domain bind to TTA. Helodimeric antibodies.

[52] A heterodimeric antibody described in

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

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

[54] the variable light chain domain has the sequence C2B8L1.113 (SEQ ID NO: XX), and The variable heavy chain domain has the sequence C2B8H1.202 (SEQ ID NO: XX) as described in

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

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

[53] .

[56]

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

[55] .

[57] A host cell containing the expression vector described in

[56] .

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

[59] the variable light chain domain has the sequence C2B8L1 (SEQ ID NO: XX), and the variable heavy chain

[58] The anti-CD20 antibody according to

[58] , wherein the domain has the sequence C2B8H1 (SEQ ID NO: XX). Body-binding domain.

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

[58] .

[61]

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

[60] .

[62] A host cell containing the expression vector described in

[61] .

[63] a) a first monomer, i) a first Fc domain; ii) an scFv variable light chain domain, an scFv linker, and an scFv variable heavy chain domain and an anti-CD31 antibody covalently linked to the N-terminus of the Fc domain using a domain linker. 3 scFv a first monomer comprising: b) a second monomer, i) a heavy chain variable domain; and ii) a heavy chain constant domain containing a second Fc domain and a second monomer comprising a heavy chain comprising: c) a light chain comprising a variable light domain and a variable light constant domain; wherein the variable light chain domain has the sequence RASSSVSYIH (SEQ ID NO: XX). vlCDR1 having the sequence ATSNLAS (SEQ ID NO: XX), and vlCDR2 having the sequence QQWTSNPPT (SEQ ID NO: XX), and The main one is vhCDR1 with the sequence SYNMH (SEQ ID NO: XX), the sequence AIYPGNG vhCDR2 with the sequence DTSYNQKFQG (SEQ ID NO: XX), and the sequence STYYGG A heterodimeric antibody comprising a vhCDR3 having DWYFNV (SEQ ID NO: XX).

[64] a) a first monomer, i) a first Fc domain; ii) an scFv variable light chain domain, an scFv linker, and an scFv variable heavy chain domain and an anti-CD31 antibody covalently linked to the N-terminus of the Fc domain using a domain linker. 3 scFv a first monomer comprising: b) a second monomer, i) a heavy chain variable domain; and ii) a heavy chain constant domain containing a second Fc domain and a second monomer comprising a heavy chain comprising: c) a light chain comprising a variable light domain and a variable light constant domain; wherein the variable light chain domain has the sequence RASSSVSYIH (SEQ ID NO: XX). vlCDR1 having the sequence ATSNLAS (SEQ ID NO: XX), and vlCDR2 having the sequence QQWTSNPPT (SEQ ID NO: XX), and The main one is vhCDR1 with the sequence SYNMH (SEQ ID NO: XX), the sequence AIYPGNG vhCDR2 with the sequence DTSYNQKFQG (SEQ ID NO: XX), and the sequence STYYGG A heterodimeric antibody comprising a vhCDR3 having DWYFNV (SEQ ID NO: XX).

[65] a) a first monomer, i) a first Fc domain; ii) an scFv variable light chain domain, an scFv linker, and an scFv variable heavy chain domain and an anti-CD31 antibody covalently linked to the N-terminus of the Fc domain using a domain linker. 3 scFv a first monomer comprising: b) a second monomer, i) a heavy chain variable domain; and ii) a heavy chain constant domain containing a second Fc domain and a second monomer comprising a heavy chain comprising: c) a light chain comprising a variable light domain and a variable light constant domain; and the variable light chain domain comprises the sequence KSSQSLLNTGNQKNYLT (SEQ ID NO: XX), vlCDR1 having the sequence WASTRES (SEQ ID NO: XX) 2, and a vlCDR3 having the sequence QNDYSYPYT (SEQ ID NO: XX), and The variable heavy domain comprises vhCDR1 having the sequence DYYMK (SEQ ID NO: XX), vhCDR2 having DIIPSNGATFYNQKFKG (SEQ ID NO: XX), and A heterodimer comprising a vhCDR3 having the sequence SHLLRASWFAY (SEQ ID NO: XX) 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 , 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 , XENP16376 and XENP16377 A nucleic acid composition comprising three nucleic acids encoding an antibody.

[68] An expression vector composition comprising three expression vectors each containing a nucleic acid, As a result, the three expression vectors were designated XENP15049, XENP15051; 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 group consisting of:

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

[67] .

[70] A host cell comprising the expression vector composition described in

[68] .

[71]

[69] or

[70] , a method for producing the heterodimeric antibody according to

[66] . Culturing the host cell according to claim 1 under conditions in which the antibody is expressed; and recovering the antibody. and

[72] A method for treating cancer, comprising administering the heterodimeric antibody according to

[66] to a patient in need thereof. The method comprises administering to a patient.

Claims

1. a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker1-scFv-linker2-CH2-CH3, where VH1 is a first variable heavy chain domain, linker1 and linker2 are a first domain linker and a second domain linker, respectively, and CH2-CH3 is a first mutant human Fc domain; b) a second monomer comprising, in N-terminal to C-terminal order, VH1-CH1-hinge-CH2-CH3, where VH1 is a first variable heavy domain and CH2-CH3 is a second variant human Fc domain; and c) a common light chain comprising the first variable light domain (VL1) Including, wherein the first variable heavy chain domain and the variable light chain domain of the first monomer, and the first variable heavy chain domain and the variable light chain domain of the second monomer each form a first antigen-binding domain, and the scFv is a second antigen-binding domain; the scFv comprises a second variable heavy domain (VH2) linked to a second variable light domain (VL2) by an scFv linker; 1) the CH1-hinge-CH2-CH3 of the second monomer comprises SEQ ID NO:471, the linker2-CH2-CH3 of the first monomer comprises SEQ ID NO:472, and the common light chain comprises SEQ ID NO:473; or 2) the CH1-hinge-CH2-CH3 of the second monomer comprises SEQ ID NO:474, the linker2-CH2-CH3 of the first monomer comprises SEQ ID NO:475, and the common light chain comprises SEQ ID NO:476; or 3) the CH1-hinge-CH2-CH3 of the second monomer comprises SEQ ID NO:477, the linker2-CH2-CH3 of the first monomer comprises SEQ ID NO:478, and the common light chain comprises SEQ ID NO:479; or 4) the CH1-hinge-CH2-CH3 of the second monomer comprises SEQ ID NO: 480, the linker2-CH2-CH3 of the first monomer comprises SEQ ID NO: 481, and the common light chain comprises SEQ ID NO: 482; Heterodimeric antibodies.

2. The heterodimeric antibody described in claim 1, wherein the first mutant human Fc domain and the second mutant human Fc domain further comprise amino acid substitutions M428L and N434S, respectively.

3. A heterodimeric antibody described in claim 1 or 2, wherein the scFv is, in order from the N-terminus to the C-terminus, VH2-scFv linker-VL2.

4. A heterodimeric antibody described in claim 1 or 2, wherein the scFv is, in order from the N-terminus to the C-terminus, VL2-scFv linker-VH2.

5. a) a first nucleic acid encoding the first monomer according to any one of claims 1 to 4; b) a second nucleic acid encoding a second monomer according to any one of claims 1 to 4, and c) a third nucleic acid encoding a common light chain according to any one of claims 1 to 4 A nucleic acid composition comprising:

6. a) a first expression vector comprising a first nucleic acid encoding the first monomer according to any one of claims 1 to 4; b) a second expression vector comprising a second nucleic acid encoding a second monomer according to any one of claims 1 to 4; and c) a third expression vector comprising a third nucleic acid encoding the common light chain according to any one of claims 1 to 4. An expression vector composition comprising:

7. A host cell comprising the nucleic acid composition described in claim 5 or the expression vector composition described in claim 6.

8. A method for producing a heterodimeric antibody described in any one of claims 1 to 4, comprising culturing a host cell described in claim 7 under conditions in which the antibody is expressed, and recovering the antibody.

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